IL-36 and IL-17A Cooperatively Induce a Psoriasis-Like Gene Expression Response in Human Keratinocytes

Psoriasis is a chronic inflammatory skin disease characterized by T helper (Th) 17 axis maintained by IL-17A and IL-17F produced by Th17 cells (Lynde et al., 2014Lynde C.W. Poulin Y. Vender R. Bourcier M. Khalil S. Interleukin 17A: toward a new understanding of psoriasis pathogenesis.J Am Acad Dermatol. 2014; 71: 141-150Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) and by keratinocyte (KC) axis stimulated by IL-17C produced by KCs (Johnston et al., 2013Johnston A. Fritz Y. Dawes S.M. Diaconu D. Al-Attar P.M. Guzman A.M. et al.Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation.J Immunol. 2013; 190: 2252-2262Crossref PubMed Scopus (193) Google Scholar; Ramirez-Carrozzi et al., 2011Ramirez-Carrozzi V. Sambandam A. Luis E. Lin Z. Jeet S. Lesch J. et al.IL-17C regulates the innate immune function of epithelial cells in an autocrine manner.Nat Immunol. 2011; 12: 1159-1166Crossref PubMed Scopus (296) Google Scholar). IL-17C has similar functions to IL-17A (Ramirez-Carrozzi et al., 2011Ramirez-Carrozzi V. Sambandam A. Luis E. Lin Z. Jeet S. Lesch J. et al.IL-17C regulates the innate immune function of epithelial cells in an autocrine manner.Nat Immunol. 2011; 12: 1159-1166Crossref PubMed Scopus (296) Google Scholar) and is a mediator in a feed-forward inflammatory program (Guttman-Yassky and Krueger, 2018Guttman-Yassky E. Krueger J.G. IL-17C: a unique epithelial cytokine with potential for targeting across the spectrum of atopic dermatitis and psoriasis.J Invest Dermatol. 2018; 138: 1467-1469Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). IL-36 family cytokines are also important mediators in a feed-forward inflammatory program (Johnston et al., 2011Johnston A. Xing X. Guzman A.M. Riblett M. Loyd C.M. Ward N.L. et al.IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression.J Immunol. 2011; 186: 2613-2622Crossref PubMed Scopus (234) Google Scholar) that includes self-amplification of IL-36 and synergy with IL-17 (Carrier et al., 2011Carrier 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: 2428-2437Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Although individual treatment of KCs with IL-36, IL-17A/A, or IL-17C induce overlapping antimicrobial proteins and innate cytokines (Hawkes et al., 2018Hawkes J.E. Yan B.Y. Chan T.C. Krueger J.G. Discovery of the IL-23/IL-17 signaling pathway and the treatment of psoriasis.J Immunol. 2018; 201: 1605-1613Crossref PubMed Scopus (205) Google Scholar; Johnston et al., 2011Johnston A. Xing X. Guzman A.M. Riblett M. Loyd C.M. Ward N.L. et al.IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression.J Immunol. 2011; 186: 2613-2622Crossref PubMed Scopus (234) Google Scholar; Ramirez-Carrozzi et al., 2011Ramirez-Carrozzi V. Sambandam A. Luis E. Lin Z. Jeet S. Lesch J. et al.IL-17C regulates the innate immune function of epithelial cells in an autocrine manner.Nat Immunol. 2011; 12: 1159-1166Crossref PubMed Scopus (296) Google Scholar), the alterations in gene signatures have not been compared directly, and the potential for cytokine interactions has been less studied (Carrier et al., 2011Carrier 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: 2428-2437Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Thus, we defined the direct and synergistic effect of IL-36 and IL-17 family cytokines on KCs. The study was conducted following the Helsinki Declaration and was approved by the institutional review boards. Written informed consent was obtained from all patients and healthy controls. The detailed methods are described in the Supplementary Materials and Methods. After confirming that IL36G expression in psoriasis skin was significantly higher than that in healthy controls (Supplementary Figure S1a), we found that IL36G expression was correlated positively with psoriasis-related gene expressions (Figure 1a). IL-36γ showed higher concentration in the upper epidermis in immunohistochemistry, whereas IL1RL2, which is a specific receptor of IL-36α, IL-36β, and IL-36γ, was equally expressed (Supplementary Figure S1b). Next, we stimulated adult normal human epidermal KCs for 24 hours with recombinant IL-36 and IL-17. IL-36α and IL-36γ self-amplified their mRNAs and enhanced their mRNAs and proteins synergistically with IL-17A (Figure 1b‒d and Supplementary Figures S2-S4). IL-17C was also increased significantly with IL-36 alone or synergistically with IL-17A (Figure 1b‒d and Supplementary Figures S2-S4). Furthermore, IL-17A and IL-36γ combinations significantly increased IL12B and IL23A expressions (Figure 1b). In