Advertisement

IL-36 Signaling Is Essential for Psoriatic Inflammation through the Augmentation of Innate Immune Responses

Open ArchivePublished:December 17, 2018DOI:https://doi.org/10.1016/j.jid.2018.12.003

      Abbreviations:

      DC (dendritic cell), Ht (heterozygous), IL-36R (IL-36 receptor), KC (keratinocyte), KO (knockout), TPA (12-O-tetradecanoylphorbol-13-acetate)
      To the Editor
      IL-36 cytokines are composed of three agonists, namely IL-36α, IL-36β, and IL-36γ, and a natural antagonist, IL-36Ra (
      • Sims J.E.
      • Smith D.E.
      The IL-1 family: regulators of immunity.
      ). IL-36 cytokines are abundantly expressed by the skin and other epithelial tissues, whereas the IL-36 receptor (IL-36R) is expressed by skin and immune cells, including dendritic cells (DCs) (
      • Gabay C.
      • Towne J.E.
      Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions.
      ,
      • Vigne S.
      • Palmer G.
      • Lamacchia C.
      • Martin P.
      • Talabot-Ayer D.
      • Rodriguez E.
      • et al.
      IL-36R ligands are potent regulators of dendritic and T cells.
      ). Earlier studies have demonstrated that IL-36 cytokines play important roles in the development of psoriasiform inflammation by enhancing the function of T helper type 17 cytokines (
      • Carrier Y.
      • Ma H.L.
      • Ramon H.E.
      • Napierata L.
      • Small C.
      • O'Toole M.
      • et al.
      Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis.
      ,
      • Tortola L.
      • Rosenwald E.
      • Abel B.
      • Blumberg H.
      • Schafer M.
      • Coyle A.J.
      • et al.
      Psoriasiform dermatitis is driven by IL-36-mediated DC-keratinocyte crosstalk.
      ). Indeed, IL-36 signaling–regulated genes in keratinocytes (KCs) are largely shared with those observed in psoriatic lesions, and they form interconnected feedback loops that potentiate IL-17 signaling and leukocyte chemotaxis (
      • Carrier Y.
      • Ma H.L.
      • Ramon H.E.
      • Napierata L.
      • Small C.
      • O'Toole M.
      • et al.
      Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis.
      ,
      • Mahil S.K.
      • Catapano M.
      • Di Meglio P.
      • Dand N.
      • Ahlfors H.
      • Carr I.M.
      • et al.
      An analysis of IL-36 signature genes and individuals with IL1RL2 knockout mutations validates IL-36 as a psoriasis therapeutic target.
      ), although the details remain largely unknown.
      Psoriatic lesions demonstrated increased IL-36α, IL36-γ, IL-36Ra, but not IL-36β, as reported previously (see Supplementary Figure S1a online). Similarly, all of the IL-36 family members were upregulated in 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced psoriatic lesions in K5.Stat3C model (see Supplementary Figure S1b), which required the IL-23/Th17 pathway (
      • Nakajima K.
      • Kanda T.
      • Takaishi M.
      • Shiga T.
      • Miyoshi K.
      • Nakajima H.
      • et al.
      Distinct roles of IL-23 and IL-17 in the development of psoriasis-like lesions in a mouse model.
      ).
      To explore the role of IL-36 signaling in KCs, we examined psoriasis-related gene expression in response to exogenous IL-36 in between control and IL-36R–/– (knockout [KO]) KCs derived from IL-36R KO mice. Following IL-36α stimulation, wild-type KCs expressed increased Il36a, S100a8, Defb3, and Il17c mRNAs, and also IL-17C protein (Figure 1a ). In contrast, the upregulation of those was not observed in IL-36R KO KCs. Similar results were obtained when we used other IL-36 cytokines, namely, IL-36β and IL-36γ (Supplementary Figure S2 online). These results strongly implicated the participation of the IL-36/IL-36R autocrine loop within KCs in psoriasis development. Our recent study revealed that in vitro IL-36 stimulation of KCs elicited Stat3 phosphorylation at the early time point (
      • Takaishi M.
      • Satoh T.
      • Akira S.
      • Sano S.
      Regnase-1, an immunomodulator, limits the IL-36/IL-36R autostimulatory loop in keratinocytes to suppress skin inflammation.
      ), suggesting that the IL-36/IL-36R autocrine loop may facilitate the Stat3-mediated innate immunity within KCs.
      Figure thumbnail gr1
