Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin

Abstract

Candida albicans is a dimorphic commensal fungus that causes severe oral infections in immunodeficient patients. Invasion of C. albicans hyphae into oral epithelium is an essential virulence trait. Interleukin-17 (IL-17) signaling is required for both innate and adaptive immunity to C. albicans During the innate response, IL-17 is produced by γδ T cells and a poorly understood population of innate-acting CD4⁺ αβ T cell receptor (TCRαβ)⁺ cells, but only the TCRαβ⁺ cells expand during acute infection. Confirming the innate nature of these cells, the TCR was not detectably activated during the primary response, as evidenced by Nur77^eGFP mice that report antigen-specific signaling through the TCR. Rather, the expansion of innate TCRαβ⁺ cells was driven by both intrinsic and extrinsic IL-1R signaling. Unexpectedly, there was no requirement for CCR6/CCL20-dependent recruitment or prototypical fungal pattern recognition receptors. However, C. albicans mutants that cannot switch from yeast to hyphae showed impaired TCRαβ⁺ cell proliferation and Il17a expression. This prompted us to assess the role of candidalysin, a hyphal-associated peptide that damages oral epithelial cells and triggers production of inflammatory cytokines including IL-1. Candidalysin-deficient strains failed to up-regulate Il17a or drive the proliferation of innate TCRαβ⁺ cells. Moreover, candidalysin signaled synergistically with IL-17, which further augmented the expression of IL-1α/β and other cytokines. Thus, IL-17 and C. albicans, via secreted candidalysin, amplify inflammation in a self-reinforcing feed-forward loop. These findings challenge the paradigm that hyphal formation per se is required for the oral innate response and demonstrate that establishment of IL-1- and IL-17-dependent innate immunity is induced by tissue-damaging hyphae.

Figure 1. Proliferation of oral TCRαβ⁺ cells following C. albicans infection

(A) Il17a^eYFP mice (17) were challenged sublingually with PBS (sham) or C. albicans. Homogenates were prepared from pooled tongues (n=2–3). YFP⁺TCRαβ⁺ or YFP⁺TCRγδ⁺ cells in the CD45⁺CD4⁺ gate were assessed by flow cytometry. Data show fold-increase versus sham, pooled from 3–4 independent experiments. (B–C) WT mice (C57BL/6J) were infected with C. albicans, and tongue homogenates prepared on days 1 or 2 p.i. Cells were gated on lymphocytes and staining of CD45 and TCRβ is shown (top). Proliferation of CD45⁺CD4⁺TCRβ⁺ cells was determined by staining for Ki67 (bottom). Data representative of 10 experiments. Graph in C: mean ± SEM of proliferating TCRαβ⁺ cells on days 1 and 2. (D) WT mice were infected with C. albicans and tongue homogenates prepared on day 2 p.i. Proliferation was determined by PCNA staining. Data representative of 3 experiments. (E) WT cervical LNs were harvested on day 2 p.i. Proliferation of CD45⁺CD4⁺TCRβ⁺ cells was determined by anti-Ki67 staining. Graph shows mean ± SEM of Ki67⁺ CD4⁺ cells in cLNs. Data are representative of 2 experiments. Statistical analyses: Student’s t test or 1-way ANOVA.

Figure 2. CCR6 is dispensable for expansion of innate TCRαβ⁺ cells in oral candidiasis

(A) WT or Ccr6^−/− mice were infected with C. albicans, and proliferation of oral TCRαβ⁺ cells was determined at day 2 p.i. Data representative of 3 independent experiments. (B) Indicated mice were infected orally with C. albicans, and fungal burden was assessed by CFU enumeration on day 4 p.i.. Bars = geometric mean. Each point represents an individual mouse. Dashed line = limit of detection (LOD, 30 CFU) (52). Data are compiled from 4 independent experiments. (C) WT mice were injected with 100 ug anti-CCL20 Abs or isotype controls on day -1 relative to infection. Proliferation of TCRαβ⁺ cells was determined on day 2 p.i. Data are representative of 2 experiments. Student’s t test or ANOVA used for statistics.

Figure 3. A primary C. albicans infection activates innate TCRαβ⁺ cells without engaging the TCR

(A) Nur77^GFP mice were sham-treated (red line) or infected with C. albicans and tongue homogenates prepared on days 1 or 2 p.i (blue line). Controls received anti-CD3 Abs (green line) to stimulate the TCR on all T cells. WT (“non-Tg”) mice were negative controls for GFP staining (grey line). Left: fluorescence intensity of GFP in oral CD45⁺CD4⁺TCRβ⁺ cells. Right: Relative mean fluorescence intensity (MFI) of GFP in CD45⁺CD4⁺TCRβ⁺ cells was assessed and normalized to sham. NS, not significant. Data from 3 independent experiments. (B) Nur77^GFP mice were infected with C. albicans. Tongue homogenates were prepared 2 d p.i. (“1° Inf”). To induce C. albicans-specific TCR signaling (“2° Inf”), mice were infected orally, rested for 6 weeks, then re-challenged with a second oral infection (8). Left: GFP fluorescence in oral CD45⁺CD4⁺TCRβ⁺ cells, with % GFP^hi cells indicated. Green line shows staining in mice administered agonistic anti-CD3 Abs, as in panel A. Right: Compiled percentage of GFP^hi cells per cohort. Data representative of 3–4 independent experiments. Graphs show mean + SEM, analyzed by student’s t test or ANOVA.

