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 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-1α/β, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R−/− mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by 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.
This article is featured in In This Issue, p.323
Mucosal barrier surfaces are equipped with multiple rapid-acting innate immune strategies to counter pathogenic, often invasive, microbes. The oral mucosa is particularly vulnerable to such pathogens, yet our understanding of the host immune arsenal in this tissue remains surprisingly limited (1). In settings of weakened immunity or immunosuppression, the oral cavity becomes permissive to the growth of the opportunistic fungal pathogen Candida albicans causing a painful condition termed oropharyngeal candidiasis (OPC, thrush) that can be associated with nutritional deficiency, failure to thrive, and an increased risk of esophageal cancer (2). To date, there are no licensed vaccines for C. albicans or, indeed, for any fungi (3, 4).
Oral epithelial cells (OECs) lining the palate, gingiva, buccal mucosa, and tongue are key “first responders” during C. albicans infection (5). In addition to providing structural integrity to the mucosal barrier, OECs are critical orchestrators of innate defenses in the mouth. OECs respond to C. albicans–mediated tissue injury by activating damage-associated immune effectors, including IL-1 family cytokines, S100A8/9 (calprotectin), and β-defensins (6, 7). The transition of commensal C. albicans cells on mucosal surfaces to invasive pathogenic cells requires hyphal formation. These hyphae secrete a pore-forming peptide toxin called Candidalysin that damages the oral epithelium during tissue invasion but also induces protective host responses, including inflammatory cytokines (6). In particular, Candidalysin-induced IL-1α/β triggers innate Type-17 immunity, which is vital for resolution of C. albicans oral infections in mice and humans (7–9). Additionally, IL-1R signaling in the oral mucosa is an important trigger for the mobilization of neutrophils to limit growth of pathogenic C. albicans (10). Thus, OEC-driven IL-1 production is a crucial host defense component of immunity to C. albicans oral infections.
Emerging data indicate that IL-36 is also an important mediator of innate immunity during microbial infections (11–15). IL-36 is part of the IL-1 superfamily and includes three agonistic cytokines (IL-36α, IL-36β, and IL-36γ) and a receptor antagonist (IL-36RA) (16). IL-36 cytokines signal through a receptor composed of the IL-36R and IL-1R accessory protein (encoded by Il1rl2 and Il1rap, respectively), which signal through the NF-κB and MAPK pathways (17). Like IL-1, IL-36 induces chemokines such as CCL2, CCL20, CXCL1, and CXCL5 in epithelial cells, which subsequently promote recruitment of neutrophils and lymphocytes (18–20). IL-36 also triggers expression of β-defensins, which exert direct antimicrobial activity (21). Recent studies showed that the Staphylococcus aureus pore-forming toxin phenol-soluble modulin α (PSMα) induces IL-36 in keratinocytes, which in turn activate a Type-17 response (14, 15). Thus, IL-36 is an integral component of mucosal and dermal host defense, but to date, little is known about the influence of IL-36 family cytokines in oral fungal infections such as oral candidiasis.
In this article, we report that the production of IL-36 cytokines by OECs is markedly induced following C. albicans infection and that IL-36R–deficient mice are highly susceptible to OPC. Susceptibility is associated with dampened antifungal innate immune responses and reduced expression of IL-23. Expression of IL-36 was not induced by hyphae per se but required the hypha-derived peptide toxin Candidalysin in both mice and human cells. Thus, our data reveal regulatory mechanisms that control IL-36 in OECs and illuminate its importance in antifungal defenses.
Materials and Methods
All mice were derived from a C57BL/6 background. Experiments were performed on both sexes, with age- and sex-matched controls. Act1−/− mice were from U. Siebenlist (National Institutes of Health); IL-36R−/− mice were provided by Amgen. IL-23R−/− mice were a kind gift from Dr. V. Kuchroo (Harvard Medical School and Brigham and Women’s Hospital, Boston, MA). IL-1R1−/− mice were purchased from The Jackson Laboratory and expanded at the University of Pittsburgh. For experiments involving IL-36R−/− mice, wild type (WT) control mice were from Taconic. For other studies, WT mice were from The Jackson Laboratory. OPC was performed by sublingual inoculation with a 2.5 mg cotton ball saturated in C. albicans for 75 min (22). Tongue homogenates were prepared on a gentleMACS (Miltenyi Biotec), and CFU were determined by plating on yeast peptone dextrose media/chloramphenicol agar. Protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All efforts were made to minimize suffering, in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Cell lines, reagent, and Candida strains
Human buccal epithelial carcinoma TR146 cell monolayers were grown in DMEM-F12/10% FBS, and experiments were performed in serum-free DMEM-F12. Inhibitors of p38-MAPK (SB203580, 10 μM), NF-κB (BAY11-7082, 2 μM), and PI3K (LY294002, 50 μM) were from Merck. Abs to phospho–c-Jun, –c-Fos, and –MKP-1 were from Cell Signaling Technology. Anti-rabbit secondary Abs were from Jackson ImmunoResearch. Fungal strains were as follows: C. albicans reference strain SC5314 (23), parental strain BWP17 (24), yeast-locked flo8Δ/Δ (25), eed1Δ/Δ and eed1Δ/Δ+EED1 (26), efg1/cph1Δ/Δ (27), and Candidalysin mutant strains ece1Δ/Δ, ece1Δ/Δ +ECE1, and ece1Δ/Δ +ECE1Δ184–279 (6).
