IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis Via Parallel Mechanisms Akash H

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IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms Akash H. Verma, Hanna Zafar, Nicole O. Ponde, Olivia W. Hepworth, Diksha Sihra, Felix E. Y. Aggor, Joseph S. This information is current as Ainscough, Jemima Ho, Jonathan P. Richardson, Bianca M. of September 28, 2021. Coleman, Bernhard Hube, Martin Stacey, Mandy J. McGeachy, Julian R. Naglik, Sarah L. Gaffen and David L. Moyes

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 11, 2018, doi:10.4049/jimmunol.1800515 The Journal of Immunology

IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms

Akash H. Verma,*,1 Hanna Zafar,†,‡,1 Nicole O. Ponde,† Olivia W. Hepworth,†,‡ Diksha Sihra,† Felix E. Y. Aggor,* Joseph S. Ainscough,x Jemima Ho,† Jonathan P. Richardson,† Bianca M. Coleman,* Bernhard Hube,{,‖,# Martin Stacey,x Mandy J. McGeachy,* Julian R. Naglik,† Sarah L. Gaffen,* and David L. Moyes†,‡

Protection against microbial infection by the induction of inﬂammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the

key players in driving these responses remain poorly deﬁned. Recent work has identiﬁed a central role played by IL-1 in inducing Downloaded from innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1a/b, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-kB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral 2/2

candidiasis in IL-36R mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by http://www.jimmunol.org/ IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17–dependent genes, indicating that this protection occurs via an alternative pathway to IL-1–driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell–driven protective pathways in immunity to oral C. albicans infection. The Journal of Immunology, 2018, 201: 000–000.

ucosal barrier surfaces are equipped with multiple Oral epithelial cells (OECs) lining the palate, gingiva, buccal rapid-acting innate immune strategies to counter mucosa, and tongue are key “ﬁrst responders” during C. albicans pathogenic, often invasive, microbes. The oral mucosa infection (5). In addition to providing structural integrity to the

M by guest on September 28, 2021 is particularly vulnerable to such pathogens, yet our under- mucosal barrier, OECs are critical orchestrators of innate defenses standing of the host immune arsenal in this tissue remains in the mouth. OECs respond to C. albicans–mediated tissue injury surprisingly limited (1). In settings of weakened immunity or by activating damage-associated immune effectors, including IL-1 immunosuppression, the oral cavity becomes permissive to the family cytokines, S100A8/9 (calprotectin), and b-defensins (6, 7). growth of the opportunistic fungal pathogen Candida albicans The transition of commensal C. albicans cells on mucosal surfaces causing a painful condition termed oropharyngeal candidiasis to invasive pathogenic cells requires hyphal formation. These (OPC, thrush) that can be associated with nutritional deﬁciency, hyphae secrete a pore-forming peptide toxin called Candidalysin failure to thrive, and an increased risk of esophageal cancer (2). To that damages the oral epithelium during tissue invasion but also date, there are no licensed vaccines for C. albicans or, indeed, for induces protective host responses, including inﬂammatory cyto- any fungi (3, 4). kines (6). In particular, Candidalysin-induced IL-1a/b triggers

*Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Research at Guys and St. Thomas’s National Health Service Foundation Trust and Pittsburgh, PA 15261; †Mucosal and Salivary Biology Division, King’s College King’s College London Biomedical Research Centre (IS-BRC-1215-20006) (to J.R.N.). London Dental Institute, London SE1 1UL, United Kingdom; ‡Centre for Host- A.H.V., H.Z., S.L.G., and D.L.M. designed the experiments and wrote the manu- Microbiome Interactions, Mucosal and Salivary Biology Division, King’s College x script. A.H.V., H.Z., N.O.P., O.W.H., D.S., J.H., F.E.Y.A., B.M.C., and J.P.R. per- London Dental Institute, London SE1 9RT, United Kingdom; Faculty of Biological formed all experiments and data analysis. J.S.A. and M.S. performed experiments Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 { and analyzed IL-36 ELISA data. M.J.M., J.R.N., and B.H. provided key reagents and 9JT, United Kingdom; Department of Microbial Pathogenicity Mechanisms, Leibniz strains. Institute for Natural Product Research and Infection Biology–Hans Knoell Institute, D-07745 Jena, Germany; ‖Friedrich Schiller University, D-07737 Jena, Germany; Address correspondence and reprint requests to Dr. David L. Moyes or Prof. Sarah L. and #Center for Sepsis Control and Care, D-07747 Jena, Germany Gaffen, King’s College London, Division of Mucosal and Salivary Biology, Floor 17, Tower Wing, Guy’s Hospital, St. Thomas Street, London SE1 9RT, U.K. (D.L.M.) or 1A.H.V. and H.Z. contributed equally to this work. University of Pittsburgh, Division of Rheumatology and Clinical Immunology, BST ORCIDs: 0000-0002-5985-5201 (A.H.V.); 0000-0002-9890-1549 (H.Z.); 0000-0002- S702, 200 Lothrop Street, Pittsburgh PA 15261 (S.L.G.). E-mail addresses: david. 0327-0095 (F.E.Y.A.); 0000-0001-9638-2725 (J.P.R.); 0000-0003-4276- [email protected] (D.L.M.) or [email protected] (S.L.G.) 7456 (B.M.C.); 0000-0002-6028-0425 (B.H.); 0000-0003-3502-5542 (M.S.); 0000- The online version of this article contains supplemental material. 0002-8072-7917 (J.R.N.); 0000-0001-8511-2041 (S.L.G.); 0000-0002-1657- 918X (D.L.M.). Abbreviations used in this article: BD2, b-defensin 2; OEC, oral epithelial cell; OPC, oropharyngeal candidiasis; p.i., postinfection; PSMa, phenol-soluble modulin a; Received for publication April 10, 2018. Accepted for publication May 16, 2018. qPCR, quantitative PCR; siRNA, small interfering RNA; WT, wild type. This work was supported by National Institutes of Health Grants DE022550 and DE023815 (to S.L.G.), and grants from the Medical Research Council (MR/ Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 M011372/1), the Biotechnology and Biological Sciences Research Council (BB/ N014677/1), the Rosetrees Trust (M680), and the National Institute for Health

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800515 2 IL-36 FUNCTIONS IN PARALLEL TO IL-1 IN ANTI-CANDIDA IMMUNITY innate Type-17 immunity, which is vital for resolution of mice were provided by Amgen. IL-23R2/2 mice were a kind gift from Dr. V. Kuchroo (Harvard Medical School and Brigham and Women’s C. albicans oral infections in mice and humans (7–9). Addi- 2/2 tionally, IL-1R signaling in the oral mucosa is an important Hospital, Boston, MA). IL-1R1 mice were purchased from The Jackson Laboratory and expanded at the University of Pittsburgh. For experiments trigger for the mobilization of neutrophils to limit growth of involving IL-36R2/2 mice, wild type (WT) control mice were from pathogenic C. albicans (10). Thus, OEC-driven IL-1 production is Taconic. For other studies, WT mice were from The Jackson Laboratory. a crucial host defense component of immunity to C. albicans oral OPC