      Figure 1IL-36 signaling is required for both keratinocytes and dendritic cells to express psoriasis signature molecules to develop psoriasiform lesions. (a) Reverse transcriptase PCR and ELISA assay using keratinocytes from WT (n = 4–5) and IL-36R KO (KO, n = 3) mice. White, untreated; black, IL-36α–treated (100 ng/ml, 24 hours). ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (b) Reverse transcriptase PCR analysis. Black bars, HKC (5 × 106 cells/ml); white bars, untreated control. WT, n = 4–7; KO, n = 3–8. P < 0.05. (c) Reverse transcriptase PCR. HKC (5 × 106 cells/ml), IL-36α (100 ng/ml, 12 hours), n = 3–7, ∗∗P < 0.01 by one-way analysis of variance with Tukey-Kramer analysis. (d, e) ELISA assay using the culture supernatant of splenic dendritic cells. White bars, untreated control, n = 4 (d, e); black bars, IL-36α–treated (1 μg/ml, 12 hours), n = 4 (d). IMQ-treated (20 μM, 12 hours), n = 4. ∗∗P < 0.01, ∗∗∗∗P < 0.0001. (f) Histology of ear skin from K5.Stat3C:IL-36R+/– (top) and K5.Stat3C:IL-36RKO mice (bottom) at day 3 of topical TPA treatment. Hematoxylin and eosin staining. Scale bars = 200 μm. (g) Ear swelling. White bar, K5.Stat3C:IL-36R controls (n = 11); black bar, K5.Stat3C:IL-36RKO mice (n = 7). ∗∗P < 0.01. (h) Reverse transcriptase PCR analysis using ear skin. White bars, K5.Stat3C:IL-36R+/– mice (n = 10); black bars, K5.Stat3C:IL-36RKO mice (n = 6). P < 0.05, ∗∗P < 0.01. (i, j) Ear swelling in chimeric K5.Stat3C mice over time (i, hours) and at 24 hours (j) of topical TPA treatment. Ht>Ht, IL-36R+/– mice reconstituted with IL-36R+/– bone marrow cells (n = 4); Ht>KO, IL-36R–/– mice with IL-36R+/– bone marrow cells (n = 7); KO>Ht, IL-36R+/–mice with IL-36R–/– bone marrow cells (n = 5); KO>KO, IL-36R–/– mice with IL-36R–/– bone marrow cells (n = 5). P < 0.05. (k) Reverse transcriptase PCR analysis using TPA–induced skin lesions of bone marrow chimeric mice of the indicated genotype combinations (n = 4–6 in each group) at 72 hours. P < 0.05, ∗∗P < 0.01. HKC, heat-killed Candida albicans; Ht, heterozygous; IMQ, imiquimod; KO, knockout; ND, not detectable; ns, not significant; TPA, 12-O-tetradecanoylphorbol-13-acetate; WT, wild-type.
      There are immunologic similarities between anti-Candida defense and psoriasis, including the IL-23/IL-17 axis (
      • Saunte D.M.
      • Mrowietz U.
      • Puig L.
      • Zachariae C.
      Candida infections in patients with psoriasis and psoriatic arthritis treated with interleukin-17 inhibitors and their practical management.
      ). Psoriasis signature molecules were upregulated in keratinocytes when treated in vitro with heat-killed Candida albicans, including IL-8 (
      • Kobayashi M.
      • Yoshiki R.
      • Sakabe J.
      • Kabashima K.
      • Nakamura M.
      • Tokura Y.
      Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes.
      ,
      • Pivarcsi A.
      • Bodai L.
      • Rethi B.
      • Kenderessy-Szabo A.
      • Koreck A.
      • Szell M.
      • et al.
      Expression and function of Toll-like receptors 2 and 4 in human keratinocytes.
      ). Interestingly, stimulation with heat-killed C. albicans resulted in the upregulation of Il36a, Il17c, and Ccl20 in wild-type KCs, but not in IL-36R KO KCs (Figure 1b). In addition, heat-killed C. albicans stimulation exerted an additive effect on IL-36 signaling in IL36a and Ccl20 expression in KCs (Figure 1c and Supplementary Figure S3 online). This result suggested that KCs could respond to C. albicans through pattern recognition receptors, leading to the transcriptional activation of Il36a, Il17c, and Ccl20 in a manner depending on the IL-36/IL-36R autocrine loop, which is shared with psoriasis and may involve the IL-23/T helper type 17 axis (
      • Saunte D.M.
      • Mrowietz U.
      • Puig L.
      • Zachariae C.
      Candida infections in patients with psoriasis and psoriatic arthritis treated with interleukin-17 inhibitors and their practical management.
      ). The role of IL-36 signaling in splenic DCs was clearly shown by the increase of IL-12/IL-23p40 and tumor necrosis factor–α following stimulation with IL-36α. This DC activation was mediated by IL-36R because DCs from IL-36RKO mice are impaired in the production of these cytokines (Figure 1d). Our previous study demonstrated that a cathepsin K inhibitor, which damped toll-like receptor 7–mediated DCs activation leading to IL-23 production, attenuated TPA–induced psoriasiform development in K5.Stat3C mice, suggesting the role of innate immunity with DCs (
      • Hirai T.
      • Kanda T.
      • Sato K.
      • Takaishi M.
      • Nakajima K.
      • Yamamoto M.
      • et al.
      Cathepsin K is involved in development of psoriasis-like skin lesions through TLR-dependent Th17 activation.