RNA, quantitative PCR, and small interfering RNA knockdown
For TR146 cells, RNA was extracted using the NucleoSpin II RNA isolation kit (MACHEREY-NAGEL). Isolated RNA was treated with the Turbo DNase Free kit (Life Technologies) to remove genomic DNA contamination. cDNA was created using the SuperScript IV Reverse Transcriptase kit (ThermoFisher Scientific). Real-time PCR was performed on cDNA using EvaGreen quantitative PCR (qPCR) MasterMix (Newmarket Scientific) on a Rotor-Gene 6000 (Corbett Life Sciences) and normalized to YWHAZ. The following primer sequences were from PrimerBank (28): IL-36α forward, 5′-GATGTGTGCTAAAGTCGGGGA-3′; IL-36α reverse, 5′-ACAGACTCGAAGGTGGAGTTC-3′; IL-36γ forward, 5′-GAAACCCTTCCTTTTCTACCGTG-3′; IL-36γ reverse, 5′- GCTGGTCTCTCTTGGAGGAG-3′.
Frozen tongue was homogenized in RLT buffer (RNeasy kit; QIAGEN) with a gentleMACS Dissociator (Miltenyi Biotec). cDNA was synthesized with a SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Relative quantification was determined by real-time PCR with SYBR Green (Quanta BioSciences, Gaithersburg, MD) normalized to Gapdh. Primers were from SABiosciences (QIAGEN). Results were analyzed on a 7300 Real-Time PCR system (Applied Biosystems).
TR146 cells were lysed with modified radioimmunoprecipitation lysis buffer (32) containing phosphatase (Sigma-Aldrich) and protease inhibitors (Perbio Science) and separated on 12% SDS-PAGE before being transferred to nitrocellulose membrane. Membranes were incubated with primary Ab overnight and developed using the Immobilon ECL reagent (Millipore) before being exposed to x-ray film.
Human IL-36γ ELISA
mAbs against human IL-36γ were generated in C57B/6 mice or Sprague Dawley rats using recombinant IL-36γ Ser18-Asp169 as Ag. Splenocytes were fused with myeloma cell lines Y3-AG 1.2.3 (rat) or SP2/O-Ag14 (mouse) as appropriate. Characterization of mAbs identified a mouse hybridoma (B5A2) as a capture Ab and a rat hybridoma (HCL17) as a detection Ab for sandwich ELISA. Abs were purified using protein A or protein G affinity chromatography. HCL17 was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin kits (ThermoFisher Scientific). ELISA plates (Life Technologies) were coated with 2 μg/ml B5A2 capture Ab, and ELISAs were performed by standard methods. Detection of cathepsin S and CCL20 were determined using ELISA kits from R&D Systems.
All data were analyzed by Student t test or one-way ANOVA with post hoc Dunnett multiple comparisons. A p value <0.05 was considered significant.
IL-36 is induced in oral candidiasis
Like immunocompetent humans, WT mice are resistant to OPC (33). Because most strains of mice do not carry C. albicans as a commensal fungus (34, 35), resistance is due to a potent innate immune response induced in the oral mucosa. This response clears the fungus within 1–2 d of infection without causing the overt symptoms of thrush (8, 36). To investigate the role of IL-36 in response to OPC, WT mice were infected with the SC5314 strain of C. albicans, and the gene expression in tongue tissue was monitored. Among the genes induced during OPC in WT mice, we observed robust induction of IL-36 family cytokines at day 2 postinfection (p.i.) (Fig. 1A). IL-36α and IL-36γ were transcribed as early as day 1 p.i., although IL-36β was detected at day 2 p.i. (Fig. 1A, 1B). These results suggested the involvement of IL-36 in promoting oral immunity to C. albicans.
