Details of the Researcher

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Villena Julio Cesar
Section
Graduate School of Agricultural Science
Job title
Professor

Research History 7

  • 2025 - Present
    Tohoku University　Laboratory of Respiratory Immunology (LaRI), Division of Animal Immunology and Omics, International Education and Research Center for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science　Distinguished Professor

  • 2023 - 2025
    National Council of Scientific and Technological Research　Reference Center for Lactobacilli　Principal Researcher

  • 2017 - 2023
    National Council of Scientific and Technological Research　Reference Center for Lactobacilli　Independent Researcher

  • 2015 - 2017
    National Council of Scientific and Technological Research　Reference Center for Lactobacilli　Adjunct Researcher

  • 2011 - 2015
    National Council of Scientific and Technological Research　Reference Center for Lactobacilli　Assistant Researcher

  • 2009 - 2011
    Tohoku University　JSPS Postdoctoral Fellowships for Research in Japan

  • 2008 - 2010
    CERELA - CONICET　CONICET Post-doctoral Fellowship

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Committee Memberships 1

  • Argentine Association of Microbiology　NOA subsidiary board of directors

    2018/05 - Present

Professional Memberships 5

  • Japan Lactic Acid Bacteria Society

    2025 - Present

  • Society of Mucosal Immunology

    2022 - Present

  • Argentina Microbiology Society

    2016 - Present

  • Latin America Societies of Immunology

    2008 - Present

  • Argentina Society of Immunology

    2008 - Present

Research Interests 6

  • Genomics

  • Immunity to infections

  • Probiotics and postbiotics

  • Lactic acid bacteria

  • Respiratory Microbiota

  • Respiratory Immunity

Research Areas 3

  • Life sciences / Genomics /

  • Life sciences / Immunology /

  • Life sciences / Applied microbiology /

Awards 13

  1. Poster Award (1st Place)

    2023/04　2nd International Hepatitis E Symposium　AN EXPERIMENTAL VACCINE FOR HEPATITIS E ADJUVANTATED WITHSAPONINS DERIVED FROM LEAVES FROM THE SOAP TREE QUILLAJABRASILIENSIS

  2. Best Oral Presentation Award

    2022/10　7th Argentine Symposium of Young Researchers in Bioinformatics　Genomic characterization of Lactiplantibacillus plantarum strains with and without antiviral immunomodulatory activity

  3. Argentinean Biochemist Association Award

    2022/05　Argentinean Biochemist Association　New mucosal vaccine against HSV-2 based on the LysM-BLP system as an antigen carrier and adjuvant

  4. Best Oral Presentation Award

    2016/10　V International Symposium on Lactic Acid Bacteria　Comparative immunotranscriptomic analysis of two immunobiotic strains with antiviral capabilities in porcine intestinal epithelial cells

  5. Best Poster Presentation Award

    2016/10　V International Symposium on Lactic Acid Bacteria　Isolation and characterization of Lactobacillus plantarum MPL16: A Wakame-utilizing immunobiotic strain from swine feces

  6. Award to the best communication

    2015/09　Japanese Dairy Science Association Symposium　Regulation of intestinal immune response by immunobiotics in virus infected mice model

  7. Best Poster presentation

    2015/08　III Congreso de Bioquímicos del Litoral, XVI Jornadas Argentinas de Microbiología　Immunomodulatory particles derived from lactic acid bacteria enhance the immune response induced by the oral rotavirus vaccine

  8. Probiotics for everyone! The novel immunobiotic Lactobacillus rhamnosus CRL1505 and the beginning of Social Probiotic Programs in Argentina

    2012/06　4th International Conference on Biotechnology for the Wellness Industry　Award to the best presented oral communication

  9. First Prize of the National Innovation Competition

    2009/10　INNOVAR 2009　Yogur probiótico: un proyecto solidario

  10. Best Poster presentation

    2006/10　II International Symposium on Lactic acid bacteria - First Argentinian LAB Net Meeting　Protective immune response against diseminated pneumococcal infection elicited by recombinant Lactococcus lactis expressing PppA protein

  11. Best Poster presentation

    2004/11　Joint Congress of Biomedical Societies　Influence of Lactobacillus casei on the recovery of hematopoietic disorders induced by malnutrition

  12. Best Student of the Faculty of Biochemestry, Chemestry and Pharmacy, Tucuman University

    2003　Faculty of Biochemestry, Chemestry and Pharmacy, Tucuman University

  13. AWARD for Research in Functional Foods and Nutraceutical Development

    Latin American Alliance for Responsible Nutrition　Immunomodulatory properties of γ-aminobutiric acid-enriched strawberry juice produced with Levilactobacillus brevis CRL2013

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Papers 201

  1. Selection and Characterization of Vaginal Lactobacilli from Healthy Algerian Women with Potential Probiotic Properties Against Urogenital Pathogens Peer-reviewed

    Asma Mammar, Hayat Trabsa, Stefania Dentice Maidana, Ammar Ayachi, Asma Abdessemed, Leonardo Albarracin, Mariano Elean, Kamel Boubakri, Ayelen A. Baillo, Haruki Kitazawa, Julio Villena

    Antibiotics　15:875　1-25　2026/09/07

    DOI： 10.3390/antibiotics15090875 　

  2. Postbiotics From Ligilactobacillus salivarius Enhance Mitochondrial Robustness Via TLR2 Signaling in Intestinal Macrophages. International-journal

    Nozomi Hariu, Fu Namai, Luciano Arellano-Arriagada, Takato Takenouchi, Hiroki Shinkai, Gong Weichen, Keita Nishiyama, Julio Villena, Hirohide Uenishi, Haruki Kitazawa

    Probiotics and antimicrobial proteins　2026/07/01

    DOI： 10.1007/s12602-026-11047-1 　

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    Postbiotics, defined as preparations of inanimate microorganisms and/or their components, have attracted increasing attention because of their stability and functional benefits. Although lactic acid bacteria (LAB) modulate immune responses and oxidative stress, their direct effects on host mitochondrial homeostasis remain poorly understood. In this study, we investigated whether postbiotics derived from Ligilactobacillus salivarius enhance mitochondrial robustness in porcine intestinal macrophages (IPIMs). To establish a model of disrupted mitochondrial redox homeostasis, mitochondrial dysfunction was induced using the mitochondrial complex III inhibitor Antimycin A (AMA). High-dose AMA induced mitochondrial reactive oxygen species (mtROS) accumulation and suppressed the expression of antioxidant genes, including SOD2. Pre-stimulation with heat-killed L. salivarius suppressed AMA-induced mtROS production in selected representative strains, with the most effective strains enhancing SOD2 expression. These mtROS-suppressive strains selectively activated Toll like receptor (TLR)1/2 and TLR2/6 signaling, whereas non-protective strains failed to induce TLR responsiveness. Pharmacological inhibition of TLR2 abolished mtROS suppression and SOD2 induction, confirming TLR2 dependency. Furthermore, Seahorse extracellular flux analysis revealed that postbiotics enhanced mitochondrial respiration, including maximal respiration and spare respiratory capacity, via TLR2 signaling. Collectively, our findings demonstrate that postbiotics function as immunometabolic modulators that reinforce mitochondrial redox homeostasis and respiratory capacity via TLR2 signaling, highlighting a novel mechanism by which microbial components contribute to intestinal immune cell homeostasis.

  3. Lactic Acid Bacteria-Derived Antimicrobial and Anti-Biofilm Strategies: Mechanisms, Functional Molecules, and Emerging Biomaterial Applications International-journal International-coauthorship Peer-reviewed

    Weichen Gong, Harum Fadhilatunnur, Miaya Kanazawa, Julio Villena, Keita Nishiyama, Haruki Kitazawa

    International Journal of Molecular Sciences　27:5749　1-20　2026/06/25

    Publisher: MDPI

    DOI： 10.3390/ijms27135749 　

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    IF = 5.6

  4. Encapsulation in Calcium Alginate Beads Regulates Growth, Release and Viability of Probiotic Bacteria Through Protective Microenvironments

    Weichen Gong, Luciano Arellano-Arriagada, Julio Villena, Keita Nishiyama, Haruki Kitazawa

    Gels　12　(6)　518-518　2026/06/10

    Publisher: MDPI AG

    DOI： 10.3390/gels12060518 　

    eISSN： 2310-2861

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    Aims: Our aim was to evaluate calcium alginate beads as a physical cultivation platform for probiotic bacteria and investigate their effects on bacterial growth localization, release dynamics, and viability under different environmental conditions. Methods and Results: Calcium alginate beads were fabricated using sodium alginate at different concentrations (1.0–2.0%, w/v), and their structural stability, permeability, and pH resistance were assessed. Lacticaseibacillus paracasei was used as the primary model organism. Among the tested formulations, 2.0% alginate exhibited the lowest permeability (~40%) compared with 1.0% and 1.5% alginate (~60%) and maintained structural integrity under alkaline conditions (pH 11–12). Encapsulated L. paracasei proliferated predominantly within the bead matrix, while less than 1% of total bacteria were released into the surrounding medium during the first 12 h and approximately 8% released after 24 h. Encapsulation also maintained higher bacterial viability over 24–48 h compared with conventional liquid culture conditions. Encapsulated cells could be efficiently recovered using sodium citrate without marked loss of viability. In addition, encapsulation enabled the growth of Bifidobacterium animalis subsp. lactis under aerobic incubation conditions, suggesting the formation of locally protective microenvironments within the gel matrix. Conclusions: Calcium alginate beads function as controllable microenvironmental regulators that influence bacterial growth, release behavior, and long-term viability. These findings suggest that alginate-based encapsulation systems may provide a simple and scalable platform for controlled microbial cultivation and probiotic-related applications.

  5. Role of C17 fengycin B from Bacillus subtilis DB9011 in enterotoxigenic Escherichia coli inhibition. International-journal

    Yuki Indo, Kosuke Shimizu, Ayane Sato, Fu Namai, Weichen Gong, Julio Villena, Chie Hikita, Keita Nishiyama, Haruki Kitazawa

    Applied and environmental microbiology　e0027626　2026/06/04

    DOI： 10.1128/aem.00276-26 　

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    Enterotoxigenic Escherichia coli (ETEC) is a leading cause of post-weaning diarrhea in piglets, and the rise of antimicrobial resistance underscores the urgent need for antibiotic alternatives in swine production. Bacillus species are promising candidates owing to their ability to produce antimicrobial secondary metabolites, particularly cyclic lipopeptides such as surfactins, iturins, and fengycins. Although Bacillus lipopeptides are known to exhibit antibacterial activity against E. coli, the specific compounds responsible for the inhibition of ETEC in probiotic-relevant Bacillus strains remain undefined. Here, we sought to identify extracellular metabolite(s) associated with the anti-ETEC activity of Bacillus subtilis DB9011. DB9011 culture supernatant strongly inhibited the growth of ETEC and other enteric pathogens, including Salmonella Typhimurium. Bioactivity-guided fractionation of the culture supernatant, followed by liquid chromatography-quadrupole time-of-flight mass spectrometry, identified C17 fengycin B as a major active metabolite enriched in inhibitory fractions, but absent from the non-inhibitory type strain NBRC13719ᵀ. A fengycin-containing fraction enriched in C17 fengycin B exhibited concentration-dependent antibacterial activity against ETEC and enhanced growth inhibition when combined with colistin, whereas incomplete inhibition at native supernatant-equivalent concentrations indicated that additional metabolites contribute to the overall antibacterial phenotype. While fengycins have been primarily recognized for antifungal activity and, more recently, for effects on gram-negative bacteria, our findings identify C17 fengycin B as a major contributor to the ETEC-inhibitory phenotype of B. subtilis DB9011. These findings provide mechanistic insights into how probiotic-relevant Bacillus strains inhibit enteric pathogens; nonetheless, further studies are required to evaluate in vivo relevance and functional performance under intestinal conditions.IMPORTANCEAntimicrobial resistance poses a major threat to livestock production, where antibiotics remain widely used to control post-weaning diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Supplementation with Bacillus subtilis DB9011 is known to reduce pathogenic E. coli colonization in piglets; however, the metabolites responsible for this protective effect remain unidentified. Here, using a bioactivity-guided fractionation approach, we identify C17 fengycin B as a major contributing metabolite associated with the anti-ETEC activity of DB9011 and show enhanced growth inhibition during co-treatment with colistin. These findings clarify the functional contribution of Bacillus-derived metabolites to pathogen inhibition and establish a mechanistic basis for strain-specific antimicrobial activity.

  6. Randomized, Double-Blind, Placebo-Controlled Clinical Trial Assessing the Efficacy of Lacticaseibacillus rhamnosus CRL 1505 in Preventing Upper Respiratory Tract Infections in Healthy Adults

    Valentina Taverniti, Ines Martinez, Beatrice Tavazzani, Carlos Baeza-Martínez, Francisco López-Garcia, Carmen Carazo-Díaz, Juan Aguera Santos, Julio Villena, Susana Salva, María Pía Taranto, Susana Álvarez, Graciela Font, Vicente Navarro-López

    Microorganisms　2026/06/04

    DOI： 10.3390/microorganisms14061270 　

  7. Live and heat-treated Lactiplantibacillus plantarum induce distinct metabolic and immune responses in intestinal epithelial cells

    Kaho Matsumoto, Yuta Takada, Yoshiya Imamura, Hina Yoshida, Kazuhiro Sonomura, Mikako Takahashi, Nobuko Moritoki, Tomoko Shindo, Junya Yamamoto, Leonardo Albarracin, Wakako Ikeda-Ohtsubo, Masatoshi Hori, Julio Villena, Fu Namai, Yuji Tsujikawa, Toyoyuki Hashimoto, Keita Nishiyama, Haruki Kitazawa

    iScience　29　(5)　115516-115516　2026/05

    Publisher: Elsevier BV

    DOI： 10.1016/j.isci.2026.115516 　

    ISSN： 2589-0042

  8. Lacticaseibacillus rhamnosus CRL1505 Ameliorates Liver Injury and Inflammation in Poly(I:C)-Induced Acute Hepatitis Peer-reviewed

    María José Lorenzo Pisarello, Ayelen Antonella Baillo, Mariano Elean, Leonardo Albarracín, Luciano Arellano-Arriagada, Yoshihito Suda, Haruki Kitazawa, Julio Villena

    Foods　15　(6)　1034-1034　2026/03/16

    Publisher: MDPI AG

    DOI： 10.3390/foods15061034 　

    eISSN： 2304-8158

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    Lacticaseibacillus rhamnosus CRL1505 enhances antiviral immunity at mucosal sites, but its capacity to modulate liver immune responses remains unclear. Therefore, this study evaluated whether this immunomodulatory bacterium protects against Toll-like receptor 3 (TLR3)-mediated acute hepatitis induced by poly(I:C), and whether this effect depends on mucosal adhesion. BALB/c mice received the wild-type CRL1505 strain or the Δmbf CRL1505 mutant lacking the mucus-binding factor gene prior to poly(I:C) challenge. Liver injury, serum transaminases, and hepatic expression of interferons (IFNs), antiviral factors, inflammatory mediators, and regulatory cytokines were evaluated 48 h later. Poly(I:C) challenge induced acute hepatitis characterized by increased ALT/AST levels, leukocyte infiltration, and elevated hepatic IFNs and proinflammatory cytokines. The CRL1505 strain administration significantly reduced TNF-α, IL-1β, and IL-6 while enhancing IFNs, antiviral factors, and the regulatory cytokines IL-10 and IL-27, resulting in improved transaminase levels and attenuated liver damage. Notably, the Δmbf CRL1505 mutant conferred protection comparable to the wild-type strain. These findings demonstrate that L. rhamnosus CRL1505 exerts immunomodulatory and hepatoprotective effects during TLR3-driven hepatitis and that mbf-mediated adhesion is not required for this protection. Overall, CRL1505 emerges as a promising preventive strategy to enhance antiviral defenses and limit inflammation-associated liver injury.

  9. Orally administered porcine intestinal lactobacilli improve the respiratory innate immune response against Streptococcus pneumoniae

    Kohtaro Fukuyama, Solange Cisterna-Vergara, Ayelen Antonella Baillo, María José Lorenzo Pisarello, Weichen Gong, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Animals　16　(5)　825-825　2026/03/06

    Publisher: MDPI AG

    DOI： 10.3390/ani16050825 　

    eISSN： 2076-2615

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    Background: Respiratory bacterial infections represent a major health challenge in swine production, highlighting the need for novel immunomodulatory strategies that enhance host resistance. In this study, we investigated whether porcine intestinal lactobacilli could modulate the gut–lung axis and improve respiratory innate immunity in a mouse model of Streptococcus pneumoniae infection, as a surrogate of Streptococcus suis pneumonia. Methods: Three strains of Ligilactobacillus salivarius (LAFF998, LAFF1071, and LAFF1095) were orally administered to Swiss mice prior to pneumococcal challenge. The resistance to the infection, the lung damage and the respiratory innate immune response were evaluated. Results: Only strain LAFF998 significantly reduced pulmonary bacterial loads, prevented bacteremia, and attenuated lung injury. This protective effect was associated with selective modulation of respiratory immunity, characterized by reduced neutrophilic inflammation, increased lymphocyte recruitment, and enhanced activation of alveolar macrophages expressing MHC-II. LAFF998 markedly increased the production of IFN-β, IFN-γ, IL-6, IL-10, and IL-27 in the respiratory tract, without inducing excessive inflammatory damage. Ex vivo and in vitro analyses confirmed that alveolar macrophages from LAFF998-treated mice exhibited a primed phenotype with heightened cytokine responses to pneumococcal stimulation. In contrast, strains LAFF1071 and LAFF1095 failed to confer protection or significantly modulate respiratory immune responses. Conclusions: These findings demonstrate a strict strain-dependent effect among porcine L. salivarius isolates and identify LAFF998 as a potent immunobiotic capable of enhancing respiratory innate immunity through the gut–lung axis. This work supports further studies of LAFF998 as an immunobiotic strategy for the prevention of respiratory infections in pigs.

  10. Pathogen Evasion of Humoral Innate Immunity: Coping with C-Reactive Protein and Serum Amyloid A Peer-reviewed

    Weichen Gong, Xuefei Cheng, Julio Villena, Haruki Kitazawa

    International Journal of Molecular Sciences　27　(2)　1072-1072　2026/01/21

    Publisher: MDPI AG

    DOI： 10.3390/ijms27021072 　

    eISSN： 1422-0067

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    C-reactive protein (CRP) and serum amyloid A (SAA) are classical acute-phase proteins that exemplify humoral innate immunity, the soluble arm of the host’s first-line defense. Beyond their traditional use as biomarkers of inflammation, both proteins function as active effectors against pathogens by binding microbial components, activating complements, and modulating inflammation. However, bacteria, viruses, and fungi have co-evolved diverse mechanisms to cope with or evade these host defenses. This review aims to summarize the current understanding of CRP and SAA as soluble innate immune effectors and to highlight pathogen strategies to counteract their antimicrobial pressure. We systematically surveyed and summarized evidence from experimental and clinical studies describing “function of CRP and SAA during infection”, “CRP and SAA in innate immune defense”, and “evasion mechanisms across bacterial, viral, and fungal pathogens”. CRP and SAA are rapidly upregulated in response to infection and contribute to pathogen recognition, opsonization, and inflammation. Pathogens, however, employ multiple coping strategies, including surface modification to block CRP binding, proteolytic degradation of acute-phase proteins, shielding within biofilms, and subversion of host signaling. These countermeasures enable microbes to reduce immune clearance and promote persistence. CRP and SAA represent central elements of humoral innate immunity, shaping the outcome of host–pathogen interactions. Pathogen adaptations to these proteins illustrate an ongoing evolutionary arms race between host defense and microbial survival. A deeper understanding of these processes may open avenues for novel therapeutic approaches, such as targeting microbial evasion factors or enhancing host acute-phase responses.

  11. Secretome Profiling of Lactiplantibacillus plantarum CRL681 Predicts Potential Molecular Mechanisms Involved in the Antimicrobial Activity Against Escherichia coli O157:H7 Peer-reviewed

    Ayelen Antonella Baillo, Leonardo Albarracín, Eliana Heredia Ojeda, Mariano Elean, Weichen Gong, Haruki Kitazawa, Julio Villena, Silvina Fadda

    Antibiotics　15　(1)　96-96　2026/01/17

    Publisher: MDPI AG

    DOI： 10.3390/antibiotics15010096 　

    eISSN： 2079-6382

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    Background/Objectives. Lactiplantibacillus plantarum CRL681 has previously demonstrated a strong antagonistic effect against Escherichia coli O157:H7 in food matrices; however, the molecular mechanisms underlying this activity remain poorly understood. Since initial interactions between beneficial bacteria and pathogens occur mainly at the cell surface and in the extracellular environment, the characterization of the bacterial secretome is essential for elucidating these mechanisms. In this study, the secretome of L. plantarum CRL681 was comprehensively characterized using an integrated in silico and in vitro approach. Methods. The exoproteome and surfaceome were analyzed by LC-MS/MS under pure culture conditions and during co-culture with E. coli O157:H7. Identified proteins were functionally annotated, classified according to subcellular localization and secretion pathways, and evaluated through protein–protein interaction network analysis. Results. A total of 275 proteins were proposed as components of the CRL681 secretome, including proteins involved in cell surface remodeling, metabolism and nutrient transport, stress response, adhesion, and genetic information processing. Co-culture with EHEC induced significant changes in the expression of proteins associated with energy metabolism, transport systems, and redox homeostasis, indicating a metabolic and physiological adaptation of L. plantarum CRL681 under competitive conditions. Notably, several peptidoglycan hydrolases, ribosomal proteins with reported antimicrobial activity, and moonlighting proteins related to adhesion were identified. Conclusions. Overall, these findings suggest that the antagonistic activity of L. plantarum CRL681 against E. coli O157:H7 would be mediated by synergistic mechanisms involving metabolic adaptation, stress resistance, surface adhesion, and the production of non-bacteriocin antimicrobial proteins, supporting its potential application as a bioprotective and functional probiotic strain.

  12. Lactiplantibacillus plantarum CRL1506 ameliorates poly(I:C)-induced acute liver injury through cell wall-dependent modulation of inflammatory and antiviral immune responses

    María José Lorenzo Pisarello, Ayelen Antonella Baillo, Mariano Elean, Leonardo Albarracín, Luciano Arellano-Arriagada, Yoshihito Suda, Haruki Kitazawa, Julio Villena

    Food & Function　2026

    DOI： 10.1039/D6FO01160H 　

  13. eDNA–Amyloid Synergistic Interactions in Bacterial Biofilms: A Hidden Driver of Antimicrobial Resistance Peer-reviewed

    Weichen Gong, Xuefei Cheng, Julio Villena, Haruki Kitazawa

    International Journal of Molecular Sciences　26　(24)　12075-12075　2025/12/15

    Publisher: MDPI AG

    DOI： 10.3390/ijms262412075 　

    eISSN： 1422-0067

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    Bacterial biofilms are critical contributors to chronic infections and antimicrobial resistance. Among the diverse extracellular matrix components, extracellular DNA (eDNA) and amyloid proteins have recently emerged as pivotal structural and functional molecules. Both individually contribute to biofilm stability and antibiotic tolerance, yet their cooperative roles remain underappreciated. This review aims to summarize current knowledge on the origins and functions of eDNA and amyloid proteins in biofilms, to highlight their molecular interactions, and to discuss how their synergistic effects promote biofilm-mediated resistance to antimicrobial agents. A comprehensive literature search was conducted using PubMed, Scopus, and Web of Science databases up to September 2025. Keywords included “biofilm”, “extracellular DNA”, “amyloid proteins”, “matrix”, and “antimicrobial resistance”. Relevant original research and review articles were systematically screened and critically analyzed to integrate emerging evidence on eDNA–amyloid interactions in bacterial biofilms. Current studies demonstrate that eDNA originates primarily from autolysis, active secretion, and host-derived DNA, while amyloid proteins are produced by multiple bacterial species, including Escherichia coli (curli), Pseudomonas aeruginosa (Fap), Bacillus subtilis (TasA), and Staphylococcus aureus (phenol-soluble modulins). Both molecules independently strengthen biofilm integrity and provide protective functions against antimicrobial agents. Importantly, recent evidence shows that eDNA can act as a nucleation template for amyloid fibrillation, while amyloid fibers stabilize and protect eDNA from degradation, creating a dense extracellular network. This synergistic eDNA–amyloid assembly enhances biofilm robustness, impedes antibiotic penetration, sequesters antimicrobial peptides, protects persister cells, and facilitates horizontal gene transfer of resistance determinants. The interplay between eDNA and amyloid proteins represents a central but underexplored mechanism driving biofilm-mediated antimicrobial resistance. Understanding this cooperative network not only deepens our mechanistic insights into bacterial pathogenesis but also highlights novel therapeutic targets. Strategies that disrupt eDNA–amyloid interactions may offer promising avenues for combating persistent biofilm-associated infections.

  14. Combined Lactiplantibacillus plantarum CRL1506 and MPL16 Nasal Priming More Effectively Modulates Respiratory Antiviral Innate Immunity than Single Strains Peer-reviewed

    Luciano Arellano-Arriagada, Leonardo Albarracin, Kohtaro Fukuyama, Solange Cisterna-Vergara, Weichen Gong, Fu Namai, Keita Nishiyama, Yoshihito Suda, Haruki Kitazawa, Julio Villena

    International Journal of Molecular Sciences　26　(20)　10079-10079　2025/10/16

    Publisher: MDPI AG

    DOI： 10.3390/ijms262010079 　

    eISSN： 1422-0067

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    This study evaluated whether the combined Lactiplantibacillus plantarum CRL1506 and MPL16 nasal priming more effectively modulated the Toll-like receptor (TLR)-3- and respiratory syncytial virus (RSV)-mediated respiratory immune responses in mice than single strains. The interaction of single and combined strains with porcine alveolar macrophages (AMs) and porcine respiratory epithelial cells (PBE cells) in the context of TLR3 activation and porcine reproductive and respiratory syndrome virus (PRRSV) was also evaluated. The in vivo studies in mice revealed that the CRL1506 + MPL16 combination was more effective than the individual strains at reducing RSV replication, protecting the lung from TLR3-mediated inflammatory injury and modulating innate antiviral responses, particularly in AMs. In vitro, lactobacilli treatment also increased the resistance of porcine AMs to PRRSV infection. Notably, the CRL1506 + MPL16 combination was not more effective than the single strains in modulating AMs antiviral immunity. Complementary assays in PBE cells revealed that L. plantarum CRL1506 induced higher production of IFN-λ than the MPL16 strain in response to TLR3 activation. Thus, the superior in vivo protection against RSV seen with the L. plantarum CRL1506–MPL16 combination likely reflects complementary actions of lactobacilli: MPL16 would efficiently modulate AMs, whereas CRL1506 would be more effective to target respiratory epithelial cells driving greater IFN-λ production that further boosts AM antiviral activity. The results from the animal models of this work furnish the scientific basis for proposing future human trials to assess the efficacy of the CRL1506 + MPL16 combination in improving respiratory antiviral immunity.