was performed by sublingual inoculation with a 2.5 mg cotton ball infections. saturated in C. albicans for 75 min (22). Tongue homogenates were prepared on a gentleMACS (Miltenyi Biotec), and CFU were determined by Emerging data indicate that IL-36 is also an important mediator plating on yeast peptone dextrose media/chloramphenicol agar. Protocols of innate immunity during microbial infections (11–15). IL-36 is were approved by the University of Pittsburgh Institutional Animal Care part of the IL-1 superfamily and includes three agonistic cytokines and Use Committee. All efforts were made to minimize suffering, in ac- (IL-36a,IL-36b, and IL-36g) and a receptor antagonist cordance with recommendations in the Guide for the Care and Use of (IL-36RA) (16). IL-36 cytokines signal through a receptor com- Laboratory Animals of the National Institutes of Health. posed of the IL-36R and IL-1R accessory protein (encoded by Cell lines, reagent, and Candida strains Il1rl2 and Il1rap, respectively), which signal through the NF-kB Human buccal epithelial carcinoma TR146 cell monolayers were grown in and MAPK pathways (17). Like IL-1, IL-36 induces chemokines DMEM-F12/10% FBS, and experiments were performed in serum-free such as CCL2, CCL20, CXCL1, and CXCL5 in epithelial cells, DMEM-F12. Inhibitors of p38-MAPK (SB203580, 10 mM), NF-kB which subsequently promote recruitment of neutrophils and lym- (BAY11-7082, 2 mM), and PI3K (LY294002, 50 mM) were from Merck. phocytes (18–20). IL-36 also triggers expression of b-defensins, Abs to phospho–c-Jun, –c-Fos, and –MKP-1 were from Cell Signaling Technology. Anti-rabbit secondary Abs were from Jackson Immuno- which exert direct antimicrobial activity (21). Recent studies Research. Fungal strains were as follows: C. albicans reference strain Downloaded from showed that the Staphylococcus aureus pore-forming toxin SC5314 (23), parental strain BWP17 (24), yeast-locked flo8D/D (25), phenol-soluble modulin a (PSMa) induces IL-36 in keratinocytes, eed1D/D and eed1D/D+EED1 (26), efg1/cph1D/D (27), and Candidalysin which in turn activate a Type-17 response (14, 15). Thus, IL-36 is mutant strains ece1D/D, ece1D/D +ECE1,andece1D/D +ECE1D184–279 (6). an integral component of mucosal and dermal host defense, but to RNA, quantitative PCR, and small interfering RNA knockdown date, little is known about the influence of IL-36 family cytokines For TR146 cells, RNAwas extracted using the NucleoSpin II RNA isolation in oral fungal infections such as oral candidiasis. http://www.jimmunol.org/ kit (MACHEREY-NAGEL). Isolated RNA was treated with the Turbo In this article, we report that the production of IL-36 cytokines by DNase Free kit (Life Technologies) to remove genomic DNA contami- OECs is markedly induced following C. albicans infection and that nation. cDNA was created using the SuperScript IV Reverse Transcriptase IL-36R–deficient mice are highly susceptible to OPC. Suscepti- kit (ThermoFisher Scientific). Real-time PCR was performed on cDNA bility is associated with dampened antifungal innate immune re- using EvaGreen quantitative PCR (qPCR) MasterMix (Newmarket Sci- entific) on a Rotor-Gene 6000 (Corbett Life Sciences) and normalized sponses and reduced expression of IL-23. Expression of IL-36 was to YWHAZ. The following primer sequences were from PrimerBank not induced by hyphae per se but required the hypha-derived (28): IL-36a forward, 59-GATGTGTGCTAAAGTCGGGGA-39; IL-36a peptide toxin Candidalysin in both mice and human cells. Thus, reverse, 59-ACAGACTCGAAGGTGGAGTTC-39;IL-36g forward, our data reveal regulatory mechanisms that control IL-36 in OECs 59-GAAACCCTTCCTTTTCTACCGTG-39;IL-36g reverse, 59-GC-

