Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis

Abstract

The commensal fungus Candida albicans causes oropharyngeal candidiasis (OPC; thrush) in settings of immunodeficiency. Although disseminated, vaginal, and oral candidiasis are all caused by C. albicans species, host defense against C. albicans varies by anatomical location. T helper 1 (Th1) cells have long been implicated in defense against candidiasis, whereas the role of Th17 cells remains controversial. IL-17 mediates inflammatory pathology in a gastric model of mucosal candidiasis, but is host protective in disseminated disease. Here, we directly compared Th1 and Th17 function in a model of OPC. Th17-deficient (IL-23p19(-/-)) and IL-17R-deficient (IL-17RA(-/-)) mice experienced severe OPC, whereas Th1-deficient (IL-12p35(-/-)) mice showed low fungal burdens and no overt disease. Neutrophil recruitment was impaired in IL-23p19(-/-) and IL-17RA(-/-), but not IL-12(-/-), mice, and TCR-alphabeta cells were more important than TCR-gammadelta cells. Surprisingly, mice deficient in the Th17 cytokine IL-22 were only mildly susceptible to OPC, indicating that IL-17 rather than IL-22 is vital in defense against oral candidiasis. Gene profiling of oral mucosal tissue showed strong induction of Th17 signature genes, including CXC chemokines and beta defensin-3. Saliva from Th17-deficient, but not Th1-deficient, mice exhibited reduced candidacidal activity. Thus, the Th17 lineage, acting largely through IL-17, confers the dominant response to oral candidiasis through neutrophils and antimicrobial factors.

Figure 1.

Model of murine OPC. (A) Timeline of infection model. (B) WT mice are resistant to OPC unless they are immunosuppressed. WT mice (n = 8) or WT mice treated with cortisone acetate (n = 16) were infected sublingually with C. albicans, and after 5 d CFU/g of tongue tissue was assessed in triplicate (log scale). These data are from at six independent experiments. (C–E) Histological evaluation of C. albicans infection. At day 5, sections of tongue from the indicated mice were stained with PAS to visualize C. albicans (Ca) or hematoxylin and eosin (H&E; which does not stain fungi) and viewed at 10X or 40X magnification. Fungal lesions, papillae, and keratinized layer are indicated. (C) WT mice subjected to sham infection. (D) WT mice without cortisone were infected orally with C. albicans. (E) WT mice treated with cortisone acetate were infected orally with C. albicans. TB, taste bud; BV, blood vessel; BL, basal layer; ker, keratinized layer; Neu, neutrophil; Eo, eosinophils; Ca, C. albicans. Data are representative of 2–3 mice per strain. Bars, 100 μm. (F) OPC induces weight loss in cortisone-treated WT mice. All mice were weighed daily, and weight (gram) on each day of infection is indicated. *, P < 0.05. Circles, Sham-infected mice; squares, C. albicans–infected mice. (G) OPC on day 5 after inoculation with C. albicans. Fungal lesions on tongue of WT mice treated with cortisone acetate.

Figure 2.

Th17-deficient mice are more susceptible to OPC than Th1-deficient mice. (A) Quantitation of C. albicans infection in Th1-deficient (IL-12^KO) and Th17-deficient (IL-23^KO) mouse strains. IL-12^KO mice (n = 13) and IL-23^KO mice (n = 9) were infected orally with C. albicans and CFU/gram tongue tissue was assessed. Each dot represents an individual mouse, and horizontal bar indicates the geometric mean, which is also indicated below. *, P < 0.05 by Mann-Whitney t test. Note, the WT mouse data are the same as that shown in Fig. 1 B. IL-12^KO mice were evaluated in three independent experiments, and IL-23^KO mice were evaluated in two independent experiments. (B) OPC-induced weight loss in Th1-deficient or Th17-deficient strains. Mice were weighed daily and percentage of weight change compared with day 0 is shown for each strain. *, P < 0.05. Triangles, Sham-infected mice; squares, C. albicans–infected mice. (C and D) Histological evaluation of C. albicans infection in IL-12^KO versus IL-23^KO mice. At day 5, sections of tongue from the indicated mice were stained with PAS or hematoxylin and eosin (H&E) and viewed at 10–40X magnification. Data are representative of two mice per strain. Bars, 100 μm. (E) Quantitation of histological evaluation of PMNs. Six sections per mouse from two mice per group were evaluated for the number of PMNs per microscopic field, either in the vicinity of fungal lesions or parallel to areas of sham-infected tongue. Evaluators were blinded to the identity of the samples. (F) OPC on day 5 after inoculation with C. albicans. Fungal lesions on tongues of representative IL-12^KO or IL-23^KO mice are shown.

Figure 3.

