Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections

Abstract

Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα(-/-), and Rag1(-/-) mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1-2 d by tongue-resident populations of γδ T cells and CD3(+)CD4(+)CD44(hi)TCRβ(+)CCR6(+) natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β(-/-) and TCR-δ(-/-) mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1(-/-), IL-7Rα(-/-), and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens.

Figure 1.

Innate immunity to OPC requires IL-17 signaling and a rearranged antigen receptor. (A) WT mice were subjected to OPC. At days 1–5, mRNA from pooled tongues (n = 5) was analyzed for il17a by qPCR in triplicate. *, P < 0.05 versus infected WT mice. (B–E) The indicated mice were subjected to OPC for 4–5 d, and fungal load was assessed by colony enumeration. Each point represents 1 mouse. Horizontal bars indicate geometric mean. WT mice were immunosuppressed with cortisone acetate (Cort.). *, P < 0.05 versus infected WT mice. Note: detection limit is 50 CFU/g. Each experiment was performed two or three independent times. (F) WT or Rag1^−/− mice were subjected to OPC and fungal loads assessed at the indicated times. Horizontal bars indicate geometric mean. *, P < 0.05 versus infected WT mice. (G) The indicated WT mice were treated with CsA daily from day −2 and subjected to OPC. Fungal loads were assessed after 5 d. Horizontal bars indicate geometric mean. *, P < 0.05 versus infected WT mice. This experiment was performed twice. (H) Tongues were isolated from WT mice (n = 5) subjected to Sham inoculation, 5 d infection (1°), or 1° infection followed by rechallenge after 6 wk (1° + 2°). The indicated genes were evaluated in triplicate by qPCR. This experiment was performed three times. Error bars indicate SD.

Figure 2.

IL-17⁺ and CD3⁺ cells in the oral mucosa after C. albicans infection. (A) IL-17^eGFP reporter mice were subjected to OPC. 2 d later, tongue was subjected to 2-photon microscopy to visualize GFP. Note that papillae appear green due to endogenous autofluorescence. Bar, 100 µm. (B) Tongue sections from sham or C. albicans–infected RORγt^+/− (surrogate for WT) mice were stained with α-CD3 or DAPI. Bar, 100 µm. (C) T cells in the tongue express CD3. Tongue homogenates from Sham- or Candida-infected WT mice were stained for CD45, TCR-β, and CD3. Red lines/boxes = TCR-β^hi cells; blue lines/boxes = TCR-β^lo cells. All experiments were performed twice.

Figure 3.

IL-17 is produced in the oral mucosa by γδT and nTh17 cells. (A) IL17^CreRosa26R^eYFP mice were subjected to OPC. 48 h later, cell homogenates from pooled tongues (n = 5) were stained for the indicated markers. Shown are CD45⁺ populations in lymphocyte gate (left), and CD44⁺ and CD4⁺ populations or SSC and YFP in the CD45⁺ gate (middle). In C. albicans–infected mice, the YFP⁺ population was further gated on CD44 and CD4 (offset, far right). Data are representative of three independent experiments. (B) Cells from WT spleen (n = 1) or pooled tongues (n = 5) were stained for TCR-β and TCR-γδ (or isotype). The percentage of TCR-β⁺ and TCR-γδ⁺ cells (as a fraction of the total lymphocyte population) is indicated in each plot. The experiment was repeated 3 times, and pooled in graph at right. Horizontal bars indicate geometric mean. (C) TCR-β^−/− and TCR-δ^−/− mice were subjected to OPC and analyzed as in Fig. 1. Data are pooled from two independent experiments. Horizontal bars indicate geometric mean. (D) IL17^CreRosa26R^eYFP (IL-17^eYFP) mice were subjected to Sham infection or to OPC. Pooled cells from tongue (n = 5) were stained for CD45 (not depicted), TCR-β, and TCR-γδ. Experiment is representative of four independent replicates. (E) WT or IL-17^eYFP mice were subjected to Sham infection or to OPC. Pooled cells from tongue (n = 5) were stained for CD45, TCR-β, and TCR-γδ and visualized for YFP (two left panels). YFP⁺ cells were analyzed for expression of TCR-β and TCR-γδ cells (right panel). The experiment is representative of two independent experiments.

Figure 4.

Phenotypic and functional characteristics of oral nTh17 cells. (A) WT or Rag1^−/− mice were subjected to Sham inoculation or infected with C. albicans, and tongue homogenates were stained for the indicated markers. CD45⁺ cells were gated on CD4/CD44. Percentage of TCR-β⁺ cells is indicated. The experiment was performed twice. (B) WT or IL-7Rα^−/− mice were infected with C. albicans, and tongue was stained for CD45 and TCR-β. The experiment was performed twice. (C) Tongue homogenates from mice housed in specific pathogen-free (SPF) or germ-free conditions was stained for CD45 and TCR-β. The experiment was performed twice. (D) WT mice were infected with C. albicans or C. glabrata, and tongue homogenates were stained for CD45 and TCR-β. Experiment was performed twice. (E) WT mice were subjected to OPC and 5 d later, mRNA was analyzed for expression of PLZF or CXCL5 as a control. The experiment was performed once. **, P < 0.01. Error bars indicate SD. (F) Tongue homogenates from WT Candida-infected mice were stained for CD45 and TCR-β and gated on CCR6. Solid = TCR-β^hi, dashed = TCR-β^lo. Experiment was performed twice. (G) Tongue homogenates from Sham- or Candida-infected WT mice were stained for the indicated markers and gated on lymphocytes. The experiment was performed twice.