9 by guest on September 28, 2021 and illuminate its importance in antifungal defenses. TGGTCTCTCTTGGAGGAG-3 . Mouse. Frozen tongue was homogenized in RLT buffer (RNeasy kit; QIAGEN) with a gentleMACS Dissociator (Miltenyi Biotec). cDNA was Materials and Methods synthesized with a SuperScript III First-Strand Synthesis System (Invi- Mice trogen, Carlsbad, CA). Relative quantiﬁcation was determined by real- time PCR with SYBR Green (Quanta BioSciences, Gaithersburg, MD) All mice were derived from a C57BL/6 background. Experiments were normalized to Gapdh. Primers were from SABiosciences (QIAGEN). performed on both sexes, with age- and sex-matched controls. Act12/2 Results were analyzed on a 7300 Real-Time PCR system (Applied mice were from U. Siebenlist (National Institutes of Health); IL-36R2/2 Biosystems).

FIGURE 1. IL-36 cytokines are induced during acute OPC. WT mice were challenged sublingually with PBS (sham) or C. albicans (OPC). (A) Tongue homogenates were prepared 2 d p.i., and total mRNA was subjected to real-time qPCR normalized to Gapdh. Graphs depict relative expression of indicated genes as mean + SEM and normalized to sham. Data are compiled from three to ﬁve mice per group from two independent experiments. (B) Relative mRNA expression of indicated genes in the tongue at 0, 12, and 24 h p.i. Data are compiled from three to ﬁve WT mice per group from a single experiment. Data were analyzed by Student t test or one- way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. IL-36 cytokines are induced during C. albicans infection of human OECs. TR146 human OECs were challenged with PBS or C. albicans. (A) IL-36 gene expression from RNA prepared at 0, 4, and 24 h p.i. normalized to YWHAZ. Graphs depict relative expression of indicated genes as mean + SEM normalized to 0-h time point. (B) IL-36g protein secretion at varying times p.i. with C. albicans determined by ELISA. (C) IL-36 gene expression from RNA prepared at 24 h p.i. of epithelial cells with WT or yeast-locked (ﬂo8D/D, efg1/cph1D/D) C. albicans strains normalized to YWHAZ. Graphs depict relative expression of indicated genes as mean + SEM normalized to PBS-treated cells. (D) IL-36 gene expression from RNA prepared at 24 h p.i. of epithelial cells with WT or ECE1 null (ece1D/D), Candidalysin null (ece1D/D+ECE1D184–279), or ECE1 reintegrant (ece1D/D+ECE1) C. albicans strains normalized to YWHAZ. Graphs depict relative expression of indicated genes as mean + SEM normalized to PBS-treated cells. (E) IL-36 gene expression from RNA prepared at 24 h posttreatment of epithelial cells with different concentrations of Candidalysin normalized to YWHAZ. Graphs depict relative expression of indicated genes as mean + SEM normalized to PBS-treated cells. (F) IL-36g protein secretion at varying times posttreatment with different concentrations of Candidalysin determined by ELISA. (G) WT mice infected with indicated strain of C. albicans and gene expression was measured at day 2 p.i. Data are mean + SEM of (A–F) three independent experiments or (G) normalized to sham, from three to ﬁve mice per group in two independent experiments. Data were analyzed by Student t test or one-way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 IL-36 FUNCTIONS IN PARALLEL TO IL-1 IN ANTI-CANDIDA IMMUNITY

RNA silencing by small interfering RNA (siRNA) was performed with the commensal fungus (34, 35), resistance is due to a potent innate HiPerFect Transfection Reagent (QIAGEN) as described: c-Jun (29), c-Fos immune response induced in the oral mucosa. This response clears (30), and MKP-1 (31). Validation by qPCR verified that targeted genes were the fungus within 1–2 d of infection without causing the overt reduced by at least 80% and maintained for at least 24 h (data not shown). symptoms of thrush (8, 36). To investigate the role of IL-36 in Western blotting response to OPC, WT mice were infected with the SC5314 strain TR146 cells were lysed with modified radioimmunoprecipitation lysis of C. albicans, and the gene expression in tongue tissue was buffer (32) containing phosphatase (Sigma-Aldrich) and protease inhibi- monitored. Among the genes induced during OPC in WT mice, we tors (Perbio Science) and separated on 12% SDS-PAGE before being observed robust induction of IL-36 family cytokines at day 2 transferred to nitrocellulose membrane. Membranes were incubated with postinfection (p.i.) (Fig. 1A). IL-36a and IL-36g were transcribed primary Ab overnight and developed using the Immobilon ECL reagent (Millipore) before being exposed to x-ray film. as early as day 1 p.i., although IL-36b was detected at day 2 p.i. (Fig. 1A, 1B). These results suggested the involvement of IL-36 in Human IL-36g ELISA promoting oral immunity to C. albicans. mAbs against human IL-36g were generated in C57B/6 mice or Sprague Dawley rats using recombinant IL-36g Ser18-Asp169 as Ag. Splenocytes IL-36 induction requires the hypha-associated fungal toxin were fused with myeloma cell lines Y3-AG 1.2.3 (rat) or SP2/O-Ag14 Candidalysin (mouse) as appropriate. Characterization of mAbs identified a mouse hybridoma (B5A2) as a capture Ab and a rat hybridoma (HCL17) as a de- OECs are the first cells to encounter C. albicans during OPC and tection Ab for sandwich ELISA. Abs were purified using protein A or regulate important early innate inflammatory responses to patho- protein G affinity chromatography. HCL17 was biotinylated using EZ-Link genic invading hyphae. To determine whether C. albicans induced Sulfo-NHS-LC-Biotin kits (ThermoFisher Scientific). ELISA plates (Life expression of IL-36 directly in OECs, we infected monolayers of Downloaded from Technologies) were coated with 2 mg/ml B5A2 capture Ab, and ELISAs were performed by standard methods. Detection of cathepsin S and CCL20 the human buccal epithelial cell line TR146 with C. albicans were determined using ELISA kits from R&D Systems. (6, 32). Expression of IL36A and IL36G, but not IL36B, mRNA (Fig. 2A) were induced within 4–6 h following C. albicans in vitro Statistics infection. Likewise, secretion of IL-36g protein was induced as All data were analyzed by Student t test or one-way ANOVA with post hoc early as 6 h p.i. (Fig. 2B). IL-36 has been reported to require , Dunnett multiple comparisons. A p value 0.05 was considered significant. cathepsin S–dependent processing for full efficacy (37), but http://www.jimmunol.org/ whereas OECs naturally secreted this enzyme, C. albicans in- Results fection did not alter its release (Supplemental Fig. 1A). IL-36 is induced in oral candidiasis Because C. albicans morphology impacts pathogenicity and Like immunocompetent humans, WT mice are resistant to OPC epithelial cell responses (32, 38, 39), we hypothesized that hypha (33). Because most strains of mice do not carry C. albicans as a formation would be required to induce IL-36. Indeed, infection of by guest on September 28, 2021