Figure 4. TLR2 and Dectin-1 are dispensable for C. albicans-induced proliferation of innate TCRαβ⁺ cells

(A–C) Indicated mice were infected with C. albicans and proliferation of CD45⁺CD4⁺TCRβ⁺ cells determined on day 2 p.i. Data representative of 2–3 independent experiments. (D) Indicated mice were infected and fungal burden quantified on day 5 p.i. Bars = geometric mean. Dashed line = LOD. Data from 2 independent experiments. Data analyzed by ANOVA, Mann Whitney correction. (E) Average % weight loss is shown.

Figure 5. Candidalysin drives proliferation of innate IL-17-producing TCRαβ⁺ cells

(A) Il17a^eYFP mice were infected with C. albicans (ece1Δ/Δ or Revertant “Rev”) and homogenates prepared 2 d p.i. Staining of CD45 and YFP in lymphocyte gate is shown. Data representative of 2 experiments. (B) WT mice were infected with the indicated strains of C. albicans, and expansion (top) and proliferation (bottom) of oral TCRαβ⁺ cells was analyzed at day 2 p.i.. Data representative of 3 experiments. (C) Fold-expansion of TCRβ⁺ cells following infection with ece1Δ/Δ or Rev strains. Data pooled from 4 experiments. (D) Fungal loads were assessed at day 2 p.i.. Bar = geometric mean. Data pooled from 2 experiments. (E–F) Tongue homogenates were prepared 2 days after infection with the indicated C. albicans strains. Total mRNA was subjected to qPCR normalized to Gapdh. Graphs show mean + SEM normalized to sham. Data compiled from 7–8 mice per group from 2 independent experiments. (G) Percentage of CD11b⁺Ly6G^hi cells in tongue was assessed at day 2 p.i. + SEM, compiled from 3 experiments. Statistics analyzed by student’s t test or ANOVA.

Figure 6. IL-1 activates innate TCRαβ⁺ T cell proliferation and anti-fungal immunity in a T cell intrinsic and T cell extrinsic manner

(A) WT mice were infected with the indicated C. albicans strains and gene expression was measured on day 2 p.i.. Data show mean + SEM normalized to sham, from 7–8 mice/group in 2 experiments. (B) Expansion and proliferation of TCRαβ⁺ cells in Il1r1^−/− mice at day 2 p.i. Data are from 3 experiments. (C) Fungal burdens in the indicated mice were quantified on day 5 p.i., from 2 experiments. (D) WT mice were administered anti-IL-1α, anti-IL-1β or isotype control Abs (1.0 mg/mouse used alone or 0.5 mg each when used together) on day -1 p.i.. Proliferation of oral TCRαβ⁺ cells was assessed at day 2 day p.i. Data representative of 2 experiments. (E) Reciprocal adoptive transfers of femoral BM were performed in WT or Il1r1^−/− mice and proliferation of oral TCRαβ⁺ cells was determined. Experimental chimera results are representative of 2 experiments; control chimera data are from 1 experiment. Data analyzed by Student’s t test or ANOVA.

Figure 7. Candidalysin and IL-17 signal synergistically or additively in oral epithelial cells

(A) TR146 OECs were untreated (“U”, grey bars) or stimulated with IL-17 (200 ng/ml, black bars). Cells were infected with WT C. albicans (Bwp17+CIp30, “Parent”), ece1Δ/Δ or the Revertant (Rev) for 24 h. Supernatants were analyzed by Luminex (IL-1β, IL-6, G-CSF) or ELISA (CCL20). Graphs indicate mean + SEM. Data are representative of 2 experiments. (B) TR146 cells were untreated (“U”, grey bars) or treated with IL-17 (200 ng/ml, black bars) or Candidalysin peptide (Clys, 15 μM) for 24 h and analyzed as in panel A. (C) TR146 cells were incubated with C. albicans ± IL-17 (200 ng/ml). LDH in supernatants was evaluated after 24 h, representative of 3 experiments. (D) TR146 cells were treated with TNFα (20 ng/ml), IL-17 (200 ng/ml) or Candidalysin (15 μM) for 5 min. Lysates were immunoblotted for phospho-IκBα and total IκBα. (E) TR146 cells were incubated with TNFα (20 ng/ml), IL-17 (200 ng/ml) or Candidalysin (15 μM) for 30 min or 2 h. Lysates were immunoblotted for c-Fos, phospho-MKP1 or Actin. Data are representative of 2 experiments. (F) TR146 cells were transfected with c-Fos siRNA and stimulated for 24 h with PBS, Clys or IL-17. Supernatants were assessed for CCL20 by ELISA. Data are representative of 2 independent experiments. All data analyzed by ANOVA and Student’s t-test.