  15. Role of the Choline‐Binding Protein A in the Ability of Porcine Ligilactobacillus salivarius FFIG58 to Modulate TLR3‐Mediated Intestinal Antiviral Immunity Peer-reviewed

    Yoshiya Imamura, Hikari Yamamuro, Mariano Elean, Lorena Paola Arce, Ryutaro Funabashi, Fernanda Raya Tonetti, Kohtaro Fukuyama, Mikado Tomokiyo, Fu Namai, Sudeb Saha, Wakako Ikeda‐Ohtsubo, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Food Frontiers　7　(1)　2025/09/25

    Publisher: Wiley

    DOI： 10.1002/fft2.70027 　

    ISSN： 2643-8429

    eISSN： 2643-8429

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    ABSTRACT The choline‐binding protein A (CbpA) was shown to mediate adhesion of probiotic Ligilactobacillus salivarius strains to the human intestinal mucosa, but studies of this protein in porcine strains were not performed before. Previously, we demonstrated that the pig‐derived L. salivarius FFIG58 beneficially modulates the Toll‐like receptor (TLR)‐3‐triggered immune responses in porcine intestinal epithelial cells (IECs) and in the gut of mice, improving antiviral immunity and reducing the intestinal inflammatory damage. In this work, we advanced in the characterization of the immunomodulatory properties of L. salivarius FFIG58 by developing a surface‐layer protein CbpA mutant (Δ cbpA ). Both wild‐type and Δ cbpA strains were evaluated in terms of their interactions with porcine IECs and macrophages in the context of TLR3‐mediated inflammation. Additionally, the immunomodulatory activities of FFIG58 and Δ cbpA strains were assessed in vivo using infant mice as a young host model considering the potential application of this bacterium to improve the resistance to postweaning viral diarrhea in piglets. The deletion of the CbpA protein reduced the ability of FFIG58 to modulate the expression of interferons (IFNs), antiviral factors, regulatory cytokines and negative regulators of the TLR signaling as well as IFN regulatory factor 3 (IRF3) and ERK activation in porcine IECs and macrophages. The Δ cbpA mutant had a diminished capacity to protect porcine IECs against rotavirus challenge compared to the FFIG58. The ability of L. salivarius FFIG58 to differentially regulate antiviral and inflammatory factors expressions, the numbers of CD8aa + NKG2D + intraepithelial lymphocytes and the TLR3‐IL‐15‐mediated damage in the intestinal mucosa of mice was also altered with the deletion of CbpA . These results allow us to conclude that the CbpA protein is a key molecule for the beneficial functions of the L. salivarius species in the immune system of distinct hosts, and particularly for L. salivarius FFIG58 in pigs. This is a step forward in the understanding of the cellular mechanisms involved in the antiviral capabilities of L. salivarius FFIG58.

  16. Effect of the Food Matrix on the Survival to the Gastrointestinal Transit of Lacticaseibacillus rhamnosus CRL1505: A Randomized, Controlled, Crossover Study Peer-reviewed

    Nicola Mangieri, Viola Termine, Giorgio Gargari, Nicolò Fornasari, Michele Isotti, Susana Salva, Julio Villena, María Pía Taranto, Valentina Taverniti, Susana Álvarez, Graciela Font, Ylenia Zanchetta, Stefania Arioli, Diego Mora

    Molecular Nutrition & Food Research　69　(23)　2025/09/20

    Publisher: Wiley

    DOI： 10.1002/mnfr.70272 　

    ISSN： 1613-4125

    eISSN： 1613-4133

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    ABSTRACT Probiotic survival is crucial for their beneficial effect, but only a few studies have assessed the impact of delivery format on probiotic viability after passage through the gastrointestinal tract.Our study aimed to investigate the survival of Lacticaseibacillus rhamnosus ( L. rhamnosus ) CRL1505 through the gastrointestinal transit in healthy volunteers after a daily consumption of at least 1 billion CFUs per day administered as freeze‐dried cells or incorporated into oat‐ and milk‐fermented drinks. The probiotic's survival was evaluated by combining culture‐based and molecular techniques. Our findings revealed that L. rhamnosus CRL1505 survival was significantly higher when the probiotic was administered as an oat‐ and as a milk‐fermented drink. The study highlighted the persistence of CRL1505 in a subset of volunteers 1 week after the last administration. The metataxonomic analysis of fecal samples did not reveal significant changes in microbiota diversity among treatment groups, as expected after probiotic administration in healthy subjects.The survival of CRL1505 through the human gastrointestinal tract was demonstrated with and without the protection of food matrices, highlighting the strain's remarkable ability to withstand harsh conditions. Our findings emphasize the importance of food matrix selection for improving probiotic viability and its potential efficacy in functional foods.

  17. Genomic Characterization of the Honeybee–Probiotic Strain Ligilactobacillus salivarius A3iob Peer-reviewed

    Mariano Elean, Alejandro Arroyo Guerra, Leonardo Albarracin, Keita Nishiyama, Haruki Kitazawa, M. Carina Audisio, Julio Villena

    Animals　15　(17)　2606　2025/09/05

    DOI： 10.3390/ani15172606 　

  18. Canine Microencapsulated Probiotic Formulations Modulate Immunity and Improve Acute Diarrhea in Dogs Peer-reviewed

    Carolina Muñoz-Flores, Felipe Sandoval, Paula Buldres, Miguel A. Gutierrez-Reinoso, Ignacio Cabezas, Carlos Riquelme, Luis Gutiérrez, Manuel García-Herreros, Diego X. Medina-Valarezo, Julio Villena, Sandra R. Quilodrán-Vega, Jorge R. Toledo

    Probiotics and Antimicrobial Proteins　2025/08/09

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1007/s12602-025-10695-z 　

    ISSN： 1867-1306

    eISSN： 1867-1314

  19. Characterisation of the cell envelope and adhesive properties of Lactobacillus johnsonii CRL1647, a probiotic from the honeybee gut Peer-reviewed

    M. Novicov-Fanciotti, S. Dentice Maidana, M. Elean, J. Villena, M.C. Audisio

    Beneficial Microbes　17　(2)　179-189　2025/07/31

    DOI： 10.1163/18762891-bja00087 　

  20. The Originally Established PBE Cell Line as a Reliable In Vitro Model for Investigating SIV Infection and Immunity Peer-reviewed

    Xi-Chen Bai, Kohtaro Fukuyama, Leonardo Albarracin, Yoshiya Imamura, Fu Namai, Weichen Gong, Wakako Ikeda-Ohtsubo, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    International Journal of Molecular Sciences　26　(12)　5764　2025/06/16

    DOI： 10.3390/ijms26125764 　

  21. Milk sialyl-oligosaccharides mediate the early colonization of gut commensal microbes in piglets Peer-reviewed

    Ryoga Hashimoto, Keita Nishiyama, Fu Namai, Kasumi Suzuki, Taiga Sakuma, Itsuko Fukuda, Yuta Sugiyama, Kenji Okano, Takafumi Shanoh, Eita Toyoshi, Ryusuke Ohgi, Sudeb Saha, Sae Tsuchida, Eri Nishiyama, Takao Mukai, Mutsumi Furukawa, Tomonori Nochi, Julio Villena, Wakako Ikeda-Ohtsubo, Gou Yoshioka, Eri Nakazaki, Yoshihito Suda, Haruki Kitazawa

    Microbiome　13　(1)　2025/05/24

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1186/s40168-025-02129-3 　

    eISSN： 2049-2618

  22. Enhancing Resistance to Enterococcus faecalis: Immunobiotic Lactiplantibacillus plantarum Strains as a Strategy for Malnourished Hosts Peer-reviewed

    María Daniela Vera, Lorena Paola Arce, Melisa Florencia Müller, Fernanda Raya Tonetti, Ramiro Ortiz Moyano, Héctor Luis Blanco, Haruki Kitazawa, María Guadalupe Vizoso-Pinto, Julio Villena

    Nutrients　17　(11)　1770　2025/05/23

    DOI： 10.3390/nu17111770 　

  23. Modulation of Macrophages TLR4-Mediated Transcriptional Response by Lacticaseibacillus rhamnosus CRL1505 and Lactiplantibacillus plantarum CRL1506 Peer-reviewed

    Masahiko Suzuki, Ayelen Baillo, Leonardo Albarracin, Mariano Elean, Rodrigo Serda, Yoshihito Suda, Fu Namai, Keita Nishiyama, Haruki Kitazawa, Julio Villena

    International Journal of Molecular Sciences　26　(6)　2688　2025/03/17

    DOI： 10.3390/ijms26062688 　

  24. An experimental chimeric hepatitis E virus vaccine elicits both local and systemic immune responses Peer-reviewed

    Melisa Florencia Müller, Jacinto Sacur, Julia Matias Brancher, María Daniela Vera, Lorena Arce, María Fernanda Raya-Tonetti, Haruki Kitazawa, Julio Villena, María Guadalupe Vizoso-Pinto

    Frontiers in Microbiology　15　(1512018)　2024/12/24

    Publisher: Frontiers Media SA

    DOI： 10.3389/fmicb.2024.1512018 　

    eISSN： 1664-302X

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    Introduction The development of a hepatitis E virus (HEV) vaccine is critical, with ORF2 capsid protein as the main target. We previously demonstrated that oral coadministration of recombinant ORF2 with immunomodulatory bacterium-like-particles (IBLP) induces a specific immune response in mice, particularly using IBLP derived from Lacticaseibacillus rhamnosus IBL027 (IBLP027), which was effective in eliciting a local humoral response. IBLP are non-live bacteria with adjuvant and carrier properties, serving as a platform for exposing proteins or antigens fused to LysM (lysine motif) domains, protein modules that bind to cell wall polysaccharides like peptidoglycan. Materials We cloned the most immunogenic domain of ORF2 (O2P2) fused to five LysM domains (LysM5O2P2) and displayed this chimeric protein on the surface of IBLP027 to create a prototype vaccine (IBLP027-LysM5O2P2). We evaluated its capacity to induce an immune response in vivo by immunizing mice with three doses of either the experimental vaccine or the chimeric protein alone, using an oral or a combined schedule with subcutaneous priming followed by oral boosting. Control groups received IBLP027. Sera and small intestine fluid were analyzed for humoral response, while Peyer’s patches and spleen immune cells were used for ex vivo stimulation with capsid protein to assess cellular response. Results The oral scheme failed to elicit an IgG response, but this was overcome by a subcutaneous priming dose followed by oral boosters, which led to increasing IgG titers in the combined scheme. The highest IgG titers were seen in the vaccine prototype group. Most groups produced significantly higher IgA levels in intestinal fluid, especially in those that received the oral scheme. Cellular response studies showed increased tumor necrosis factor (TNF)-α, interferon (IFN)-γ interleukin (IL)-4, and IL-17 levels in groups receiving the chimeric protein via oral or combined schedules. Conclusion Further and continuous research is needed to better understand both the needs and expectations of students and supervisors in different academic realities, including in Veterinary Medicine schools, from which the information available on the subject is scarce.

  25. Orally Administered Lactobacilli Strains Modulate Alveolar Macrophages and Improve Protection Against Respiratory Superinfection Peer-reviewed

    Leonardo Albarracin, Stefania Dentice Maidana, Kohtaro Fukuyama, Mariano Elean, Julio Nicolás Argañaraz Aybar, Yoshihito Suda, Keita Nishiyama, Haruki Kitazawa, Julio Villena

    Biomolecules　14　(12)　1600　2024/12/14

    DOI： 10.3390/biom14121600 　

  26. Modulation of the Gut–Lung Axis by Water Kefir and Kefiran and Their Impact on Toll-like Receptor 3-Mediated Respiratory Immunity Peer-reviewed

    Stefania Dentice Maidana, Julio Nicolás Argañaraz Aybar, Leonardo Albarracin, Yoshiya Imamura, Luciano Arellano-Arriagada, Fu Namai, Yoshihito Suda, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Biomolecules　14　(11)　1457　2024/11/17

    DOI： 10.3390/biom14111457 　

  27. Effect of Lactiplantibacillus plantarum cell-free culture on bacterial pathogens isolated from cystic fibrosis patients: in vitro and in vivo studies Peer-reviewed

    Carla Luciana Abán, Silvia Orosco, Julio Nicolás Argañaraz Aybar, Leonardo Albarracín, Analía Venecia, Liliana Perret, Sonia Ortiz Mayor, Keita Nishiyama, Juan Carlos Valdéz, Haruki Kitazawa, Julio Villena, Nadia Gobbato

    Frontiers in Microbiology　15　(1440090)　2024/09/16

    Publisher: Frontiers Media SA

    DOI： 10.3389/fmicb.2024.1440090 　

    eISSN： 1664-302X

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    This study aimed to investigate the effects of the cell-free supernatant of Lactiplantibacillus plantarum ATCC® 10241TM on the biofilm-forming capacity of Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients. In addition, the study evaluated the in vivo potential of the cell-free supernatant to modulate inflammation and reduce lung damage in mice infected with P. aeruginosa strains or co-challenged with P. aeruginosa and the Streptococcus milleri group (SMG). The results showed that CF-derived P. aeruginosa strains can infect the respiratory tract of adult mice, inducing local inflammation and lung damage. The severity of these infections was exacerbated when P. aeruginosa was co-administered with SMG. Notably, nebulization with the cell-free supernatant of L. plantarum produced beneficial effects, reducing respiratory infection severity and inflammatory responses induced by P. aeruginosa, both alone or in combination with SMG. Reduced bacterial loads and lung damage were observed in supernatant-treated mice compared to controls. Although further mechanistic studies are necessary, the results show that the cell-free supernatant of L. plantarum ATCC® 10241TM is an interesting adjuvant alternative to treat P. aeruginosa respiratory infections and superinfections in CF patients.

  28. Role of immunomodulatory probiotics in alleviating bacterial diarrhea in piglets: a systematic review Peer-reviewed

    Sudeb Saha, Fu Namai, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Journal of Animal Science and Biotechnology　15　(112)　2024/08/12

    DOI： 10.1186/s40104-024-01070-z 　

  29. Modulation of the Toll-like Receptor 3-Mediated Intestinal Immune Response by Water Kefir Peer-reviewed

    Stefania Dentice Maidana, Ramiro Ortiz Moyano, Mariano Elean, Yoshiya Imamura, Leonardo Albarracín, Fu Namai, Yoshihito Suda, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Microbiology Research　15　(3)　1239-1250　2024/07/20

    DOI： 10.3390/microbiolres15030083 　

  30. Antagonistic Effects of Corynebacterium pseudodiphtheriticum 090104 on Respiratory Pathogens Peer-reviewed

    Ramiro Ortiz Moyano, Stefania Dentice Maidana, Yoshiya Imamura, Mariano Elean, Fu Namai, Yoshihito Suda, Keita Nishiyama, Vyacheslav Melnikov, Haruki Kitazawa, Julio Villena

    Microorganisms　12　(7)　1295　2024/06/26

    DOI： 10.3390/microorganisms12071295 　

  31. Immunobiotic Bacteria Attenuate Hepatic Fibrosis through the Modulation of Gut Microbiota and the Activation of Aryl‐Hydrocarbon Receptors Pathway in Non‐Alcoholic Steatohepatitis Mice Peer-reviewed

    Paulraj Kanmani, Julio Villena, Soo‐kyoung Lim, Eun‐Ji Song, Young‐Do Nam, Hojun Kim

    Molecular Nutrition & Food Research　68　(14)　2024/06/20

    Publisher: Wiley

    DOI： 10.1002/mnfr.202400227 　

    ISSN： 1613-4125

    eISSN： 1613-4133

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    Scope Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease worldwide that can progress to liver fibrosis (LF). Probiotics have beneficial roles in reducing intestinal inflammation and gut‐associated diseases, but their effects and mechanisms beyond the gut in attenuating the progression of LF are remained unclear. Methods and results In a mouse model of NASH/LF induced by a methionine‐choline deficient (MCD) diet, immunobiotics are administered to investigate their therapeutic effects. Results show that the MCD diet leads to liver inflammation, steatosis, and fibrosis, which are alleviated by immunobiotics. Immunobiotics reduces serum endotoxin and inflammatory markers while increasing regulatory cytokines and liver weight. They also suppress Th17 cells, known for producing inflammatory cytokines. Furthermore, immunobiotics mitigate collagen deposition and fibrogenic signaling in the liver, while restoring gut‐barrier integrity and microbiota composition. Additionally, immunobiotics enhance the activation of the aryl hydrocarbon receptor (AhR) pathway in both colonic and liver tissues. Conclusions Overall, these results demonstrate a novel insight into the mechanisms through which immunobiotic administration improves the gut health which in turn increases the AhR pathway and inhibits HSCs activation and fibrosis progression beyond the gut in the liver tissue of NASH/LF mice.

  32. Comprehensive characterization of γ-aminobutyric acid (GABA) production by Levilactobacillus brevis CRL 2013: insights from physiology, genomics, and proteomics Peer-reviewed

    Pablo G. Cataldo, María Paulina Urquiza Martínez, Julio Villena, Haruki Kitazawa, Lucila Saavedra, Elvira M. Hebert

    Frontiers in Microbiology　15　(1408624)　2024/06/19

    Publisher: Frontiers Media SA

    DOI： 10.3389/fmicb.2024.1408624 　

    eISSN： 1664-302X

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    Introduction Levilactobacillus brevis CRL 2013, a plant-derived lactic acid bacterium (LAB) with immunomodulatory properties, has emerged as an efficient producer of γ-aminobutyric acid (GABA). Notably, not all LAB possess the ability to produce GABA, highlighting the importance of specific genetic and environmental conditions for GABA synthesis. This study aimed to elucidate the intriguing GABA-producing machinery of L. brevis CRL 2013 and support its potential for safe application through comprehensive genome analysis. Methods A comprehensive genome analysis of L. brevis CRL 2013 was performed to identify the presence of antibiotic resistance genes, virulence markers, and genes associated with the glutamate decarboxylase system, which is essential for GABA biosynthesis. Then, an optimized chemically defined culture medium (CDM) was supplemented with monosodium glutamate (MSG) and yeast extract (YE) to analyze their influence on GABA production. Proteomic and transcriptional analyses were conducted to assess changes in protein and gene expression related to GABA production. Results The absence of antibiotic resistance genes and virulence markers in the genome of L. brevis CRL 2013 supports its safety for potential probiotic applications. Genes encoding the glutamate decarboxylase system, including two gad genes (gadA and gadB) and the glutamate antiporter gene (gadC), were identified. The gadB gene is located adjacent to gadC, while gadA resides separately on the chromosome. The transcriptional regulator gadR was found upstream of gadC, with transcriptional analyses demonstrating cotranscription of gadR with gadC. Although MSG supplementation alone did not activate GABA synthesis, the addition of YE significantly enhanced GABA production in the optimized CDM containing glutamate. Proteomic analysis revealed minimal differences between MSG-supplemented and non-supplemented CDM cultures, whereas YE supplementation resulted in significant proteomic changes, including upregulation of GadB. Transcriptional analysis confirmed increased expression of gadB and gadR upon YE supplementation, supporting its role in activating GABA production. Conclusion These findings provide valuable insights into the influence of nutrient composition on GABA production. Furthermore, they unveil the potential of L. brevis CRL 2013 as a safe, nonpathogenic strain with valuable biotechnological traits which can be further leveraged for its probiotic potential in the food industry.

  33. The Mucus-Binding Factor Mediates Lacticaseibacillus rhamnosus CRL1505 Adhesion but Not Immunomodulation in the Respiratory Tract Peer-reviewed

    Binghui Zhou, Mariano Elean, Lorena Arce, Kohtaro Fukuyama, Kae Tomotsune, Stefania Dentice Maidana, Sudeb Saha, Fu Namai, Keita Nishiyama, María Guadalupe Vizoso-Pinto, Julio Villena, Haruki Kitazawa

    Microorganisms　12　(6)　1209　2024/06

    DOI： 10.3390/microorganisms12061209 　

  34. Insights into the Anti-Adipogenic and Anti-Inflammatory Potentialities of Probiotics against Obesity Peer-reviewed

    A. K. M. Humayun Kober, Sudeb Saha, Mutamed Ayyash, Fu Namai, Keita Nishiyama, Kazutoyo Yoda, Julio Villena, Haruki Kitazawa

    Nutrients　16　(9)　1373　2024/04/30

    DOI： 10.3390/nu16091373 　

  35. Bacterium-like Particles from Corynebacterium pseudodiphtheriticum as Mucosal Adjuvant for the Development of Pneumococcal Vaccines Peer-reviewed

    Ramiro Ortiz Moyano, Fernanda Raya Tonetti, Mariano Elean, Yoshiya Imamura, Kohtaro Fukuyama, Yoshihito Suda, Vyacheslav Melnikov, Alexander Suvorov, María Guadalupe Vizoso-Pinto, Haruki Kitazawa, Julio Villena

    Vaccines　12　(4)　412-412　2024/04/12

    Publisher: MDPI AG

    DOI： 10.3390/vaccines12040412 　

    eISSN： 2076-393X

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    Previously, it was shown that intranasally (i.n.) administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or CP-derived bacterium-like particles (BLPs) improve the immunogenicity of the pneumococcal conjugate vaccine (PCV). This work aimed to deepen the characterization of the adjuvant properties of Cp and CP-derived BLPs for their use in the development of pneumococcal vaccines. The ability of Cp and CP-derived BLPs to improve both the humoral and cellular specific immune responses induced by i.n. administered polysaccharide-based commercial pneumococcal vaccine (Pneumovax 23®) and the chimeric recombinant PSPF (PsaA-Spr1875-PspA-FliC) protein was evaluated, as well as the protection against Streptococcus pneumoniae infection in infant mice. Additionally, whether the immunization protocols, including Cp and CP-derived BLPs, together with the pneumococcal vaccines can enhance the resistance to secondary pneumococcal pneumonia induced after inflammatory lung damage mediated by the activation of Toll-like receptor 3 (TLR3) was assessed. The results showed that both Cp and CP-derived BLPs increased the immunogenicity and protection induced by two pneumococcal vaccines administered through the nasal route. Of note, the nasal priming with the PSPF T-dependent antigen co-administered with Cp or CP-derived BLPs efficiently stimulated humoral and cellular immunity and increased the resistance to primary and secondary pneumococcal infections. The CP-derived BLPs presented a stronger effect than live bacteria. Given safety concerns associated with live bacterium administration, especially in high-risk populations, such as infants, the elderly, and immunocompromised patients, BLPs emerge as an attractive mucosal adjuvant to improve the host response to pneumococcal infections and to enhance the vaccines already in the market or in development.

  36. In vitro evaluation of the immunomodulatory and wakame assimilation properties of Lactiplantibacillus plantarum strains from swine milk Peer-reviewed

    Ryusuke Ohgi, Sudeb Saha, Binghui Zhou, Taiga Sakuma, Mitsuki Sakurai, Yuhka Nakano, Fu Namai, Wakako Ikeda-Ohtsubo, Yoshihito Suda, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Frontiers in Microbiology　15　(1324999)　2024/01/26

    Publisher: Frontiers Media SA

    DOI： 10.3389/fmicb.2024.1324999 　

    eISSN： 1664-302X

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    The emergence and spread of antibiotic resistance threat forced to explore alternative strategies for improving the resistance to pathogens in livestock production. Probiotic lactic acid bacteria represent an alternative for this objective. In this study, seven Lactiplantibacillus plantarum strains from porcine colostrum and milk were isolated, identified and characterized in terms of their abilities to modulate immunity in porcine intestinal epithelial (PIE) cells. Then, two potential immunoregulatory strains were studied in terms of their ability to utilize and grow in wakame (Undaria pinnafida). Isolates were identified by 16S rRNA gene and evaluated by studying their interaction with PIE cells. The expressions of peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain (NODs), host defense peptides (pBD), and type I interferons (IFNs) were evaluated by RT-qPCR. The strain 4M4417 showed a remarkable capacity to differentially regulate the expression of PGRP1, PGRP3, NOD1, NOD2, and pBD1 in PIE cells. On the other hand, the strain 4M4326 was the most efficient to improve the expression of IFN-α and IFN-β in PIE cells challenged with poly (I:C). Both L. plantarum 4M4326 and 4M4417 were characterized in terms of their ability to utilize wakame. Results demonstrated that both strains efficiently grew in wakame-based broth. Our results suggest that L. planatrum 4M4326 and 4M4417 are interesting candidates to develop immunomodulatory feeds based on wakame utilization. These new immunosynbiotic feeds could help to reduce severity of intestinal infections and improve immune health status in pigs.

  37. Establishment of porcine fecal-derived <i>ex vivo</i> microbial communities to evaluate the impact of livestock feed on gut microbiome Peer-reviewed

    Yuji TSUJIKAWA, Keita NISHIYAMA, Fu NAMAI, Yoshiya IMAMURA, Taiga SAKUMA, Sudeb SAHA, Masahiko SUZUKI, Mitsuki SAKURAI, Ryo IWATA, Kengo MATSUO, Hironori TAKAMORI, Yoshihito SUDA, Binghui ZHOU, Itsuko FUKUDA, Julio VILLENA, Iwao SAKANE, Ro OSAWA, Haruki KITAZAWA

    Bioscience of Microbiota, Food and Health　43　(2)　100-109　2024

    Publisher: BMFH Press

    DOI： 10.12938/bmfh.2023-085 　

    eISSN： 2186-3342

  38. Development of an intestinal epithelial cell line and organoids derived from the same swine and characterization of their antiviral responses Peer-reviewed

    Kaho MATSUMOTO, Fu NAMAI, Ayako MIYAZAKI, Yoshiya IMAMURA, Kohtaro FUKUYAMA, Wakako IKEDA-OHTSUBO, Keita NISHIYAMA, Julio VILLENA, Kohtaro MIYAZAWA, Haruki KITAZAWA

    Bioscience of Microbiota, Food and Health　43　(4)　342-351　2024

    Publisher: BMFH Press

    DOI： 10.12938/bmfh.2024-0046 　

    eISSN： 2186-3342

  39. Lacticaseibacillus rhamnosus CRL1505 Peptidoglycan Modulates the Inflammation-Coagulation Response Triggered by Poly(I:C) in the Respiratory Tract Peer-reviewed

    Hortensia Zelaya, Luciano Arellano-Arriagada, Kohtaro Fukuyama, Kaho Matsumoto, Gabriela Marranzino, Fu Namai, Susana Salva, Susana Alvarez, Graciela Agüero, Haruki Kitazawa, Julio Villena

    International Journal of Molecular Sciences　24　(23)　16907-16907　2023/11/29

    Publisher: MDPI AG

    DOI： 10.3390/ijms242316907 　

    eISSN： 1422-0067

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    Lacticaseibacillus rhamnosus CRL1505 beneficially modulates the inflammation-coagulation response during respiratory viral infections. This study evaluated the capacity of the peptidoglycan obtained from the CRL1505 strain (PG-Lr1505) to modulate the immuno-coagulative response triggered by the viral pathogen-associated molecular pattern poly(I:C) in the respiratory tract. Adult BALB/c mice were nasally treated with PG-Lr1505 for two days. Treated and untreated control mice were then nasally challenged with poly(I:C). Mice received three doses of poly(I:C) with a 24 h rest period between each administration. The immuno-coagulative response was studied after the last administration of poly(I:C). The challenge with poly(I:C) significantly increased blood and respiratory pro-inflammatory mediators, decreased prothrombin activity (PT), and increased von Willebrand factor (vWF) levels in plasma. Furthermore, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and thrombomodulin (TM) expressions were increased in the lungs. PG-Lr1505-treated mice showed significant modulation of hemostatic parameters in plasma (PT in %, Control = 71.3 ± 3.8, PG-Lr1505 = 94.0 ± 4.0, p < 0.01) and lungs. Moreover, PG-Lr1505-treated mice demonstrated reduced TF in F4/80 cells from lungs, higher pro-inflammatory mediators, and increased IL-10 compared to poly(I:C) control mice (IL-10 in pg/mL, Control = 379.1 ± 12.1, PG-Lr1505 = 483.9 ± 11.3, p < 0.0001). These changes induced by PG-Lr1505 correlated with a significant reduction in lung tissue damage. Complementary in vitro studies using Raw 264.7 cells confirmed the beneficial effect of PG-Lr1505 on poly(I:C)-induced inflammation, since increased IL-10 expression, as well as reduced damage, production of inflammatory mediators, and hemostatic parameter expressions were observed. In addition, protease-activated receptor-1 (PAR1) activation in lungs and Raw 264.7 cells was observed after TLR3 stimulation, which was differentially modulated by PG-Lr1505. The peptidoglycan from L. rhamnosus CRL1505 is able to regulate inflammation, the procoagulant state, and PAR1 activation in mice and macrophages in the context of the activation of TLR3 signaling pathways, contributing to a beneficial modulation of inflammation-hemostasis crosstalk.