FIGURE 3. IL-36 gene expression is regulated by NF-kB, MAPK, and particularly by PI3K signaling. (A) IL-36 gene expression from RNA prepared at 0, 4, and 24 h p.i. with C. albicans of TR146 epithelial cells pretreated with signaling pathway inhibitors normalized to YWHAZ. Graphs depict percentage inhibition of indicated normalized gene expression relative to the DMSO vehicle control. (B) Effect of siRNA knockdown in TR146 epithelial cells on protein expression 2 h p.i. with C. albicans.(C) Impact of siRNA knockdown of c-Jun or c-Fos on gene expression of IL-36 genes 24 h p.i. with C. albicans. Graphs depict percentage inhibition of indicated normalized gene expression relative to sham-transfected cells. (D) Impact of siRNA knockdown of MKP1 on gene expression of IL-36 genes 24 h p.i. with C. albicans. Graphs depict percentage inhibition of indicated normalized gene expression relative to sham-transfected cells. Data are (B) representative or (A, C,andD) mean + SEM of three independent experiments and analyzed by one-way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 5

TR146 OECs with C. albicans yeast-locked mutants that cannot form hyphae [flo8D/D (25)] and [efg1/cph1D/D (27)] resulted in reduced expression of IL36A and IL36G compared with the parental WT strain (BWP17) (Fig. 2C). IL-1 induction during OPC requires signals from Candidalysin, a pore-forming peptide that is secreted by hyphae and generated by processing of the Ece1p protein (6, 7, 40). To determine whether IL-36 production is regulated by Candidalysin, TR146 OECs were infected with mutants lacking the entire ECE1 gene (ece1D/D), just the Can- didalysin peptide encoding region (ece1D/D+ECE1D184–279), or an ECE1 control reintegrant strain (ece1D/D+ECE1) (6). Infection of OECs with the BWP17 or the ece1D/D+ECE1 reintegrant strains stimulated IL36A and IL36G gene expression, whereas neither the ece1D/D nor ece1D/D+ECE1D184–279 mutants induced these cytokines appreciably (Fig. 2D). To determine whether Candidalysin alone was sufficient for induction of IL-36, TR146 cells were treated with lytic (.15 mM) and sublytic doses (,15 mM) of synthetic Candidalysin peptide. Both IL36A and IL36G gene expression (Fig. 2E) and IL-36g protein (Fig. 2F) were induced by Downloaded from Candidalysin concentrations as low as 3 mM. Thus, Candidalysin induces production of IL-36 family cytokines in OECs. To determine whether IL-36 is regulated by Candidalysin during OPC in vivo, WT mice were challenged with a C. albicans strain lacking the Candidalysin encoding gene ECE1 (ece1D/D) or the reintegrant control (ece1D/D+ECE1), and correlates of infection http://www.jimmunol.org/ were assessed. Consistent with the findings in TR146 cells, expression of all three IL-36 cytokines was markedly reduced following infection with an ece1D/D mutant (Fig. 2G). Together, FIGURE 4. IL-36 synergizes with C. albicans to induce CCL20 pro- these data demonstrate that IL-36 is induced in OECs following duction. Induction of CCL20 release in response to IL-36g (100 ng/ml) infection with invasive hyphae in a Candidalysin-dependent stimulation with and without C. albicans infection after 6 h treatment and/ manner. or infection. IL-36g stimulation is reversed by cotreatment with IL-36RA (1 mg/ml). Data are the mean + SEM of three independent experiments and Functional role of NF-kB, MAPK, and PI3K signaling analyzed by one-way ANOVA. ****p , 0.0001 relative to C. albicans + IL-36g, ####p , 0.0001 relative to PBS control. pathways in IL-36 family gene expression by guest on September 28, 2021 We next sought to define the C. albicans–induced epithelial signaling pathways that regulate IL-36 cytokine production. We first C. albicans (32, 41). Inhibition of NF-kB signaling with