IL-36 induction requires the hypha-associated fungal toxin Candidalysin
OECs are the first cells to encounter C. albicans during OPC and regulate important early innate inflammatory responses to pathogenic invading hyphae. To determine whether C. albicans induced expression of IL-36 directly in OECs, we infected monolayers of the human buccal epithelial cell line TR146 with C. albicans (6, 32). Expression of IL36A and IL36G, but not IL36B, mRNA (Fig. 2A) were induced within 4–6 h following C. albicans in vitro infection. Likewise, secretion of IL-36γ protein was induced as early as 6 h p.i. (Fig. 2B). IL-36 has been reported to require cathepsin S–dependent processing for full efficacy (37), but whereas OECs naturally secreted this enzyme, C. albicans infection did not alter its release (Supplemental Fig. 1A).
Because C. albicans morphology impacts pathogenicity and epithelial cell responses (32, 38, 39), we hypothesized that hypha formation would be required to induce IL-36. Indeed, infection of TR146 OECs with C. albicans yeast-locked mutants that cannot form hyphae [flo8Δ/Δ (25)] and [efg1/cph1Δ/Δ (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 (ece1Δ/Δ), just the Candidalysin peptide encoding region (ece1Δ/Δ+ECE1Δ184–279), or an ECE1 control reintegrant strain (ece1Δ/Δ+ECE1) (6). Infection of OECs with the BWP17 or the ece1Δ/Δ+ECE1 reintegrant strains stimulated IL36A and IL36G gene expression, whereas neither the ece1Δ/Δ nor ece1Δ/Δ+ECE1Δ184–279 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 μM) and sublytic doses (<15 μM) of synthetic Candidalysin peptide. Both IL36A and IL36G gene expression (Fig. 2E) and IL-36γ protein (Fig. 2F) were induced by Candidalysin concentrations as low as 3 μM. 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 (ece1Δ/Δ) or the reintegrant control (ece1Δ/Δ+ECE1), and correlates of infection were assessed. Consistent with the findings in TR146 cells, expression of all three IL-36 cytokines was markedly reduced following infection with an ece1Δ/Δ mutant (Fig. 2G). Together, these data demonstrate that IL-36 is induced in OECs following infection with invasive hyphae in a Candidalysin-dependent manner.
Functional role of NF-κB, MAPK, and PI3K signaling pathways in IL-36 family gene expression
We next sought to define the C. albicans–induced epithelial signaling pathways that regulate IL-36 cytokine production. We first focused on the p38-MAPK pathway, because p38-MAPK–induced c-Fos is a key driver of the epithelial innate danger response to Candidalysin and is required to induce other innate cytokines such as IL-1β and CCL20 (6, 32). To that end, TR146 cells were infected with C. albicans in conjunction with pharmacological inhibitors of MAPK. Blockade of p38-MAPK with SB203580 impaired IL36A (48%) and IL36G (40%) gene expression in infected cells (Fig. 3A). In contrast, neither JNK-MAPK nor ERK1/2-MAPK inhibition affected gene expression of IL-36 genes (data not shown). We next interrogated the role of the p38-MAPK–induced transcription factors c-Jun and c-Fos (AP-1) using siRNA knockdown (32) (Fig. 3B). Knockdown of c-Fos led to a modest, but statistically significant, reduction in IL36A (20%) and IL36G (32%) gene expression (Fig. 3C). In contrast, knockdown of c-Jun led to a significant increase in IL36A gene expression (365%) but had no effect on IL36G expression (Fig. 3C). The MAPK phosphatase MKP1 (DUSP1) is a negative regulator of the p38- and JNK-MAPK pathways, and both production and stabilization of this phosphatase are triggered by C. albicans hyphae. Knockdown of MKP1 caused a significant increase in IL36A (229%) and IL36G (155%) expression (Fig. 3D), consistent with our prior findings for G-CSF and GM-CSF (32). Together, these data support a positive role for p38-MAPK/c-Fos in triggering IL-36 gene induction and MKP-1 as a negative feedback regulator of MAPK-induced signaling.
Because the p38-MAPK/c-Fos pathway did not fully account for induction of IL-36, we also evaluated the role of NF-κB and PI3K pathways, which are implicated in the OEC response to C. albicans (32, 41). Inhibition of NF-κB signaling with BAY11-7082 resulted in a reduction in both IL36A (43%) and IL36G (43%) gene expression (Fig. 3A). Strikingly, inhibition of PI3K signaling using LY294002 resulted in almost complete inhibition of IL-36 gene expression, with expression levels of both IL36A (87%) and IL36G (89%) reduced to resting levels (Fig. 3A). Collectively, these data suggest that although p38-MAPK and NF-κB contribute to induction of IL-36 expression, PI3K plays the predominant role.