  40. Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae Peer-reviewed

    Mariano Elean, Fernanda Raya Tonetti, Kohtaro Fukuyama, Luciano Arellano-Arriagada, Fu Namai, Yoshihito Suda, Nadia Gobbato, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    International Journal of Molecular Sciences　24　(21)　15773　2023/10/30

    DOI： 10.3390/ijms242115773 　

  41. In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii Peer-reviewed

    Mariano Elean, Leonardo Albarracin, Julio Villena, Haruki Kitazawa, Lucila Saavedra, Elvira M. Hebert

    International Journal of Molecular Sciences　24　(14)　11309　2023/07/11

    DOI： 10.3390/ijms241411309 　

  42. Establishment of a porcine bronchial epithelial cell line and its application to study innate immunity in the respiratory epithelium Peer-reviewed

    Kohtaro Fukuyama, Tao Zhuang, Eita Toyoshi, Fernanda Raya Tonetti, Sudeb Saha, Binghui Zhou, Wakako Ikeda-Ohtsubo, Keita Nishiyama, Hisashi Aso, Julio Villena, Haruki Kitazawa

    Frontiers in Immunology　14　(1117102)　2023/07/03

    Publisher: Frontiers Media SA

    DOI： 10.3389/fimmu.2023.1117102 　

    eISSN： 1664-3224

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    In vitro culture models that precisely mirror the porcine respiratory epithelium are needed to gain insight into how pathogens and host interact. In this study, a new porcine bronchial epithelial cell line, designated as PBE cells, was established from the respiratory tract of a neonatal pig. PBE cells assumed a cobblestone-epithelial like morphology with close contacts between the cells when they reached confluence. The PBE cell line was characterized in terms of its expression of pattern recognition receptors (PRRs) and its ability to respond to the activation of the Toll-like receptor 3 (TLR3) and TLR4 signaling pathways, which are key PRRs involved in the defense of the respiratory epithelium against pathogens. PBE cells stimulated with poly(I:C) were able to up-regulate the expression of IFN-β, IFN-λ1 (IL-29), IFN-λ3 (IL-28B), the antiviral factors Mx1, OAS1, and PKR, as well as the viral PRRs RIG-1 and MDA5. The expression kinetics studies of immune factors in PBE cells allow us to speculate that this cell line can be a useful in vitro tool to investigate treatments that help to potentiate antiviral immunity in the respiratory epithelium of the porcine host. In addition, poly(I:C) and LPS treatments increased the expression of the inflammatory cytokines TNF-α, IL-6, IL-8, and MCP-1/CCL2 and differentially modulated the expression of negative regulators of the TLR signaling pathways. Then, PBE cells may also allow the evaluation of treatments that can regulate TLR3- and TLR4-mediated inflammatory injury in the porcine airway, thereby protecting the host against harmful overresponses.

  43. Partial Characterization and Immunomodulatory Effects of Exopolysaccharides from Streptococcus thermophilus SBC8781 during Soy Milk and Cow Milk Fermentation Peer-reviewed

    Hajime Nakata, Yoshiya Imamura, Sudeb Saha, René Emanuel Lobo, Shugo Kitahara, Shota Araki, Mikado Tomokiyo, Fu Namai, Masanori Hiramitsu, Takashi Inoue, Keita Nishiyama, Julio Villena, Haruki Kitazawa

    Foods　12　(12)　2374　2023/06/15

    Publisher: MDPI AG

    DOI： 10.3390/foods12122374 　

    eISSN： 2304-8158

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    The immunomodulatory properties of exopolysaccharides (EPSs) produced by Streptococcus thermophilus have not been explored in depth. In addition, there are no comparative studies of the functional properties of EPSs produced by streptococci in different food matrices. In this work, EPSs from S. thermophilus SBC8781 were isolated after soy milk (EPS-s) or cow milk (EPS-m) fermentation, identified, and characterized in their abilities to modulate immunity in porcine intestinal epithelial cells. Fresh soy milk and cow milk were inoculated with S. thermophilus SBC8781 (7 log CFU/mL) and incubated at 37 °C for 24 h. The extraction of EPSs was performed by the ethanol precipitation method. Analytical techniques, including NMR, UV-vis spectroscopy, and chromatography, identified and characterized both biopolymer samples as polysaccharides with high purity levels and similar Mw. EPS-s and EPS-m had heteropolysaccharide structures formed by galactose, glucose, rhamnose, ribose, and mannose, although with different monomer proportions. On the other hand, EPS-s had higher quantities of acidic polymer than EPS-m. The biopolymer production of the SBC8781 strain from the vegetable culture broth was 200–240 mg/L, which was higher than that produced in milk, which reached concentrations of 50–70 mg/L. For immunomodulatory assays, intestinal epithelial cells were stimulated with 100 µg/mL of EPS-s or EPS-m for 48 h and then stimulated with the Toll-like receptor 3 agonist poly(I:C). EPS-s significantly reduced the expression of IL-6, IFN-β, IL-8, and MCP-1 and increased the negative regulator A20 in intestinal epithelial cells. Similarly, EPS-m induced a significant reduction of IL-6 and IL-8 expressions, but its effect was less remarkable than that caused by EPS-s. Results indicate that the structure and the immunomodulatory activity of EPSs produced by the SBC8781 strain vary according to the fermentation substrate. Soy milk fermented with S. thermophilus SBC8781 could be a new immunomodulatory functional food, which should be further evaluated in preclinical trials.

  44. Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infect Human Intestinal Epithelial Cells and Induce Moderate Inflammation Peer-reviewed

    Stefania Dentice Maidana, Mariano Elean, Kohtaro Fukuyama, Yoshiya Imamura, Leonardo Albarracín, Sudeb Saha, Yoshihito Suda, Shoichiro Kurata, María Ángela Jure, Haruki Kitazawa, Julio Villena

    International Journal of Molecular Sciences　24　(10)　8804　2023/05/15

    Publisher: MDPI AG

    DOI： 10.3390/ijms24108804 　

    eISSN： 1422-0067

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    Klebsiella pneumoniae is an opportunistic pathogen that can produce moderate and severe infections in immunosuppressed hosts. In recent years, an increase in the isolation of hypermucoviscous carbapenem-resistant K. pneumoniae with sequence type 25 (ST25) in hospitals in Norwest Argentina was observed. This work aimed to study the virulence and inflammatory potential of two K. pneumoniae ST25 strains (LABACER01 and LABACER27) in the intestinal mucosa. The human intestinal Caco-2 cells were infected with the K. pneumoniae ST25 strains, and their adhesion and invasion rates and changes in the expression of tight junction and inflammatory factors genes were evaluated. ST25 strains were able to adhere and invade Caco-2 cells, reducing their viability. Furthermore, both strains reduced the expression of tight junction proteins (occludin, ZO-1, and claudin-5), altered permeability, and increased the expression of TGF-β and TLL1 and the inflammatory factors (COX-2, iNOS, MCP-1, IL-6, IL-8, and TNF-α) in Caco-2 cells. The inflammatory response induced by LABACER01 and LABACER27 was significantly lower than the one produced by LPS or other intestinal pathogens, including K. pneumoniae NTUH-K2044. No differences in virulence and inflammatory potential were found between LABACER01 and LABACER27. In line with these findings, no major differences between the strains were found when the comparative genomic analysis of virulence factors associated with intestinal infection/colonization was performed. This work is the first to demonstrate that hypermucoviscous carbapenem-resistant K. pneumoniae ST25 infects human intestinal epithelial cells and induces moderate inflammation.

  45. Oral Administration of Lacticaseibacillus rhamnosus CRL1505 Modulates Lung Innate Immune Response against Klebsiella pneumoniae ST25 Peer-reviewed

    Stefania Dentice Maidana, Yoshiya Imamura, Mariano Elean, Leonardo Albarracín, Keita Nishiyama, Yoshihito Suda, Shoichiro Kurata, María Ángela Jure, Haruki Kitazawa, Julio Villena

    Microorganisms　11　(5)　1148　2023/04/28

    DOI： 10.3390/microorganisms11051148 　

  46. The Respiratory Commensal Bacterium Corynebacterium pseudodiphtheriticum as a Mucosal Adjuvant for Nasal Vaccines Peer-reviewed

    Ramiro Ortiz Moyano, Fernanda Raya Tonetti, Kohtaro Fukuyama, Mariano Elean, Mikado Tomokiyo, Yoshihito Suda, Vyacheslav Melnikov, Haruki Kitazawa, Julio Villena

    Vaccines　11　(3)　611　2023/03/08

    DOI： 10.3390/vaccines11030611 　

  47. Bioprotective Lactic Acid Bacteria and Lactic Acid as a Sustainable Strategy to Combat Escherichia coli O157:H7 in Meat Peer-reviewed

    Ayelen A. Baillo, Lucia Cisneros, Julio Villena, Graciela Vignolo, Silvina Fadda

    Foods　12　(2)　231　2023/01/04

    DOI： 10.3390/foods12020231 　

  48. Isolation, characterization, and immunomodulatory activity evaluation of probiotic strains from colostrum and canine milk. International-journal Peer-reviewed

    Sandra Rayén Quilodrán-Vega, Carolina Muñoz-Flores, Ana Pino, Paula Buldres, Felipe Sandoval, Alex Aguirre, Brígida Portillo, Natalie Parra, Claudia Altamirano, Leonardo Albarracín, Julio Villena, Jorge R Toledo

    Frontiers in veterinary science　10　1266064　2023

    DOI： 10.3389/fvets.2023.1266064 　

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    BACKGROUND: This study aimed to characterize potential probiotic strains for use in dogs to prevent infectious enteropathies. Lactic acid bacteria (LAB) isolated from canine milk and colostrum were characterized according to their functional properties, including their resistance to gastrointestinal conditions, inhibitory effect against pathogens, and intestinal adhesion. METHODS: The immunomodulatory effects of the strains were also analyzed in in vitro and in vivo studies. Among the strains evaluated, two LAB strains (TUCO-16 and TUCO-17) showed remarkable resistance to pH 3.0, bile salts, and pancreatin, as well as inhibitory effects against pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. RESULTS: The TUCO-16 and TUCO-17 strains induced a significant increase in the expression of TNF-α, IL-8, and TLR2 in canine macrophages. The oral administration of TUCO-16 and TUCO-17 strains to mice significantly augmented their resistance to pathogenic E. coli or Salmonella intestinal infections. Both canine strains reduced intestinal damage and pathogen counts in the liver and spleen and avoided their dissemination into the bloodstream. These protective effects were related to the ability of TUCO-16 and TUCO-17 strains to differentially modulate the production of IFN-γ, IFN-β, TNF-α, IL-6, KC, MCP-1, and IL-10 in the intestinal mucosa. CONCLUSION: Both strains, TUCO-16 and TUCO-17, are potential probiotic candidates for improving intestinal health in dogs, particularly for their ability to inhibit the growth of Gram-negative pathogens common in gastrointestinal infections and modulate the animal's immune response. Further studies are required to effectively demonstrate the beneficial effects of TUCO-16 and TUCO-17 strains in dogs.

  49. Post-immunobiotics increase resistance to primary respiratory syncytial virus infection and secondary pneumococcal pneumonia Peer-reviewed

    F. Raya Tonetti, M. Tomokiyo, K. Fukuyama, M. Elean, R. Ortiz Moyano, H. Yamamuro, R. Shibata, S. Quilodran-Vega, S. Kurata, J. Villena, H. Kitazawa

    Beneficial Microbes　14　(3)　209-221　2023

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2022.0118 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    Previously, we demonstrated that post-immunobiotics derived from Lactobacillus gasseri TMT36, TMT39, and TMT40 strains (HK36, HK39 and HK40, respectively) differentially regulated Toll-like receptor 3 (TLR3)-mediated antiviral respiratory immunity in infant mice. In this work, we investigated whether the HK36, HK39 and HK40 nasal treatments were able to improve the resistance against primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. Our results demonstrated that the three treatments increased the resistance to primary viral infection by reducing variations in body weight, RSV titers and lung damage of infected infant mice. Post-immunobiotics significantly enhanced the expressions of interferon (IFN)-λ, IFN-β, IFN-γ, interleukin(IL) - 1β, IL-6, IL-27, Mx1, RNAseL and 2’-5’-oligoadenylate synthetase 1 (OAS1) genes and decreased tumour necrosis factor (TNF)-α in alveolar macrophages of RSV-challenged mice. In addition, the studies in the model of RSV-Streptococcus pneumoniae superinfection showed that the HK39 and HK40 treatments were capable of reducing lung damage, lung bacterial cell counts, and the dissemination of S. pneumoniae into the blood of infant mice. The protective effect was associated with increases in IFN-β, IFN-γ, IL-10, and IL-27 in the respiratory tract. This study demonstrates that the nasal application of the post-immunobiotics HK39 and HK40 stimulates innate respiratory immunity and enhances the defences against primary RSV infection and secondary pneumococcal pneumonia offering an alternative to combat respiratory superinfections in children, which can be fatal.

  50. Lactiplantibacillus plantarum Strains Modulate Intestinal Innate Immune Response and Increase Resistance to Enterotoxigenic Escherichia coli Infection

    Ayelen Baillo, Julio Villena, Leonardo Albarracín, Mikado Tomokiyo, Mariano Elean, Kohtaro Fukuyama, Sandra Quilodrán-Vega, Silvina Fadda, Haruki Kitazawa

    Microorganisms　2022/12/25

    DOI： 10.3390/microorganisms11010063 　

  51. Immunomodulatory Effects of Probiotics: A Novel Preventive Approach for the Control of Bovine Mastitis

    A. K. M. Humayun Kober, Sudeb Saha, Md. Aminul Islam, Muhammad Shahid Riaz Rajoka, Kohtaro Fukuyama, Hisashi Aso, Julio Villena, Haruki Kitazawa

    Microorganisms　2022/11/14

    DOI： 10.3390/microorganisms10112255 　

  52. The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites International-journal

    Kae Tomotsune, Fernanda Raya Tonetti, Hiroya Mizuno, Mariano Elean, Kohtaro Fukuyama, Binghui Zhou, Wakako Ikeda-Ohtsubo, Keita Nishiyama, Akihiro Yamamura, Hideaki Karasawa, Shinobu Ohnuma, Akira Horii, Tadao Saito, Haruki Kitazawa, Julio Villena

    International Journal of Molecular Sciences　23　(22)　2022/11

    DOI： 10.3390/ijms232214357 　

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    Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-β and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505.

  53. The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic International-journal

    Fernanda Raya Tonetti, Patricia Clua, Kohtaro Fukuyama, Guillermo Marcial, Jacinto Sacur, Gabriela Marranzino, Mikado Tomokiyo, Guadalupe Vizoso-Pinto, Apolinaria Garcia-Cancino, Shoichiro Kurata, Haruki Kitazawa, Julio Villena

    Microorganisms　10　(11)　2022/11

    DOI： 10.3390/microorganisms10112185 　

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    Previously, we demonstrated that the non-viable strain Lacticaseibacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulated the respiratory innate antiviral immune response triggered by Toll-like receptor (TLR)-3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In this work, we evaluated the effect of other non-viable L. rhamnosus strains and their peptidoglycans on the respiratory immune response and their impact on primary and secondary respiratory infections. In addition, the duration of the protective effect induced by NV1505 and PG1505 as well as their ability to protect against different Streptococcus pneumoniae serotypes were evaluated. Our results showed that among the five selected L. rhamnosus strains (CRL1505, CRL498, CRL576, UCO25A and IBL027), NV1505 and NVIBL027 improved the protection against viral and pneumococcal infections by modulating the respiratory immune response. Of note, only the PG1505 presented immunomodulatory activities when compared with the other purified peptidoglycans. Studies on alveolar macrophages showed that NV1505 and PG1505 differentially modulated the expression of IL-6, IFN-γ, IFN-β, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1β, TNF-α, CCL2, CXCL2, CXCL10 and IL-27 in response to pneumococcal challenge. Furthermore, we demonstrated that NV1505 and PG1505 treatments protected mice against secondary pneumococcal pneumonia produced by different serotypes of S. pneumoniae until 30 days after stimulation with poly(I:C). This work advances the characterization of the protective effect of NV1505 and PG1505 by demonstrating that they increase resistance against the pneumococcal serotypes 3, 6B, 14 and 19F, with an effect that lasts up to 30 days after the primary viral inflammation. The results also confirm that the immunomodulatory properties of NV1505 and PG1505 are unique and are not shared by other members of this species, and suggest the existence of a capacity to stimulate trained immunity in alveolar macrophages.

  54. Lactiplantibacillus plantarum LOC1 Isolated from Fresh Tea Leaves Modulates Macrophage Response to TLR4 Activation

    Masahiko Suzuki, Leonardo Albarracin, Yuji Tsujikawa, Kohtaro Fukuyama, Iwao Sakane, Julio Villena, Haruki Kitazawa

    Foods　2022/10/18

    DOI： 10.3390/foods11203257 　

  55. Respiratory Commensal Bacteria Increase Protection against Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infection

    Stefania Dentice Maidana, Ramiro Ortiz Moyano, Juan Vargas, Kohtaro Fukuyama, Shoichiro Kurata, Vyacheslav Melnikov, María Jure, Haruki Kitazawa, Julio Villena

    Pathogens　2022/09/19

    DOI： 10.3390/pathogens11091063 　

  56. Modulation of Alveolar Macrophages by Postimmunobiotics: Impact on TLR3-Mediated Antiviral Respiratory Immunity International-journal

    Mikado Tomokiyo, Fernanda Raya Tonetti, Hikari Yamamuro, Ryoko Shibata, Kohtaro Fukuyama, Nadia Gobbato, Leonardo Albarracin, Muhammad Shahid Riaz Rajoka, A. K. M. Humayun Kober, Wakako Ikeda-Ohtsubo, Julio Villena, Haruki Kitazawa

    Cells　11　(19)　2022/09

    DOI： 10.3390/cells11192986 　

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    Beneficial microbes with immunomodulatory capacities (immunobiotics) and their non-viable forms (postimmunobiotics) could be effectively utilized in formulations towards the prevention of respiratory viral infections. In this study, novel immunobiotic strains with the ability to increase antiviral immunity in porcine alveolar macrophages were selected from a library of Lactobacillus gasseri. Postimmunobiotics derived from the most remarkable strains were also evaluated in their capacity to modulate the immune response triggered by Toll-like receptor 3 (TLR3) in alveolar macrophages and to differentially regulate TLR3-mediated antiviral respiratory immunity in infant mice. We provide evidence that porcine alveolar macrophages (3D4/31 cells) are a useful in vitro tool for the screening of new antiviral immunobiotics and postimmunobiotics by assessing their ability to modulate the expression IFN-β, IFN-λ1, RNAseL, Mx2, and IL-6, which can be used as prospective biomarkers. We also demonstrate that the postimmunobiotics derived from the Lactobacillus gasseri TMT36, TMT39 and TMT40 (HK36, HK39 or HK40) strains modulate the innate antiviral immune response of alveolar macrophages and reduce lung inflammatory damage triggered by TLR3 activation in vivo. Although our findings should be deepened and expanded, the results of the present work provide a scientific rationale for the use of nasally administered HK36, HK39 or HK40 to beneficially modulate TLR3-triggerd respiratory innate immune response.

  57. Genomic Characterization of Lactiplantibacillus plantarum Strains Possessing Differential Antiviral Immunomodulatory Activities Peer-reviewed

    Leonardo Albarracin, Fernanda Raya Tonetti, Kohtaro Fukuyama, Yoshihito Suda, Binghui Zhou, Ayelén A. Baillo, Silvina Fadda, Lucila Saavedra, Shoichiro Kurata, Elvira M. Hebert, Haruki Kitazawa, Julio Villena

    Bacteria　1　(3)　136-160　2022/07/06

    Publisher: MDPI AG

    DOI： 10.3390/bacteria1030012 　

    eISSN： 2674-1334

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    Lactiplantibacillus plantarum strains are used in the food industry for their probiotic properties. Some of these bacteria have immunomodulatory effects on the host and are able to improve resistance against different pathogens, including viruses. However, to date, the bacterial genes involved in the immunomodulatory effect are not known. In this work, the complete genomes of L. plantarum MPL16, CRL1506, CRL681 and TL2766 were used to perform comparative genomics with the aim of identifying the genes involved in their differential immunomodulatory effects. L. plantarum WCFS1, a strain with proven probiotic activity, was also used for comparisons. The analysis of the genes involved in the metabolic pathways of the five strains did not reveal differences in the metabolism of amino acids, lipids, nucleotides, cofactors and vitamins, nor in the genes associated with energy metabolism or the biosynthesis of lipoproteins and teichoic acids. However, differences were found between the five strains when considering carbohydrate metabolism pathways, particularly in the presence/absence of glycosylhydrolases and glycosyltransferases. In addition, a great variability was detected in the predicted surface proteins of each L. plantarum strain. These results suggest that the surface molecules expressed in the different strains of L. plantarum could be involved in their differential ability to modulate the innate antiviral immune response.

  58. Genomic and Immunological Characterization of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Isolates from Northwest Argentina International-journal

    Leonardo Albarracin, Ramiro Ortiz Moyano, Juan Martin Vargas, Bruno G. N. Andrade, Juan Cortez Zamar, Stefania Dentice Maidana, Kohtaro Fukuyama, Shoichiro Kurata, María Ángela Jure, Haruki Kitazawa, Julio Villena

    International Journal of Molecular Sciences　23　(13)　2022/07

    DOI： 10.3390/ijms23137361 　

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    In recent years, an increase in the prevalence hypermucoviscous carbapenem-resistant Klebsiella pneumoniae with sequence type 25 (ST25) was detected in hospitals of Tucuman (Northwest Argentina). In this work, the virulence and the innate immune response to two K. pneumoniae ST25 strains (LABACER 01 and LABACER 27) were evaluated in a murine model after a respiratory challenge. In addition, comparative genomics was performed with K. pneumoniae LABACER01 and LABACER27 to analyze genes associated with virulence. Both LABACER01 and LABACER27 were detected in the lungs of infected mice two days after the nasal challenge, with LABACER01 counts significantly higher than those of LABACER27. Only LABACER01 was detected in hemocultures. Lactate dehydrogenase (LDH) and albumin levels in bronchoalveolar lavage (BAL) samples were significantly higher in mice challenged with LABACER01 than in LABACER27-infected animals, indicating greater lung tissue damage. Both strains increased the levels of neutrophils, macrophages, TNF-α, IL-1β, IL-6, KC, MCP-1, IFN-γ, and IL-17 in the respiratory tract and blood, with the effect of LABACER01 more marked than that of LABACER27. In contrast, LABACER27 induced higher levels of IL-10 in the respiratory tract than LABACER01. Genomic analysis revealed that K. pneumoniae LABACER01 and LABACER27 possess virulence factors found in other strains that have been shown to be hypervirulent, including genes required for enterobactin (entABCDEF) and salmochelin (iroDE) biosynthesis. In both strains, the genes of toxin-antitoxin systems, as well as regulators of the expression of virulence factors and adhesion genes were also detected. Studies on the genetic potential of multiresistant K. pneumoniae strains as well as their cellular and molecular interactions with the host are of fundamental importance to assess the association of certain virulence factors with the intensity of the inflammatory response. In this sense, this work explored the virulence profile based on genomic and in vivo studies of hypermucoviscous carbapenem-resistant K. pneumoniae ST25 strains, expanding the knowledge of the biology of the emerging ST25 clone in Argentina.

  59. Alterations of Blood Immune Cells in Elderly Patients with COVID-19 and with or without Pre-Existing Type 2 Diabetes Peer-reviewed

    Susana Salva, Yanina Kolling, Nicolás Argañaraz, María Pía Rocchia Novillo, María del Milagro Rocchia Novillo, Leonardo Albarracin, Jacqueline Charubi, Alejandra Casen, Nadia Gobbato, Mirta Rachid, Susana Alvarez, Haruki Kitazawa, Julio Villena, Juan Carlos Valdez

    Open Journal of Immunology　12　(02)　41-63　2022/06

    Publisher: Scientific Research Publishing, Inc.

    DOI： 10.4236/oji.2022.122003 　

    ISSN： 2162-450X

    eISSN： 2162-4526

  60. Topical Administration of Lactiplantibacillus plantarum Accelerates the Healing of Chronic Diabetic Foot Ulcers through Modifications of Infection, Angiogenesis, Macrophage Phenotype and Neutrophil Response International-journal Peer-reviewed

    Julio Nicolás Argañaraz Aybar, Sonia Ortiz Mayor, Luis Olea, Juan José Garcia, Sebastian Nisoria, Yanina Kolling, Constanza Melian, Mirta Rachid, Rafael Torres Dimani, Cecilia Werenitzky, Cecilia Lorca, Susana Salva, Nadia Gobbato, Julio Villena, Juan C. Valdez

    Microorganisms　10　(3)　2022/03

    DOI： 10.3390/microorganisms10030634 　

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    This work aimed to evaluate the adjuvant treatment to surgical debridement using topical applications of Lactiplantibacillus plantarum ATCC 10241 cultures in complicated diabetic foot ulcers as compared to diabetic foot ulcers receiving surgical wound debridement. A randomised controlled trial was performed involving 22 outpatients with complicated diabetic foot ulcers that either received surgical debridement (SuDe, n = 12) or surgical debridement plus topical applications of L. plantarum cultures (SuDe + Lp, n = 10) every week during a 12 week treatment period. Compared to patients receiving SuDe, patients treated with SuDe + Lp exhibited significantly increased fibroplasia and angiogenesis, as determined by Masson’s trichrome staining and the study of CD34 cells, α-smooth muscle actin to semi-quantify vascular area, number of vessels and endothelial cells. In addition, a promotion of the polarisation of macrophages from M1 (CD68) to M2 (CD163) phenotype was observed in SuDe + Lp patients with remarkable differences in the tissue localisation. Bacterial counts were significantly diminished in the SuDe + Lp group compared to the SuDe group. Ex vivo assays, using polymorphonuclears isolated from peripheral blood of patients with diabetes and healthy individuals and challenged with Staphylococcus aureus demonstrated that the addition of L. plantarum supernatants significantly improved the phagocytosis of these cells. L. plantarum-secreted components increased the neutrophils bactericidal activity and regulated the netosis induced by S. aureus. At day 49, the average wound area reduction with SuDe + Lp was 73.5% compared with 45.8% for SuDe (p < 0.05). More patients progressed to closure with SuDe + Lp compared with SuDe treatment, indicating the ability of L. plantarum to accelerate the healing. At day 60, 60% of patients treated with SuDe + Lp achieved 100% of wound area reduction compared with 40% for SuDe. We propose that SuDe + Lp could be an effective adjuvant to surgical debridement when SuDe is not satisfactory for patients with complicated diabetic foot ulcers. The treatment is cheap and easy to apply and the product is easy to obtain.