BAY11- focused on the p38-MAPK pathway, because p38-MAPK–induced 7082 resulted in a reduction in both IL36A (43%) and IL36G c-Fos is a key driver of the epithelial innate danger response to (43%) gene expression (Fig. 3A). Strikingly, inhibition of PI3K Candidalysin and is required to induce other innate cytokines such signaling using LY294002 resulted in almost complete inhibition as IL-1b and CCL20 (6, 32). To that end, TR146 cells were in- of IL-36 gene expression, with expression levels of both IL36A fected with C. albicans in conjunction with pharmacological in- (87%) and IL36G (89%) reduced to resting levels (Fig. 3A). hibitors of MAPK. Blockade of p38-MAPK with SB203580 Collectively, these data suggest that although p38-MAPK and impaired IL36A (48%) and IL36G (40%) gene expression in in- NF-kB contribute to induction of IL-36 expression, PI3K plays fected cells (Fig. 3A). In contrast, neither JNK-MAPK nor ERK1/ the predominant role. 2-MAPK inhibition affected gene expression of IL-36 genes (data not shown). We next interrogated the role of the p38-MAPK–in- IL-36g synergizes with C. albicans to induce CCL20 duced transcription factors c-Jun and c-Fos (AP-1) using siRNA IL-36g has previously been found to induce IL-17– and Type- knockdown (32) (Fig. 3B). Knockdown of c-Fos led to a modest, 17–related cytokines in response to both Aspergillus fumigatus but statistically significant, reduction in IL36A (20%) and IL36G and S. aureus infections (13–15), as well as antimicrobial pep- (32%) gene expression (Fig. 3C). In contrast, knockdown of c-Jun tides from keratinocytes (42, 43). Thus, we investigated whether led to a significant increase in IL36A gene expression (365%) but IL-36g induced the production of antimicrobial peptides (human had no effect on IL36G expression (Fig. 3C). The MAPK phos- b-defensin 2 [BD2] and LL-37) or the Type-17–recruiting che- phatase MKP1 (DUSP1) is a negative regulator of the p38- and mokine CCL20 (a ligand of CCR6) from OECs upon C. albicans JNK-MAPK pathways, and both production and stabilization of infections. TR146 epithelial cells were stimulated with IL-36g 6 this phosphatase are triggered by C. albicans hyphae. Knockdown the IL-36 receptor antagonist (IL-36Ra) in the presence of of MKP1 caused a significant increase in IL36A (229%) and C. albicans, and supernatants were evaluated for BD2, LL-37, IL36G (155%) expression (Fig. 3D), consistent with our prior and CCL20 release. IL-36g did not induce secretion of the findings for G-CSF and GM-CSF (32). Together, these data sup- antimicrobial peptides HBD2 and LL-37, either with or without port a positive role for p38-MAPK/c-Fos in triggering IL-36 gene C. albicans infection at 6 or 24 h p.i. (data not shown), and induction and MKP-1 as a negative feedback regulator of MAPK- IL-36g alone did not induce CCL20 secretion (Fig. 4). However, induced signaling. levels of CCL20 were markedly higher in supernatants from Because the p38-MAPK/c-Fos pathway did not fully account OECs infected with C. albicans in the presence of IL-36g than for induction of IL-36, we also evaluated the role of NF-kBand in supernatants from OECs infected with C. albicans without PI3K pathways, which are implicated in the OEC response to IL-36g at 6 h p.i. (Fig. 4). This effect was reversed by addition 6 IL-36 FUNCTIONS IN PARALLEL TO IL-1 IN ANTI-CANDIDA IMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. IL-36R2/2 mice are susceptible to acute oral candidiasis. (A) Indicated mice were orally infected with C. albicans. Oral fungal load (top) was assessed at day 5 p.i.; bars represent geometric mean CFU; dashed line is the limit of detection. Weight loss (bottom) measured throughout the course of oral infection. Each group contains three