      ). Notably, stimulation of toll-like receptor 7 with imiquimod resulted in an increase of tumor necrosis factor–α and IL-12/IL-23p40 production by control DCs, but not by IL-36R KO DCs (Figure 1e). This result highlights the role of IL-36 signaling to enhance the innate immune response through toll-like receptor 7 in DCs. We next sought to address the in vivo effect of IL-36R deficiency on psoriasiform inflammation in K5.Stat3C mice. Deficiency of IL-36R protected from TPA–induced skin lesions in K5.Stat3C mice (Figure 1f, g), while those with IL-36R+/– background (heterozygous [Ht]) developed lesions similar to IL-36R+/+ (see Supplementary Figure S4 online). Further, the upregulation of psoriasis signature genes, including Il36a, Tnfa, Il23a, Il17c, Ccl20, and Cxcl2, were all attenuated by IL-36R deficiency (Figure 1h). To dissect the roles of KCs and DCs in IL-36 signaling, IL-36R KO or control bone marrow cells were transferred to lethally irradiated recipient K5.Stat3C mice in a criss-cross manner. Skin lesions were dramatically attenuated in Ht:K5.Stat3C mice reconstituted with IL-36R KO bone marrow (KO>Ht) cells compared with mice with the opposite combination (Ht>KO), whose lesions were only marginally affected, comparable to the positive controls (Ht>Ht) at the later time point (Figure 1i, j, and Supplementary Figure S5 online); however, at the early time point (6 hours), both chimeric mice with Ht>KO and KO>Ht showed impaired ear swelling like KO>KO chimeras. Analysis of gene expression of skin lesions revealed that Il17c mRNA levels were significantly down-modulated in mice with Ht>KO, but not with KO>Ht chimeras (Figure 1k), supporting that IL-17C was produced by the radio-resistant epidermis via IL-36 signaling (Figure 1a). However, Il36 mRNA levels were decreased in lesions of mice with both Ht>KO and KO>Ht chimeras (Figure 1k). Collectively, these results suggested that bona fide psoriasiform lesions depended on IL-36 signaling both in the epidermis and in bone marrow–derived cells, including DCs. In contrast, the previous study showed that IL-36R expression on radio-resistant skin-resident cells, but not on bone marrow–derived cells, was essential for pathology upon imiquimod treatment (
      • Tortola L.
      • Rosenwald E.
      • Abel B.
      • Blumberg H.
      • Schafer M.
      • Coyle A.J.
      • et al.
      Psoriasiform dermatitis is driven by IL-36-mediated DC-keratinocyte crosstalk.
      ). The discrepancy of the results in the present and previous studies might be due to the difference in mouse models, although the underlying mechanism remains unknown.
      Given that psoriasis develops via IL-36 signaling, the inhibition of that pathway should attenuate psoriatic inflammation. To explore this possibility, we treated K5.Stat3C mice and ex vivo tissue cultures of human plaque psoriasis with recombinant IL-36Ra. Subcutaneous injection of IL-36Ra attenuated the development of TPA–induced skin lesions in K5.Stat3C mice (Figure 2a, b , and Supplementary Figure S6 online). In addition, the epidermal thickness of psoriasis lesions in ex vivo cultures was attenuated by the addition of IL-36Ra (Figure 2c, d), which also downregulated IL8, CCL20, and IL17A mRNAs (Figure 2e). In conclusion, these results strongly suggest that the inhibition of IL-36 signaling would be relevant for new treatments of psoriasis.
      Figure thumbnail gr2
      Figure 2Treatment with IL-36R antagonists attenuate mouse and human psoriasiform lesions. (a) TPA–induced psoriasiform lesions in K5.Stat3C mice intradermally treated with PBS (left panel) or IL-36Ra (right panel). Hematoxylin and eosin staining. Scale bars = 200 μm. (b) TPA–induced ear swelling of K5.Stat3C at day 3. White bar, PBS control (n = 5); black bar, IL-36Ra-treated (n = 5). P < 0.05. (c) Representative views of histopathology of ex vivo organ culture of lesional skins in the elbow from a patient at 24 hours after treatment with 5 ng/ml BSA (left panel) or human IL-36Ra (right panel). Hematoxylin and eosin staining. Scale bars = 200 μm. (d) Epidermal thickness in psoriatic lesions treated with BSA (white bar, n = 3) or IL-36Ra (black bar, n = 3). P < 0.05. (e) Changes in the mRNA levels in IL-36Ra–treated psoriatic lesions (black bars, n = 3) versus BSA controls (white bars, n = 3) that are set to 100. P < 0.05, ∗∗∗P < 0.001. IL-36R, IL-36 receptor; ns, not significant; PBS, phosphate buffered saline; TPA, 12-O-tetradecanoylphorbol-13-acetate.
      Supplementary Figure S7 online illustrates the role of IL-36 signaling (red jagged mark) both in KCs and in DCs in the augmentation of innate immune responses, which leads to the development of psoriasis through activation of the IL-23/IL-17 axis.
      All experimental procedures performed on mice were approved by the Institutional Animal Care and Use Committee of the Kochi Medical School. The written informed consent and approval of the ethics committee were obtained from all patients.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank Reiko Kamijima for her help with the histopathology, and JE Sims, JE Towne, and Amgen Inc for providing us with IL-36RKO mice.
      This work was supported in part by a Grant-in-Aid for Scientific Research (26461695) from the Ministry of Education, Culture, Sports, Science and Technology of Japan .