  61. Soymilk-fermented with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L improves immune-health in pigs Peer-reviewed

    Y. Suda, K. Kagawa, K. Fukuyama, M. Elean, B. Zhou, M. Tomokiyo, M. Aminul Islam, M.S.R. Rajoka, A.K.M. Humayun Kober, T. Shimazu, S. Egusa, Y. Terashima, H. Aso, W. Ikeda-Ohtsubo, J. Villena, H. Kitazawa

    Beneficial Microbes　13　(1)　61-72　2022/02/28

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2021.0068 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    Lactobacillus delbrueckii subsp. delbrueckii TUA4408L has the ability to grow and ferment soymilk and is able to modulate the innate immune response of intestinal epithelial cells in vitro. These two properties prompt us to evaluate whether the soymilk fermented with the TUA4408L strain can induce beneficial immunomodulatory effects in vivo. For this purpose, pigs were selected as a preclinical model. The studies performed here demonstrated that the L. delbrueckii subsp. delbrueckii TUA4408L-fermented soymilk (TUA4408L FSM) reduced blood markers of inflammation and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These immunological changes induced by the TUA4408L FSM were associated to an enhanced resistance to pathogenic Escherichia coli and an improved grow performance and meat quality of pigs. The experiments and analysis in our study indicate that the immunobiotic TUA4408L FSM could be an interesting non-dairy functional food to beneficially modulate the intestinal immune system, improve protection against pathogens and reduce inflammatory damage. The preclinical study carried out here in pigs could have a better correlation in humans, compared to a rodent model. However, the clinical relevance of these findings still needs to be confirmed by further research, for example, in controlled human challenge studies.

  62. Immunomodulation Potential of Probiotics: A Novel Strategy for Improving Livestock Health, Immunity, and Productivity

    A. K. M. Humayun Kober, Muhammad Shahid Riaz Rajoka, Hafiza Mahreen Mehwish, Julio Villena, Haruki Kitazawa

    Microorganisms　2022/02/07

    DOI： 10.3390/microorganisms10020388 　

  63. 粘膜免疫防御機構：有用微生物によるCOVID-19感染・重症化の軽減ネットワーク Invited

    Julio Villena, 北澤 春樹

    腎と透析　92　(1)　54-61　2022/01

  64. The role of respiratory microbiota in the protection against viral diseases: respiratory commensal bacteria as next-generation probiotics for COVID-19 Peer-reviewed

    Bruno G. N. ANDRADE, Rafael R. C. CUADRAT, Fernanda Raya TONETTI, Haruki KITAZAWA, Julio VILLENA

    Bioscience of Microbiota, Food and Health　41　(3)　94-102　2022

    Publisher: BMFH Press

    DOI： 10.12938/bmfh.2022-009 　

    eISSN： 2186-3342

  65. Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal Epithelial Cells and Macrophages

    Mariano Elean, Leonardo Albarracin, Kohtaro Fukuyama, Binghui Zhou, Mikado Tomokiyo, Shugo Kitahara, Shota Araki, Yoshihito Suda, Lucila Saavedra, Julio Villena, Elvira M. Hebert, Haruki Kitazawa

    Microorganisms　2021/11/27

    DOI： 10.3390/microorganisms9122449 　

  66. Novel LysM motifs for antigen display on lactobacilli for mucosal immunization. International-journal

    Fernanda Raya-Tonetti, Melisa Müller, Jacinto Sacur, Haruki Kitazawa, Julio Villena, Maria Guadalupe Vizoso-Pinto

    Scientific reports　11　(1)　21691-21691　2021/11/04

    DOI： 10.1038/s41598-021-01087-8 　

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    We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

  67. Immunobiotic Lactobacilli Improve Resistance of Respiratory Epithelial Cells to SARS-CoV-2 Infection

    Md. Aminul Islam, Leonardo Albarracin, Mikado Tomokiyo, Juan Carlos Valdez, Jacinto Sacur, Maria Guadalupe Vizoso-Pinto, Bruno G. N. Andrade, Rafael R. C. Cuadrat, Haruki Kitazawa, Julio Villena

    Pathogens　2021/09/15

    DOI： 10.3390/pathogens10091197 　

  68. Draft genome sequences of two hypermucoviscous carbapenem-resistant ST25 Klebsiella pneumoniae strains causing respiratory and systemic infections

    María Angela Jure, Leonardo Albarracin, Juan Martin Vargas, Stefania Dentice Maidana, Juan Cortez Zamar, Haruki Kitazawa, Julio Villena

    Journal of Global Antimicrobial Resistance　26　174-176　2021/09

    Publisher: Elsevier BV

    DOI： 10.1016/j.jgar.2021.05.018 　

    ISSN： 2213-7165

  69. Characterization of Weissella viridescens UCO-SMC3 as a Potential Probiotic for the Skin: Its Beneficial Role in the Pathogenesis of Acne Vulgaris

    Marcela Espinoza-Monje, Jorge Campos, Eduardo Alvarez Villamil, Alonso Jerez, Stefania Dentice Maidana, Mariano Elean, Susana Salva, Haruki Kitazawa, Julio Villena, Apolinaria García-Cancino

    Microorganisms　2021/07/13

    DOI： 10.3390/microorganisms9071486 　

  70. The Respiratory Commensal Bacterium Dolosigranulum pigrum 040417 Improves the Innate Immune Response to Streptococcus pneumoniae

    Fernanda Raya Tonetti, Mikado Tomokiyo, Ramiro Ortiz Moyano, Sandra Quilodrán-Vega, Hikari Yamamuro, Paulraj Kanmani, Vyacheslav Melnikov, Shoichiro Kurata, Haruki Kitazawa, Julio Villena

    Microorganisms　2021/06/18

    DOI： 10.3390/microorganisms9061324 　

  71. Evaluation of Porcine Intestinal Epitheliocytes as an In vitro Immunoassay System for the Selection of Probiotic Bifidobacteria to Alleviate Inflammatory Bowel Disease. International-journal

    Nana Sato, Mao Yuzawa, Md Islam Aminul, Mikado Tomokiyo, Leonardo Albarracin, Valeria Garcia-Castillo, Wakako Ideka-Ohtsubo, Noriyuki Iwabuchi, Jin-Zhong Xiao, Apolinaria Garcia-Cancino, Julio Villena, Haruki Kitazawa

    Probiotics and antimicrobial proteins　13　(3)　824-836　2021/06

    DOI： 10.1007/s12602-020-09694-z 　

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    The use of in vitro systems that allow efficient selection of probiotic candidates with immunomodulatory properties could significantly minimize the use of experimental animals. In this work, we generated an in vitro immunoassay system based on porcine intestinal epithelial (PIE) cells and dextran sodium sulfate (DSS) administration that could be useful for the selection and characterization of potential probiotic strains to be used in inflammatory bowel disease (IBD) patients. Our strategy was based on two fundamental pillars: on the one hand, the capacity of PIE cells to create a monolayer by attaching to neighboring cells and efficiently mount inflammatory responses and, on the other hand, the use of two probiotic bifidobacteria strains that have been characterized in terms of their immunomodulatory capacities, particularly in mouse IBD models and patients. Our results demonstrated that DSS administration can alter the epithelial barrier created in vitro by PIE cells and induce a potent inflammatory response, characterized by increases in the expression levels of several inflammatory factors including TNF-α, IL-1α, CCL4, CCL8, CCL11, CXCL5, CXCL9, CXCL10, SELL, SELE, EPCAM, VCAM, NCF2, and SAA2. In addition, we demonstrated that Bifidobacterium breve M-16V and B. longum BB536 are able to regulate the C-jun N-terminal kinase (JNK) intracellular signalling pathway, reducing the DSS-induced alterations of the in vitro epithelial barrier and differentially regulating the inflammatory response in a strain-dependent fashion. The good correlation between our in vitro findings in PIE cells and previous studies in animal models and IBD patients shows the potential value of our system to select new probiotic candidates in an efficient way.

  72. Dolosigranulum pigrum Modulates Immunity against SARS-CoV-2 in Respiratory Epithelial Cells

    Md. Aminul Islam, Leonardo Albarracin, Vyacheslav Melnikov, Bruno G. N. Andrade, Rafael R. C. Cuadrat, Haruki Kitazawa, Julio Villena

    Pathogens　2021/05/21

    DOI： 10.3390/pathogens10060634 　

  73. Immunobiotic Feed Developed with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and the Soymilk By-Product Okara Improves Health and Growth Performance in Pigs International-journal

    Yoshihito Suda, Nana Sasaki, Kyoma Kagawa, Mariano Elean, Binghui Zhou, Mikado Tomokiyo, Md. Aminul Islam, Muhammad Shahid Riaz Rajoka, A. K. M. Humayun Kober, Tomoyuki Shimazu, Shintaro Egusa, Yuji Terashima, Hisashi Aso, Wakako Ikeda-Ohtsubo, Julio Villena, Haruki Kitazawa

    Microorganisms　9　(5)　2021/04

    DOI： 10.3390/microorganisms9050921 　

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    Lactobacillus delbrueckii subsp. delbrueckii TUA4408L is able to differentially modulate the innate immune response of porcine intestinal epithelial cells triggered by TLR4 activation. This strain also has a remarkable ability to grow on plant substrates. These two immunological and biotechnological characteristics prompted us to evaluate whether the soymilk by-product okara fermented with the TUA4408L strain can serve as an immunobiotic feed with the ability to beneficially modulate the intestinal immunity of piglets after weaning to improve their productivity. Our in vivo studies demonstrated that the administration of immunobiotic TUA4408L-fermented okara feed significantly increased piglet growth performance and meat quality. These positive effects were associated with the ability of the TUA4408L-fermented okara feed to beneficially modulate both intestinal microbiota and immunity in pigs. The immunobiotic feed improved the abundance of the beneficial bacteria Lactobacillus and Lactococcus in the gut of pigs, reduced blood markers of inflammation, and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These findings indicate that the immunobiotic TUA4408L-fermented okara feed could be an economical and environmentally friendly option to improve the growth performance and immune health of pigs.

  74. Lactiplantibacillus plantarum as a Potential Adjuvant and Delivery System for the Development of SARS-CoV-2 Oral Vaccines

    Julio Villena, Chang Li, Maria Guadalupe Vizoso-Pinto, Jacinto Sacur, Linzhu Ren, Haruki Kitazawa

    Microorganisms　2021/03/26

    DOI： 10.3390/microorganisms9040683 　

  75. Effect of Dietary Supplementation of Immunobiotic Lactiplantibacillusplantarum N14 Fermented Rakkyo (Allium chinense) Pickled Juice on the Immunocompetence and Production Performance of Pigs

    Md. Aminul Islam, Kenji Hashiguchi, A.K.M. Humayun Kober, Kyoko Morie, Binghui Zhou, Mikado Tomokiyo, Tomoyuki Shimazu, Hisashi Aso, Julio Villena, Yoshihito Suda, Haruki Kitazawa

    Animals　2021/03/09

    DOI： 10.3390/ani11030752 　

  76. Ligilactobacillus salivarius Strains Isolated From the Porcine Gut Modulate Innate Immune Responses in Epithelial Cells and Improve Protection Against Intestinal Viral-Bacterial Superinfection. International-journal

    Yuhki Indo, Shugo Kitahara, Mikado Tomokiyo, Shota Araki, Md Aminul Islam, Binghui Zhou, Leonardo Albarracin, Ayako Miyazaki, Wakako Ikeda-Ohtsubo, Tomonori Nochi, Takato Takenouchi, Hirohide Uenishi, Hisashi Aso, Hideki Takahashi, Shoichiro Kurata, Julio Villena, Haruki Kitazawa

    Frontiers in immunology　12　652923-652923　2021

    DOI： 10.3389/fimmu.2021.652923 　

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    Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-β expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-β, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-β, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets.

  77. Lactobacillus casei CRL431 modulates hemostatic activation induced by protein malnourishment and pneumococcal respiratory infection. International-journal

    Hortensia Zelaya, Jonathan Laiño, Julio Villena, Gabriela Marranzino, Susana Alvarez, Graciela Agüero

    Applied microbiology and biotechnology　104　(24)　10669-10683　2020/12

    DOI： 10.1007/s00253-020-10957-6 　

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    Previously, we demonstrated that Lactobacillus casei CRL431, a well-known immunomodulatory bacterium, beneficially regulates coagulation activation, fibrin formation in lung, and the pro-inflammatory state induced by protein malnourishment and pneumococcal infection. In this study, we deepen in the understanding of the mechanisms involved in the immunoregulatory activity of L. casei CRL431 during a nutritional repletion process by evaluating (a) platelet and endothelial activation, (b) tissue factor (TF) expression, and (c) protease-activated receptor (PAR) activation in an experimental bacterial respiratory infection model in malnourished mice. Our findings demonstrate for the first time that the repletion diet supplemented with L. casei CRL431 was effective to normalize platelet counts in blood, modulate platelet activation and their recruitment into the lung, and regulate local and systemic TF expression and endothelial activation, which were affected by malnourishment. Streptococcus pneumoniae challenge induced local and systemic increase of platelet counts, PARs activation, P-selectin and TF expression, as well as endothelial activation in both well-nourished and malnourished mice. Malnourished animals evidenced the highest alterations of the parameters evaluated while the mice fed with the probiotic bacterium had similar behavior to normal controls but with lower PAR activation in lung. These results demonstrate that supplementation of repletion diet with L. casei CRL431 is effective to modulate alterations induced by malnourishment and pneumococcal infection, restraining coagulation activation, the inflammatory process, and lung damage. These observations contribute to set the basis for the application of probiotic functional foods to modulate the inflammation-hemostasis interactions altered by malnourishment or bacterial respiratory infections. KEY POINTS: • Pneumococcal infection increases pro-coagulant state induced by protein malnourishment. • Repletion with L. casei CRL431 modulates platelet, TF, and endothelial activation. • L. casei CRL431 improves immune-coagulative response in protein malnourishment.

  78. Improvement of Disease Resistance in Livestock: Application of Immunogenomics and CRISPR/Cas9 Technology

    Md. Aminul Islam, Sharmin Aqter Rony, Mohammad Bozlur Rahman, Mehmet Ulas Cinar, Julio Villena, Muhammad Jasim Uddin, Haruki Kitazawa

    Animals　2020/11/28

    DOI： 10.3390/ani10122236 　

  79. Selection of Immunobiotic Ligilactobacillus salivarius Strains from the Intestinal Tract of Wakame-Fed Pigs: Functional and Genomic Studies International-journal

    Binghui Zhou, Leonardo Albarracin, Yuhki Indo, Lorena Arce, Yuki Masumizu, Mikado Tomokiyo, Md. Aminul Islam, Valeria Garcia-Castillo, Wakako Ikeda-Ohtsubo, Tomonori Nochi, Hidetoshi Morita, Hideki Takahashi, Shoichiro Kurata, Julio Villena, Haruki Kitazawa

    Microorganisms　8　(11)　2020/10

    DOI： 10.3390/microorganisms8111659 　

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    In this article, Ligilactobacillus salivarius FFIG strains, isolated from the intestinal tract of wakame-fed pigs, are characterized according to their potential probiotic properties. Strains were evaluated by studying their interaction with porcine intestinal epithelial (PIE) cells in terms of their ability to regulate toll-like receptor (TLR)-3- or TLR4-mediated innate immune responses, as well as by assessing their adhesion capabilities to porcine epithelial cells and mucins. These functional studies were complemented with comparative genomic evaluations using the complete genome sequences of porcine L. salivarius strains selected from subgroups that demonstrated different "immune" and "adhesion" phenotypes. We found that their immunomodulatory and adhesion capabilities are a strain-dependent characteristic. Our analysis indicated that the differential immunomodulatory and adhesive activities of FFIG strains would be dependent on the combination of several surface structures acting simultaneously, which include peptidoglycan, exopolysaccharides, lipoteichoic acid, and adhesins. Of note, our results indicate that there is no correlation between the immunomodulatory capacity of the strains with their adhesion ability to mucins and epithelial cells. Therefore, in the selection of strains destined to colonize the intestinal mucosa and modulate the immunity of the host, both properties must be adequately evaluated. Interestingly, we showed that L. salivarius FFIG58 functionally modulated the innate immune responses triggered by TLR3 and TLR4 activation in PIE cells and efficiently adhered to these cells. Moreover, the FFIG58 strain was capable of reducing rotavirus replication in PIE cells. Therefore, L. salivarius FFIG58 is a good candidate for further in vivo studying the protective effect of lactobacilli against intestinal infections in the porcine host. We also reported and analyzed, for the first time, the complete genome of several L. salivarius strains that were isolated from the intestine of pigs after the selective pressure of feeding the animals with wakame. Further genomic analysis could be of value to reveal the metabolic characteristics and potential of the FFIG strains in general and of the FFIG58 strain, in particular, relating to wakame by-products assimilation.

  80. Draft Genome Sequence of Ligilactobacillus salivarius FFIG58, Isolated from the Intestinal Tract of Wakame-Fed Pig

    Binghui Zhou, Leonardo Albarracin, Yuki Masumizu, Yuhki Indo, M. Aminul Islam, Valeria Garcia-Castillo, Wakako Ikeda-Ohtsubo, Yoshihito Suda, Hisashi Aso, Julio Villena, Haruki Kitazawa

    Microbiology Resource Announcements　2020/08/20

    DOI： 10.1128/MRA.00839-20 　

  81. New immunobiotics from highly proteolytic Lactobacillus delbrueckii strains: their impact on intestinal antiviral innate immune response Peer-reviewed

    M. Elean, L. Albarracín, P.G. Cataldo, A. Londero, H. Kitazawa, L. Saavedra, J. Villena, E.M. Hebert

    Beneficial Microbes　11　(4)　375-390　2020/08/12

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2019.0198 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    Proteolytic starter cultures with intrinsic immunomodulatory activities are desirably features for the development of functional foods, which would significantly reduce the cost of their production (one-strain starter) having an additional beneficial effect on the host. In this work, Lactobacillus delbrueckii strains were selected according to their ability to efficiently hydrolyse β-casein and to modulate the immune system. Among 36 strains evaluated, the highest proteolytic activities were found for L. delbrueckii subsp. lactis CRL581 and L. delbrueckii subsp. bulgaricus CRL656. The immunomodulatory effect of both strains and their β-casein hydrolysates (CRL581 and CRL656 hydrolysates, respectively) were studied in a murine model. Balb/c mice were fed lactobacilli or their hydrolysates for three days. One day after the last lactobacilli or hydrolysate treatments, mice were challenged with the Toll-like receptor 3 (TLR3) agonist poly(I:C) by intraperitoneal injection. Before and after poly(I:C) challenge the phagocytic and microbicidal activity of peritoneal macrophages, intestinal immunoglobulin A (IgA), cytokine profile, and histological analysis of the intestine were analysed. L. delbrueckii subsp. lactis CRL581 significantly increased the activation of peritoneal macrophages as well as the levels of intestinal IgA, interleukin (IL)-10 and interferon (IFN)-γ when compared to untreated controls. In addition, the CRL581 strain was able to significantly reduce the intestinal inflammatory damage triggered by TLR3 activation. L. delbrueckii CRL581 increased the levels of IL-10, IFN-γ and IFN-β, and reduced tumour necrosis factor alpha and IL-6 concentrations in the intestine of poly(I:C)-challenged mice. No immunomodulatory effects were observed for the CRL656 strain or for the CRL581 or CRL656 hydrolysates. The results of this work show that the technologically relevant and high proteolytic strain L. delbrueckii CRL581 is able to beneficially modulate the intestinal innate antiviral immune response. Although further studies with the CRL581 strain are required to corroborate and deepen its immunological effects, this bacterium is an interesting alternative for the development of new functional foods with antiviral capabilities.

  82. Draft Genome Sequence of Ligilactobacillus salivarius TUCO-L2, Isolated from Lama glama Milk

    Leonardo Albarracin, Sandra Quilodrán-Vega, Kohtaro Fukuyama, Mikado Tomokiyo, M. Aminul Islam, Lorena Arce, Haruki Kitazawa, Julio Villena

    Microbiology Resource Announcements　2020/08/06

    DOI： 10.1128/MRA.00784-20 　

  83. Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics. International-journal

    Asuka Tada, Akm Humayun Kober, Md Aminul Islam, Manami Igata, Michihiro Takagi, Masahiko Suzuki, Hisashi Aso, Wakako Ikeda-Ohtsubo, Kazutoyo Yoda, Kenji Miyazawa, Fang He, Hideki Takahashi, Julio Villena, Haruki Kitazawa

    Cells　9　(7)　2020/07/17

    DOI： 10.3390/cells9071715 　

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    The degree of fat accumulation and adipokine production are two major indicators of obesity that are correlated with increased adipose tissue mass and chronic inflammatory responses. Adipocytes have been considered effector cells for the inflammatory responses due to their capacity to express Toll-like receptors (TLRs). In this study, we evaluated the degree of fat accumulation and adipokine production in porcine intramuscular preadipocyte (PIP) cells maintained for in vitro differentiation over a long period without or with stimulation of either TNF-α or TLR2-, TLR3-, or TLR4-ligands. The cytosolic fat accumulation was measured by liquid chromatography and the expression of adipokines (CCL2, IL-6, IL-8 and IL-10) were quantified by RT-qPCR and ELISA at several time points (0 to 20 days) of PIP cells differentiation. Long-term adipogenic differentiation (LTAD) induced a progressive fat accumulation in the adipocytes over time. Activation of TLR3 and TLR4 resulted in an increased rate of fat accumulation into the adipocytes over the LTAD. The production of CCL2, IL-8 and IL-6 were significantly increased in unstimulated adipocytes during the LTAD, while IL-10 expression remained stable over the studied period. An increasing trend of adiponectin and leptin production was also observed during the LTAD. On the other hand, the stimulation of adipocytes with TLRs agonists or TNF-α resulted in an increasing trend of CCL2, IL-6 and IL-8 production while IL-10 remained stable in all four treatments during the LTAD. We also examined the influences of several immunoregulatory probiotic strains (immunobiotics) on the modulation of the fat accumulation and adipokine production using supernatants of immunobiotic-treated intestinal immune cells and the LTAD of PIP cells. Immunobiotics have shown a strain-specific ability to modulate the fat accumulation and adipokine production, and differentiation of adipocytes. Here, we expanded the utility and potential application of our in vitro PIP cells model by evaluating an LTAD period (20 days) in order to elucidate further insights of chronic inflammatory pathobiology of adipocytes associated with obesity as well as to explore the prospects of immunomodulatory intervention for obesity such as immunobiotics.

  84. Erratum: Fukuyama, K., et al. Evaluation of the Immunomodulatory Ability of Lactic Acid Bacteria Isolated from Feedlot Cattle Against Mastitis Using a Bovine Mammary Epithelial Cells In Vitro Assay. Pathogens 2020, 9, 410 International-journal

    Kohtaro Fukuyama, Md. Aminul Islam, Michihiro Takagi, Wakako Ikeda-Ohtsubo, Shoichiro Kurata, Hisashi Aso, Maria Elena Fatima Nader-Macías, Graciela Vignolo, Julio Villena, Haruki Kitazawa

    Pathogens　9　(7)　2020/07/16

    DOI： 10.3390/pathogens9070574 　

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    The authors would like to make the following corrections about the published paper [...].

  85. The Role of Alveolar Macrophages in the Improved Protection against Respiratory Syncytial Virus and Pneumococcal Superinfection Induced by the Peptidoglycan of Lactobacillus rhamnosus CRL1505 International-journal

    Patricia Clua, Mikado Tomokiyo, Fernanda Raya Tonetti, Md. Aminul Islam, Valeria García Castillo, Guillermo Marcial, Susana Salva, Susana Alvarez, Hideki Takahashi, Shoichiro Kurata, Haruki Kitazawa, Julio Villena

    Cells　9　(7)　2020/07

    DOI： 10.3390/cells9071653 　

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    The nasal priming with nonviable Lactobacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulates the respiratory innate immune response in infant mice, improving their resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, it was found that NV1505 or PG1505 significantly enhance the numbers of CD11c+SiglecF+ alveolar macrophages (AMs) producing interferon (IFN)-β. In this work, we aimed to further advance in the characterization of the beneficial effects of NV1505 and PG1505 in the context of a respiratory superinfection by evaluating whether their immunomodulatory properties are dependent on AM functions. Macrophage depletion experiments and a detailed study of their production of cytokines and antiviral factors clearly demonstrated the key role of this immune cell population in the improvement of both the reduction of pathogens loads and the protection against lung tissue damage induced by the immunobiotic CRL1505 strain. Studies at basal conditions during primary RSV or S. pneumoniae infections, as well as during secondary pneumococcal pneumonia, brought the following five notable findings regarding the immunomodulatory effects of NV1505 and PG1505: (a) AMs play a key role in the beneficial modulation of the respiratory innate immune response and protection against RSV infection, (b) AMs are necessary for improved protection against primary and secondary pneumococcal pneumonia, (c) the generation of activated/trained AMs would be essential for the enhanced protection against respiratory pathogens, (d) other immune and nonimmune cell populations in the respiratory tract may contribute to the protection against bacterial and viral infections, and (e) the immunomodulatory properties of NV1505 and PG1505 are strain-specific. These findings significantly improve our knowledge about the immunological mechanisms involved in the modulation of respiratory immunity induced by beneficial microbes.

  86. The Modulation of Mucosal Antiviral Immunity by Immunobiotics: Could They Offer Any Benefit in the SARS-CoV-2 Pandemic? Peer-reviewed

    Julio Villena, Haruki Kitazawa

    Frontiers in Physiology　11　2020/06/16

    Publisher: Frontiers Media SA

    DOI： 10.3389/fphys.2020.00699 　

    eISSN： 1664-042X

  87. Nasal Priming with Lactobacillus rhamnosus CRL1505 Stimulates Mononuclear Phagocytes of Immunocompromised Malnourished Mice: Improvement of Respiratory Immune Response. International-journal

    Natalia Barbieri, Susana Salva, Matías Herrera, Julio Villena, Susana Alvarez

    Probiotics and antimicrobial proteins　12　(2)　494-504　2020/06

    DOI： 10.1007/s12602-019-09551-8 　

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    The effect of Lactobacillus rhamnosus CRL1505 (Lr) on macrophages (Ma) and dendritic cells (DC) in the orchestration of anti-pneumococcal immunity was studied using malnutrition and pneumococcal infection mouse models. Monocytes (Mo), Ma, and DC in two groups of malnourished mice fed with balanced diet (BCD) were studied through flow cytometry; one group was nasally administered with Lr (BCD+Lr group), and the other group was not (BCD group). Well-nourished (WNC) and malnourished (MNC) mice were used as controls.Malnutrition affected the number of respiratory and splenic mononuclear phagocytes. The BCD+Lr treatment, unlike BCD, was able to increase and normalize lung Mo and Ma. The BCD+Lr mice were also able to upregulate the expression of the activation marker MHC II in lung DC and to improve this population showing a more significant effect on CD11b+ DC subpopulation. At post-infection, lung Mo values were higher in BCD+Lr mice than in BCD mice and similar to those obtained in WNC group. Although both repletion treatments showed similar values of lung Ma post-infection, the Ma activation state in BCD+Lr mice was higher than that in BCD mice. Furthermore, BCD+Lr treatment was able to normalize the number and activation of splenic Ma and DC after the challenge.Lr administration stimulates respiratory and systemic mononuclear phagocytes. Stimulation of Ma and DC populations would increase the microbicide activity and improve the adaptive immunity through its antigen-presenting capacity. Thus, Lr contributes to improved outcomes of pneumococcal infection in immunocompromised hosts.

