Aberrant type 1 immunity drives susceptibility to mucosal fungal infections

Abstract

Human monogenic disorders have revealed the critical contribution of type 17 responses in mucosal fungal surveillance. We unexpectedly found that in certain settings, enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. Notably, in mice and humans with AIRE deficiency, an autoimmune disease characterized by selective susceptibility to mucosal but not systemic fungal infection, mucosal type 17 responses are intact while type 1 responses are exacerbated. These responses promote aberrant interferon-γ (IFN-γ)- and signal transducer and activator of transcription 1 (STAT1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility. Concordantly, genetic and pharmacologic inhibition of IFN-γ or Janus kinase (JAK)-STAT signaling ameliorates mucosal fungal disease. Thus, we identify aberrant T cell-dependent, type 1 mucosal inflammation as a critical tissue-specific pathogenic mechanism that promotes mucosal fungal infection susceptibility in mice and humans.

Fig. 1.. Aire deficiency causes mucosal fungalspecific infection susceptibility without a defect in mucosal IL-17R/IL-22–dependent immune responses.

(A) Progression of fungal burden in wild-type and Aire^−/− tongue tissue after oral C. albicans infection (N = 9 to 16 per group; three experiments). (B) Representative histological micrographs of periodic acid-Schiff staining in wild-type and Aire^−/− tongue tissue at day 5 after infection. Scale bars, 100 μm (N = 8 per group; two experiments). (C) Fungal burden in wild-type and Aire^−/− kidney tissue at day 4 after intravenous C. albicans challenge (N = 6 to 8 per group; two experiments). (D to F) Frequencies of indicated lymphoid cells with (D) IL-17A–, (E) IL-17F–, and (F) IL-22–producing potential in wild-type and Aire^−/− oral gingival mucosal tissue at day 1 after infection (N = 5 to 13 per group; two to six experiments). ILCs, innate lymphoid cells. (G to I) Concentrations of IL-17A (G), IL-17F (H), and IL-22 (I) in wild-type and Aire^−/− tongue tissue homogenates before and after infection (N = 6 to 10 per group; two to three experiments). (J to M) Relative mRNA expression of S100a8 (J), S100a9 (K), Defb3 (L), and Defb1 (M) in wild-type and Aire^−/− tongue tissue before and after infection (N = 4 to 9 per group; two experiments). (N) Protein concentration of Ccl20 in wild-type and Aire^−/− tongue tissue homogenates before and after infection (N = 6 to 10 per group; two experiments). (O) Saliva was harvested after pilocarpine administration in wild-type and Aire^−/− mice over 20 min. The percentage of C. albicans that remained alive relative to the input inoculum after 1 hour or 3 hours of incubation ex vivo with salivary secretions harvested from wild-type and Aire^−/− mice was calculated (N = 6 to 9 per group; two experiments). (P) The tongues of wild-type and Aire^−/− mice were harvested at day 1 after infection. Epithelial cells were isolated by FACS. mRNA was extracted and RNA-seq was then performed. The heat map shows expression of selected IL-17R–regulated genes curated by Ingenuity Pathway Analysis (IPA) in RNA-seq of oral epithelial cells of Aire^+/+ (N = 3) and Aire^−/− (N = 3) mice with oral candidiasis. (Q and R) Fungal burden in Aire^−/− tongue tissue at day 4 after infection following antibody-mediated depletion of IL-17A and IL-17F (Q) or IL-22 (R) relative to control antibody (N = 5 to 7 per group; two experiments). All quantitative data are means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 as calculated using unpaired t test [(A), day 1, (O), and (Q)] or Mann-Whitney U test [(A), days 3 and 5, (C), (N), and (R)]; ns, not significant.

Fig. 2.. APECED patients have intact mucosal type 17 immune responses.

(A) Frequencies of indicated lymphoid cells with IL-17A–producing potential in healthy donor (N = 8) or APECED patient (N = 3) oral gingival mucosal tissue. Each dot represents an individual patient. ILCs, innate lymphoid cells. No IL-17A production by CD8⁺ T cells was observed in any of the healthy donors or APECED patients. (B and C) Relative mRNA expression of IL17A, IL17F, and IL22 (B) and S100A9 (C) in healthy donor (N = 3) and APECED patient (N = 6) oral gingival mucosal tissue. (D) S100A8 and S100A9 concentrations in saliva of healthy donors (N = 33 to 38) and APECED patients (N = 75 to 77). (E) Oral gingival mucosal tissue was obtained from uninfected healthy donors (HD; N = 4) and uninfected APECED patients (N = 5), mRNA was extracted, and RNA-seq was performed. The heat map shows expression of IL-17R–regulated genes, curated from an earlier study (19), in RNA-seq of oral gingival mucosal tissue from APECED patients versus healthy donors. All quantitative data are means ± SEM.