IL-36γ synergizes with C. albicans to induce CCL20
IL-36γ has previously been found to induce IL-17– and Type-17–related cytokines in response to both Aspergillus fumigatus and S. aureus infections (13–15), as well as antimicrobial peptides from keratinocytes (42, 43). Thus, we investigated whether IL-36γ induced the production of antimicrobial peptides (human β-defensin 2 [BD2] and LL-37) or the Type-17–recruiting chemokine CCL20 (a ligand of CCR6) from OECs upon C. albicans infections. TR146 epithelial cells were stimulated with IL-36γ ± the IL-36 receptor antagonist (IL-36Ra) in the presence of C. albicans, and supernatants were evaluated for BD2, LL-37, and CCL20 release. IL-36γ did not induce secretion of the antimicrobial peptides HBD2 and LL-37, either with or without C. albicans infection at 6 or 24 h p.i. (data not shown), and IL-36γ alone did not induce CCL20 secretion (Fig. 4). However, levels of CCL20 were markedly higher in supernatants from OECs infected with C. albicans in the presence of IL-36γ than in supernatants from OECs infected with C. albicans without IL-36γ at 6 h p.i. (Fig. 4). This effect was reversed by addition of IL-36Ra (Fig. 4). We further tested the ability of IL-36γ to synergize with IL-17 and synthetic Candidalysin. No discernible additive or synergistic induction of downstream genes was observed when TR146 cells were simultaneously incubated with IL-36γ and IL-17A or IL-36γ with Candidalysin (Supplemental Fig. 1B). Thus, in contrast to previous reports for IL-1 (7), IL-36γ triggers the release of CCL20 from epithelial cells in the presence of C. albicans but not with Candidalysin alone or with IL-17.
IL-36R–deficient mice are susceptible to acute oral candidiasis
To determine whether IL-36 receptor signaling is required for immunity to OPC, we challenged IL-36R−/− mice with C. albicans and assessed oral fungal burdens 5 d p.i. IL-36R−/− mice exhibited higher fungal loads and greater weight loss than WT controls (Fig. 5A). Notably, the fungal burden in IL-36R−/− mice was similar to that seen previously in Il1r1−/− mice (Fig. 5A) (7) but was not as severe as in mice with defective IL-17 signaling (Act1−/−) (44). Thus, there is a nonredundant requirement for IL-36 in driving immunity to oral mucosal candidiasis.
To identify immune responses that are dysregulated in IL-36R−/− mice during OPC, we evaluated the expression of a panel of known antifungal genes at the peak of the immune response (day 2 p.i.) (8, 36). Surprisingly, the expression levels of IL-17 cytokines (Il17a, Il17f), multiple IL-17–dependent genes (Defb3, S100a9), the Type-17 cytokine IL-22, and IFN-γ were comparable between WT and IL-36R−/− mice (Fig. 5B, Supplemental Fig. 1C). Despite this, the expression of Il23a was significantly and consistently impaired in IL-36R−/− mice (Fig. 5B), and Il23r−/− mice failed to clear C. albicans from the oral cavity (Fig. 5C). In sharp contrast to IL-36R−/− mice, IL-1R1−/− mice displayed reduced expression of IL-17 and IL-17–dependent genes, but not IL-23 (Fig. 5D). Thus, C. albicans–driven IL-36 responses protect against oral candidiasis in a parallel but independent fashion to the classical IL-1 cytokines by inducing IL-23, which is essential for controlling oral infection, but not IL-17 or IL-17–driven genes, the canonical innate anti-Candida response previously described (7, 36).
The ability of the mucosal epithelium to discriminate between the commensal and pathogenic states of microbes is a crucial component of host barrier defenses. Emerging data indicate that the IL-36 cytokine family bolsters epithelial immunity at multiple mucosal sites (45). In addition to shaping adaptive immune responses (13, 16), IL-36 cytokines are important mediators of innate antimicrobial immunity in the epithelium (21). However, little is known about the role of IL-36 in the oral mucosa. The current study was prompted by our discovery of a signaling circuit in OPC, whereby the fungal toxin Candidalysin drives IL-1α/β release, which in turn triggers innate antifungal immune responses (7). In this study, we show that an analogous response also occurs via IL-36 family cytokines, which contribute nonredundantly to host defense in OPC; thus, oral candidiasis responses form part of a wider spectrum of IL-36 involvement in antimicrobial immunity.