  88. Modulation of Toll-like receptor-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle Peer-reviewed

    F. Mansilla, M. Takagi, V. Garcia-Castillo, H. Aso, M.E. Nader-Macias, G. Vignolo, H. Kitazawa, J. Villena

    Beneficial Microbes　11　(3)　269-282　2020/05/11

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2019.0189 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    The ability of lactobacilli isolated from feedlot cattle environment to differentially modulate the innate immune response triggered by Toll-like receptors (TLRs) activation in bovine intestinal epithelial (BIE) cells was evaluated. BIE cells were stimulated with Lactobacillus mucosae CRL2069, Lactobacillus acidophilus CRL2074, Lactobacillus fermentum CRL2085 or Lactobacillus rhamnosus CRL2084 and challenged with heat-stable pathogen associated molecular patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) to induce the activation of TLR4 or with polyinosinic:polycytidylic acid (poly(I:C)) to activate TLR3. Type I interferons, cytokines, chemokines and negative regulators of TLR signalling were studied by RT-PCR. L. mucosae CRL2069 significantly reduced the expression of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 in BIE cells in the context of TLR3 activation. L. mucosae CRL2069 also reduced the expression of tumour necrosis factor-α, IL-β, MCP-1, and IL-8 in heat-stable ETEC PAMPs-challenged BIE cells. In addition, reduced expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for the CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR negative regulators. L. mucosae CRL2069 significantly improved the expression of A20-binding inhibitor of NFκ-B activation 3 (ABIN-3), interleukin-1 receptor-associated kinase M (IRAK-M) and mitogen-activated protein kinase 1 (MKP-1) while L. rhamnosus CRL2084 augmented ABIN-3 expression in BIE cells. The results of this work suggest that among the studied strains, L. mucosae CRL2069 was able to regulate TLR3-mediated innate immune response and showed a remarkable capacity to modulate TLR4-mediated inflammation in BIE cells. The CRL2069 strain induce the up-regulation of three TLR negative regulators that would influence nuclear factor kB and mitogen-activated protein kinases signalling pathways while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting immunobiotic candidate for the protection of the bovine host against TLR-mediated intestinal inflammatory damage.

  89. The Ability of Respiratory Commensal Bacteria to Beneficially Modulate the Lung Innate Immune Response Is a Strain Dependent Characteristic International-journal

    Ramiro Ortiz Moyano, Fernanda Raya Tonetti, Mikado Tomokiyo, Paulraj Kanmani, María Guadalupe Vizoso-Pinto, Hojun Kim, Sandra Quilodrán-Vega, Vyacheslav Melnikov, Susana Alvarez, Hideki Takahashi, Shoichiro Kurata, Haruki Kitazawa, Julio Villena

    Microorganisms　8　(5)　2020/05

    DOI： 10.3390/microorganisms8050727 　

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    We investigated whether the ability of commensal respiratory bacteria to modulate the innate immune response against bacterial and viral pathogens was a shared or strain-specific characteristic. Bacterial strains belonging to the Corynebacterium pseudodiphtheriticum and Dolosigranulum pigrum species were compared by studying their influence in the Toll-like receptor (TLR)-2- and TLR3-triggered immune responses in the respiratory tract, as well as in the resistance to Respiratory Syncytial Virus (RSV) and Streptococcus pneumoniae infections. We demonstrated that nasally administered C. pseudodiphteriticum 090104 or D. pigrum 040417 were able to modulate respiratory immunity and increase the resistance against pathogens, while other strains of the same species did not influence the respiratory immune responses, demonstrating a clear strain-dependent immunomodulatory effect of respiratory commensal bacteria. We also reported here that bacterium-like particles (BLP) and cell walls derived from immunomodulatory respiratory commensal bacteria are an interesting alternative for the modulation of the respiratory immune system. Our study is a step forward in the positioning of certain strains of respiratory commensal bacteria as next-generation probiotics for the respiratory tract.

  90. The Exopolysaccharide of Lactobacillus fermentum UCO-979C Is Partially Involved in Its Immunomodulatory Effect and Its Ability to Improve the Resistance against Helicobacter pylori Infection

    Valeria Garcia-Castillo, Guillermo Marcial, Leonardo Albarracín, Mikado Tomokiyo, Patricia Clua, Hideki Takahashi, Haruki Kitazawa, Apolinaria Garcia-Cancino, Julio Villena

    Microorganisms　2020/03/27

    DOI： 10.3390/microorganisms8040479 　

  91. Transcriptome Analysis of The Inflammatory Responses of Bovine Mammary Epithelial Cells: Exploring Immunomodulatory Target Genes for Bovine Mastitis. International-journal

    Md Aminul Islam, Michihiro Takagi, Kohtaro Fukuyama, Ryoya Komatsu, Leonardo Albarracin, Tomonori Nochi, Yoshihito Suda, Wakako Ikeda-Ohtsubo, Victor Rutten, Willem van Eden, Julio Villena, Hisashi Aso, Haruki Kitazawa

    Pathogens (Basel, Switzerland)　9　(3)　2020/03/09

    DOI： 10.3390/pathogens9030200 　

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    Bovine mastitis is the inflammatory reaction of the mammary gland and is commonly caused by bacterial infections in high-yielding dairy cows. The detailed investigation of the immunotranscriptomic response of bovine mammary epithelial (BME) cells to pattern recognition receptors (PRRs) activation by microbial-associated molecular patterns (MAMPs) can be of great importance for understanding the innate immune defense mechanisms, and for exploring the immunomodulatory candidate genes. In this work, we investigated the transcriptome modifications of BME cells after the in vitro stimulation with Escherichia coli derived lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus JE2 and S. aureus SA003. In addition, the effect of Pam3CSK4 (a synthetic triacylated lipopeptide that activates Toll-like receptor 2 (TLR2)), and the intracellular chemotactic protein cyclophilin A (CyPA), which is secreted by BME cells during mastitis, in the expression changes of selected cytokines and chemokines were evaluated by qPCR. Microarray analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in the BME cells after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. A major differential response in the inflammatory gene expression was noticed between the stimulation of LPS and S. aureus strains. Unlike the S. aureus strains, LPS stimulation resulted in significant upregulation of CCL2, CXCL2, CXCL3, CXCL8, IL1 and IL1, which were confirmed by qPCR analysis. Pam3CSK4 was not able to induce significant changes in the expression of cytokines and chemokines in challenged BME cells. The exogenous CyPA administration was able to upregulate CXCL2, CXCL3, CXCL8, IL1 and IL1 expression in BME cells indicating its ability to promote inflammation. The identification of transcriptional markers of mastitis specific for individual inflammatory factors such as LPS, Pam3CSK4 or CyPA, which can be evaluated in vitro in BME cells, may enable the development of novel diagnostics and/or immunomodulatory treatments, providing new tools for the effective management of mastitis in dairy cows. The results of this work are an advance in this regard.

  92. Immunomodulatory Properties of Bacterium-Like Particles Obtained From Immunobiotic Lactobacilli: Prospects for Their Use as Mucosal Adjuvants. International-journal

    Fernanda Raya Tonetti, Lorena Arce, Susana Salva, Susana Alvarez, Hideki Takahashi, Haruki Kitazawa, Maria Guadalupe Vizoso-Pinto, Julio Villena

    Frontiers in immunology　11　15-15　2020

    DOI： 10.3389/fimmu.2020.00015 　

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    Non-viable lactic acid bacteria (LAB) have been proposed as antigen delivery platforms called bacterium-like particles (BLPs). Most studies have been performed with Lactococcus lactis-derived BLPs where multiple antigens were attached to the peptidoglycan surface and used to successfully induce specific immune responses. It is well-established that the immunomodulatory properties of LAB are strain dependent and therefore, the BLPs derived from each individual strain could have different adjuvant capacities. In this work, we obtained BLPs from immunomodulatory (immunobiotics) and non-immunomodulatory Lactobacillus rhamnosus and Lactobacillus plantarum strains and comparatively evaluated their ability to improve the intestinal and systemic immune responses elicited by an attenuated rotavirus vaccine. Results demonstrated that orally administered BLPs from non-immunomodulatory strains did not induce significant changes in the immune response triggered by rotavirus vaccine in mice. On the contrary, BLPs derived from immunobiotic lactobacilli were able to improve the levels of anti-rotavirus intestinal IgA and serum IgG, the numbers of CD24+B220+ B and CD4+ T cells in Peyer's patches and spleen as well as the production of IFN-γ by immune cells. Interestingly, among immunobiotics-derived BLPs, those obtained from L. rhamnosus CRL1505 and L. rhamnosus IBL027 enhanced more efficiently the intestinal and systemic humoral immune responses when compared to BLPs from other immunobiotic bacteria. The findings of this work indicate that it is necessary to perform an appropriate selection of BLPs in order to find those with the most efficient adjuvant properties. We propose the term Immunobiotic-like particles (IBLPs) for the BLPs derived from CRL1505 and IBL027 strains that are an excellent alternative for the development of mucosal vaccines.

  93. Nasal priming with immunobiotic lactobacilli improves the adaptive immune response against influenza virus. International-journal

    Fernanda Raya Tonetti, Md Aminul Islam, Maria Guadalupe Vizoso-Pinto, Hideki Takahashi, Haruki Kitazawa, Julio Villena

    International immunopharmacology　78　106115-106115　2020/01

    DOI： 10.1016/j.intimp.2019.106115 　

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    The nasal priming with Lactobacillus rhamnosus CRL1505 modulates the respiratory antiviral innate immune response and improves protection against influenza virus (IFV) challenge in mice. However, the potential beneficial effect of the CRL1505 strain on the adaptive immune response triggered by IFV infection or vaccination was not evaluated before. In this work, we demonstrated that nasally administered L. rhamnosus CRL1505 is able to improve both the humoral and cellular adaptive immune responses induced by IFV infection or vaccination. Higher levels of IFV-specific IgA and IgG as well as IFN-γ were found in the serum and the respiratory tract of CRL1505-treated mice after IFV challenge. Lactobacilli treated mice also showed reduced concentrations of IL-17 and improved levels of IL-10 during IFV infection. The differential balance of inflammatory and regulatory cytokines induced by L. rhamnosus CRL1505 contributed to the protection against IFV by favoring an effective effector immune response without inducing inflammatory-mediated lung damage. The optimal immunomodulatory effect of the CRL1505 strain was achieved with viable bacteria. However, non-viable L. rhamnosus CRL1505 was also efficient in improving the adaptive immune responses generated by IFV challenges and therefore, emerged as an interesting alternative for vaccination of immunocompromised hosts. Similar to other immunomodulatory properties of lactobacilli, it was shown here that the adjuvant effect in the context of IFV vaccination was a strain dependent ability, since differences were found when L. rhamnosus CRL1505 and the immunomodulatory strain L. rhamnosus IBL027 were compared. This investigation represents a thorough exploration of the role of immunobiotic lactobacilli in improving humoral and cellular adaptive immune responses against IFV in the context of both infection and vaccination.

  94. Efficient Selection of New Immunobiotic Strains With Antiviral Effects in Local and Distal Mucosal Sites by Using Porcine Intestinal Epitheliocytes. International-journal

    Leonardo Albarracin, Valeria Garcia-Castillo, Yuki Masumizu, Yuhki Indo, Md Aminul Islam, Yoshihito Suda, Apolinaria Garcia-Cancino, Hisashi Aso, Hideki Takahashi, Haruki Kitazawa, Julio Villena

    Frontiers in immunology　11　543-543　2020

    DOI： 10.3389/fimmu.2020.00543 　

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    Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNβ, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-β and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells.

  95. Lipoteichoic Acid Is Involved in the Ability of the Immunobiotic Strain Lactobacillus plantarum CRL1506 to Modulate the Intestinal Antiviral Innate Immunity Triggered by TLR3 Activation. International-journal

    Hiroya Mizuno, Lorena Arce, Kae Tomotsune, Leonardo Albarracin, Ryutaro Funabashi, Daniela Vera, Md Aminul Islam, Maria Guadalupe Vizoso-Pinto, Hideki Takahashi, Yasuko Sasaki, Haruki Kitazawa, Julio Villena

    Frontiers in immunology　11　571-571　2020

    DOI： 10.3389/fimmu.2020.00571 　

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    Studies have demonstrated that lipoteichoic acid (LTA) is involved in the immunomodulatory properties of some immunobiotic lactobacilli. The aim of this work was to evaluate whether LTA contributes to the capacity of Lactobacillus plantarum CRL1506 in modulating the intestinal innate antiviral immune response. A D-alanyl-lipoteichoic acid biosynthesis protein (dltD) knockout CRL1506 strain (L. plantarumΔdltD) was obtained, and its ability to modulate Toll-like receptor (TLR)-3-mediated immune response was evaluated in vitro in porcine intestinal epithelial (PIE) cells and in vivo in Balb/c mice. Wild-type (WT) CRL1506 (L. plantarum WT) was used as positive control. The challenge of PIE cells with the TLR3 agonist poly(I:C) significantly increased interferon (IFN)-β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 expressions. PIE cells pretreated with L. plantarumΔdltD or L. plantarum WT showed higher levels of IFN-β while only L. plantarum WT significantly reduced the expression of IL-6 and MCP-1 when compared with poly(I:C)-treated control cells. The oral administration of L. plantarum WT to mice prior the intraperitoneal injection of poly(I:C) significantly increased IFN-β and IL-10 and reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6, and IL-15) in the intestinal mucosa. Similar to the WT strain, L. plantarumΔdltD-treated mice showed enhanced levels of IFN-β after poly(I:C) challenge. However, treatment of mice with L. plantarumΔdltD was not able to increase IL-10 or reduce CD3+NK1.1+CD8αα+ cells, TNF-α, IL-6, or IL-15 in the intestine. These results indicate that LTA would be a key molecule in the anti-inflammatory effect induced by the CRL1506 strain in the context of TLR3-mediated inflammation.

  96. Exopolysaccharides From Streptococcus thermophilus ST538 Modulate the Antiviral Innate Immune Response in Porcine Intestinal Epitheliocytes. International-journal

    Hiroya Mizuno, Kae Tomotsune, Md Aminul Islam, Ryutaro Funabashi, Leonardo Albarracin, Wakako Ikeda-Ohtsubo, Hisashi Aso, Hideki Takahashi, Katsunori Kimura, Julio Villena, Yasuko Sasaki, Haruki Kitazawa

    Frontiers in microbiology　11　894-894　2020

    DOI： 10.3389/fmicb.2020.00894 　

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    It was reported that exopolysaccharides (EPSs) from lactobacilli are able to differentially modulate mucosal antiviral immunity. Although research has described the ability of EPSs derived from Streptococcus thermophilus to modulate the mucosal immune system, their impact on antiviral immunity was less explored. In this work, we investigated the capacity of the EPS-producing S. thermophilus ST538 to modulate the innate antiviral immune response triggered by the activation of the Toll-like receptor 3 (TLR3) in porcine intestinal epitheliocytes (PIE cells). Moreover, in order to study the immunomodulatory potential of S. thermophilus ST538 EPS, we successfully developed two mutant strains through the knockout of the epsB or epsC genes. High-performance liquid chromatography and scanning electron microscopy studies demonstrated that the wild type (WT) strain produced as high as 595 μg/ml of EPS in the skim milk medium, while none of the mutant strains (S. thermophilus ΔepsB and ΔepsC) were able to produce EPS. Studies in PIE cells demonstrated that the EPS of S. thermophilus ST538 is able to significantly improve the expression of interferon β (IFN-β), interleukin 6 (IL-6), and C-X-C motif chemokine 10 (CXCL10) in response to TLR3 stimulation. The role of EPS in the modulation of antiviral immune response in PIE cells was confirmed by comparative studies of cell free culture supernatants and fermented skim milks obtained from S. thermophilus ΔepsB and ΔepsC. These results suggest that S. thermophilus ST538 could be used as an immunobiotic strain for the development of new immunologically functional foods, which might contribute to improve resistance against viral infections.

  97. Immunobiotic Lactobacillus jensenii TL2937 Alleviates Dextran Sodium Sulfate-Induced Colitis by Differentially Modulating the Transcriptomic Response of Intestinal Epithelial Cells. International-journal

    Nana Sato, Valeria Garcia-Castillo, Mao Yuzawa, Md Aminul Islam, Leonardo Albarracin, Mikado Tomokiyo, Wakako Ikeda-Ohtsubo, Apolinaria Garcia-Cancino, Hideki Takahashi, Julio Villena, Haruki Kitazawa

    Frontiers in immunology　11　2174-2174　2020

    DOI： 10.3389/fimmu.2020.02174 　

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    Immunobiotics have emerged as a promising intervention to alleviate intestinal damage in inflammatory bowel disease (IBD). However, the beneficial properties of immunobiotics are strain dependent and, therefore, each strain has to be evaluated in order to demonstrate its potential application in IBD. Our previous in vitro and in vivo studies demonstrated that Lactobacillus jensenii TL2937 attenuates gut acute inflammatory response triggered by Toll-like receptor 4 activation. However, its effect on colitis has not been evaluated before. In this work, we studied whether the TL2937 strain was able to protect against the development of colitis in a dextran sodium sulfate (DSS)-induced mouse model and we delved into the mechanisms of action by evaluating the effect of the immunobiotic bacteria on the transcriptomic response of DSS-challenged intestinal epithelial cells. L. jensenii TL2937 was administered to adult BALB/c mice before the induction of colitis by the administration of DSS. Colitis and the associated inflammatory response were evaluated for 14 days. Mice fed with L. jensenii TL2937 had lower disease activity index and alterations of colon length when compared to control mice. Reduced myeloperoxidase activity, lower production of pro-inflammatory (TNF-α, IL-1, CXCL1, MCP-1, IL-15, and IL-17), and higher levels of immunoregulatory (IL-10 and IL-27) cytokines were found in the colon of TL2937-treated mice. In addition, the treatment of porcine intestinal epithelial (PIE) cells with L. jensenii TL2937 before the challenge with DSS differentially regulated the activation of the JNK pathway, leading to an increase in epithelial cell integrity and to a differential immunotranscriptomic response. TL2937-treated PIE cells had a significant reduction in the expression of inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6, IL-15), chemokines (CCL2, CCL4, CCL8, CXCL4, CXCL5, CXCL9, CXCL10), adhesion molecules (SELE, SELL, EPCAM), and other immune factors (NCF1, NCF2, NOS2, SAA2) when compared to control cells after the challenge with DSS. The findings of this work indicate that (a) L. jensenii TL2937 is able to alleviate DSS-induced colitis suggesting a potential novel application for this immunobiotic strain, (b) the modulation of the transcriptomic response of intestinal epithelial cells would play a key role in the beneficial effects of the TL2937 strain on colitis, and (c) the in vitro PIE cell immunoassay system could be of value for the screening and selection of new immunobiotic strains for their application in IBD.

  98. Alveolar Macrophages Are Key Players in the Modulation of the Respiratory Antiviral Immunity Induced by Orally Administered Lacticaseibacillus rhamnosus CRL1505. International-journal

    Valeria Garcia-Castillo, Mikado Tomokiyo, Fernanda Raya Tonetti, Md Aminul Islam, Hideki Takahashi, Haruki Kitazawa, Julio Villena

    Frontiers in immunology　11　568636-568636　2020

    DOI： 10.3389/fimmu.2020.568636 　

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    The oral administration of Lacticaseibacillus rhamnosus CRL1505 differentially modulates the respiratory innate antiviral immune response triggered by Toll-like receptor 3 (TLR3) activation in infant mice, improving the resistance to Respiratory Syncytial Virus (RSV) infection. In this work, by using macrophages depletion experiments and a detailed study of their production of cytokines and antiviral factors we clearly demonstrated the key role of this immune cell population in the improvement of both viral elimination and the protection against lung tissue damage induced by the CRL1505 strain. Orally administered L. rhamnosus CRL1505 activated alveolar macrophages and enhanced their ability to produce type I interferons (IFNs) and IFN-γ in response to RSV infection. Moreover, an increased expression of IFNAR1, Mx2, OAS1, OAS2, RNAseL, and IFITM3 was observed in alveolar macrophages after the oral treatment with L. rhamnosus CRL1505, which was consistent with the enhanced RSV clearance. The depletion of alveolar macrophages by the time of L. rhamnosus CRL1505 administration abolished the ability of infant mice to produce increased levels of IL-10 in response to RSV infection. However, no improvement in IL-10 production was observed when primary cultures of alveolar macrophages obtained from CRL1505-treated mice were analyzed. Of note, alveolar macrophages from the CRL1505 group had an increased production of IL-6 and IL-27 suggesting that these cells may play an important role in limiting inflammation and protecting lung function during RSV infection, by increasing the maturation and activation of Treg cells and their subsequent production of IL-10. In addition, we provided evidence of the important role of CD4+ cells and IFN-γ in the activation of alveolar macrophages highlighting a putative pathway through which the intestinal and respiratory mucosa are communicated under the influence of L. rhamnosus CRL1505.

  99. Functional and Genomic Characterization of Ligilactobacillus salivarius TUCO-L2 Isolated From Lama glama Milk: A Promising Immunobiotic Strain to Combat Infections. International-journal

    Sandra Quilodrán-Vega, Leonardo Albarracin, Flavia Mansilla, Lorena Arce, Binghui Zhou, Md Aminul Islam, Mikado Tomokiyo, Imad Al Kassaa, Yoshihito Suda, Haruki Kitazawa, Julio Villena

    Frontiers in microbiology　11　608752-608752　2020

    DOI： 10.3389/fmicb.2020.608752 　

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    Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

  100. Immunomodulatory Properties of a γ-Aminobutyric Acid-Enriched Strawberry Juice Produced by Levilactobacillus brevis CRL 2013. International-journal

    Pablo G Cataldo, Julio Villena, Mariano Elean, Graciela Savoy de Giori, Lucila Saavedra, Elvira M Hebert

    Frontiers in microbiology　11　610016-610016　2020

    DOI： 10.3389/fmicb.2020.610016 　

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    Gamma-aminobutyric acid (GABA) plays a key role in mammals as the major inhibitory neurotransmitter of the central nervous system. Although GABA may not be able to cross the human blood-brain barrier, it was approved as a food ingredient because of its benefits to the host after oral administration including anti-hypertensive, anti-depressant and anti-inflammatory activities. Considering the current trend toward the development of new functional and natural products and that microbial fermentation is one of the most promising methods to produce this non-protein amino acid, the in situ production of GABA through fermentation of strawberry and blueberry juices by the efficient GABA producer strain, Levilactobacillus brevis (formerly known as Lactobacillus brevis) CRL 2013, was evaluated. A high GABA production (262 mM GABA) was obtained after fermenting strawberry juice supplemented with yeast extract for 168 h, being GABA yield significantly higher in strawberry juices than in the blueberry ones. Thus, GABA-enriched fermented strawberry juice (FSJ) was selected to carry out in vivo and in vitro studies. The in vitro functional analysis of the GABA-enriched FSJ demonstrated its ability to significantly decrease the expression of cox-2 gene in LPS stimulated RAW 264.7 macrophages. In addition, in vivo studies in mice demonstrated that both, L. brevis CRL 2013 and the GABA-enriched FSJ were capable of reducing the levels of peritoneal, intestinal and serum TNF-α, IL-6, and CXCL1, and increasing IL-10 and IFN-γ in mice exposed to an intraperitoneal challenge of LPS. Of note, the GABA-enriched FSJ was more efficient than the CRL 2013 strain to reduce the pro-inflammatory factors and enhance IL-10 production. These results indicated that the CRL 2013 strain exerts anti-inflammatory effects in the context of LPS stimulation and that this effect is potentiated by fermentation. Our results support the potential use of L. brevis CRL 2013 as an immunomodulatory starter culture and strawberry juice as a remarkable vegetable matrix for the manufacture of GABA-enriched fermented functional foods capable of differentially modulating the inflammatory response triggered by TLR4 activation.

  101. Transcriptome Modifications in the Porcine Intramuscular Adipocytes during Differentiation and Exogenous Stimulation with TNF-α and Serotonin International-journal

    Asuka Tada, Md Aminul Islam, AKM Humayun Kober, Kohtaro Fukuyama, Michihiro Takagi, Manami Igata, Leonardo Albarracin, Wakako Ikeda-Ohtsubo, Kenji Miyazawa, Kazutoyo Yoda, Fang He, Hideki Takahashi, Julio Villena, Hisashi Aso, Haruki Kitazawa

    International Journal of Molecular Sciences　21　(2)　2020/01

    DOI： 10.3390/ijms21020638 　

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    Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the 'PPAR signaling' and 'insulin resistance' pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of 'GPCR ligand-binding', 'cell chemotaxis', 'blood coagulation and complement', 'metabolism of lipid and lipoproteins', 'regulation of lipid metabolism by PPARA', and 'lipid digestion, mobilization and transport' pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.

  102. Oral Vaccination with Hepatitis E Virus Capsid Protein and Immunobiotic Bacterium-Like Particles Induce Intestinal and Systemic Immunity in Mice Peer-reviewed

    L. P. Arce, M. F. Raya Tonetti, M. P. Raimondo, M. F. Müller, S. Salva, S. Álvarez, A. Baiker, J. Villena, M. G. Vizoso Pinto

    Probiotics and Antimicrobial Proteins　12　(3)　961-972　2019/10/19

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1007/s12602-019-09598-7 　

    ISSN： 1867-1306

    eISSN： 1867-1314

  103. 豚小腸由来の抗ウイルスイムノバイオティクス(Anti-viral immunobiotics from porcine small intestine) Peer-reviewed

    犬童 優樹, Zhou Binghui, Islam Md. Aminul, 宮崎 綾子, 大坪 和香子, 須田 義人, 麻生 久, Villena Julio, 北澤 春樹

    日本畜産学会大会講演要旨集　126回　85-85　2019/09

    Publisher: (公社)日本畜産学会

    ISSN： 1342-4688

  104. Characterization of the immunomodulatory and anti-Helicobacter pylori properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A. International-journal

    Valeria Garcia-Castillo, Ana María Marín-Vega, Alejandra Ilabaca, Leonardo Albarracín, Guillermo Marcial, Haruki Kitazawa, Apolinaria Garcia-Cancino, Julio Villena

    Biofouling　35　(8)　922-937　2019/09

    DOI： 10.1080/08927014.2019.1675153 　

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    The ability to form biofilms and the potential immunomodulatory properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A were characterized in vitro. It was demonstrated that L. rhamnosus UCO-25A is able to form biofilms on abiotic and cell surfaces, and to modulate the inflammatory response triggered by Helicobacter pylori infection in gastric epithelial cells and THP-1 macrophages. L. rhamnosus UCO-25A exhibited a substantial anti-inflammatory effect in both cell lines and improved IL-10 levels produced by challenged macrophages. Additionally, UCO-25A protected AGS cells against H. pylori infection with a higher pathogen inhibition when a biofilm was formed. Given the importance of inflammation in H. pylori-mediated diseases, the differential modulation of the inflammatory response in the gastric mucosa by an autochthonous strain is an attractive alternative for improving H. pylori eradication and reducing the severity of the diseases that arise from the resulting chronic inflammation.

  105. Paraimmunobiotic Bifidobacteria Modulate the Expression Patterns of Peptidoglycan Recognition Proteins in Porcine Intestinal Epitheliocytes and Antigen Presenting Cells. International-journal

    Hikaru Iida, Masanori Tohno, Md Aminul Islam, Nana Sato, Hisakazu Kobayashi, Leonardo Albarracin, Akm Humayun Kober, Wakako Ikeda-Ohtsubo, Yoshihito Suda, Hisashi Aso, Tomonori Nochi, Ayako Miyazaki, Hirohide Uenishi, Noriyuki Iwabuchi, Jin-Zhong Xiao, Julio Villena, Haruki Kitazawa

    Cells　8　(8)　2019/08/14

    DOI： 10.3390/cells8080891 　

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    Peptidoglycan recognition proteins (PGLYRPs) are a family of pattern recognition receptors (PRRs) that are able to induce innate immune responses through their binding to peptidoglycan (PGN), lipopolysaccharide, or lipoteichoic acid, or by interacting with other PRR-ligands. Recently, progress has been made in understanding the immunobiology of PGLYRPs in human and mice, however, their functions in livestock animals have been less explored. In this study, we characterized the expression patterns of PGLYRPs in porcine intestinal epithelial (PIE) cells and antigen-presenting cells (APCs) and their modulation by the interactions of host cells with PRR-ligands and non-viable immunomodulatory probiotics referred to as paraimmunobiotics. We demonstrated that PGLYRP-1, -2, -3, and -4 are expressed in PIE cells and APCs from Peyer's patches, being PGLYPR-3 and -4 levels higher than PGLYRP-1 and -2. We also showed that PGLYRPs expression in APCs and PIE cells can be modulated by different PRR agonists. By using knockdown PIE cells for TLR2, TLR4, NOD1, and NOD2, or the four PGLYRPs, we demonstrated that PGLYRPs expressions would be required for activation and functioning of TLR2, TLR4, NOD1, and NOD2 in porcine epitheliocytes, but PGLYRPs activation would be independent of those PRR expressions. Importantly, we reported for the first time that PGLYRPs expression can be differentially modulated by paraimmunobiotic bifidobacteria in a strain-dependent manner. These results provide evidence for the use of paraimmunobiotic bifidobacteria as an alternative for the improvement of resistance to intestinal infections or as therapeutic tools for the reduction of the severity of inflammatory damage in diseases in which a role of PGLYRPs-microbe interaction has been demonstrated.