      Supplementary Material

      References

        • Carrier Y.
        • Ma H.L.
        • Ramon H.E.
        • Napierata L.
        • Small C.
        • O'Toole M.
        • et al.
        Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis.
        J Invest Dermatol. 2011; 131: 2428-2437
        • Gabay C.
        • Towne J.E.
        Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions.
        J Leukoc Biol. 2015; 97: 645-652
        • Hirai T.
        • Kanda T.
        • Sato K.
        • Takaishi M.
        • Nakajima K.
        • Yamamoto M.
        • et al.
        Cathepsin K is involved in development of psoriasis-like skin lesions through TLR-dependent Th17 activation.
        J Immunol. 2013; 190: 4805-4811
        • Kobayashi M.
        • Yoshiki R.
        • Sakabe J.
        • Kabashima K.
        • Nakamura M.
        • Tokura Y.
        Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes.
        Br J Dermatol. 2009; 160: 297-304
        • Mahil S.K.
        • Catapano M.
        • Di Meglio P.
        • Dand N.
        • Ahlfors H.
        • Carr I.M.
        • et al.
        An analysis of IL-36 signature genes and individuals with IL1RL2 knockout mutations validates IL-36 as a psoriasis therapeutic target.
        Sci Transl Med. 2017; 9
        • Nakajima K.
        • Kanda T.
        • Takaishi M.
        • Shiga T.
        • Miyoshi K.
        • Nakajima H.
        • et al.
        Distinct roles of IL-23 and IL-17 in the development of psoriasis-like lesions in a mouse model.
        J Immunol. 2011; 186: 4481-4489
        • Pivarcsi A.
        • Bodai L.
        • Rethi B.
        • Kenderessy-Szabo A.
        • Koreck A.
        • Szell M.
        • et al.
        Expression and function of Toll-like receptors 2 and 4 in human keratinocytes.
        Int Immunol. 2003; 15: 721-730
        • Saunte D.M.
        • Mrowietz U.
        • Puig L.
        • Zachariae C.
        Candida infections in patients with psoriasis and psoriatic arthritis treated with interleukin-17 inhibitors and their practical management.
        Br J Dermatol. 2017; 177: 47-62
        • Sims J.E.
        • Smith D.E.
        The IL-1 family: regulators of immunity.
        Nat Rev Immunol. 2010; 10: 89-102
        • Takaishi M.
        • Satoh T.
        • Akira S.
        • Sano S.
        Regnase-1, an immunomodulator, limits the IL-36/IL-36R autostimulatory loop in keratinocytes to suppress skin inflammation.
        J Invest Dermatol. 2018; 138: 1439-1442
        • Tortola L.
        • Rosenwald E.
        • Abel B.
        • Blumberg H.
        • Schafer M.
        • Coyle A.J.
        • et al.
        Psoriasiform dermatitis is driven by IL-36-mediated DC-keratinocyte crosstalk.
        J Clin Invest. 2012; 122: 3965-3976
        • Vigne S.
        • Palmer G.
        • Lamacchia C.
        • Martin P.
        • Talabot-Ayer D.
        • Rodriguez E.
        • et al.
        IL-36R ligands are potent regulators of dendritic and T cells.
        Blood. 2011; 118: 5813-5823