to five mice from at least two independent experiments. (B) Relative mRNA expression of indicated genes in the tongue measured at 2 d p.i. Data are compiled from three to five mice per group from three independent experiments. Bars are mean + SEM normalized to WT sham. (C) Oral fungal burden of indicated mice at day 5 p.i. Data for WT group compiled from historical controls. Bars represent the geometric mean, data from one experiment. (D) Relative mRNA expression of indicated genes analyzed at 2 d p.i. in WT and IL-1R12/2 mice. Data are compiled from 10 mice per group from two independent experiments. Bars are mean + SEM normalized to WT sham. Data were analyzed by Student t test or ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. of IL-36Ra (Fig. 4). We further tested the ability of IL-36g to thepresenceofC. albicans but not with Candidalysin alone or synergize with IL-17 and synthetic Candidalysin. No discernible with IL-17. additive or synergistic induction of downstream genes was observed when TR146 cells were simultaneously incubated with IL-36R–deficient mice are susceptible to acute oral candidiasis IL-36g and IL-17A or IL-36g with Candidalysin (Supplemental To determine whether IL-36 receptor signaling is required for im- Fig. 1B). Thus, in contrast to previous reports for IL-1 (7), munity to OPC, we challenged IL-36R2/2 mice with C. albicans IL-36g triggers the release of CCL20 from epithelial cells in and assessed oral fungal burdens 5 d p.i. IL-36R2/2 mice exhibited The Journal of Immunology 7 higher fungal loads and greater weight loss than WT controls these two arms are regulated independently by IL-36 and IL-1 (Fig. 5A). Notably, the fungal burden in IL-36R2/2 mice was cytokines, respectively (Supplemental Fig. 2). similar to that seen previously in Il1r12/2 mice (Fig. 5A) (7) but We recently described an epithelial signaling circuit that senses was not as severe as in mice with defective IL-17 signaling invasive fungal hyphae via Candidalysin activity to induce local (Act12/2) (44). Thus, there is a nonredundant requirement for innate immune responses, especially IL-1 (7). This study reveals IL-36 in driving immunity to oral mucosal candidiasis. that a Candidalysin-activated danger–response pathway also trig- To identify immune responses that are dysregulated in IL-36R2/2 gers the release of IL-36 from OECs. A similar scenario occurs mice during OPC, we evaluated the expression of a panel of known in the skin, as described in two recent studies showing an anti- antifungal genes at the peak of the immune response (day 2 p.i.) (8, microbial defense mechanism that is activated by a secreted 36). Surprisingly, the expression levels of IL-17 cytokines (Il17a, S. aureus toxin, PSMa (14, 15). Just as Candidalysin-deficient Il17f), multiple IL-17–dependent genes (Defb3, S100a9), the Type- C. albicans strains fail to trigger Type-17 responses in the 17 cytokine IL-22, and IFN-g were comparable between WT and mouth, PSMa-deficient S. aureus bacteria do not induce dermal IL-36R2/2 mice (Fig. 5B, Supplemental Fig. 1C). Despite this, the IL-17. Together, these findings indicate that the host barrier sur- expression of Il23a was significantly and consistently impaired faces are programmed to discriminate between benign and path- in IL-36R2/2 mice(Fig.5B),andIl23r2/2 mice failed to clear ogenic forms of commensal and opportunistic microbes by C. albicans from the oral cavity (Fig. 5C). In sharp contrast to sensing tissue-destructive microbial toxins. IL-36R2/2 mice, IL-1R12/2 mice displayed reduced expression The epithelial signaling pathways by which C. albicans induces of IL-17 and IL-17–dependent genes, but not IL-23 (Fig. 5D). IL-36 expression share both common and distinct features from