  106. Isolation and Immunocharacterization of Lactobacillus salivarius from the Intestine of Wakame-Fed Pigs to Develop Novel "Immunosynbiotics". International-journal

    Yuki Masumizu, Binghui Zhou, A K M Humayun Kober, Md Aminul Islam, Hikaru Iida, Wakako Ikeda-Ohtsubo, Yoshihito Suda, Leonardo Albarracin, Tomonori Nochi, Hisashi Aso, Keiichi Suzuki, Julio Villena, Haruki Kitazawa

    Microorganisms　7　(6)　2019/06/06

    DOI： 10.3390/microorganisms7060167 　

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    Emerging threats of antimicrobial resistance necessitate the exploration of effective alternatives for healthy livestock growth strategies. 'Immunosynbiotics', a combination of immunoregulatory probiotics and prebiotics with synergistic effects when used together in feed, would be one of the most promising candidates. Lactobacilli are normal residents of the gastrointestinal tract of pigs, and many of them are able to exert beneficial immunoregulatory properties. On the other hand, wakame (Undaria pinnafida), an edible seaweed, has the potential to be used as an immunoregulatory prebiotic when added to livestock feed. Therefore, in order to develop a novel immunosynbiotic, we isolated and characterized immunoregulatory lactobacilli with the ability to utilize wakame. Following a month-long in vivo wakame feeding trial in 8-week-old Landrace pigs (n = 6), sections of intestinal mucous membrane were processed for bacteriological culture and followed by identification of pure colonies by 16S rRNA sequence. Each isolate was characterized in vitro in terms of their ability to assimilate to the wakame and to differentially modulate the expression of interleukin-6 (IL-6) and interferon beta (IFN-β) in the porcine intestinal epithelial (PIE) cells triggered by Toll-like receptor (TLR)-4 and TLR-3 activation, respectively. We demonstrated that feeding wakame to pigs significantly increased the lactobacilli population in the small intestine. We established a wakame-component adjusted culture media that allowed the isolation and characterization of a total of 128 Lactobacilli salivarius colonies from the gut of wakame-fed pigs. Interestingly, several L. salivarius isolates showed both high wakame assimilation ability and immunomodulatory capacities. Among the wakame assimilating isolates, L. salivarius FFIG71 showed a significantly higher capacity to upregulate the IL-6 expression, and L. salivarius FFIG131 showed significantly higher capacity to upregulate the IFN-β expression; these could be used as immunobiotic strains in combination with wakame for the development of novel immunologically active feeds for pigs.

  107. Draft Genome Sequence of Weissella viridescens UCO-SMC3, Isolated from the Slime of Helix aspersa Müller Snails International-journal Peer-reviewed

    Apolinaria Garcia-Cancino, Leonardo Albarracin, Marcela Espinoza-Monje, Jorge Campos-Martin, Valeria Garcia-Castillo, Yuka Nakano, Wakako Ikeda-Ohtsubo, Cristian Guitierrez-Zamorano, Hidetoshi Morita, Haruki Kitazawa, Julio Villena

    Microbiology Resource Announcements　8　(11)　2019/03/14

    Publisher: American Society for Microbiology

    DOI： 10.1128/mra.01654-18 　

    eISSN： 2576-098X

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    This report describes the draft genome sequence of <named-content content-type="genus-species">Weissella viridescens</named-content> UCO-SMC3, isolated from <named-content content-type="genus-species">Helix aspersa</named-content> Müller slime. The reads were generated by a whole-genome sequencing (WGS) strategy on an Illumina MiSeq sequencer and were assembled into contigs with a total estimated size of 1,612,814 bp.

  108. Deciphering the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of bovine intestinal epitheliocytes by transcriptomic analysis Peer-reviewed

    L. Albarracin, R. Komatsu, V. Garcia-Castillo, H. Aso, N. Iwabuchi, J.-Z. Xiao, F. Abe, H. Takahashi, J. Villena, H. Kitazawa

    Beneficial Microbes　10　(2)　199-210　2019/03/13

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2018.0024 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    Previously, we reported that the non-viable immunomodulatory Bifidobacterium infantis MCC12 and Bifidobacterium breve MCC1274 strains (paraimmunobiotic bifidobacteria) were able to increase the protection against rotavirus infection in bovine intestinal epithelial (BIE) cells. In order to gain insight into the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of BIE cells, their effect on the transcriptomic response triggered by Toll-like receptor 3 (TLR3) activation was investigated. By using microarray technology and qPCR analysis, we obtained a global overview of the immune genes involved in the innate antiviral immune response in BIE cells. Activation of TLR3 by poly(I:C) in BIE cells significantly increased the expression of interferon (IFN)-α and IFN-β, several interferon-stimulated genes, cytokines, and chemokines. It was also observed that both paraimmunobiotic bifidobacteria differently modulated immune genes expression in poly(I:C)-challenged BIE cells. Most notable changes were found in genes involved in antiviral defence (IFN-β, MX1, OAS1X, MDA5, TLR3, STAT2, STAT3), cytokines (interleukin (IL)-6), and chemokines (CCL2, CXCL2, CXCL6) that were significantly increased in bifidobacteria-treated BIE cells. B. infantis MCC12 and B. breve MCC1274 showed quantitative and qualitative differences in their capacities to modulate the innate antiviral immune response in BIE cells. B. breve MCC1274 was more efficient than the MCC12 strain to improve the production of type I IFNs and antiviral factors, an effect that could be related to its higher ability to protect against rotavirus replication in BIE cells. Interestingly, B. infantis MCC12 showed a remarkable anti-inflammatory effect. The MCC12 strain was more efficient to reduce the expression of inflammatory cytokines and chemokines (IL-16, IL-20, CX3CL1) when compared with B. breve MCC1274. These results provided valuable information for the deeper understanding of the antiviral immune response of intestinal epithelial cells as well as the host-paraimmunobiotic interaction in the bovine host.

  109. 抗下痢イムノバイオティクスを選択するin vitro評価システムの進歩(Advanced in vitro evaluation system for selecting anti-diarrheal immunobiotics) Peer-reviewed

    小松 稜弥, 飯田 ひかる, Albarracn Leonardo, Islam Aminul Md., 大坪 和香子, 麻生 久, 岩渕 紀介, 清水 金忠[肖], 須田 義人, 齋藤 忠夫, Villena Julio, 北澤 春樹

    日本畜産学会大会講演要旨集　125回　121-121　2019/03

    Publisher: (公社)日本畜産学会

    ISSN： 1342-4688

  110. 乳房炎病原菌にて刺激した牛乳腺上皮細胞のマイクロアレイ解析(Microarray analysis of bovine mammary epithelial cells stimulated with mastitis pathogens) Peer-reviewed

    高木 理宏, 小松 稜弥, Villena Julio, Albarracin Leonardo, Islam Md. Aminul, 須田 義人, 大坪 和香子, 野地 智法, 麻生 久, 北澤 春樹

    日本畜産学会大会講演要旨集　125回　200-200　2019/03

    Publisher: (公社)日本畜産学会

    ISSN： 1342-4688

  111. 豚イムノバイオティクスライブラリーの構築(Construction of porcine immunobiotics library) Peer-reviewed

    中野 祐香, 舛水 由季, 扇 隆介, Albarracin Leonardo, 大坪 和香子, Islam Md Aminul, 須田 義人, 麻生 久, Villena Julio, 北澤 春樹

    日本畜産学会大会講演要旨集　125回　200-200　2019/03

    Publisher: (公社)日本畜産学会

    ISSN： 1342-4688

  112. Immunobiotics Beneficially Modulate TLR4 Signaling Triggered by Lipopolysaccharide and Reduce Hepatic Steatosis In Vitro. International-journal

    Paulraj Kanmani, AbuZar Ansari, Julio Villena, Hojun Kim

    Journal of immunology research　2019　3876896-3876896　2019

    DOI： 10.1155/2019/3876896 　

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    Hepatic inflammation and injury may result from the translocation of pathological bacteria and their proinflammatory mediators. Probiotics attenuate hepatic diseases related to inflammation by exhibiting immunoregulatory effects. Therefore, this study was conducted to evaluate lipid reduction and immunoregulatory potentials of probiotic bacteria in vitro. HepG2 cells treated with total cellular fluid (TCF) of LABs reduced lipid accumulation. Moreover, cells responded to lipopolysaccharide (LPS) by producing higher levels of IL-6, IL-8, MCP-1, and TNF-α. TCF of LABs treatment showed remarkably diminished levels of the expression of these cytokines via modulation of the expression of TLR-negative regulators, as well as MAPK and NF-κB pathways. Moreover, heat-killed LABs were able to diminish TGF-β, IL-1β, and IL-6 and to increase IL-10 and TLR4 levels in THP-1 cells. LABs also decreased the protein level of TNF-α. These results demonstrated that immunobiotics exhibit potent immunoregulatory activity and may be used as effective therapeutic agents to alleviate inflammatory response.

  113. Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation. International-journal

    Manami Igata, Md Aminul Islam, Asuka Tada, Michihiro Takagi, A K M Humayun Kober, Leonardo Albarracin, Hisashi Aso, Wakako Ikeda-Ohtsubo, Kenji Miyazawa, Kazutoyo Yoda, Fang He, Hideki Takahashi, Julio Villena, Haruki Kitazawa

    Frontiers in immunology　10　1180-1180　2019

    DOI： 10.3389/fimmu.2019.01180 　

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    Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.

  114. Evaluation of the Immunomodulatory Activities of the Probiotic Strain Lactobacillus fermentum UCO-979C. International-journal

    Valeria Garcia-Castillo, Ryoya Komatsu, Patricia Clua, Yuhki Indo, Michihiro Takagi, Susana Salva, Md Aminul Islam, Susana Alvarez, Hideki Takahashi, Apolinaria Garcia-Cancino, Haruki Kitazawa, Julio Villena

    Frontiers in immunology　10　1376-1376　2019

    DOI： 10.3389/fimmu.2019.01376 　

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    Lactobacillus fermentum UCO-979C, a strain isolated from a human stomach, was previously characterized by its potential probiotic properties. The UCO-979C strain displayed the ability to beneficially regulate the innate immune response triggered by Helicobacter pylori infection in human gastric epithelial cells. In this work, we conducted further in vitro studies in intestinal epithelial cells (IECs) and in vivo experiments in mice in order to characterize the potential immunomodulatory effects of L. fermentum UCO-979C on the intestinal mucosa. Results demonstrated that the UCO-979C strain is capable to differentially modulate the immune response of IECs triggered by Toll-like receptor 4 (TLR4) activation through the modulation of TLR negative regulators' expression. In addition, we demonstrated for the first time that L. fermentum UCO-979C is able to exert its immunomodulatory effect in the intestinal mucosa in vivo. The feeding of mice with L. fermentum UCO-979C significantly increased the production of intestinal IFN-γ, stimulated intestinal and peritoneal macrophages and increased the number of Peyer's patches CD4+ T cells. In addition, L. fermentum UCO-979C augmented intestinal IL-6, reduced the number of immature B220+CD24high B cells from Peyer's patches, enhanced the number of mature B B220+CD24low cells, and significantly increased intestinal IgA content. The results of this work revealed that L. fermentum UCO-979C has several characteristics making it an excellent candidate for the development of immunobiotic functional foods aimed to differentially regulate immune responses against gastric and intestinal pathogens.

  115. Screening and Characterization of Immunobiotic Lactic Acid Bacteria with Porcine Immunoassay Systems

    Valeria Garcia-Castillo, Leonardo Albarracin, Haruki Kitazawa, Julio Villena

    Methods in Molecular Biology　131-144　2018/12/01

    Publisher: Springer New York

    DOI： 10.1007/978-1-4939-8907-2_12 　

    ISSN： 1064-3745

    eISSN： 1940-6029

  116. Nasal immunization with recombinant chimeric pneumococcal protein and cell wall from immunobiotic bacteria improve resistance of infant mice to Streptococcus pneumoniae infection

    Jonathan Laiño, Julio Villena, Alexander Suvorov, Hortensia Zelaya, Ramiro Ortiz Moyano, Susana Salva, Susana Alvarez

    PLOS ONE　2018/11/05

    DOI： 10.1371/journal.pone.0206661 　

  117. Lactobacillus fermentum UCO-979C beneficially modulates the innate immune response triggered by Helicobacter pylori infection in vitro Peer-reviewed

    V. Garcia-Castillo, H. Zelaya, A. Ilabaca, M. Espinoza-Monje, R. Komatsu, L. Albarracín, H. Kitazawa, A. Garcia-Cancino, J. Villena

    Beneficial Microbes　9　(5)　829-842　2018/09/14

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2018.0019 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    Helicobacter pylori infection is associated with important gastric pathologies. An aggressive proinflammatory immune response is generated in the gastric tissue infected with H. pylori, resulting in gastritis and a series of morphological changes that increase the susceptibility to cancer development. Probiotics could present an alternative solution to prevent or decrease H. pylori infection. Among them, the use of immunomodulatory lactic acid bacteria represents a promising option to reduce the severity of chronic inflammatory-mediated tissue damage and to improve protective immunity against H. pylori. We previously isolated Lactobacillus fermentum UCO-979C from human gastric tissue and demonstrated its capacity to reduce adhesion of H. pylori to human gastric epithelial cells (AGS cells). In this work, the ability of L. fermentum UCO-979C to modulate immune response in AGS cells and PMA phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 (human monocytic leukaemia) macrophages in response to H. pylori infection was evaluated. We demonstrated that the UCO-979C strain is able to differentially modulate the cytokine response of gastric epithelial cells and macrophages after H. pylori infection. Of note, L. fermentum UCO-979C was able to significantly reduce the production of inflammatory cytokines and chemokines in AGS and THP-1 cells as well as increase the levels of immunoregulatory cytokines, indicating a remarkable anti-inflammatory effect. These findings strongly support the probiotic potential of L. fermentum UCO-979C and provide evidence of its beneficial effects against the inflammatory damage induced by H. pylori infection. Although our findings should be proven in appropriate experiments in vivo, in both H. pylori infection animal models and human trials, the results of the present work provide a scientific rationale for the use of L. fermentum UCO-979C to prevent or reduce H. pylori-induced gastric inflammation in humans.

  118. Receptors and signaling pathways for recognition of bacteria in livestock and crops: prospects for beneficial microbes in healthy growth strategies Peer-reviewed

    Villena, J, H. Kitazawa, S. C. M, Van Wees, C. M. J. Pieterse, H. Takahashi

    9　(2223)　1-15　2018/09

    DOI： 10.3389/fimmu.2018.02223 　

  119. Exopolysaccharides from Lactobacillus delbrueckii OLL1073R-1 modulate innate antiviral immune response in porcine intestinal epithelial cells. International-journal

    Paulraj Kanmani, Leonardo Albarracin, Hisakazu Kobayashi, Hikaru Iida, Ryoya Komatsu, A K M Humayun Kober, Wakako Ikeda-Ohtsubo, Yoshihito Suda, Hisashi Aso, Seiya Makino, Hiroshi Kano, Tadao Saito, Julio Villena, Haruki Kitazawa

    Molecular immunology　93　253-265　2018/01

    DOI： 10.1016/j.molimm.2017.07.009 　

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    Previous studies demonstrated that the extracellular polysaccharides (EPSs) produced by Lactobacillus delbrueckii OLL1073R-1 (LDR-1) improve antiviral immunity, especially in the systemic and respiratory compartments. However, it was not studied before whether those EPSs are able to beneficially modulate intestinal antiviral immunity. In addition, LDR-1-host interaction has been evaluated mainly with immune cells while its interaction with intestinal epithelial cells (IECs) was not addressed before. In this work, we investigated the capacity of EPSs from LDR-1 to modulate the response of porcine IECs (PIE cells) to the stimulation with the Toll-like receptor (TLR)-3 agonist poly(I:C) and the role of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effect. We showed that innate immune response triggered by TLR3 activation in porcine IECs was differentially modulated by EPS from LDR-1. EPSs treatment induced an increment in the expression of interferon (IFN)-α and IFN-β in PIE cells after the stimulation with poly(I:C) as well as the expression of the antiviral factors MxA and RNase L. Those effects were related to the reduced expression of A20 in EPS-treated PIE cells. EPS from LDR-1 was also able to reduce the expression of IL-6 and proinflammatory chemokines. Although further in vivo studies are needed, our results suggest that these EPSs or a yogurt fermented with LDR-1 have potential to improve intestinal innate antiviral response and protect against intestinal viruses.

  120. Are the immunomodulatory properties of Lactobacillus rhamnosus CRL1505 peptidoglycan common for all Lactobacilli during respiratory infection in malnourished mice? International-journal

    Yanina Kolling, Susana Salva, Julio Villena, Susana Alvarez

    PloS one　13　(3)　e0194034　2018

    DOI： 10.1371/journal.pone.0194034 　

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    Previously, we reported that Lactobacillus rhamnosus CRL1505 peptidoglycan (PG05) improves the innate immune response in immunocompromised-malnourished mice after Streptococcus pneumoniae infection. This study extends those previous findings by demonstrating that the dietary recovery of malnourished mice with nasal administration of PG05 improves not only the innate immune response but the respiratory and systemic adaptive humoral response as well. PG05 enhanced the Th2 response, the recovery of B cells, and the concentration and opsonophagocytic activity of anti-pneumococcal antibodies. In addition, by performing comparative studies with the peptidoglycans from lactobacilli of the same species (L. rhamnosus CRL534) or with similar immunomodulatory properties (L. plantarum CRL1506), we demonstrated here that PG05 has unique immunomodulatory properties that cannot be extended to peptidoglycans from other probiotic strains. However, the knowledge of the molecular characteristics of PG05 is indispensable to understand immunomodulatory abilities of L. rhamnosus CRL1505.

  121. Genomic Characterization of Lactobacillus delbrueckii TUA4408L and Evaluation of the Antiviral Activities of its Extracellular Polysaccharides in Porcine Intestinal Epithelial Cells. International-journal

    Paulraj Kanmani, Leonardo Albarracin, Hisakazu Kobayashi, Elvira Maria Hebert, Lucila Saavedra, Ryoya Komatsu, Brian Gatica, Ayako Miyazaki, Wakako Ikeda-Ohtsubo, Yoshihito Suda, Hisashi Aso, Shintaro Egusa, Takashi Mishima, Alexis Salas-Burgos, Hideki Takahashi, Julio Villena, Haruki Kitazawa

    Frontiers in immunology　9　2178-2178　2018

    DOI： 10.3389/fimmu.2018.02178 　

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    In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-β, Myxovirus resistance gene A (MxA) and RNaseL.

  122. Editorial: immunobiotics-interactions of Beneficial Microbes with the immune System Peer-reviewed

    Julio Villena, Haruki Kitazawa

    FRONTIERS IN IMMUNOLOGY　8　(1580)　1-2　2017/11

    DOI： 10.3389/fimmu.2017.01580 　

    ISSN： 1664-3224

  123. Peptidoglycan from Immunobiotic Lactobacillus rhamnosus Improves Resistance of Infant Mice to Respiratory Syncytial Viral Infection and Secondary Pneumococcal Pneumonia Peer-reviewed

    Patricia Clua, Paulraj Kanmani, Hortensia Zelaya, Asuka Tada, A. K. M. Humayun Kober, Susana Salva, Susana Alvarez, Haruki Kitazawa, Julio Villena

    Frontiers in Immunology　8　2017/08/10

    Publisher: Frontiers Media SA

    DOI： 10.3389/fimmu.2017.00948 　

    eISSN： 1664-3224

  124. Respiratory Commensal Bacteria Corynebacterium pseudodiphtheriticum Improves Resistance of Infant Mice to Respiratory Syncytial Virus and Streptococcus pneumoniae Superinfection Peer-reviewed

    Paulraj Kanmani, Patricia Clua, Maria G. Vizoso-Pinto, Cecilia Rodriguez, Susana Alvarez, Vyacheslav Melnikov, Hideki Takahashi, Haruki Kitazawa, Julio Villena

    Frontiers in Microbiology　8　2017/08

    Publisher: Frontiers Media {SA}

    DOI： 10.3389/fmicb.2017.01613 　

    ISSN： 1664-302X

  125. Exopolysaccharide-producing Streptococcus thermophilus CRL1190 reduces the inflammatory response caused by Helicobacter pylori Peer-reviewed

    G. Marcial, J. Villena, G. Faller, A. Hensel, G. Font de Valdéz

    Beneficial Microbes　8　(3)　451-462　2017/05/30

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2016.0186 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    This work evaluated the ability of the probioticStreptococcus thermophilusCRL1190 strain and its exopolysaccharides to adhere to gastric mucosa. Probiotic bacteria attachment to the human stomach epithelium was confirmed in human stomach tissue samples and the gastric epithelial cell line AGS. In addition, it was demonstrated thatS. thermophilusCRL1190 strain reducedHelicobacter pyloriadhesion and attenuated inflammatory response in AGS cells. This is the first demonstration of the capacity ofS. thermophilusCRL1190 to adhere to the stomach gastric mucosa, and improve protection againstH. pylorithrough the reduction of its adhesion and the modulation of the inflammatory response. Therefore,S. thermophilusCRL1190 fermented milk is a good candidate for furtherin vivostudying of the protective effect of functional food againstH. pyloriinfection and gastric inflammatory damage.

  126. Lactobacillus rhamnosus CRL1505 nasal administration improves recovery of T-cell mediated immunity against pneumococcal infection in malnourished mice Peer-reviewed

    N. Barbieri, M. Herrera, S. Salva, J. Villena, S. Alvarez

    Beneficial Microbes　8　(3)　393-406　2017/05/30

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2016.0152 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    Immunobiotic lactic acid bacteria have become an interesting alternative for the prevention of respiratory infections. Previously, we demonstrated that the nasal administration of Lactobacillus rhamnosus CRL1505, during repletion of malnourished mice, resulted in diminished susceptibility to the challenge with the respiratory pathogen Streptococcus pneumoniae. Considering the known alterations induced by malnutrition on T lymphocytes and the importance of this cell population on the protection against respiratory pathogens, we aimed to study the effect of L. rhamnosus CRL1505 nasal administration on the recovery of T cell-mediated defences against pneumococcal infection in malnourished mice under nutritional recovery. Malnourished mice received a balanced conventional diet (BCD) for seven days or BCD for seven days with nasal L. rhamnosus CRL1505 supplementation during last two days of the treatment. After the treatments mice were infected with S. pneumoniae. Flow cytometry studies were carried out in bone marrow, thymus, spleen and lung to study T cells, and Th1/Th2 cytokine profiles were determined in broncho-alveolar lavages and serum. The administration of CRL1505 strain to malnourished mice under recovery reduced quantitative and qualitative alterations of CD4+ T cells in the bone marrow, thymus, spleen and lung induced by malnutrition. In addition, CRL1505 treatment augmented Th2-cytokines (interleukin 10 and 4) in respiratory and systemic compartments after pneumococcal infection. These results show that modulation of CD4+ T lymphocytes induced by L. rhamnosus CRL1505 has an important role in the beneficial effect induced by this strain on the recovery of malnourished mice. These data also indicate that nasally administered L. rhamnosus CRL1505 may represent a non-invasive alternative to modulate and improve the T cell-mediated immunity against respiratory pathogens in immunocompromised malnourished hosts.

  127. Safety Assessment of the Antimicrobial Lipopeptides Synthesized by Bacillus subtilis Subsp. subtilis CBMDC3f Peer-reviewed

    Maria Julia Torres, Julio Villena, Paola Barroso, Mariana Novicov Fanciotti, Marcela Carina Audisio

    EC Microbiology　8　(3)　113-122　2017/05/17

  128. Development of an in vitro immunobiotic evaluation system against rotavirus infection in bovine intestinal epitheliocytes Peer-reviewed

    H. Kobayashi, P. Kanmani, T. Ishizuka, A. Miyazaki, J. Soma, L. Albarracin, Y. Suda, T. Nochi, H. Aso, N. Iwabuchi, J.-Z. Xiao, T. Saito, J. Villena, H. Kitazawa

    Beneficial Microbes　8　(2)　309-322　2017/04/26

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2016.0155 　

    ISSN： 1876-2883

    eISSN： 1876-2891

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    The bovine intestinal epithelial cell line (BIE cells) expresses the Toll-like receptor (TLR)3 and is able to mount an antiviral immune response after the stimulation with poly(I:C). In the present study, we aimed to further characterise the antiviral defence mechanisms in BIE cells by evaluating the innate immune response triggered by rotavirus (RV) infection. In addition, we attempted to determine whether immunobiotic bifidobacteria are able to confer protection of BIE cells against RV infection by beneficially modulating the antiviral immune response. RV OSU (porcine) and UK (bovine) effectively infected BIE cells, while a significant lower capacity to infect BIE cells was observed for human (Wa) and murine (EW) RV. We observed that viral infection in BIE cells triggered TLR3/RIG-I-mediated immune responses with activation of IRF3 and TRAF3, induction of interferon beta (IFN-β) and up-regulation of inflammatory cytokines. Our results also demonstrated that preventive treatments with Bifidobacterium infantis MCC12 or Bifidobacterium breve MCC1274 significantly reduced RV titres in infected BIE cells and differentially modulated the innate immune response. Of note, both strains significantly improved the production of the antiviral factor IFN-β in RV-infected BIE cells. In conclusion, this work provides comprehensive information on the antiviral immune response of BIE cells against RV, that can be further studied for the development of strategies aimed to improve antiviral defences in bovine intestinal epithelial cells. Our results also demonstrate that BIE cells could be used as a newly immunobiotic evaluation system against RV infection for application in the bovine host.

  129. Draft Genome Sequence of Lactobacillus plantarum MPL16, a Wakame-Utilizing Immunobiotic Strain Isolated from Swine Feces. International-journal Peer-reviewed

    Julio Villena, Lucila Saavedra, Elvira Maria Hebert, Yoshihito Suda, Yuki Masumizu, Leonardo Albarracin, Patricia Clua, Wakako Ikeda-Ohtsubo, Haruki Kitazawa

    Genome announcements　5　(10)　1-3　2017/03/09

    DOI： 10.1128/genomeA.00006-17 　

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    The genome of the immunomodulatory Lactobacillus plantarum MPL16, a strain able to ferment wakame (Undaria pinnatifida), is described here. The reads were assembled into contigs with a total size 3,278,495 bp. The genome information will be useful for further specific genetic studies of this strain that evaluate its immunomodulatory and biotechnological properties.