addition, IL-36γ uniquely induced the inflammatory genes CCL5, MMP9, MMP12, ICAM1, and interferon response‒related factors CXCL9, CXCL10, and CXCL11 as well as IL32 and CCL27 (Supplementary Figures S3 and S4). Of note, IL-36 and IL-17A induced similar genes in KCs, such as Th17-regulated molecules CCL20 and CXCL1; IL-1 family IL36RN; and antimicrobial peptides DEFB4A, S100A7, and S100A12 (Figure 1b and Supplementary Figures S2‒S5). Similarly, CCL20 and CXCL8 proteins were significantly increased by IL-36 alone or synergistically with IL-17A (Figure 1c and d). We performed blocking studies using two IL1RL2-blocking antibodies to exclude that IL-17A triggered inflammatory gene expressions through IL-36 induction. Both antibodies failed to inhibit the IL17C, IL36G, CXCL8, and DEFB4A expressions induced by IL-17A (Figure 1e and f), whereas control experiments blocked IL-36‒induced genes (Supplementary Figure S6). IL-17C and IL-36 combinations had similar mRNA profiles to those of IL-17A and IL-36 combinations, but the response was relatively weak (Supplementary Figure S7). We further profiled the transcriptional responses to IL-17A and IL-36γ alone or in combination using Affymetrix arrays. IL-17A and IL-36γ individually induced over 1,000 genes, and about 700 mRNAs were coinduced by both cytokines, including many feed-forward IL-17 pathway mRNAs that typify psoriasis (Figure 2a and b and Supplementary Table S1). However, these also had unique transcriptional effects of 500 mRNAs induced only by IL-17A and 750 mRNAs induced only by IL-36γ (Figure 2a‒c and Supplementary Tables S2 and S3). For gene sets previously associated with psoriasis transcriptomes or IL-17A and TNF-α effects on KCs as judged by Gene Set Variation Analysis, both cytokines showed about equal induction (Figure 2d). Whereas genes induced uniquely by IL-17A were largely noninflammatory in nature (Supplementary Table S2), those induced by IL-36γ were inflammatory with increases in CCL5, CXCL9, and CXCL11 (29-fold, 15-fold, and 6-fold increases, respectively; false discovery rate < 0.01; Supplementary Table S3). In addition, the cytokines LTB, IL1A, IL32, EBI3, and CCL27 and GFs TGFA, IGF2, and PDGFB were induced significantly by IL-36γ (Supplementary Table S3). When combined, IL-17A and IL-36γ more than doubled the number of differentially expressed genes and increased mRNAs in additive and synergistic manners (Figure 2b and c and Supplementary Table S4). The Gene Set Variation Analysis also showed a strong representation of psoriasis—IL-17A and TNF-α gene sets by IL-17A and IL-36γ combination—which was confirmed by the second pathway analysis (Figure 2d and e). Differentially expressed genes in the KCs stimulated with IL-36 significantly overlapped with genes altered in psoriasis skin, including CXCL1 and CXCL8, which are key regulators of neutrophilic infiltration (Johnston et al., 2011Johnston A. Xing X. Guzman A.M. Riblett M. Loyd C.M. Ward N.L. et al.IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression.J Immunol. 2011; 186: 2613-2622Crossref PubMed Scopus (234) Google Scholar; Mahil et al., 2017Mahil 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: eaan2514Crossref PubMed Scopus (81) Google Scholar). KCs response to IL-17A/A alone can encompass over 600 mRNAs with increased C/EBP-β expression mediating IL-17‒stimulated gene expression in the upper spinous or granular layers (Chiricozzi et al., 2014Chiricozzi A. Nograles K.E. Johnson-Huang L.M. Fuentes-Duculan J. Cardinale I. Bonifacio K.M. et al.IL-17 induces an expanded range of downstream genes in reconstituted human epidermis model.PLoS One. 2014; 9: e90284Crossref PubMed Scopus (115) Google Scholar). Our direct comparison of these two cytokines showed that IL-17A and IL-36γ independently expressed the same genes that typify psoriasis and that genes induced uniquely by IL-36 have inflammatory features. Our data suggest that the ability of IL-36 to amplify the IL-17A‒induced psoriasis response may be more critical in vivo in the epidermis because IL-36 was concentrated in the upper spinous and/or granular epidermis. The extent that IL-36 can self-amplify and cross-induce with IL-17C, IL-17A and IL-36, and IL-17C and IL-36 combinations may interact to promote the inflammatory phenotype in psoriasis. Indeed, IL-36 regulation of IL-17C may provide feedback to Th17 cells because IL-17C is an activator of Th17 cells, whereas T cells do not bear IL-36 receptors (Hawkes et al., 2018Hawkes J.E. Yan B.Y. Chan T.C. Krueger J.G. Discovery of the IL-23/IL-17 signaling pathway and the treatment of psoriasis.J Immunol. 