Immunological Consequences of Intestinal Fungal Dysbiosis

Abstract

Compared to bacteria, the role of fungi within the intestinal microbiota is poorly understood. In this study we investigated whether the presence of a "healthy" fungal community in the gut is important for modulating immune function. Prolonged oral treatment of mice with antifungal drugs resulted in increased disease severity in acute and chronic models of colitis, and also exacerbated the development of allergic airway disease. Microbiota profiling revealed restructuring of fungal and bacterial communities. Specifically, representation of Candida spp. was reduced, while Aspergillus, Wallemia, and Epicoccum spp. were increased. Oral supplementation with a mixture of three fungi found to expand during antifungal treatment (Aspergillus amstelodami, Epicoccum nigrum, and Wallemia sebi) was sufficient to recapitulate the exacerbating effects of antifungal drugs on allergic airway disease. Taken together, these results indicate that disruption of commensal fungal populations can influence local and peripheral immune responses and enhance relevant disease states.

Figure 1. Antifungal treatment exacerbates colitis

Mice were given fluconazole in their drinking water for 3 weeks, followed by treatment with DSS for 7 days to induce acute colitis. Body weight (A), histology score on H&E stained colon sections (B), and the percentage of IL-17- and IFN-γ-producing colonic lamina propria CD4⁺ T cells (C) were determined upon sacrifice. (D) H&E stained colonic sections from Rag1^−/− mice adoptively transferred with CD4⁺CD45RB^high T cells and treated with fluconazole for 6 weeks. (E) Total CD4⁺ T cell numbers in colonic LP and MLNs. Dot plots (F) and bar graphs (G) show the percentage of IL-17- and IFN-γ-producing CD4⁺ T cells isolated from colonic lamina propria 6 weeks after naïve T cell transfer. Each symbol represents a different mouse. One of several independent experiments with similar outcome is shown. Error bars, s.d., * P < 0.05, ** P < 0.01 Student’s t-test.

Figure 2. Antifungal treatment exacerbates allergic airway disease

(A) Experimental setup for antifungal treatment and induction of allergic airway disease. Lung histology (H&E) (B), BAL cell counts (C), and serum antibody titers (D) from mice treated with (filled black bars) and without (open bars) fluconazole and immunized with HDM or not (PBS). (E) Representative intracellular cytokine staining (left panel) and quantification of cytokine producing CD4⁺ T cells (right panel) from HDM re-stimulated mediastinal lymph nodes from control and fluconazole treated mice. (F-H) Allergic airway disease in amphotericin-B-treated mice. H&E lung histology (F), representative FACS gating strategy showing eosinophil frequency (left panel) and quantification of total eosinophils (right panel) in the BAL (G), and serum antibody titers (H) in control (open bars) and amphotericin-treated (filled black bars) mice immunized with HDM. Data are representative of 4 (A-E) and 2 (F-H) independent experiments. Each dot represents an individual mouse. *P<0.05, **P<0.01 Student’s t-test. See also Figure S1.

Figure 3. Antifungal treatment alters the composition of commensal fungal populations of the gut

(A) Quantitative PCR for fungal 18S rDNA in feces of mice before and after 3 weeks treatment with fluconazole. (B) Bar graphs show relative abundance of specific fungal genera sequences before and after treatment with fluconazole assessed by ITS1 amplicon sequencing. (C) PCR quantification of Candida and Aspergillus DNA, relative to total 18S fungal rDNA, in the feces of mice before and after fluconazole treatment. (D) PCoA analysis of fungal communities in control mice and mice treated with fluconazole or amphotericin. (E) Taxonomic distribution of most abundant fungal species in control mice and mice treated with fluconazole or amphotericin. LEfSe analysis on samples from control mice and mice treated with fluconazole or amphotericin showed significant decrease (F) of some or expansion (G) of other fungal species. The horizontal straight lines in the panels (F, G) indicate the group means, and the dotted lines indicate the group medians. Each bar represents an individual mouse. Data are representative of three independent sequencing experiments with 5 mice/group for each condition. See also Figure S2.

Figure 4. Allergic airway disease in mice supplemented with fungi that expand during antifungal treatment

(A) Species-specific PCR quantification (relative to the amount of input DNA in PCR reaction) of indicated fungi in feces of mice treated with or without fluconazole for 3 weeks. (B) Experimental setup and time course of oral fungal inoculation and HDM immunization. (C and E) Quantification of cell counts in the BAL of HDM-immunized mice treated (filled black bars) or not (open bars) with the fungal cocktail (C) or P. brevicompactum (E). (D and F) Serum levels of IgE and HDM-specific IgG1 in HDM-immunized mice treated (filled black bars) or not (open bars) with the fungal cocktail (D) or P. brevicompactum (F). Each dot represents and individual mouse. Data are representative to two independent experiments. *P<0.05, **P<0.01 Student’s t-test. See also Figure S3 and S4.