Fig. 3.. Pathogenic TCRαβ⁺ cells drive mucosal fungal infection susceptibility in Aire deficiency.

(A and B) Numbers of CD4⁺ (A) and CD8⁺ (B) T cells in wild-type and Aire^−/− tonguetissue before and after oral C. albicans infection (N = 6 to 10 per group; two experiments). (C and D) Representative contour plots of CD44 and CD69 expression (left, day 0) and proportions of CD44^hiCD69⁺ cells (right, days 0 and 1) within CD4⁺ (C) and CD8⁺ (D) T cells in wild-type and Aire^−/− tongue tissue (N = 6 to 10 per group; two experiments). (E and F) Representative immunofluorescence images of CD4 and CD8 staining [(E), top and bottom, respectively] and quantification of CD4⁺ and CD8⁺ T cells (F) within epithelial and submucosal layers of wild-type and Aire^−/− tongue tissue at day 1 after infection. Scale bars, 100 μm (N = 6 or 7 per group; two experiments). (G) Immunohistochemical analysis of CD4 (brown) and CD8 (red) in healthy donor and APECED patient oral gingival mucosal tissue. Scale bars, 1200 μm (top), 200 μm (bottom). Shown are images from one of two examined healthy donors and one of three examined APECED patients; images from all patients are shown in fig. S15. (H and I) Aire^−/−Tcra^−/− mice control mucosal fungal infection better than do Aire^−/− mice, as indicated by fungal burden analysis in tongue tissue (H) and representative histological micrographs of periodic acid-Schiff staining in wild-type, Aire^−/−, and Aire^−/−Tcra^−/− tongue tissue at day 5 after infection (I). Scale bars, 200 μm (N = 6 to 16 per group; three experiments). (J and K) In Aire^−/− mice, antibody-mediated depletion of CD4⁺ T cells [(J); N = 8 or 9 per group, three experiments] or CD8⁺ T cells [(K); N = 4 to 6 per group, two experiments] results in decreased fungal burden in tongue tissue. (L) Fungal burden in tongue tissue of Tcra^−/− recipient mice 10 weeks after adoptive transfer of the indicated T cells derived from wild-type or Aire^−/− mice (N = 6 to 10 per group; three experiments). All quantitative data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated using unpaired t test [(A), days 1 and 3, (B), days 0 and 5, (C), day 0, and (D)], Mann-Whitney U test [(A), days 0 and 5, (B), days 1 and 3, (C), day 1, (F), (J), and (K)], one-way ANOVA with Holm-Šidák multiple-comparisons test (H), or Kruskal-Wallis H test with Dunn’s multiple-comparisons test (L).

Fig. 4.. Aire deficiency results in IFN-γ–driven mucosal interferonopathy.

