BACKGROUND: Cytokine receptor-like factor 2 (CRLF2) is a subunit of the receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation (insEIM) in the transmembrane domains of CRLF2 has been identified in acute lymphocytic leukemia (ALL), and Glu-Ile-Met (EIM) CRLF2 induces constitutive activation of signals. However, the signaling mechanism remains unclear. METHODS: HEK293 cells were transfected with expression vectors encoding wild-type (WT), insEIM CRLF2, or their mutants which N-glycosylation site was replaced with a glutamine. Cell surface expression of CRLF2 was assessed by flow cytometry. Total CRLF2 and phosphorylated signal transducer and activator of transcription 5 (STAT5) were detected by western blotting. RESULTS: Three major species of CRLF2 (53-, 57- and 58-kDa) were identified. Deglycosylation analysis revealed that they were modified with complex-type and oligomannose-type glycans. The expression of both WT and EIM CRLF2 decreased in N-acetylglucosaminyltransferase (GnT)-I (MGAT1) knockout (KO) cells and slightly decreased in α1,6-fucosyltransferase (Fut8) KO cells compared to that in the control cells. In GnT-I or Fut8 KO cells, WT CRLF2 did not induce ligand-independent activation. Both WT and EIM CRLF2 contained four N-glycosylation sites. N55 of CRLF2 was required for the cell surface expression and activation by EIM CRLF2. CONCLUSIONS: We found that N-glycosylation of CRLF2 plays crucial roles for its cell surface expression and signaling. However, N-glycan processing in the Golgi apparatus does not seem to be essential for ligand-independent activation of EIM CRLF2. GENERAL SIGNIFICANCE: Our studies provide a crucial role of glycosylation in the cell surface expression of receptors.

Atopic dermatitis (AD) is an allergic skin disease, triggered by excessive type 2 immune reactions. Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine that induces type 2 immune response through dendritic cell activation. Therefore, TSLP inhibitors may serve as novel antiallergic drugs. Hypoxia-inducible factor (HIF) activation in the epithelia contributes to several homeostatic phenomena, such as re-epithelialization. However, the effects of HIF activation on TSLP production and immune activation in the skin remain unclear. In this study, we found that selective HIF prolyl hydroxylase inhibitors (PHD inhibitors), which induce HIF activation, suppressed TSLP production in a mouse ovalbumin (OVA) sensitization model. PHD inhibitors also suppressed the production of tumor necrosis factor-alpha (TNF-α), which is a major inducer of TSLP production, in this mouse model and in a macrophage cell line. Consistent with these findings, PHD inhibitors suppressed OVA-specific IgE levels in the serum and OVA-induced allergic responses. Furthermore, we found a direct suppressive effect on TSLP expression in a human keratinocyte cell line mediated by HIF activation. Taken together, our findings suggest that PHD inhibitors exert antiallergic effects by suppressing TSLP production. Controlling the HIF activation system has therapeutic potential in AD.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) has been shown to be able to amplify Tregs. Thus, TSLP induction has the potential to induce endogenous Tregs and control autoimmunity. In the previous research, we found that a new compound named 02F04 can induce TSLP production while simultaneously activating the liver X receptor (LXR). Because LXR activation leads to a decrease in Treg, we attempted to find a 02F04-derivative, druggable lead compound with a basic skeleton that induces TSLP production without activating LXR. As the results, we found HA-7 and HA-19 and, in this study, examined the molecular mechanisms in TSLP production. METHODS: A murine keratinocyte cell line PAM 212 was stimulated with HA-7 and HA-19, and then the expressions of cytokines were examined via ELISA and real-time fluorescence quantitative PCR. RESULTS: HA-7 and HA-19 induced TSLP production but almost not the expression of TNF-α, IL-13, IL-25, and IL-33 in PAM212 cells. These compounds inhibited LXR activities. The TSLP expression induced by HA-7 and HA-19 was inhibited by the Gq/11 inhibitor YM-254890, ROCK inhibitor Y-27632, and ERK inhibitor U0126. HA-7 and HA-19 also induced the formation of stress fiber and ERK phosphorylation, which were inhibited by YM-254890 and Y-27632. CONCLUSIONS: Our findings indicated that HA-7 and HA-19 selectively induced TSLP production in PAM212 via Gq/11, Rho/ROCK and ERK pathways. Our findings also indicated that TSLP expression was differentially regulated from other cytokines, and the selective expression could be induced with low-molecular-weight compounds such as HA-7 and HA-19.