  130. Transcriptomic Analysis of the Innate Antiviral Immune Response in Porcine Intestinal Epithelial Cells: Influence of Immunobiotic Lactobacilli Peer-reviewed

    Leonardo Albarracin, Hisakazu Kobayashi, Hikaru Iida, Nana Sato, Tomonori Nochi, Hisashi Aso, Susana Salva, Susana Alvarez, Haruki Kitazawa, Julio Villena

    Frontiers in Immunology　8　2017/02/02

    Publisher: Frontiers Media SA

    DOI： 10.3389/fimmu.2017.00057 　

    eISSN： 1664-3224

  131. Characterization of Lactobacillus fermentum UCO-979C, a probiotic strain with a potent anti-Helicobacter pylori activity Peer-reviewed

    Apolinaria García, Karen Navarro, Enrique Sanhueza, Susana Pineda, Edgar Pastene, Manuel Quezada, Karem Henríquez, Andrey Karlyshev, Julio Villena, Carlos González

    Electronic Journal of Biotechnology　25　75-83　2017/01

    Publisher: Elsevier {BV}

    DOI： 10.1016/j.ejbt.2016.11.008 　

    ISSN： 0717-3458

  132. Modulation of porcine intestinal epitheliocytes immunetranscriptome response by Lactobacillus jensenii TL2937 Peer-reviewed

    H. Kobayashi, L. Albarracin, N. Sato, P. Kanmani, A.K.M.H. Kober, W. Ikeda-Ohtsubo, Y. Suda, T. Nochi, H. Aso, S. Makino, H. Kano, S. Ohkawara, T. Saito, J. Villena, H. Kitazawa

    Beneficial Microbes　7　(5)　769-782　2016/11/30

    Publisher: Walter de Gruyter GmbH

    DOI： 10.3920/bm2016.0095 　

    ISSN： 1876-2883

    eISSN： 1876-2891

    More details Close

    In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs.

  133. Draft Genome Sequence of Lactobacillus plantarum TL2766, a Strain with the Ability To Ferment Wakame. International-journal Peer-reviewed

    Julio Villena, Lucila Saavedra, Elvira Maria Hebert, Yuki Masumizu, Nana Sato, A K M Humayun Kober, Leonardo Albarracin, Wakako Ikeda-Ohtsubo, Seiya Makino, Katsunori Kimura, Sou Ohkawara, Haruki Kitazawa

    Genome announcements　4　(6)　e01328-16　2016/11/23

    DOI： 10.1128/genomeA.01328-16 　

    More details Close

    The genome sequence of Lactobacillus plantarum TL2766, a strain with the ability to ferment wakame (Undaria pinnatifida), is described here. The reads were assembled into contigs, with a total size of 3,310,195 bp. The genome information will be useful for further specific genetic studies of this strain and for its biotechnological applications.

  134. Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells Peer-reviewed

    Jonathan Laiño, Julio Villena, Paulraj Kanmani, Haruki Kitazawa

    Microorganisms　4　(3)　27-27　2016/08/15

    Publisher: {MDPI} {AG}

    DOI： 10.3390/microorganisms4030027 　

    ISSN： 2076-2607

  135. Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells Peer-reviewed

    Kohichiro Tsukida, Takuya Takahashi, Hikaru Iida, Paulraj Kanmani, Yoshihito Suda, Tomonori Nochi, Shuichi Ohwada, Hisashi Aso, Sou Ohkawara, Seiya Makino, Hiroshi Kano, Tadao Saito, Julio Villena, Haruki Kitazawa

    BMC Immunology　17　(21)　2016/06/24

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1186/s12865-016-0160-1 　

    eISSN： 1471-2172

  136. Immunobiotic Lactobacillus strains reduce small intestinal injury induced by intraepithelial lymphocytes after Toll-like receptor 3 activation Peer-reviewed

    Asuka Tada, Hortensia Zelaya, Patricia Clua, Susana Salva, Susana Alvarez, Haruki Kitazawa, Julio Villena

    Inflammation Research　65　771-783　2016/06/09

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1007/s00011-016-0957-7 　

    ISSN： 1023-3830

    eISSN： 1420-908X

  137. Isolation of lactic acid bacteria from swine milk and characterization of potential probiotic strains with antagonistic effects against swine-associated gastrointestinal pathogens Peer-reviewed

    Sandra Rayén Quilodrán-Vega, Julio Villena, José Valdebenito, María José Salas, Cristian Parra, Alvaro Ruiz, Haruki Kitazawa, Apolinaria García

    Canadian Journal of Microbiology　62　(6)　514-524　2016/06

    Publisher: Canadian Science Publishing

    DOI： 10.1139/cjm-2015-0811 　

    ISSN： 0008-4166

  138. Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling Peer-reviewed

    Takamasa Ishizuka, Paulraj Kanmani, Hisakazu Kobayashi, Ayako Miyazaki, Junichi Soma, Yoshihito Suda, Hisashi Aso, Tomonori Nochi, Noriyuki Iwabuchi, Jin-zhong Xiao, Tadao Saito, Julio Villena, Haruki Kitazawa

    PLOS ONE　11　(3)　e0152416-e0152416　2016/03/29

    Publisher: Public Library of Science (PLoS)

    DOI： 10.1371/journal.pone.0152416 　

    eISSN： 1932-6203

  139. Draft Genome Sequence ofLactobacillus plantarumCRL1506, an Immunomodulatory Strain Isolated from Goat Milk Peer-reviewed

    Lucila Saavedra, Elvira María Hebert, Leonardo Albarracin, Susana Salva, Susana Alvarez, Haruki Kitazawa, Julio Villena

    Genome Announcements　4　(2)　e00108-16　2016/03/10

    Publisher: American Society for Microbiology

    DOI： 10.1128/genomea.00108-16 　

    ISSN： 2169-8287

  140. EVALUATION OF LACTOBACILLUS PROBIOTICS AS ADJUVANTS FOR NASAL IMMUNIZATION WITH CHIMERIC PNEUMOCOCCAL VACCINE Peer-reviewed

    G. F. Leontieva, T. A. Kramskaya, K. B. Grabovskaya, V. Yu. Filimonova, J. Laiñ o, J. Villena, S. Alvares, V. N. Danilenko, A. N. Suvorov

    Medical Immunology (Russia)　18　(6)　545-554　2016/01/01

    Publisher: SPb RAACI

    DOI： 10.15789/1563-0625-2016-6-545-554 　

    ISSN： 1563-0625

    eISSN： 2313-741X

  141. Dietary Supplementation with Probiotic Strain Improves Immune-Health in Aged Mice Peer-reviewed

    Verónica Molina, Marta Médici, Julio Villena, Graciela Font, María Pía Taranto

    Open Journal of Immunology　06　(03)　73-78　2016

    Publisher: Scientific Research Publishing, Inc.

    DOI： 10.4236/oji.2016.63008 　

    ISSN： 2162-450X

    eISSN： 2162-4526

  142. Intestinal Innate Antiviral Immunity and Immunobiotics: Beneficial Effects against Rotavirus Infection Peer-reviewed

    Julio Villena, Maria Guadalupe Vizoso-Pinto, Haruki Kitazawa

    Frontiers in Immunology　7　2016

    Publisher: Frontiers Media {SA}

    DOI： 10.3389/fimmu.2016.00563 　

    ISSN： 1664-3224

  143. Respiratory Antiviral Immunity and Immunobiotics: Beneficial Effects on Inflammation-Coagulation Interaction during Influenza Virus Infection Peer-reviewed

    Hortensia Zelaya, Susana Alvarez, Haruki Kitazawa, Julio Villena

    Frontiers in Immunology　7　2016

    Publisher: Frontiers Media {SA}

    DOI： 10.3389/fimmu.2016.00633 　

    ISSN： 1664-3224

  144. Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation–coagulation interactions and reduces influenza virus-associated pulmonary damage

    Hortensia Zelaya, Asuka Tada, Maria Guadalupe Vizoso-Pinto, Susana Salva, Paulraj Kanmani, Graciela Agüero, Susana Alvarez, Haruki Kitazawa, Julio Villena

    Inflammation Research　64　(8)　589-602　2015/06/14

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1007/s00011-015-0837-6 　

    ISSN： 1023-3830

    eISSN： 1420-908X

  145. Non-viable immunobiotic Lactobacillus rhamnosus CRL1505 and its peptidoglycan improve systemic and respiratory innate immune response during recovery of immunocompromised-malnourished mice

    Yanina Kolling, Susana Salva, Julio Villena, Gabriela Marranzino, Susana Alvarez

    International Immunopharmacology　25　(2)　474-484　2015/04

    Publisher: Elsevier BV

    DOI： 10.1016/j.intimp.2015.02.006 　

    ISSN： 1567-5769

  146. The toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of exopolysaccharides from Lactobacillus plantarum N14

    Yo Murofushi, Julio Villena, Kyoko Morie, Paulraj Kanmani, Masanori Tohno, Tomoyuki Shimazu, Hisashi Aso, Yoshihito Suda, Kenji Hashiguchi, Tadao Saito, Haruki Kitazawa

    Molecular Immunology　64　(1)　63-75　2015/03

    Publisher: Elsevier BV

    DOI： 10.1016/j.molimm.2014.10.027 　

    ISSN： 0161-5890

  147. Advanced Application of Porcine Intramuscular Adipocytes for Evaluating Anti-Adipogenic and Anti-Inflammatory Activities of Immunobiotics

    Masahiko Suzuki, Asuka Tada, Paulraj Kanmani, Hitoshi Watanabe, Hisashi Aso, Yoshihito Suda, Tomonori Nochi, Kenji Miyazawa, Kazutoyo Yoda, Fang He, Masataka Hosoda, Tadao Saito, Julio Villena, Haruki Kitazawa

    PLOS ONE　10　(3)　2015/03

    Publisher: Public Library of Science ({PLoS})

    DOI： 10.1371/journal.pone.0119644 　

    ISSN： 1932-6203

  148. Recent Advances and Future Perspective in Microbiota and Probiotics

    Haruki Kitazawa, Susana Alvarez, Alexander Suvorov, Vyacheslav Melnikov, Julio Villena, Borja Sánchez

    BioMed Research International　2015　1-2　2015

    Publisher: Hindawi Limited

    DOI： 10.1155/2015/275631 　

    ISSN： 2314-6133

  149. Soluble Factors from Lactobacillus reuteri CRL1098 Have Anti-Inflammatory Effects in Acute Lung Injury Induced by Lipopolysaccharide in Mice

    Milagros Griet, Hortensia Zelaya, Melina Valeria Mateos, Susana Salva, Guillermo Esteban Juarez, Graciela Font de Valdez, Julio Villena, Gabriela Alejandra Salvador, Ana Virginia Rodriguez

    PLoS ONE　9　(10)　2014/10

    Publisher: Public Library of Science ({PLoS})

    DOI： 10.1371/journal.pone.0110027 　

    ISSN： 1932-6203

  150. Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide-treated mice

    Susana Salva, Gabriela Marranzino, Julio Villena, Graciela Agüero, Susana Alvarez

    International Immunopharmacology　22　(1)　209-221　2014/09

    Publisher: Elsevier BV

    DOI： 10.1016/j.intimp.2014.06.017 　

    ISSN： 1567-5769

  151. Lactobacillus delbrueckii TUA4408L and its extracellular polysaccharides attenuate enterotoxigenic Escherichia coli-induced inflammatory response in porcine intestinal epitheliocytes via Toll-like receptor-2 and 4

    Satoshi Wachi, Paulraj Kanmani, Yohsuke Tomosada, Hisakazu Kobayashi, Toshihito Yuri, Shintaro Egusa, Tomoyuki Shimazu, Yoshihito Suda, Hisashi Aso, Makoto Sugawara, Tadao Saito, Takashi Mishima, Julio Villena, Haruki Kitazawa

    Molecular Nutrition & Food Research　58　(10)　2080-2093　2014/08/05

    Publisher: Wiley

    DOI： 10.1002/mnfr.201400218 　

    ISSN： 1613-4125

    eISSN： 1613-4133

    More details Close

    Scope Immunobiotics are known to modulate intestinal immune responses by regulating Toll‐like receptor (TLR) signaling pathways, which are responsible for the induction of cytokines and chemokines in response to microbial‐associated molecular patterns. However, little is known about the immunomodulatory activity of compounds or molecules from immunobiotics. Methods and results We evaluated whether Lactobacillus delbrueckii subsp. delbrueckii TUA4408L (Ld) or its extracellular polysaccharide (EPS): acidic EPS (APS) and neutral EPS (NPS), modulated the response of porcine intestinal epitheliocyte (PIE) cells against Enterotoxigenic Escherichia coli (ETEC) 987P. The roles of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effects were also studied. ETEC‐induced inflammatory cytokines were downregulated when PIE cells were prestimulated with both Ld or EPSs. Ld, APS, and NPS inhibited ETEC mediated mitogen‐activated protein kinase (MAPK) and nuclear factor‐κB (NF‐κB) activation by upregulating TLR negative regulators. The capability of Ld to suppress inflammatory cytokines was diminished when PIE cells were blocked with anti‐TLR2 antibody, while APS failed to suppress inflammatory cytokines when cells were treated with anti‐TLR4 antibody. Induction of Ca2+ fluxes in TLR knockdown cells confirmed that TLR2 plays a principal role in the immunomodulatory action of Ld, while the activity of APS is mediated by TLR4. In addition, NPS activity depends on both TLR4 and TLR2. Conclusion Ld and its EPS have the potential to be used for the development of anti‐inflammatory functional foods to prevent intestinal diseases in both humans and animals.

  152. Modulation of the inflammation-coagulation interaction during pneumococcal pneumonia by immunobioticLactobacillus rhamnosusCRL1505: Role of Toll-like receptor 2

    Hortensia Zelaya, Julio Villena, Andres Gramajo Lopez, Susana Alvarez, Graciela Agüero

    Microbiology and Immunology　58　(7)　416-426　2014/07

    Publisher: Wiley-Blackwell

    DOI： 10.1111/1348-0421.12163 　

    ISSN： 0385-5600

  153. Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs Peer-reviewed

    Yoshihito Suda, Julio Villena, Yu Takahashi, Shoichi Hosoya, Yohsuke Tomosada, Kohichiro Tsukida, Tomoyuki Shimazu, Hisashi Aso, Masanori Tohno, Mitsuharu Ishida, Seiya Makino, Shuji Ikegami, Haruki Kitazawa

    BMC Immunology　15　(1)　2014/06/19

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1186/1471-2172-15-24 　

    eISSN： 1471-2172

  154. Modulation of Respiratory TLR3-Anti-Viral Response by Probiotic Microorganisms: Lessons Learned from Lactobacillus rhamnosus CRL1505

    Haruki Kitazawa, Julio Villena

    Frontiers in Immunology　5　2014/05

    Publisher: Frontiers Media {SA}

    DOI： 10.3389/fimmu.2014.00201 　

    ISSN： 1664-3224

  155. Dietary Supplementation with Lactobacilli Improves Emergency Granulopoiesis in Protein-Malnourished Mice and Enhances Respiratory Innate Immune Response

    Matias Herrera, Susana Salva, Julio Villena, Natalia Barbieri, Gabriela Marranzino, Susana Alvarez

    PLoS ONE　9　(4)　e90227-e90227　2014/04/01

    Publisher: Public Library of Science (PLoS)

    DOI： 10.1371/journal.pone.0090227 　

    eISSN： 1932-6203

  156. Immunobiotic lactobacilli reduce viral-associated pulmonary damage through the modulation of inflammation–coagulation interactions

    Hortensia Zelaya, Kohichiro Tsukida, Eriko Chiba, Gabriela Marranzino, Susana Alvarez, Haruki Kitazawa, Graciela Agüero, Julio Villena

    International Immunopharmacology　19　(1)　161-173　2014/03

    Publisher: Elsevier {BV}

    DOI： 10.1016/j.intimp.2013.12.020 　

    ISSN： 1567-5769

  157. Evaluation of the Immunoregulatory Capacities of Feed Microbial Materials in Porcine Intestinal Immune and Epithelial Cells

    Naosuke Kumagae, Julio Villena, Yohsuke Tomosada, Hisakazu Kobayashi, Paulraj Kanmani, Hisashi Aso, Takashi Sasaki, Motohiko Yoshida, Hiroshi Tanabe, Isao Shibata, Tadao Saito, Haruki Kitazawa

    Open Journal of Veterinary Medicine　04　(03)　15-28　2014

    Publisher: Scientific Research Publishing, Inc.

    DOI： 10.4236/ojvm.2014.43003 　

    ISSN： 2165-3356

    eISSN： 2165-3364

  158. Bifidobacterium breve MCC-117 Induces Tolerance in Porcine Intestinal Epithelial Cells: Study of the Mechanisms Involved in the Immunoregulatory Effect

    Kozue MURATA, Yohsuke TOMOSADA, Julio VILLENA, Eriko CHIBA, Tomoyuki SHIMAZU, Hisashi ASO, Noriyuki IWABUCHI, Jin-zhong XIAO, Tadao SAITO, Haruki KITAZAWA

    Bioscience of Microbiota, Food and Health　33　(1)　1-10　2014

    Publisher: BMFH Press

    DOI： 10.12938/bmfh.33.1 　

    eISSN： 2186-3342

  159. Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells

    Julio Villena, Eriko Chiba, Maria Vizoso-Pinto, Yohsuke Tomosada, Takuya Takahashi, Takamasa Ishizuka, Hisashi Aso, Susana Salva, Susana Alvarez, Haruki Kitazawa

    BMC Microbiology　14　(1)　126-126　2014

    Publisher: Springer Nature

    DOI： 10.1186/1471-2180-14-126 　

    ISSN： 1471-2180

  160. Modulation of Intestinal TLR4-Inflammatory Signaling Pathways by Probiotic Microorganisms: Lessons Learned from Lactobacillus jensenii TL2937

    Julio Villena, Haruki Kitazawa

    Frontiers in Immunology　4　2014

    Publisher: Frontiers Media {SA}

    DOI： 10.3389/fimmu.2013.00512 　

    ISSN： 1664-3224

  161. Advanced application of porcine intestinal epithelial cells for the selection of immunobiotics modulating toll-like receptor 3-mediated inflammation Peer-reviewed

    Shoichi Hosoya, Julio Villena, Eriko Chiba, Tomoyuki Shimazu, Yoshihito Suda, Hisashi Aso, Tadao Saito, Haruki Kitazawa

    Journal of Microbiology, Immunology and Infection　46　(6)　474-481　2013/12

    Publisher: Elsevier BV

    DOI： 10.1016/j.jmii.2012.04.005 　

    ISSN： 1684-1182

  162. Effect of human milk on blood and bone marrow cells in a malnourished mice model; comparative study with cow milk Peer-reviewed

    Isabel García, Susana Salva, Hortensia Zelaya, Julio Villena, Graciela Agüero

    28　(6)　2157-2164　2013/11

  163. Lactobacillus reuteri CRL1101 beneficially modulate lipopolysaccharide-mediated inflammatory response in a mouse model of endotoxic shock

    Guillermo E. Juarez, Julio Villena, Susana Salva, Graciela Font de Valdez, Ana V. Rodriguez

    Journal of Functional Foods　5　(4)　1761-1773　2013/10

    Publisher: Elsevier BV

    DOI： 10.1016/j.jff.2013.08.002 　

    ISSN： 1756-4646

  164. Lactobacillus rhamnosus CRL1505 enhances systemic and respiratory innate immune response in immunocompromised malnourished mice

    Matias Herrera, Susana Salva, Julio Villena, Natalia Barbieri, Susana Alvarez

    Journal of Functional Foods　5　(4)　1693-1704　2013/10

    Publisher: Elsevier BV

    DOI： 10.1016/j.jff.2013.07.014 　

    ISSN： 1756-4646

  165. Lactobacillus rhamnosus CRL1505 beneficially modulates the immuno-coagulative response after pneumococcal infection in immunocompromised malnourished mice Peer-reviewed

    Hortensia Zelaya, Jonathan Laiño, Julio Villena, Susana Alvarez, Graciela Agüero

    Canadian Journal of Microbiology　59　(10)　684-693　2013/10

    Publisher: Canadian Science Publishing

    DOI： 10.1139/cjm-2013-0361 　

    ISSN： 0008-4166

    eISSN： 1480-3275

    More details Close

    This work evaluated the effect of orally or nasally administered Lactobacillus rhamnosus CRL1505 on the resistance of immunocompromised protein-malnourished mice to pneumococcal infection. In particular, we aimed to gain knowledge of the mechanism involved in the immunomodulatory effect of L. rhamnosus CRL1505 in malnourished hosts by evaluating its impact on the immuno-coagulative response. Malnutrition significantly increased lung tissue damage caused by Streptococcus pneumoniae infection. Lung damage was associated with a deregulated activation of coagulation and an altered inflammatory response. Pneumococcal colonization of lung and bacteremia were significantly reduced (p &lt; 0.05) in malnourished mice receiving the CRL1505 strain. Moreover, mice repleted with supplemental L. rhamnosus CRL1505 showed the least alteration of the alveolar–capillary barrier and cell damage in lungs after the infectious challenge, especially when the CRL1505 strain was administered by nasal route. Besides, mice treated with L. rhamnosus CRL1505 showed an improved respiratory innate immune response and a lower activation of coagulation. The results of this work indicate that L. rhamnosus CRL1505 is able to beneficially modulate the inflammation–coagulation interaction after respiratory infections in malnourished hosts.

  166. Immunobiotic Lactobacillus rhamnosus improves resistance of infant mice against respiratory syncytial virus infection Peer-reviewed

    Eriko Chiba, Yohsuke Tomosada, Maria Guadalupe Vizoso-Pinto, Susana Salva, Takuya Takahashi, Kohichiro Tsukida, Haruki Kitazawa, Susana Alvarez, Julio Villena

    International Immunopharmacology　17　(2)　373-382　2013/10

    Publisher: Elsevier BV

    DOI： 10.1016/j.intimp.2013.06.024 　

    ISSN： 1567-5769

  167. Bifidobacteria Upregulate Expression of Toll-Like Receptor Negative Regulators Counteracting Enterotoxigenic &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; Mediated Inflammation in Bovine Intestinal Epitheliocytes Peer-reviewed

    Kozue Murata, Julio Villena, Yohsuke Tomosada, Risa Hara, Eriko Chiba, Tomoyuki Shimazu, Hisashi Aso, Yoshihito Suda, Noriyuki Iwabuchi, Jin-Zhong Xiao, Tadao Saito, Haruki Kitazawa

    Open Journal of Veterinary Medicine　03　(02)　143-155　2013/06

    Publisher: Scientific Research Publishing, Inc.

    DOI： 10.4236/ojvm.2013.32023 　

    ISSN： 2165-3356

    eISSN： 2165-3364

  168. Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation Peer-reviewed

    Naoya Takanashi, Yohsuke Tomosada, Julio Villena, Kozue Murata, Takuya Takahashi, Eriko Chiba, Masanori Tohno, Tomoyuki Shimazu, Hisashi Aso, Yoshihito Suda, Shuji Ikegami, Hiroyuki Itoh, Yasushi Kawai, Tadao Saito, Susana Alvarez, Haruki Kitazawa

    BMC Microbiology　13　(1)　2013/03/07

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1186/1471-2180-13-54 　

    eISSN： 1471-2180

    More details Close

    Abstract Background Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). Results All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1α and MCP-1 in BIE cells by activating both NF-κB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-κB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3). Conclusions BIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.

  169. Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

    Yohsuke Tomosada, Julio Villena, Kozue Murata, Eriko Chiba, Tomoyuki Shimazu, Hisashi Aso, Noriyuki Iwabuchi, Jin-zhong Xiao, Tadao Saito, Haruki Kitazawa

    PLoS ONE　8　(3)　2013/03

    Publisher: Public Library of Science ({PLoS})

    DOI： 10.1371/journal.pone.0059259 　

    ISSN： 1932-6203

  170. Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection

    Yohsuke Tomosada, Eriko Chiba, Hortensia Zelaya, Takuya Takahashi, Kohichiro Tsukida, Haruki Kitazawa, Susana Alvarez, Julio Villena

    BMC Immunology　14　(1)　40-40　2013

    Publisher: Springer Nature

    DOI： 10.1186/1471-2172-14-40 　

    ISSN： 1471-2172

  171. Lactobacillus reuteri CRL1098 soluble factors modulate tumor necrosis factor alpha production in peripheral blood mononuclear cells: Involvement of lipid rafts Peer-reviewed

    Mónica A. Mechoud, Melina V. Mateos, Graciela Font de Valdez, Julio Villena, Gabriela A. Salvador, Ana V. Rodriguez

    International Immunopharmacology　14　(4)　446-453　2012/12

    Publisher: Elsevier BV

    DOI： 10.1016/j.intimp.2012.08.020 　

    ISSN： 1567-5769

  172. A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle Peer-reviewed

    Eriko Chiba, Julio Villena, Shoichi Hosoya, Naoya Takanashi, Tomoyuki Shimazu, Hisashi Aso, Masanori Tohno, Yoshihito Suda, Yasushi Kawai, Tadao Saito, Kenji Miyazawa, Fang He, Haruki Kitazawa

    Research in Veterinary Science　93　(2)　688-694　2012/10

    Publisher: Elsevier BV

    DOI： 10.1016/j.rvsc.2011.10.002 　

    ISSN： 0034-5288

  173. Stimulation of macrophages by immunobioticLactobacillusstrains: influence beyond the intestinal tract

    Gabriela Marranzino, Julio Villena, Susana Salva, Susana Alvarez

    Microbiology and Immunology　56　(11)　771-781　2012/10

    Publisher: Wiley-Blackwell

    DOI： 10.1111/j.1348-0421.2012.00495.x 　

    ISSN： 0385-5600

  174. Probiotics for Everyone! The Novel Immunobiotic Lactobacillus rhamnosus CRL1505 and the Beginning of Social Probiotic Programs in Argentina Peer-reviewed

    Julio Villena, Susana Salva, Martha Nuñez, Jose Luis Corzo, Rene Tolaba, Julio Faedda, Graciela Fon, Susana Alvarez

    International Journal of Biotechnology for Wellness Industries　1　189-198　2012/09/19

    Publisher: Lifescience Global

    DOI： 10.6000/1927-3037/2012.01.03.05 　

    eISSN： 1927-3037

  175. Immunobiotic Lactobacillus jensenii Modulates the Toll-Like Receptor 4-Induced Inflammatory Response via Negative Regulation in Porcine Antigen-Presenting Cells Peer-reviewed

    Julio Villena, Rie Suzuki, Hitomi Fujie, Eriko Chiba, Takuya Takahashi, Yohsuke Tomosada, Tomoyuki Shimazu, Hisashi Aso, Shyuichi Ohwada, Yoshihito Suda, Shuji Ikegami, Hiroyuki Itoh, Susana Alvarez, Tadao Saito, Haruki Kitazawa

    Clinical and Vaccine Immunology　19　(7)　1038-1053　2012/05/09

    Publisher: American Society for Microbiology

    DOI： 10.1128/cvi.00199-12 　

    ISSN： 1556-6811

    eISSN： 1556-679X

    More details Close

    ABSTRACT Previously, we demonstrated thatLactobacillus jenseniiTL2937 attenuates the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial cells. In view of the critical importance of antigen-presenting cell (APC) polarization in immunoregulation, the objective of the present study was to examine the effect of strain TL2937 on the activation patterns of APCs from swine Peyer's patches (PPs). We demonstrated that direct exposure of porcine APCs toL. jenseniiin the absence of inflammatory signals increased expression of interleukin-10 (IL-10) and transforming growth factor β in CD172a+APCs and caused them to display tolerogenic properties. In addition, pretreatment of CD172a+APCs withL. jenseniiresulted in differential modulation of the production of pro- and anti-inflammatory cytokines in response to TLR4 activation. The immunomodulatory effect of strain TL2937 was not related to a downregulation of TLR4 but was related to an upregulation of the expression of three negative regulators of TLRs: single immunoglobulin IL-1-related receptor (SIGIRR), A20, and interleukin-1 receptor-associated kinase M (IRAK-M). Our results also indicated that TLR2 has an important role in the anti-inflammatory activity ofL. jenseniiTL2937, since anti-TLR2 antibodies blocked the upregulation of SIGIRR and IRAK-M in CD172a+APCs and the production of IL-10 in response to TLR4 activation. We performed, for the first time, a precise functional characterization of porcine APCs from PPs, and we demonstrated that CD172a+cells were tolerogenic. Our findings demonstrate that adherent cells and isolated CD172a+cells harvested from swine PPs were useful forin vitrostudy of the inflammatory responses in the porcine gut and the immunomodulatory effects of immunobiotic microorganisms.