Thus, C. albicans–driven IL-36 responses protect against oral those pathways previously shown to be induced by Candidalysin. Downloaded from candidiasis in a parallel but independent fashion to the classical We have previously shown that C. albicans hyphae acting through IL-1 cytokines by inducing IL-23, which is essential for con- Candidalysin induce activation of the p38/c-Fos and ERK1/2– trolling oral infection, but not IL-17 or IL-17–driven genes, the MKP1 signaling pathways, whereas NF-kB is not activated di- canonical innate anti-Candida response previously described rectly by Candidalysin (5, 7, 32, 38). However, whereas IL-36 (7, 36). induction does partially involve p38-MAPk, it also involves

NF-kB, and particularly PI3K, with the latter playing a dominant http://www.jimmunol.org/ Discussion role. Both p38-MAPK and NF-kB have been identified as key sig- The ability of the mucosal epithelium to discriminate between the naling pathways in eliciting IL-36 (50), but a role for PI3K has not commensal and pathogenic states of microbes is a crucial com- so far been described. In contrast to a prior study that implicated ponent of host barrier defenses. Emerging data indicate that the both c-Jun and c-Fos in regulating IL-36 (50), we observed that IL-36 cytokine family bolsters epithelial immunity at multiple c-Fos is the only AP-1 family member that induces its transcrip- mucosal sites (45). In addition to shaping adaptive immune re- tion in OECs. Interestingly, c-Jun appears to inhibit the production sponses (13, 16), IL-36 cytokines are important mediators of in- of IL-36a by OECs, although the significance of this in OPC nate antimicrobial immunity in the epithelium (21). However, remains unclear. Thus, c-Fos joins IRF6, IRAK1, and C/EBPb little is known about the role of IL-36 in the oral mucosa. The as key factors regulating IL-36 gene expression. Future studies by guest on September 28, 2021 current study was prompted by our discovery of a signaling circuit will focus on understanding the interplay and cell-type specific in OPC, whereby the fungal toxin Candidalysin drives IL-1a/b activities of these molecules controlling IL-1 family cytokines. release, which in turn triggers innate antifungal immune responses In conclusion, our findings provide fresh insights into the pro- (7). In this study, we show that an analogous response also occurs tective role of IL-36 in oral mucosal antifungal immunity. The via IL-36 family cytokines, which contribute nonredundantly to complexity of the IL-1 family in shaping acute immune responses host defense in OPC; thus, oral candidiasis responses form part of is highlighted by the unexpected differences in how IL-1 and IL-36 a wider spectrum of IL-36 involvement in antimicrobial immunity. appear to act in this context. Both of these cytokines are current or Even though IL-36 is crucial for controlling oral C. albicans potential targets of biologic therapies and understanding their infections, it was unexpected that IL-36–driven immunity appears activities with respect to microbial opportunistic infection will be to be independent of IL-17 and IL-17–dependent genes. This important for predicting adverse side effects of such treatments. contrasts with the role of IL-1 in OPC, which promotes prolifer- ation of innate IL-17–producing T cells, and induction of IL-17 Acknowledgments and IL-17–dependent genes (7). Instead, IL-36 deficiency led to We thank Amgen for kindly providing the IL-36R2/2 mice. We also thank reduced gene expression of IL23a, which is vital for defense Dr. Duncan Wilson (University of Aberdeen) for constructing and provid- against C. albicans (8). IL-36 is known to be a strong inducer of ing WT (BWP17), ece1D/D, ece1D/D+ECE1, and ece1D/D+ECE1D184–279 IL-23 in macrophages isolated from psoriasis patients (46), strains. whereas IL-36 is known to be a key driver of the disease. So, it is probable that the same mechanism is in effect in this study. Disclosures Specifically, epithelial-produced IL-36 drives IL-23 secretion The authors have no financial conflicts of interest. from macrophages and other myeloid cells, leading to protection against C. albicans in an IL-17– and IFN-g–independent mecha- References nism. Both IL-17 signaling and IL-23 are required for immunity to 1. Moutsopoulos, N. M., and J. E. Konkel. 2017. Tissue-specific immunity at the OPC, but there is evidence that in some circumstances, their ac- oral mucosal barrier. Trends Immunol. 39: 276–287. tivities may be uncoupled. For example, IL-23 was shown to 2. Koo, S., D. Kejariwal, T. Al-Shehri, A. Dhar, and D. Lilic. 2017. Oesophageal regulate gut epithelial integrity differently from IL-17 (47, 48). As candidiasis and squamous cell cancer in patients with gain-of-function STAT1 gene mutation. United European Gastroenterol. J. 5: 625–631. such, blockade of IL-17 exacerbates inflammatory bowel disease 3. Brown, G. D., D. W. Denning, N. A. Gow, S. M. Levitz, M. G. Netea, and in humans, whereas anti–IL-23 therapy is beneficial (49). Hence, T. C. White. 2012. Hidden killers: human fungal infections. Sci. Transl. Med. 4: 165rv13. we speculate that in OPC the host may use IL-23–driven responses 4. Fidel, P. L., Jr., and J. E. Cutler. 2011. Prospects for development of a vaccine to to complement Type-17 immunity in the oral mucosa and that prevent and control vaginal candidiasis. Curr. Infect. Dis. Rep. 13: 102–107. 8 IL-36 FUNCTIONS IN PARALLEL TO IL-1 IN ANTI-CANDIDA IMMUNITY

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