To prevent the onset and aggravation of allergic diseases, it is necessary to modulate excessive Th2-type immune responses. It is well accepted that thymic stromal lymphopoietin (TSLP) plays important roles in the change of Th1/Th2 balance to Th2 dominance and would be a druggable target. In this study, using a drug repositioning strategy, we identified 6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one (FK3453) as a novel inhibitor of TSLP production. FK3453 inhibited constitutive production of TSLP in the KCMH-1 mouse keratinocyte cell line and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced one in PAM212 cells. FK3453 also inhibited TSLP mRNA expression induced by a mixture of tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, fibroblast-stimulation lipopeptide-1, and protease activated-receptor agonist and TPA in normal human epidermal keratinocytes (NHEKs). Although FK3453 inhibited TPA-induced IL-33 expression in NHEKs in addition to TSLP, it did not inhibit TNF-α and IL-6 production. In addition, FK3453 did not inhibit MAP kinase (ERK) phosphorylation. We have confirmed that topical treatment with FK3453 inhibited TSLP production in the lipopolysaccharide-induced air pouch-type inflammation model. FK3453 could be a lead compound for a novel type of medicine which prevents the onset and aggravation of allergic diseases.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that plays an important role in the regulation of skin immunity and is upregulated in the lesions of atopic dermatitis patients. Therefore, inhibitors of TSLP production are effective as therapeutic agents for atopic dermatitis. In our laboratory, we found that hypoxia condition and DMOG, a PHD inhibitor, suppressed the expression of TSLP in keratinocytes. In this study, we analyzed the mechanism by which PHD inhibitors suppress TSLP expression. TSLP mRNA expression was induced in human keratinocyte cell line HaCaT cells by combined stimulation with TNFα, IL-4, FSL-1, and PAR2 agonist (T4FP stimulation), which was inhibited by PHD inhibitors Roxadustat and Enarodustat. The knockdown of HIF1α by siRNA abolished the inhibitory effect of Enarodustat on TSLP expression. Chromatin immunoprecipitation assay suggested that HIF1α binds to the hypoxia response elements (HRE) on the TSLP promoter. In conclusion, this study demonstrated that PHD inhibitors suppressed TSLP mRNA expression induced by T4FP stimulation in HaCaT cells. The suppression of TSLP expression was found to be HIF1α-dependent. In addition, we found that HIF1α binds to the HRE on the TSLP promoter, suggesting that HIF1α may affect transcription by binding to the TSLP promoter.

Although treatments for allergic diseases have improved, side effects and treatment resistance remain as challenges. New therapeutic drugs for allergic diseases are urgently required. Thymic stromal lymphopoietin (TSLP) is a cytokine target for prevention and treatment of allergic diseases. Since TSLP is produced from epithelial cells in allergic diseases, TSLP inhibitors may be new anti-allergic drugs. We previously identified a new inhibitor of TSLP production, named 16D10. However, its target of action remained unclarified. In this study, we found proteins binding to 16D10 from 24,000 human protein arrays by AlphaScreen-based high-throughput screening and identified bromodomain and extra-terminal (BET) family proteins as targets. We also clarified the detailed mode of interaction between 16D10 and a BET family protein using X-ray crystallography. Furthermore, we confirmed that inhibitors of BET family proteins suppressed TSLP induction and IL-33 and IL-36γ expression in both mouse and human keratinocyte cell lines. Taken together, our findings suggest that BET family proteins are involved in the suppression of TSLP production by 16D10. These proteins can contribute to the pathology of atopic dermatitis via TSLP regulation in keratinocytes and have potential as therapeutic targets in allergic diseases.