  176. Potentiality of standardized extract and isolated flavonoids from Zuccagnia punctata for the treatment of respiratory infections by Streptococcus pneumoniae: In vitro and in vivo studies Peer-reviewed

    I.C. Zampini, J. Villena, S. Salva, M. Herrera, M.I. Isla, S. Alvarez

    Journal of Ethnopharmacology　140　(2)　287-292　2012/03

    Publisher: Elsevier BV

    DOI： 10.1016/j.jep.2012.01.019 　

    ISSN： 0378-8741

  177. Immunobiotic Lactobacillus jensenii Elicits Anti-Inflammatory Activity in Porcine Intestinal Epithelial Cells by Modulating Negative Regulators of the Toll-Like Receptor Signaling Pathway Peer-reviewed

    Tomoyuki Shimazu, Julio Villena, Masanori Tohno, Hitomi Fujie, Shoichi Hosoya, Takeshi Shimosato, Hisashi Aso, Yoshihito Suda, Yasushi Kawai, Tadao Saito, Seiya Makino, Shuji Ikegami, Hiroyuki Itoh, Haruki Kitazawa

    Infection and Immunity　80　(1)　276-288　2012/01

    Publisher: American Society for Microbiology

    DOI： 10.1128/iai.05729-11 　

    ISSN： 0019-9567

    eISSN： 1098-5522

    More details Close

    ABSTRACT The effect ofLactobacillus jenseniiTL2937 on the inflammatory immune response triggered by enterotoxigenicEscherichia coli(ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated thatL. jenseniiTL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated thatL. jenseniiTL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action.L. jenseniiTL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

  178. Orally administered Lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(I:C)

    Julio Villena, Eriko Chiba, Yohsuke Tomosada, Susana Salva, Gabriela Marranzino, Haruki Kitazawa, Susana Alvarez

    BMC Immunology　13　(1)　53-53　2012

    Publisher: Springer Nature

    DOI： 10.1186/1471-2172-13-53 　

    ISSN： 1471-2172

  179. Nasally Administered Lactobacillus rhamnosus Accelerate the Recovery of Humoral Immunity in B Lymphocyte-Deficient Malnourished Mice1–3

    Natalia Barbieri, Julio Villena, Matias Herrera, Susana Salva, Susana Alvarez

    The Journal of Nutrition　143　(2)　227-235　2012

    Publisher: Oxford University Press ({OUP})

    DOI： 10.3945/jn.112.165811 　

    ISSN： 0022-3166

  180. Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells Peer-reviewed

    Shoichi Hosoya, Julio Villena, Tomoyuki Shimazu, Masanori Tohno, Hitomi Fujie, Eriko Chiba, Takeshi Shimosato, Hisashi Aso, Yoshihito Suda, Yasushi Kawai, Tadao Saito, Susana Alvarez, Shuji Ikegami, Hiroyuki Itoh, Haruki Kitazawa

    Veterinary Research　42　(111)　2011/11/03

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1186/1297-9716-42-111 　

    eISSN： 1297-9716

    More details Close

    Abstract This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.

  181. Lactic acid bacteria in the prevention of pneumococcal respiratory infection: Future opportunities and challenges

    Julio Villena, Maria Leonor S. Oliveira, Patricia C.D. Ferreira, Susana Salva, Susana Alvarez

    International Immunopharmacology　11　(11)　1633-1645　2011/11

    Publisher: Elsevier {BV}

    DOI： 10.1016/j.intimp.2011.06.004 　

    ISSN： 1567-5769

  182. Toll-like receptor-2-activating bifidobacteria strains differentially regulate inflammatory cytokines in the porcine intestinal epithelial cell culture system: finding new anti-inflammatory immunobiotics Peer-reviewed

    Hitomi Fujie, Julio Villena, Masanori Tohno, Kyoko Morie, Tomoyuki Shimazu, Hisashi Aso, Yoshihito Suda, Takeshi Shimosato, Noriyuki Iwabuchi, Jin-Zhong Xiao, Tomoko Yaeshima, Keiji Iwatsuki, Tadao Saito, Muneo Numasaki, Haruki Kitazawa

    FEMS Immunology &amp; Medical Microbiology　63　(1)　129-139　2011/10/01

    Publisher: Oxford University Press (OUP)

    DOI： 10.1111/j.1574-695x.2011.00837.x 　

    ISSN： 0928-8244

    eISSN： 1574-695X

  183. Development of a fermented goats' milk containing Lactobacillus rhamnosus: in vivo study of health benefits Peer-reviewed

    Susana Salva, Martha Nuñez, Julio Villena, Adriana Ramón, Graciela Font, Susana Alvarez

    Journal of the Science of Food and Agriculture　91　(13)　2355-2362　2011/05/20

    Publisher: Wiley

    DOI： 10.1002/jsfa.4467 　

    ISSN： 0022-5142

    eISSN： 1097-0010

    More details Close

    Abstract BACKGROUND: Lactobacillus rhamnosus CRL1505, a strain of goats' milk origin, is able to stimulate mucosal immunity and protect immunocompetent mice from intestinal and respiratory infections. RESULTS: In this work we developed and characterized a fermented goats' milk containing L. rhamnosus CRL1505, and we demonstrated in a model of immunosuppression in mice that the final dairy product preserves the immunomodulatory properties of the strain. L. rhamnosus CRL1505 survived the manufacturing process of fermented milk and maintained a viability of 106 cfu g−1 during storage. The fermented goats' milk was accepted by 90.48% of the panelists and was considered as having an acid taste and pleasant aroma. We also demonstrated that the developed product, used as a supplement during the repletion of immunocompromised malnourished mice, was effective in accelerating the recovery of clinical parameters altered by malnutrition and to induce increased resistance against intestinal and respiratory infections. CONCLUSION: Goats' milk fermented with L. rhamnosus CRL1505 could be manufactured as an alternative probiotic dairy product since this new food has the ability to stimulate the common mucosal immune system and to improve defenses against respiratory and intestinal infections. Copyright © 2011 Society of Chemical Industry

  184. Immunomodulatory and protective effect of probiotic Lactobacillus casei against Candida albicans infection in malnourished mice

    Julio Villena, Susana Salva, Graciela Agüero, Susana Alvarez

    Microbiology and Immunology　55　(6)　434-445　2011/05

    Publisher: Wiley-Blackwell

    DOI： 10.1111/j.1348-0421.2011.00334.x 　

    ISSN： 0385-5600

  185. Lactococcus lactisas an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

    Marcela Susana Medina, Elisa Ofelia Vintiñi, Julio Villena, Raul R. Raya, Susana Gladis Alvarez

    Bioengineered Bugs　1　(5)　313-325　2010/09

    Publisher: Informa {UK} Limited

    DOI： 10.4161/bbug.1.5.12086 　

    ISSN： 1949-1018

  186. Immunomodulatory activity of Lactobacillus rhamnosus strains isolated from goat milk: Impact on intestinal and respiratory infections Peer-reviewed

    Susana Salva, Julio Villena, Susana Alvarez

    International Journal of Food Microbiology　141　(1-2)　82-89　2010/06/30

    Publisher: Elsevier BV

    DOI： 10.1016/j.ijfoodmicro.2010.03.013 　

    ISSN： 0168-1605

  187. Resistance of Young Mice to Pneumococcal Infection can be Improved by Oral Vaccination with Recombinant Lactococcus lactis

    Julio Villena, Marcela Medina, Silvia Racedo, Susana Alvarez

    Journal of Microbiology, Immunology and Infection　43　(1)　1-10　2010/02

    Publisher: Elsevier {BV}

    DOI： 10.1016/s1684-1182(10)60001-1 　

    ISSN： 1684-1182

  188. Administration of a probiotic associated with nasal vaccination with inactivated Lactococcus lactis-PppA induces effective protection against pneumoccocal infection in young mice Peer-reviewed

    E Vintiñi, J Villena, S Alvarez, M Medina

    Clinical and Experimental Immunology　159　(3)　351-362　2009/12/04

    Publisher: Oxford University Press (OUP)

    DOI： 10.1111/j.1365-2249.2009.04056.x 　

    ISSN： 0009-9104

    eISSN： 1365-2249

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    Summary Streptococcus pneumoniae is a serious public health problem, especially in developing countries, where available vaccines are not part of the vaccination calendar. We evaluated different respiratory mucosa immunization protocols that included the nasal administration of Lactococcus lactis-pneumococcal protective protein A (PppA) live, inactivated, and in association with a probiotic (Lc) to young mice. The animals that received Lc by the oral and nasal route presented the highest levels of immunoglobulin (Ig)A and IgG anti-PppA antibodies in bronchoalveolar lavages (BAL) and IgG in serum, which no doubt contributed to the protection against infection. However, only the groups that received the live and inactivated vaccine associated with the oral administration of the probiotic were able to prevent lung colonization by S. pneumoniae serotypes 3 and 14 in a respiratory infection model. This would be related to a preferential stimulation of the T helper type 1 (Th1) cells at local and systemic levels and with a moderate Th2 and Th17 response, shown by the cytokine profile induced in BAL and by the results of the IgG1/IgG2a ratio at local and systemic levels. Nasal immunization with the inactivated recombinant strain associated with oral Lc administration was able to stimulate the specific cellular and humoral immune response and afford protection against the challenge with the two S. pneumoniae serotypes. The results obtained show the probiotic-inactivated vaccine association as a valuable alternative for application to human health, especially in at-risk populations, and are the first report of a safe and effective immunization strategy using an inactivated recombinant strain.

  189. Enhanced immune response to pneumococcal infection in malnourished mice nasally treated with heat‐killed Lactobacillus casei Peer-reviewed

    Julio Villena, Natalia Barbieri, Susana Salva, Matías Herrera, Susana Alvarez

    Microbiology and Immunology　53　(11)　636-646　2009/10/23

    Publisher: Wiley

    DOI： 10.1111/j.1348-0421.2009.00171.x 　

    ISSN： 0385-5600

    eISSN： 1348-0421

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    ABSTRACT The present study analyzed whether nasal administration of viable and non‐viable Lactobacillus casei CRL 431 to immunocompromised mice was capable of increasing resistance against Streptococcus pneumoniae. Weaned mice were malnourished after consuming a PFD for 21 days. Malnourished mice were fed a BCD for 7 days or BCD for 7 days with viable or non‐viable L. casei nasal treatments on day 6 and day 7 (BCD+LcV and BCD+LcN, respectively). The MNC group received PFD whereas the WNC mice consumed BCD. MNC mice showed greater lung colonization, more severe lung injuries, impaired leukocyte recruitment and reduced antibodies and cytokine production when compared with WNC mice. Administration of L. casei increased the resistance of malnourished mice to the infection. Both BCD+LcV and BCD+LcN treatments prevented the dissemination of the pathogen to the blood and induced its lung clearance. BCD+LcV or BCD+LcN groups showed improved production of TNF‐α and activity of phagocytes in the respiratory tract, an effect that was not observed in the BCD control group. In addition, IL‐4 and IL‐10 were significantly increased in BCD+LcV and BCD+LcN groups, which correlated with the increase in the levels of specific respiratory IgA. The nasal treatments with L. casei were also effective at stimulating the production of specific IgG at both the systemic and the respiratory levels. The comparative study between the viable and the non‐viable bacteria demonstrated that viability would be an important factor to achieve maximum protective effects. However, the results from this study suggest that heat‐killed lactic acid bacteria are also effective in the immunomodulation of the systemic and respiratory immune system.

  190. Influence of yogurt consumption on the respiratory immune response Peer-reviewed

    Silvia Racedo, Julio Villena, Susana Salva, Susana Alvarez

    Food and Agricultural Immunology　20　(3)　231-244　2009/09/03

    Publisher: Informa UK Limited

    DOI： 10.1080/09540100903061659 　

    ISSN： 0954-0105

    eISSN： 1465-3443

  191. Lactobacillus casei modulates the inflammation-coagulation interaction in a pneumococcal pneumonia experimental model

    Cecilia Haro, Julio Villena, Hortensia Zelaya, Susana Alvarez, Graciela Agüero

    Journal of Inflammation　6　(1)　28-28　2009

    Publisher: Springer Nature

    DOI： 10.1186/1476-9255-6-28 　

    ISSN： 1476-9255

  192. Oral immunization with recombinant Lactococcus lactis confers protection against respiratory pneumococcal infection

    Julio Villena, Marcela Medina, Raúl Raya, Susana Alvarez

    Canadian Journal of Microbiology　54　(10)　845-853　2008/10

    Publisher: Canadian Science Publishing

    DOI： 10.1139/w08-077 　

    ISSN： 0008-4166

  193. Lactobacillus caseiaddition to a repletion diet-induced early normalisation of cytokine profils during a pneumococcal infection in malnourished mice

    Susana Salva, Julio Villena, Silvia Racedo, Susana Alvarez, Graciela Agüero

    Food and Agricultural Immunology　19　(3)　195-211　2008/08/15

    Publisher: Informa UK Limited

    DOI： 10.1080/09540100802247243 　

    ISSN： 0954-0105

    eISSN： 1465-3443

  194. Stimulation of respiratory immunity by oral administration of Lactococcus lactis

    Julio Villena, Marcela Medina, Elisa Vintiñi, Susana Alvarez

    Canadian Journal of Microbiology　54　(8)　630-638　2008/08

    Publisher: Canadian Science Publishing

    DOI： 10.1139/w08-052 　

    ISSN： 0008-4166

  195. Nasal administration ofLactococcus lactisimproves local and systemic immune responses againstStreptococcus pneumoniae

    Marcela Medina, Julio Villena, Susana Salva, Elisa Vintiñi, Philippe Langella, Susana Alvarez

    Microbiology and Immunology　52　(8)　399-409　2008/08

    Publisher: Wiley-Blackwell

    DOI： 10.1111/j.1348-0421.2008.00050.x 　

    ISSN： 0385-5600

  196. Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice Peer-reviewed

    Marcela Medina, Julio Villena, Elisa Vintiñi, Elvira María Hebert, Raúl Raya, Susana Alvarez

    Infection and Immunity　76　(6)　2696-2705　2008/06

    Publisher: American Society for Microbiology

    DOI： 10.1128/iai.00119-08 　

    ISSN： 0019-9567

    eISSN： 1098-5522

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    ABSTRACT Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis : it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages.

  197. Lactobacillus casei administration reduces lung injuries in a Streptococcus pneumoniae infection in mice

    Silvia Racedo, Julio Villena, Marcela Medina, Graciela Agüero, Virginia Rodríguez, Susana Alvarez

    Microbes and Infection　8　(9-10)　2359-2366　2006/08

    Publisher: Elsevier {BV}

    DOI： 10.1016/j.micinf.2006.04.022 　

    ISSN： 1286-4579

  198. Beneficial immunomodulatory activity of Lactobacillus casei in malnourished mice pneumonia: effect on inflammation and coagulation

    Graciela Agüero, Julio Villena, Silvia Racedo, Cecilia Haro, Susana Alvarez

    Nutrition　22　(7-8)　810-819　2006/07

    Publisher: Elsevier {BV}

    DOI： 10.1016/j.nut.2006.03.013 　

    ISSN： 0899-9007

  199. Yoghurt accelerates the recovery of defence mechanisms against Streptococcus pneumoniae in protein-malnourished mice

    Julio Villena, Silvia Racedo, Graciela Agüero, Susana Alvarez

    British Journal of Nutrition　95　(03)　591-591　2006/03

    Publisher: Cambridge University Press ({CUP})

    DOI： 10.1079/bjn20051663 　

    ISSN： 0007-1145

  200. Lactobacillus casei Improves Resistance to Pneumococcal Respiratory Infection in Malnourished Mice

    Julio Villena, Silvia Racedo, Graciela Agüero, Elena Bru, Marcela Medina, Susana Alvarez

    The Journal of Nutrition　135　(6)　1462-1469　2005/06/01

    Publisher: Oxford University Press ({OUP})

    DOI： 10.1093/jn/135.6.1462 　

    ISSN： 0022-3166

  201. Biological effects of goat milk administration in a malnutrition model Peer-reviewed

    Aguero Graciela, Lazarte Sandra, Salva Susana, VillenaJulio, Gonzalez Silvia Nelina, Alvarez Susana

    Milchwissenschaft　59　266-270　2004/01

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Misc. 3

  1. PIP細胞におけるTLRを介する脂質/免疫関連因子のトランスクリプトーム解析

    井形 愛美, Kober A.K.M. Humayun, 多田 明日翔, Albarracin Leonardo, 大坪 和香子, 麻生 久, 宮澤 賢司, 依田 一豊, 何 方, 齋藤 忠夫, Villena Julio, 北澤 春樹

    ミルクサイエンス　66　(2)　167-167　2017/08

    Publisher: 日本酪農科学会

    ISSN： 1343-0289

  2. ブタ腸管上皮細胞におけるビフィズス菌のToll様受容体を介する抗炎症機構

    友定 洋介, Villena Julio, 島津 朋之, 麻生 久, 岩淵 紀之, 清水 金忠[肖], 齋藤 忠夫, 北澤 春樹

    日本畜産学会大会講演要旨集　118回　114-114　2014/03

    Publisher: (公社)日本畜産学会

    ISSN： 1342-4688

  3. ブタ対応型イムノバイオティクスの腸管モデル共培養系による抗ウイルス性免疫評価

    千葉 絵里子, 細矢 翔一, Villena Julio, 藤江 瞳, 島津 朋之, 麻生 久, 川井 泰, 齋藤 忠夫, 池上 秀二, 伊藤 裕之, 北澤 春樹

    日本畜産学会大会講演要旨集　115回　119-119　2012/03

    Publisher: (公社)日本畜産学会

    ISSN： 1342-4688

Books and Other Publications 7

  1. La microbiota del tracto respiratorio y las bacterias comensales respiratorias como probióticos de nueva generación

    Julio Villena, M. Elean, L. Albarracin

    Ediciones UNL Ciencia y Tecnología　2024

  2. La microbiota intestinal y su efecto en el tracto respiratorio: el eje intestino–pulmón

    Julio Villena, M. Elean

    Ediciones UNL Ciencia y Tecnología　2024

  3. Lactic acid bacteria and respiratory health: their beneficial effects on viral infections

    Julio Villena, L. Albarracin, M. Elean, H. Kitazawa

    CRC Press　2023

  4. Immunobiotics: Interactions of Beneficial Microbes with the Immune System

    Julio Villena

    Frontiers Research Topics　2018

    DOI： 10.3389/978-2-88945-382-5 　

    ISBN: 9782889453825

  5. Editorial: Immunobiotics—Interactions of Beneficial Microbes with the Immune System

    Julio Villena, Haruki Kitazawa

    Frontiers Media {SA}　2017/11

    DOI： 10.3389/fimmu.2017.01580 　

  6. Probiotic Microorganisms: A Closer Look

    Julio Villena, Haruki Kitazawa

    {MDPI} {AG}　2017/04/08

    DOI： 10.3390/microorganisms5020017 　

  7. Probiotics : immunobiotics and immunogenics

    北澤, 春樹, Villena, Julio, Alvarez, Susana A.

    CRC Press　2013

    ISBN: 9781482206845

Show all Show first 5

Presentations 23

  1. Opening remarks: Food Design and One Health Invited

    Julio Villena

    International Kickoff Symposium Institute for Food Design and One Health Research Interdisciplinary Cluster for Cutting Edge Research　2026/02/19

  2. La taxifolina inhibe selectivamente a Clostridium perfringens al inducir una adaptación al estrés asociada con la limitación de hierro. International-presentation International-coauthorship

    Harum Fadhilatunnur, Weichen Gong, Haruna Sato, Eiji Hishinuma, Naomi Matsukawa, Fu Namai, Wakako-Ikeda Ohtsubo, Julio Villena, Hui-Yu Huang, Hiroaki Komatsu, Keita Nishiyama, Haruki Kitazawa

    ACLAB15　2025/11/19

  3. The oral administration of combined immunobiotic Lactiplantibacillus plantarum strains improve the respiratory TLR3-mediated antiviral immunity International-presentation

    Leonardo Albarracin, Luciano Arellano-Arriagada, Fu Namai, Weichen Gong, Keita Nishiyama, Haruki Kitazawa, Julio Villena

    ACLAB15　2025/11/20

  4. Identification of a novel seaweed polysaccharides-utilizing Lacticaseibacillus paracasei strain and its potential role in enhancing gut immunity in pigs International-presentation

    Weichen Gong, Fu Namai, Riku Ozawa, Wakako Ikeda-Ohtsubo, Julio Villena, Keita Nishiyama, Haruki Kitazawa

    ACLAB15　2025/11/20

  5. Mitochondrial Regulation by Limocilactobacillus salivarius in Porcine Intestinal Macrophages International-presentation

    Nozomi Hariu, Fu Namai, Wakako Ikeda-Ohtsubo, Julio Villena, Keita Nishiyama, Haruki Kitazawa

    ACLAB15　2025/11/20

  6. Immunobiotic Lactiplantibacillus plantarum strains beneficially modulate the intestinal antiviral immunity in malnourished immunocompromised mice International-presentation Invited

    Leonardo Albarracin, Luciano Arellano-Arriagada, Fu Namai, Weichen Gong, Keita Nishiyama, Haruki Kitazawa, Julio Villena

    ACLAB15　2025/11/20

  7. Potential of Postimmunobiotics to Modulate Immunity to Infections: Mechanistic Insights International-presentation International-coauthorship Invited

    Haruki Kitazawa, Julio Villena

    ACLAB15　2025/11/20

  8. Modulation of the gut-lung axis by probiotic lactobacilli and their beneficial impact on the resistance to respiratory infections Invited

    Julio Villena

    15th Asian Conference of Lactic Acid Bacteria　2025/11/20

  9. The originally established PBE cell line as an in vitro model for investigating immunobiotics against swine influenza virus International-presentation International-coauthorship

    Xi-Chen Bai, L. Albarracin, F. Namai, W. Gong, K. Nishiyama, J. Villena, H. Kitazawa

    The 11th Beneficial Microbes Conference

  10. Modulation of lung immune responses by immunobiotic respiratory commensal bacteria Invited

    Julio Villena

    CFAI 10th Anniversary Symposium　2025/10/23

  11. Modulation of antiviral immunity by probiotic lactic acid bacteria: their beneficial effects in immunocompromised hosts Invited

    Julio Villena

    Japanese Society for Lactic Acid Bacteria Annual Meeting 2025　2025/07/04

  12. Modulation of the innate immune memory in the respiratory mucosa by the local microbiota Invited

    Julio Villena

    14th Latin American and Caribbean Immunology Congress　2024/11/07

  13. Becoming a principal investigator in a globalized world

    Julio Villena

    Future Principal Investigator Program　2024/10/20

  14. Development of functional foods with the capacity to improve respiratory health: impact on resistance to viral infections Invited

    Julio Villena

    Special Seminar International Education and Research Centre for Food and Agricultural Immunology (CFAI)　2024/10/07

  15. Modulation of the gut-lung axis by the microbiota and probiotics: impact on the respiratory trained immunity and the resistance to infections Invited

    Julio Villena

    Tohoku Forum for Creativity: Designing Foods for the Future　2024/09/19

  16. The interplay between microbiota and the immune system in the respiratory tract: its impact on the resistance to infection Invited

    Julio Villena

    LXXI Annual Meeting of the Argentinean Society of Immunology　2023/11/09

  17. Respiratory commensal bacteria as next generation immunobiotics for the protection of lung infections Invited

    Julio Villena

    Special Seminar International Education and Research Centre for Food and Agricultural Immunology　2022/11/25

  18. The role of microbiota in the protection of lung infections: respiratory commensal bacteria as next generation immunobiotics Invited

    Julio Villena

    Special Seminar Shinshu University　2022/11/11

  19. La microbiota respiratoria como aliada para combatir infecciones virales: su potencial rol en la COVID-19 International-presentation International-coauthorship Invited

    Julio Villena

    XXIII Congreso y XLI Reunión Anual de la Sociedad de Biologia de Rosario　2021/11/23

  20. Respiratory commensal bacteria as next-generation immunobiotics International-presentation International-coauthorship Invited

    Julio Villena

    LXVII Annual Scientific Meeting of the Argentine Society of Immunology　2019/10/09

  21. Immunobiotechnological applications of beneficial microorganisms for the improvement of antiviral immunity in humans and animals Invited

    Julio Villena

    Morinaga Milk Industry Meeting　2018/10

  22. Evidence on the use of probiotics in social programs International-presentation Invited

    Julio Villena

    Probiotics, their role in nutrition and health. A view from the Southern Cone.　2018/04

  23. Interaction of the microbiota with the host and its impact on the immune system International-presentation Invited

    Julio Villena

    Russian Institute of Experimental Medicine Meeting “Advances in Microbiota”　2017/10

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Research Projects 5

  1. Commensal bacteria from nasopharynx as next-generation probiotics to potentiate the respiratory immunity: role of epithelial cells, macrophages, and innate immune memory

    VILLENA JULIO

    Offer Organization: FONCyT

    Institution: CERELA-CONICET

    2023 - 2027

  2. Beneficial microorganisms as modulators of immune responses mediated by innate lymphopid cells and thier impact on chronic respiratory diseases.

    Villena Julio

    Offer Organization: FONCyT

    Institution: CERELA-CONICET

    2019 - 2023

  3. Modulación of mucosal antiviral immunity in malnourished immunocompromised hosts using immunobiotic lactic acid bacteria. Study of the mechanisms of action with genomics tools

    Villena Julio

    Offer Organization: FONCyT

    Institution: CERELA-CONICET

    2017 - 2020

  4. Prevention of respiratory superinfection using non-viable lactic acid bacteria. Role of pattern recognition receptors and signalling pathways

    Villena Julio

    Offer Organization: FONCyT

    Institution: CERELA-CONICET

    2013 - 2015

  5. パターン認識受容体を介する腸管免疫調節性プロバイオティクスの食品免疫学的利用性

    北澤 春樹, VILLENA J.C.

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業

    Category: 特別研究員奨励費

    Institution: 東北大学

    2009 - 2011

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    【研究の目的】 本研究は、ヒトモデル系として期待されるブタにおいて、下痢症に関わる免疫パラメータを指標として、イムノバイオティクスの選抜から、その実証研究を行うことを目的としている。本年度は、昨年度までの成果を基礎として、イムノバイオティック機構の解析を進め、選抜イムノバイオティクスの有用性について検討した。 【研究成果】 1.イムノバイオティクスのPIE細胞における抗炎症シグナル伝達機構:選抜イムノバイオティクスによる細胞内シグナル分子の活性調節について解析したところ、MAPKのリン酸化とIkB分解の抑制が認められ、それらに関連するネガティブ調節因子の発現誘導が見られた。2.腸管パイエル板モデル培養系による抗炎症機能解析:PIE細胞と腸管パイエル板由来免疫担当細胞との共培養系において、抗炎症性発現における細胞間のサイトカインネットワークの特徴が把握できた。これらの結果から、選抜イムノバイオティクスの腸管抗炎症に関わる調節機構の一端が解明できた。今後、インビボ実証試験を続けることにより、選抜イムノバイオティクスの有用性が検証できる。

Teaching Experience 3

  1. Invited Full Professor Position. Invitational Fellowship Program for Collaborative Research at Tohoku University (2022)　Graduate School of Agricultural Science- Tohoku University.

  2. Invited Full Professor Position. Tohoku University Invitation for Foreign Researchers (Short-term)　Graduate School of Agricultural Science - Tohoku University

  3. Invited Full Professor Position. Tohoku University (Short-term)　Graduate School of Agricultural Science - Tohoku University