IL-17 signaling is essential for host defense against OPC. (A) IL-17RA^KO mice are sensitive to OPC. The indicated mouse strains (IL-17RA^KO, n = 8; IL-22^KO, n = 7) were infected with C. albicans and CFU/g tongue was assessed by colony enumeration. *, P < 0.05. Geometric mean is indicated by horizontal bars and below. Note, data from WT mice are the same as shown in Fig. 1 B. IL-17RA^KO mice data are representative of three independent experiments, and IL-22^KO data were from one experiment. (B) OPC-induced weight loss in susceptible strains. Percentage of weight change compared with day 0 is shown. (C) IL-17⁺ cells are induced after C. albicans infection. Cells were isolated from the draining cervical lymph nodes of infected IL-17RA^KO mice. Cells were incubated ± anti-CD3 + anti-CD28 for 48 h, and percentage of cells staining positive for IL-17 was assessed by intracellular flow cytometry. **, P < 0.01. n.s. not statistically significant. (D and E) Histological evaluation of C. albicans infection in IL-17RA^KO or IL-22^KO mice. At day 5, sections of tongue from the indicated mice were stained with PAS or H&E and viewed at 10X-40X magnification. Data are representative of two mice per strain. Scale bars indicate 100 microns. (F) Quantitation of histological evaluation of PMNs. Six sections per mouse from 2 mice per group were evaluated for the number of PMNs per microscopic field, either in the vicinity of fungal lesions or parallel areas of sham-infected tongue. Evaluators were blinded to the identity of the samples. * P < 0.05. (G) IL-12^KO but not IL-17RA^KO mice can recover from oral C. albicans infection. WT (n = 4), IL-17RA^KO (n = 12) and IL-12^KO (n = 12) mice were infected with C. albicans. On the indicated days (ranging from 1–17), 1–3 mice per group were killed and C. albicans CFU per gram of tongue was evaluated by colony enumeration. Geometric mean for each day is shown. Time course data are from one experiment. (H) Weight loss correlates with degree of OPC susceptibility. Weight changes in IL-12^KO and IL-17RA^KO mice over time are shown.

Figure 4.

Host defense against oral C. albicans is mediated mainly by αβ-T cells, rather than γδ-T cells. Mice deficient in αβ-T cells (n = 14), γδ-T cells (n = 13), or CD8 (n = 8) were inoculated with C. albicans and fungal burden (CFU/g tissue) in tongue (A) and daily weight (B) were assessed. CD8^KO mice were evaluated in one experiment, and the γδ^KO and αβ^KO mice were evaluated in three independent experiments.

Figure 5.

Mechanism of IL-17–mediated host defense against OPC. (A) Oral inoculation with C. albicans induces Th17 signature genes in oral mucosal tissue, which are suppressed in IL-17RA^KO mice. Tongue tissue from female WT or IL-17RA^KO mice (n = 2, from independent experiments) was taken at baseline (day 0) or 24 h after oral C. albicans inoculation (day 1), and cDNA was prepared and hybridized to Affymetrix genechips. Shown are Th17-related genes found to be induced on day 1 compared with day 0 from WT mice (red bars) or IL-17RA^KO mice (white bars). (B) Differentially expressed genes in WT versus IL-17RA^KO mice determined by microarray analysis. Fold change of genes induced in WT mouse tongue compared with IL-17RA^KO tongue on day 1 are indicated. * indicates genes previously connected to IL-17 or Th17 cells. (C–J) OPC-induced expression of selected genes. Tongue mRNA from WT or IL-17RA^KO mice on day 0 or day 1 (mRNA from two mice from independent experiments) was subjected to quantitative real-time RT-PCR analysis in triplicate. Data are presented as fold induction, normalized to GAPDH. *, P < 0.05 compared with WT Day 0; ‡, P < 0.05 compared with IL-17RA^KO Day 0.

Figure 6.

Anticandidal factors and Th17 deficiency. (A) IL-17RA^KO mice show decreased expression of mBD3 mRNA. Quantitative real-time RT-PCR analysis was performed in triplicate on tongue cDNA from WT or IL-17RA^KO mice (n = 2–4) at baseline (day 0) or day 1 or 5 after C. albicans infection. *, P < 0.05 compared with Day 0; ‡, P < 0.05 compared with WT sample at Day 5. (B) IL-17RA^KO mice show decreased expression of mBD3 protein. Tongue tissue from WT mice or IL-17RA^KO mice at day 0 or 5 (n = 4 for each condition) were applied to nitrocellulose in slot blots and probed with antibodies to mBD3. Signals were quantified by densitometry. *, P < 0.05. ns = not significant. Sample slot blot samples are shown below (note: all were taken from the same film at the same exposure level). (C) Candidacidal activity of BD3 and CCL20. Recombinant BD3 (0.1, 0.5 or 25 µM) and CCL20 (25 µM) were evaluated for fungicidal activity by incubation with 10⁶ C. albicans cells for 1.5 h in triplicate. Human histatin-5 (7 µM) was used as a positive control (37). Yeast were diluted and spread on YPD agar for colony enumeration. Data were compared by unpaired Student's t test (*, P < 0.05 compared with WT controls). Data are representative of three independent experiments. (D) Reduced candidacidal activity in IL-17RA-deficient and Th17-deficient (IL-23^KO) saliva. Saliva was collected from the indicated mice (n = 3–6) after carbachol injection and assayed for candidacidal activity in triplicate as in B. Experiments were performed twice with similar results.

Figure 7.

IL-12, but not IL-23 or IL-17RA, limit dissemination of C. albicans from the oral cavity. The indicated KO mice (γδ-T^KO, n = 13; αβ-T^KO, n = 14; IL-12^KO, n = 14; IL-23^KO, n = 4; IL-17RA^KO, n = 9) were infected orally with C. albicans as in Fig. 1, and kidneys were isolated on day of sacrifice. After homogenization, CFU/gram kidney was determined by colony enumeration in triplicate. Data were collected from all experiments in Figs. 2–4.