Elution of Ni ions from medical devices induces inflammation and toxicity. We previously reported that elution of Ni ions from Ni wires induced COX-2 expression and increased lactate production, but whether lactate is involved in the further elution of Ni ions remains unclear. In this study, using KMST-6, a human fibroblast cell line, we examined the molecular mechanisms by which Ni ions increase lactate release and the role of lactate in enhancing the elution of Ni ions. When KMST-6 cells were incubated on a Ni plate or stimulated with NiCl2 (1 mM), the expression of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), and the release of lactate were enhanced. The NiCl2 (1 mM)-induced expression of these genes was inhibited by a hypoxia-inducible factor-1α (HIF-1α) inhibitor, PX-478 (10-25 μM). Stimulation of cells with a prolyl hydroxylase domain (PHD) inhibitor, roxadustat, increased the expression of these genes, lactate release, and elution of Ni ions at 10 μM. A monocarboxylate transporter-4 (MCT4) inhibitor, syrosingopine, inhibited lactate release from roxadustat-treated cells and reduced the elution of Ni ions by the cells at 10 μM. Finally, syrosingopine (10 μM) reduced the elution of Ni ions by the cells from the Ni plate. These results suggest that elution of Ni ions from metals promotes the production of lactate via HIF-1α-mediated gene expression and causes further Ni elution. Thus, Ni ions show a positive feedback mechanism of Ni elution, and this step may be potentially targeted to protect against metal elution from metal devices.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived immunostimulatory factor, which activates several immune cells such as dendritic cells, T cells, and mast cells. Recently, epithelial cell-derived TSLP has gained immense attention as a cytokine that induces allergic immune responses. Therefore, understanding the regulation of TSLP production is an important step in uncovering the pathophysiology of allergic diseases. Moreover, the compounds that regulate TSLP production can be used as therapeutic drugs for the treatment of allergic diseases. We aim to elucidate the detailed regulation of TSLP production from epithelial cells, and in doing so discovered new regulating factors and an inhibitor of TSLP production. This review article explains the role of TSLP in allergic diseases, its regulation, and our research results.

Thymic stromal lymphopoietin (TSLP) is a key epithelial-derived factor that aggravates allergic diseases. Therefore, TSLP inhibitors are candidate compounds for the treatment of allergic diseases. Previously, we reported that KCMH-1, a mouse keratinocyte cell line, constitutively produces TSLP. In this study, we tried to identify inhibitors of TSLP by screening 2169 compounds in KCMH-1 cells and found one such chalcone derivative (code no. 16D10). 16D10 inhibited TSLP expression and TSLP promoter activation in HaCaT cells, a human keratinocyte cell line. Although nuclear factor kappa-B (NF-κB) is a key transcription factor for the induction of TSLP, 16D10 did not inhibit the activation pathway of NF-κB, such as degradation of inhibitor of κB (IκB) and p65 nuclear translocation. 16D10 activated the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system, although this system was not involved in the inhibitory effect of 16D10. 16D10 also inhibited TSLP production in a lipopolysaccharide (LPS)- or ovalbumin (OVA)-induced air-pouch-type inflammation model. Further, repeated 16D10 administration diminished serum immunoglobulin G1 (IgG1) and IgE concentration in an OVA-induced air-pouch-type sensitization model. Taken together, these results indicate that 16D10 is an inhibitor of TSLP production and has an anti-allergic effect. This inhibitory effect is independent of the activation of NF-κB and the Keap1-Nrf2 system. Therefore, 16D10 could be a new type of candidate drug for allergic diseases.

