IL-17 Receptor Signaling in Oral Epithelial Cells Is Critical for Protection against Oropharyngeal Candidiasis

Abstract

Signaling through the IL-17 receptor (IL-17R) is required to prevent oropharyngeal candidiasis (OPC) in mice and humans. However, the IL-17-responsive cell type(s) that mediate protection are unknown. Using radiation chimeras, we were able to rule out a requirement for IL-17RA in the hematopoietic compartment. We saw remarkable concordance of IL-17-controlled gene expression in C. albicans-infected human oral epithelial cells (OECs) and in tongue tissue from mice with OPC. To interrogate the role of the IL-17R in OECs, we generated mice with conditional deletion of IL-17RA in superficial oral and esophageal epithelial cells (Il17ra^ΔK13). Following oral Candida infection, Il17ra^ΔK13 mice exhibited fungal loads and weight loss indistinguishable from Il17ra^-/- mice. Susceptibility in Il17ra^ΔK13 mice correlated with expression of the antimicrobial peptide β-defensin 3 (BD3, Defb3). Consistently, Defb3^-/- mice were susceptible to OPC. Thus, OECs dominantly control IL-17R-dependent responses to OPC through regulation of BD3 expression.

Figure 1. IL-17RA expressed on hematopoietic cells is dispensable for protection against oral candidiasis

Reciprocal adoptive transfers of femoral BM were performed in WT or Il17ra^−/− mice followed by oral infection with C. albicans. A. After 5 d, fungal loads were assessed by CFU enumeration of tongue homogenates. Bars show geometric mean. Each point is 1 mouse. ***P<0.01 by ANOVA and t-test with Mann-Whitney correction. B. Weight was assessed daily, and is shown as mean % of starting weight. C. Defb3 was evaluated on d 5 by qPCR. Data relative to Gapdh + SEM. ***P<0.01 by ANOVA and post-hoc Tukey’s test. N.S., not significant. All experiments were performed at least twice.

Figure 2. C. albicans induces an antifungal gene response in human OECs that parallels the IL-17-dependent response to murine OPC

A. OKF6/TERT2 human OECs were treated with IL-17+TNFα for 24 h. Cells were exposed to C. albicans yeast for 5 h and subjected to RNA-Seq. Data show normalized RPKM values of genes whose orthologues were previously shown to be induced in an IL-17R-dependent manner in murine OPC (Conti et al., 2009). Yellow = high and blue = low relative expression. B. Ingenuity Pathway Analysis of RNA-Seq data, showing C. albicans-exposed OKF6/TERT2 cells (untreated relative to IL-17+TNFα-treated) compared to murine OPC (C. albicans-infected WT tongue relative to uninfected). Data indicate z-scores of predicted regulation by infection. Red = predicted activation. Blue = predicted repression. White = no predicted regulation.

Figure 3. The Keratin 13 promoter shows specific activity in oral epithelium

A. The K13 promoter in the pGL3-Luc vector was transfected into oral epithelial IMOK cells, and Luc activity assessed after 24 h. Data show mean + SEM. ***P<0.01 by student’s t-Test. EV, empty vector. B. K13^LacZ mice were analyzed for β-galactosidase activity by Xgal staining. C. K13-Cre cassette used to create K13^CRE mice. D. Indicated tissues from K13^CRE founder #176 were analyzed for Krt13 and Cre by qPCR. SMG, submandibular salivary gland; cLN, cervical lymph node. Arrow = specific PCR product. * = unincorporated PCR primers. E. Tissues from K13^CRE-Rosa26^LacZ mice were stained with Xgal to assess β-galactosidase activity. Data are from 2–4 independent experiments.

Figure 4. IL-17RA in oral epithelial cells is necessary for immunity to OPC

A. Expression of IL-17RA was assessed by IHC. BL, suprabasal layer (n=3). B. Indicated mice were subjected to OPC as in Fig 1. Left: Mean fungal loads + SD at the indicated days post infection (left). Il17ra^−/− (green, n= 3–6), Cre⁺ (blue, n=3–7), Cre⁻ (black, n= 3–4). Right: Mean % weight change relative to day 0 + SD. Data are from 2–4 experiments per time point. C. Saliva was incubated with C. albicans cells for 1 h at 37°C and CFU was assessed by plating. Data show mean + SEM. Experiment was done twice.

Figure 5. Tissue changes in Il17ra^K13 compared to Il17ra^−/− mice following C. albicans infection

A. 20X images from H&E-stained tongue sections from the indicated C. albicans-infected mice at days 1 or 5. Black arrow indicates division between supra- and sub-basal layers. White arrows indicate PMNs. Scale bar is 100 μM. B. Quantification of PMNs in H&E images (n=4 mice/group, minimum of 6 sections per mouse). *P<0.05 WT (Cre⁻) vs. Il17ra^−/− ‡ P<0.05 Il17ra^K13 vs. WT (Cre⁻) by ANOVA with Kruskal-Wallis correction. Data indicate means + SEM. C. Hyperplasia is indicated as thickening of epithelium between basal cell layer to superficial keratinized layer. Arrows indicate distance measured in microns using ImageJ. D. Quantification of hyperplasia by blinded assessors (n=4 mice/group, minimum 4 sections per mouse). * P<0.05 compared to WT. ‡P<0.05 compared to Il17ra^ΔK13 by ANOVA. Data indicate means + SEM. E. MPO staining in infected mice. Arrows indicate areas of high MPO staining. Experiments were performed on sections from 3–5 mice per strain. All experiments were done twice.

Figure 6. C. albicans-induced gene expression changes require epithelial IL-17RA and demonstrate a role for BD3

A. RNA-seq was performed 24 h after infection (n=3). Heatmap shows genes that are differentially expressed after infection of WT mice for 24 h and whose regulation is altered in infected Il17ra^−/− or Il17ra^K13 mice. Values = log (base 2) fold change. Red bar indicates genes whose C. albicans-induced regulation required IL-17RA in oral epithelium. B. Tongue RNA from C. albicans-infected mice was assessed by qPCR (n= 6–9). Bars indicate mean + SEM. *P<0.05 by ANOVA and student’s t-test. C. Tongue sections were stained with α-BD3 Abs or 2° Ab. Arrows indicate suprabasal areas (n=3). D. TR146 cells were incubated with IL-17 for 20 h ± IL-17A. BD2 in supernatants was assessed by ELISA. Bars indicate mean + SEM. ** P<0.01. E. TR146 cells were infected with C. albicans yeast at an MOI of 0.01 ± IL-17A (200 ng/ml) for 24 h. Secreted LDH was assessed in triplicate. F. Indicated mice were subjected to OPC and oral fungal loads assessed after 5 d. Bars indicate geometric mean. **P<0.01. **** P<0.0001. All experiments were done at least twice. See also Fig S1 and Table S1.