(A and B) Relative mRNA expression of Ifng [(A); N = 4 to 9 per group; two experiments] and concentration of IFN-γ [(B); N = 5 to 20 per group; two experiments] in wild-type and Aire^−/− tongue tissue before and after infection. (C and D) Representative contour plots of IFN-γ expression (left; day 0) and frequencies of IFN-γ–producing cells (right; days 0 and 1) within CD4⁺ (C) and CD8⁺ (D) T cells in wild-type and Aire^−/− oral gingival mucosal tissue (N = 4 to 8 per group; two experiments). (E) Relative mRNA expression of Stat1 in wild-type and Aire^−/− tongue tissue homogenates before and after infection (N = 4 to 13 per group; two experiments). (F and G) Concentrations of IFN-γ–inducible CXCL9 (F) and CXCL10 (G) in wild-type and Aire^−/− tongue tissue homogenates before and after infection (N = 6 to 10 per group; two to three experiments). (H) Epithelial layers of wild-type, Aire^−/−, and Aire^−/−Ifng^−/− tongue tissues were harvested at day 1 after infection for immunoblot analysis of phospho-Stat1 (Tyr⁷⁰¹), total Stat1, and β-actin as loading control. Top, quantification of protein immunoblot data; bottom, representative protein immunoblot images (N = 8 to 10 per group; three experiments). (I to K) Tongues of wild-type and Aire^−/− mice were harvested at day 1 after infection. Epithelial cells were isolated by FACS. mRNA was extracted and RNA-seq was then performed. (I) Principal components analysis of RNA-seq in wild-type (N = 3) and Aire^−/− (N = 3) oral epithelial cells at day 1 after infection. (J) Pathway analysis using differentially expressed genes between wild-type and Aire^−/− oral epithelial cells was performed using Enrichr and graphed according to enrichment score for significant Reactome biological processes. (K) Volcano plot of RNA-seq demonstrating differential gene expression in wild-type and Aire^−/− oral epithelial cells at day 1 after infection. Shown are IFN-γ–dependent genes. Each dot represents an individual mouse. All quantitative data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated using unpaired t test [(A), (C), (D), day 0, and (G), day 0], unpaired t test with Welch’s correction [(B), day 3, and (E), days 0, 3, and 5], Mann-Whitney U test [(B), days 0, 1, and 5, (D), day 1, (E), day 1, (F), and (G), days 1, 3, and 5] or one-way ANOVA with Holm-Šidák multiple-comparisons test (H).

Fig. 5.. APECED patients exhibit IFN-γ/STAT1–associated mucosal interferonopathy.

(A and B) Representative contour plots of IFN-γ expression within CD4⁺ (A) and CD8⁺ (B) T cells in healthy donor and APECED patient oral gingival mucosal tissue. (C and D) Frequencies of CD4⁺ (C) and CD8⁺ (D) T cells with IFN-γ–producing potential in healthy donor (N = 8) or APECED patient (N = 3) oral gingival mucosal tissue. (E and F) Concentrations of IFN-γ–inducible CXCL9 (E) and CXCL10 (F) in saliva of healthy donors (N = 28 to 31) and APECED patients (N = 73 to 79). (G) Immunohistochemical analysis of phospho-STAT1 in healthy donor and APECED patient oral gingival mucosal tissue. Scale bars, 400 μm. (H to K) Oral gingival mucosal tissue was obtained from uninfected healthy donors (N = 4) and uninfected APECED patients (N = 5), mRNA was extracted, and RNA-seq was performed. (H) Volcano plot comparing RNA-seq of oral gingival mucosal tissue from APECED patients (N = 5) versus healthy donors (HD; N = 4). Highlighted in red are differentially expressed genes [DEGs; fold change (FC) ≥ ±2 at FDR < 0.05]. Marked are select IFN-γ–regulated genes. (I) Top five enriched MSigDB hallmark gene sets in DEGs from (H). Shown are −log₁₀ FDR q-value (bottom) and fold enrichment (FE; top). (J) Heat map showing expression of the 11 enriched IFN-γ response genes from (I) in each sample. (K) Ingenuity Pathway Analysis (IPA)–predicted upstream regulators of DEGs in humans (APECED patients versus HD) and mice (Aire^−/− versus Aire^+/+). The heat map shows activation Z-scores of the top five predicted cytokines or transcription factors. All quantitative data are means ± SEM. **P < 0.01, ****P < 0.0001 as calculated using unpaired t test [(C) and (D)] or Mann-Whitney U test [(E) and (F)].

Fig. 6.. Excessive IFN-γ in Aire-deficient mice drives oral epithelial barrier defects.