Even though IL-36 is crucial for controlling oral C. albicans infections, it was unexpected that IL-36–driven immunity appears to be independent of IL-17 and IL-17–dependent genes. This contrasts with the role of IL-1 in OPC, which promotes proliferation of innate IL-17–producing T cells, and induction of IL-17 and IL-17–dependent genes (7). Instead, IL-36 deficiency led to reduced gene expression of IL23a, which is vital for defense against C. albicans (8). IL-36 is known to be a strong inducer of IL-23 in macrophages isolated from psoriasis patients (46), 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. Specifically, epithelial-produced IL-36 drives IL-23 secretion from macrophages and other myeloid cells, leading to protection against C. albicans in an IL-17– and IFN-γ–independent mechanism. Both IL-17 signaling and IL-23 are required for immunity to OPC, but there is evidence that in some circumstances, their activities may be uncoupled. For example, IL-23 was shown to regulate gut epithelial integrity differently from IL-17 (47, 48). As such, blockade of IL-17 exacerbates inflammatory bowel disease in humans, whereas anti–IL-23 therapy is beneficial (49). Hence, we speculate that in OPC the host may use IL-23–driven responses to complement Type-17 immunity in the oral mucosa and that these two arms are regulated independently by IL-36 and IL-1 cytokines, respectively (Supplemental Fig. 2).
We recently described an epithelial signaling circuit that senses invasive fungal hyphae via Candidalysin activity to induce local innate immune responses, especially IL-1 (7). This study reveals that a Candidalysin-activated danger–response pathway also triggers the release of IL-36 from OECs. A similar scenario occurs in the skin, as described in two recent studies showing an antimicrobial defense mechanism that is activated by a secreted S. aureus toxin, PSMα (14, 15). Just as Candidalysin-deficient C. albicans strains fail to trigger Type-17 responses in the mouth, PSMα-deficient S. aureus bacteria do not induce dermal IL-17. Together, these findings indicate that the host barrier surfaces are programmed to discriminate between benign and pathogenic forms of commensal and opportunistic microbes by sensing tissue-destructive microbial toxins.
The epithelial signaling pathways by which C. albicans induces IL-36 expression share both common and distinct features from those pathways previously shown to be induced by Candidalysin. We have previously shown that C. albicans hyphae acting through Candidalysin induce activation of the p38/c-Fos and ERK1/2–MKP1 signaling pathways, whereas NF-κB is not activated directly by Candidalysin (5, 7, 32, 38). However, whereas IL-36 induction does partially involve p38-MAPk, it also involves NF-κB, and particularly PI3K, with the latter playing a dominant role. Both p38-MAPK and NF-κB have been identified as key signaling pathways in eliciting IL-36 (50), but a role for PI3K has not so far been described. In contrast to a prior study that implicated both c-Jun and c-Fos in regulating IL-36 (50), we observed that c-Fos is the only AP-1 family member that induces its transcription in OECs. Interestingly, c-Jun appears to inhibit the production of IL-36α by OECs, although the significance of this in OPC remains unclear. Thus, c-Fos joins IRF6, IRAK1, and C/EBPβ as key factors regulating IL-36 gene expression. Future studies will focus on understanding the interplay and cell-type specific activities of these molecules controlling IL-1 family cytokines.
In conclusion, our findings provide fresh insights into the protective role of IL-36 in oral mucosal antifungal immunity. The complexity of the IL-1 family in shaping acute immune responses is highlighted by the unexpected differences in how IL-1 and IL-36 appear to act in this context. Both of these cytokines are current or potential targets of biologic therapies and understanding their activities with respect to microbial opportunistic infection will be important for predicting adverse side effects of such treatments.
We thank Amgen for kindly providing the IL-36R−/− mice. We also thank Dr. Duncan Wilson (University of Aberdeen) for constructing and providing WT (BWP17), ece1Δ/Δ, ece1Δ/Δ+ECE1, and ece1Δ/Δ+ECE1Δ184–279 strains.
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/M011372/1), the Biotechnology and Biological Sciences Research Council (BB/N014677/1), the Rosetrees Trust (M680), and the National Institute for Health Research at Guys and St. Thomas’s National Health Service Foundation Trust and King’s College London Biomedical Research Centre (IS-BRC-1215-20006) (to J.R.N.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
oral epithelial cell
phenol-soluble modulin α
small interfering RNA
The authors have no financial conflicts of interest.