The expression of thymic stromal lymphopoietin (TSLP), a cytokine which greatly contributes to the induction of type I allergy, is upregulated in chronic inflammation such as atopic dermatitis and psoriasis. As hypoxia in the epidermis is important for maintaining skin homeostasis, we examined the regulation of TSLP expression by hypoxic conditions in normal skin epithelial tissues. TNF-α-induced expression of TSLP in human keratinocyte HaCaT and in mouse keratinocyte PAM212 cell lines were inhibited under hypoxic condition (1% O2), although the mRNA expressions of TNF-α, IL-6, IL-8, MCP-1, and VEGF-A were not inhibited. Hypoxia-mimicking conditions, which include NiCl2, CoCl2, and DMOG, an inhibitor of 2-oxoglutarate-dependent enzymes, also selectively inhibited TNF-α-induced TSLP expression. These results suggested that inactivation of prolyl hydroxylase by hypoxia and hypoxia-mimicking conditions is involved in the repression of TNF-α-induced TSLP expression. Interestingly, the inhibition of TSLP production by hypoxic treatment was significantly reversed by treatment with the HIF-2α antagonist but not with the HIF-1α inhibitor. DMOG-induced inhibition of TSLP promoter activity was dependent on the -71 to +185 bp promoter region, suggesting that the binding of HIF-2 to hypoxia response element (HRE) in this region repressed the TSLP expression. These results indicated that hypoxia and hypoxia-mimicking conditions inhibited TSLP expression via HIF-2 and HRE-dependent mechanisms. Therefore, PHD and HIF-2α could be a new strategy for treatment of atopic dermatitis and psoriasis.

Many classical vaccines contain whole pathogens and, thus, may occasionally induce adverse effects, such as inflammation. Vaccines containing purified rAgs resolved this problem, but, owing to their low antigenicity, they require adjuvants. Recently, the use of several cytokines, including thymic stromal lymphopoietin (TSLP), has been proposed for this purpose. However, it is difficult to use cytokines as vaccine adjuvants in clinical practice. In this study, we examined the effects of all-trans retinoic acid (atRA) on TSLP production and Ag-induced Ab production. Application of atRA onto the ear lobes of mice selectively induced TSLP production without inducing apparent inflammation. The effects appeared to be regulated via retinoic acid receptors γ and α. Treatment with atRA was observed to enhance OVA-induced specific Ab production; however, this effect was completely absent in TSLP receptor-knockout mice. An enhancement in Ab production was also observed when recombinant hemagglutinin was used as the Ag. In conclusion, atRA was an effective adjuvant through induction of TSLP production. Therefore, we propose that TSLP-inducing low m.w. compounds, such as atRA, may serve as effective adjuvants for next-generation vaccines.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine involved in the pathology of inflammatory skin diseases, such as atopic dermatitis and psoriasis. Tumor necrosis factor (TNF)-α, a key cytokine in inflammatory skin diseases, is a known TSLP inducer. TNF-α activates NF-κB and induces transactivation of epidermal growth factor receptor (EGFR) in epithelial cells. However, the detailed mechanism of TSLP induction by TNF-α has remained unclear. OBJECTIVE: We investigated the involvement of TNF-α-induced EGFR transactivation in TSLP expression. METHODS: HaCaT cells were stimulated with TNF-α or EGF in the presence or absence of an EGFR kinase inhibitor or other signaling inhibitors. The expression of TSLP mRNA was analyzed by RT-PCR and the phosphorylation level of signal proteins was analyzed by western blot. TSLP promoter and NF-κB transcription activities were analyzed by luciferase assay. RESULTS: TNF-α-induced TSLP expression was inhibited by the EGFR kinase inhibitor AG1478. While TSLP expression was induced by EGF, it was inhibited by the MEK inhibitor, U0126. Inhibitors of p38 and ADAM proteases suppressed the TNF-α-induced TSLP expression and EGFR phosphorylation, but not the EGF-induced expression. CONCLUSION: TNF-α-induced EGFR transactivation results in TSLP induction through ERK activation. The activation of p38 and ADAM proteases mediates TNF-α-induced EGFR phosphorylation. These findings suggested that the TNF-α-induced EGFR transactivation pathway could be a target for the treatment of inflammatory skin diseases.