(A to E) Oral epithelial cells from tongue tissue of Aire^−/− mice exhibit decreased survival relative to wild-type and Aire^−/−Ifng^−/− mice. (A) Representative contour plots of dead cell dye–positive oral epithelial cells; (B) frequencies of dead cell dye–positive oral epithelial cells; (C) numbers of live oral epithelial cells at day 1 after infection (N = 6 to 8 per group, two experiments); representative images (D) and frequencies (E) of propidium iodide (PI)–positive oral epithelial cells from wild-type, Aire^−/−, and Aire^−/−Ifng^−/− mice as assessed by two-photon confocal imaging at day 1 after infection. Scale bars, 50 μm (N = 17 to 25 per group, three experiments). (F and G) Epithelial layers of wild-type, Aire^−/−, and Aire^−/−Ifng^−/− tongue tissues were harvested at day 1 after infection for immunoblot analyses. Shown are quantification of protein immunoblot data and representative protein immunoblot images of γ-H2A.X (phosphorylated Ser¹³⁹) [(F); N = 10 or 11 per group, three experiments] and Ki-67 [(G); N = 12 to 15 per group, three experiments]. β-Actin or α/β-tubulin were used as loading controls. (H and I) Immunohistochemical analysis of Ki-67 in the oral epithelium of tongue tissue of wild-type, Aire^−/−, and Aire^−/−Ifng^−/− mice at day 1 after infection. (H) Summary histology score data; (I) representative immunohistochemistry images. Scale bars, 150 μm (N = 7 to 13 per group, three experiments). (J) Increased oral mucosal barrier permeability in Aire^−/− mice, as shown by quantification of the amount of FITC-dextran measured in the serum of wild-type, Aire^−/−, and Aire^−/−Ifng^−/− mice at day 1 after infection following topical application of FITC-dextran onto the mouse tongue (N = 10 or 11 per group, three experiments). The dotted horizontal line indicates the fluorescence values obtained in the serum of mice in which FITC-dextran was not applied onto the mouse tongue. (K) Epithelial layers of wild-type, Aire^−/−, and Aire^−/−Ifng^−/− tongue tissues were harvested at day 1 after infection for immunoblot analyses. Left, quantification of protein immunoblot data; right, representative protein immunoblot images of claudin-1 with β-actin as loading control (N = 14 to 18 per group, four experiments). Each dot represents an individual mouse or an individual field of view of mouse tongue tissue imaged using two-photon confocal microscopy (E). All quantitative data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated using one-way ANOVA with Newman-Keuls multiple comparisons test [(C) and (G)], one-way ANOVA with Holm-Šidák multiple comparisons test [(B), (E), (F), (H), and (J)], or Kruskal-Wallis H test with Dunn’s multiple-comparisons test (K).

Fig. 7.. Prolonged IFN-γ exposure impairs the viability and barrier integrity of human oral epithelial cells.

OKF6/TERT-2 human oral epithelial cells were exposed for 72 hours to the indicated concentrations of IFN-γ (U, units). (A) Lactate dehydrogenase (LDH) release was assessed after changing the culture media, 8 hours after C. albicans infection, with a starting point of similar confluency (N = 8 or 9 per group; three experiments). (B) Permeability of confluent cells assessed by measuring fluorescent counts of FITC-dextran in receiver wells (N = 6 to 9 per group; three experiments). (C) Quantification of protein immunoblot data (top) and representative protein immunoblot images (bottom) of claudin-1 with GAPDH as loading control (N = 3 per group; three experiments). (D) Representative images of claudin-1 expression and localization in human oral epithelial cells. Scale bar, 20 mm. Shown are mean (±SEM) fluorescent values of claudin-1 obtained from three randomly selected high-power fields per experiment (three independent experiments). All quantitative data are means ± SEM. ***P < 0.001, ****P < 0.0001 as calculated using one-way ANOVA with Dunnett’s post hoc multiple-comparisons test.

Fig. 8.. Inhibition of IFN-γ or JAK-STAT signaling in Aire-deficient mice ameliorates mucosal fungal infection susceptibility.

(A) Fungal burden in Aire^−/− and Aire^−/−Ifng^−/− tongue tissue after oral C. albicans infection (N = 9 or 10 per group; three experiments). (B) Representative histological micrographs of periodic acid-Schiff staining in Aire^−/− and Aire^−/−Ifng^−/− tongue tissue at day 5 after infection. Scale bars, 150 μm (N = 5 or 6 per group; two experiments). (C) Fungal burden in tongue tissue of Tcra^−/− recipient mice 10 weeks after adoptive transfer of the indicated T cells derived from wild-type, Aire^−/−, or Aire^−/−Ifng^−/− mice (N = 7 to 14 per group; five experiments). (D and E) Fungal burden in tongue tissue of Aire^−/− mice at day 5 after infection following (D) antibody-mediated neutralization of IFN-γ (N = 6 per group; two experiments) or (E) ruxolitinib administration (N = 9 to 11 per group; three experiments). All quantitative data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated using Mann-Whitney U test [(A), (D), and (E)] or Kruskal-Wallis H test with Dunn’s multiple-comparisons test (C).