2018; 201: 1605-1613Crossref PubMed Scopus (205) Google Scholar; Johnston et al., 2011Johnston A. Xing X. Guzman A.M. Riblett M. Loyd C.M. Ward N.L. et al.IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression.J Immunol. 2011; 186: 2613-2622Crossref PubMed Scopus (234) Google Scholar). A further argument is that IL-17A and IL-36γ combinations alter over 1,900 mRNAs in KCs, resulting in a stronger psoriasis molecular phenotype and, potentially, an in vitro disease model. The strong correlations between IL-36 and their products also suggested an in vivo role for IL-36 agonists in regulating key psoriasis-pathway mRNAs. Considering that IL12B and IL23A expressions were significantly increased synergistically with IL-36γ and IL-17A, adequate concentrations of both cytokines are required to act synergistically for KCs to efficiently produce IL-12 and IL-23, potentially activating Th1 and Th17 cells. Therapeutic studies with IL-36 antagonists are needed to determine whether IL-36 is a direct contributor to the pathogenic phenotype of psoriasis vulgaris or an amplifier of the IL-17 program. The microarray datasets of psoriasis skin are deposited in the Gene Expression Omnibus (accession number GSE106992). The microarray datasets using adult normal human epidermal keratinocytes are deposited in the Gene Expression Omnibus (accession number GSE160904). Other data that support the findings of this study are available from the corresponding author, JGK, on reasonable request. Shunsuke Miura: https://orcid.org/0000-0001-7259-2242 Sandra Garcet: https://orcid.org/0000-0002-4465-8547 Charissa Salud-Gnilo: https://orcid.org/0000-0002-1623-5141 Juana Gonzalez: https://orcid.org/0000-0001-7933-7017 Xuan Li: https://orcid.org/0000-0001-7021-8054 Mika Murai-Yamamura: https://orcid.org/0000-0002-0739-3551 Kazuhiko Yamamura: https://orcid.org/0000-0001-9216-945X Darshna Rambhia: https://orcid.org/0000-0002-9789-8077 Norma Kunjravia: https://orcid.org/0000-0003-0308-1048 Emma Guttman-Yassky: https://orcid.org/0000-0002-9363-324X James G. Krueger: https://orcid.org/0000-0002-3775-1778 EGY is an employee of Mount Sinai; has received research funds (grants paid to the institution) from AbbVie, Almirall, Amgen, AnaptysBio, Asana Biosciences, AstraZeneca, Boerhinger Ingelhiem, Celgene, Dermavant, DS Biopharma, Eli Lilly, Galderma, Glenmark/Ichnos Sciences, Innovaderm, Janssen, Kiniksa, Kyowa Kirin, Leo Pharma, Novan, Novartis, Pfizer, Ralexar, Regeneron Pharmaceuticals, Sienna Biopharma, UCB, and UNION Therapeutics/Antibiotx; and is a consultant for AbbVie, Aditum Bio, Almirall, Alpine, Amgen, Arena, Asana Biosciences, AstraZeneca, Bluefin Biomedicine, Boehringer Ingelheim, Boston Pharmaceuticals, Botanix, Bristol-Meyers Squibb, Cara Therapeutics, Celgene, Clinical Outcome Solutions, DBV Technologies, Dermavant, Dermira, Douglas Pharmaceutical, DS Biopharma, Eli Lilly, EMD Serono, Evelo Bioscience, Evidera, FIDE, Galderma, GlaxoSmithKline, Haus Bioceuticals, Ichnos Sciences, Incyte, Kyowa Kirin, Larrk Bio, Leo Pharma, Medicxi, Medscape, Neuralstem, Noble Insights, Novan, Novartis, Okava Pharmaceuticals, Pandion Therapeutics, Pfizer, Principia Biopharma, RAPT Therapeutics, Realm, Regeneron Pharmaceuticals, Sanofi, SATO Pharmaceutical, Sienna Biopharma, Seanegy Dermatology, Seelos Therapeutics, Serpin Pharma, Siolta Therapeutics, Sonoma Biotherapeutics, Sun Pharma, Target RWE, UNION Therapeutics, Vanda Pharmaceuticals, Ventyx Biosciences, and Vimalan. JGK has received research support (grants paid to institution) from AbbVie, Amgen, Bristol-Myers Squibb, Boehringer Ingelheim, EMD Serono, Innovaderm, Kineta, LEO Pharma, Novan, Novartis, Paraxel, Pfizer, Regeneron, and Vitae and personal fees from AbbVie, Acros, Allergan, Aurigne, BiogenIdec, Boehringer Ingelheim, Escalier, Janssen, Lilly, Novartis, Pfizer, Roche, and Valeant. The remaining authors state no conflict of interest. This work was supported in part by a fellowship from the Uehara Memorial Foundation, Japan (award number: 201830050). Conceptualization: SM, JGK; Data Curation: SM, SG, XL; Formal Analysis: SM, SG; Funding Acquisition: SM, JGK; Investigation: SM, JG, XL; Methodology: SM; Project Administration: JGK; Resources: SM, EGY, JGK; Supervision: JGK; Validation: SM; Visualization: SM, SG; Writing - Original Draft Preparation: SM; Writing - Review and Editing: JG, XL, MMY, KY, DR, CSG, NK, EGY, JGK Download .pdf (2.2 MB) Help with pdf files Supplementary Data Download .xlsx (.08 MB) Help with xlsx files Table S1 Download .xlsx (.03 MB) Help with xlsx files Table S2 Download .xlsx (.04 MB) Help with xlsx files Table S3 Download .xlsx (1.37 MB) Help with xlsx files Table S4