Nickel ions (Ni2+) eluted from biomedical devices cause inflammation and Ni allergy. Although Ni2+ and Co2+ elicit common effects, Ni2+ induces a generally stronger inflammatory reaction. However, the molecular mechanism by which Ni2+ and Co2+ induce such different responses remains to be elucidated. In the present study, we compared the effects of Ni2+ and Co2+ on the expression of interleukin (IL)-8 in human monocyte THP-1 cells. We report that NiCl2 but not CoCl2 induced the expression of IL-8; in contrast, CoCl2 elicited a higher expression of hypoxia-inducible factor-1α (HIF-1α). The NiCl2-induced expression of IL-8 in late phase was blocked by a HIF-1α inhibitor, PX-478, indicating that NiCl2 targets additional factors responsible for activating HIF-1α. To identify such targets, proteins that bound preferentially to Ni-NTA beads were analyzed by LC/MS/MS. The analysis yielded heat shock protein 90β (HSP90β) as a possible candidate. Furthermore, Ni2+ reduced the interaction of HSP90β with HIF-1α, and instead promoted the interaction between HIF-1α and HIF-1β, as well as the nuclear localization of HIF-1α. Using various deletion variants, we showed that Ni2+ could bind to the linker domain on HSP90β. These results suggest that HSP90β plays important roles in Ni2+-induced production of IL-8 and could be a potential target for the regulation of Ni2+-induced inflammation.

Nickel ions (Ni2+) are eluted from various metallic materials, such as medical devices implanted in human tissues. Previous studies have shown that Ni2+ enters inflammatory cells inducing inflammation. However, the regulation of Ni2+ uptake in cells has not yet been reported in detail. In the present study, we investigated the effects of various divalent cations on Ni2+ uptake and Ni2+-induced interleukin (IL)-8 production in the human monocytic cell line, THP-1. We demonstrated that ZnCl2, MnCl2, and CoCl2 inhibited the Ni2+ uptake, while CuCl2, FeCl2, MgCl2, and divalent metal transporter (DMT)-1 inhibitor, Chlorazol Black, did not. Furthermore, ZnCl2 inhibited Ni2+-induced IL-8 production, correlating with the inhibition of Ni2+ uptake. These results suggested that Ni2+ uptake occurred through Zn2+, Mn2+, and Co2+-sensitive transporters and that the inhibition of Ni2+ uptake resulted in the inhibition of IL-8 production. Furthermore, using an Ni wire-implanted mouse model, we found that Ni wire-induced expression of mouse macrophage inflammatory protein-2 (MIP-2) and cyclooxygenase-2 (COX-2) mRNA in the skin tissue surrounding the wire were enhanced by low Zn conditions. These results suggested that the physiological concentration of Zn2+ modulates Ni2+ uptake by inflammatory cells, and a Zn deficient state might increase sensitivity to Ni.

Thymic stromal lymphopoietin (TSLP) plays critical roles in inducing and exacerbating allergic diseases. Chemical compounds that induce TSLP production can enhance sensitization to antigens and exacerbate allergic inflammation. Hence, identifying such chemicals will be important to prevent an increase in allergic diseases. In the present study, we found, for the first time, that a steroid alkaloid derivative, code no. 02F04, concentration and time dependently induced mRNA expression and production of TSLP in a mouse keratinocyte cell line, PAM212. In particular, the activity of 02F04 was selective to TSLP. As an analogue of the liver X receptor (LXR) endogenous ligand, 02F04 rapidly increased ATP-binding cassette transporter A1 (ABCA1) expression by regulating the nuclear receptor of LXR. However, instead of being inhibited by the LXR antagonist, 02F04-induced TSLP production was delayed and markedly suppressed by inhibitors of phospholipase C (PLC), pan-protein kinase C (PKC), PKCδ, Rho-associated protein kinase (ROCK), extracellular signal-regulated kinase (ERK) 1/2, and IκΒ kinase 2 (IKK2). Treatment with 02F04 caused the formation of F-actin filaments surrounding the nucleus of PAM212 cells, which then disappeared following addition of ROCK inhibitor. 02F04 also induced phosphorylation of ERK1/2 from 2h after treatment, with a maximum at 24h, and increased nuclear factor-κB (NF-κB) promoter activity by 1.3-fold. Taken together, these results indicate that 02F04-induced TSLP production is regulated via distinct signal transduction pathways, including PLC, PKC, ROCK, ERK1/2, and NF-κB but not nuclear receptors. 02F04, with a unique skeletal structure in inducing TSLP production, can represent a potential new tool for investigating the role of TSLP in allergic diseases.