Antifungal drugs induce dysbiosis in which some fungi are reduced and others expand

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.

Here we provide evidence for the role of a healthy intestinal fungal community in regulation of immune responses in mice. Disruption of intestinal fungi by antifungal drug treatment increases disease severity in chemically induced and T cell transfer-mediated models of experimental colitis. It also exacerbates development of allergic airway disease induced by house dust mite (HDM) challenge. High-throughput rDNA sequencing analysis of fecal material demonstrated that antifungal treatment resulted in restructuring of both the fungal and bacterial commensal communities. Surprisingly, antifungal treatment led to a relative increase in several specific fungal species that are present at low levels or absent in healthy untreated mice. Oral supplementation of mice with a mixture of three of these fungi (A. amstelodami, E. nigrum, and W. sebi) was sufficient to recapitulate the exacerbated features of allergic airway disease that were observed with antifungal drug treatment. These results suggest that in addition to bacteria, a healthy fungal microbiome is important for regulation of immune responses.

Intestinal commensal bacteria are integral to immune homeostasis within the intestines as well as at distant sites such as the lungs (). Studies with antibiotic-treated and germ-free mice have demonstrated that lack of innate immune stimulation by commensal bacteria predisposes mice to allergic diseases such as asthma, owing to a general Th2 skewing of the immune system (), and also results in more severe disease in chemically induced models of colitis due to compromised intestinal epithelial barrier integrity (). In humans, epidemiological data have indicated that early-life treatment with broad-spectrum antibiotics is associated with an increased risk of developing allergic diseases such as asthma () and may also contribute to the development of inflammatory bowel disease (). Little is presently known about the immunological effects of disrupting the healthy intestinal fungal community and whether this could have beneficial or detrimental effects on inflammatory disease development.

Antibiotics associated with increased risk of new-onset Crohn’s disease but not ulcerative colitis: a meta-analysis.

Assessing the association of early life antibiotic prescription with asthma exacerbations, impaired antiviral immunity, and genetic variants in 17q21: a population-based birth cohort study.

Microbial communities associated with mucosal surfaces of the body play essential roles in diverse biological processes ranging from digestion to behavior and are increasingly recognized as integral components of normal physiology (). Research over the last several decades has revealed that intestinal bacteria are critical for regulating homeostatic and protective immune responses; however, in recent years it has become evident that additional players such as fungi and viruses also have the potential to influence these processes (). We and others have utilized culture-independent sequencing approaches to define the composition of fungal populations associated with mucosal surfaces including the intestinal tract, oral cavity, skin, and lungs (). We previously showed that deficiency in the antifungal innate immune receptor Dectin-1 leads to more severe disease in an experimental model of colitis and that this is accompanied by fungal invasion of the colonic mucosa and expansion of opportunistic fungi such as Candida and Trichosporon spp. (). In support of this, pathogenic Candida spp. have been reported to be enriched in the intestines of some IBD patients (), and a polymorphism in the gene for Dectin-1 is associated with increased disease severity in ulcerative colitis patients (). These observations suggest that fungal dysbiosis could be an important factor in development or progression of inflammatory diseases. However, little is known about the importance of a healthy fungal microbiota in regulation of immune responses.

The interplay between the intestinal microbiota and the brain.

The impact of the gut microbiota on human health: an integrative view.

To test whether expansion of these fungi would be sufficient to influence allergic airway disease, we fed mice by repeated oral gavage with a mixture of A. amstelodami, E. nigrum, and W. sebi and immunized with HDM as described ( Figure 4 B). Similar to treatment of mice with antifungal drugs, supplementation of mice with A. amstelodami, E. nigrum, and W. sebi was sufficient to cause exaggerated HDM-mediated allergic airway disease as measured by an increase in the number of BAL eosinophils and lymphocytes, as well as elevated serum IgE and HDM-specific IgG1 levels ( Figures 4 C and 4D). Oral supplementation with similar amounts of Penicillium brevicompactum, which does not expand with antifungal treatment ( Figure 3 F), did not exacerbate airway disease ( Figures 4 E and 4F). We did not observe exacerbated DSS colitis in mice supplemented with these fungi, indicating that antifungal drug treatment modulates intestinal disease through a mechanism that is independent of expansion of these specific fungi ( Figures S4 A–S4F). Together, these results support the hypothesis that manipulation of the intestinal fungal community significantly impacts peripheral immune responses, and suggests that expansion of certain fungal populations by antifungal treatment might be a contributing factor.

The exacerbated immune responses to challenge (colitis or asthma) that we observed after fungal community dysbiosis induced by antifungal drug treatment could have been a consequence of the general reduction in fungal burden, reduction in specific fungi, or the relative expansion of specific fungi. Having unexpectedly observed that several fungi (A. amstelodami, E. nigrum, and W. sebi) appeared to expand during treatment with either drug, we hypothesized that this expansion might be sufficient to influence systemic immunity. Species-specific quantitative PCR of fecal DNA revealed that fluconazole treatment resulted in an increase in A. amstelodami, E. nigrum, and W. sebi rDNA ( Figure 4 A), indicating that levels of these species were truly increased and did not simply become more common in the context of a total reduction in fungal burden. We confirmed that all three fungi were resistant to fluconazole in vitro and showed little (A. amstelodami, E. nigrum) or no (W. sebi) susceptibility to amphotericin-B compared to Candida albicans ( Figure S3 A). It is unlikely that the effect of antifungal treatment is due to alterations of fungal populations within the lung, as we consistently recovered less fungal DNA from an entire lobe of lung tissue compared to a single fecal pellet and were unable to detect the presence of the fungi of interest in the mouse lung by quantitative PCR ( Figures S3 B and S3C).

(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 of two independent experiments.p < 0.05,p < 0.01, Student’s t test. See also Figures S3 and S4

(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).

(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.

Oral Supplementation of Mice with Fungi Found to Expand during Antifungal Treatment Exacerbates Development of Allergic Airway Disease

Fungal and bacterial populations occupy similar spaces in the gut and can influence each other (). We therefore wondered if disruption of commensal fungi could have an effect on bacterial populations that could impact disease. We sequenced bacterial 16S rDNA regions and found that antifungal treatment did not affect overall bacterial diversity, but did affect specific bacterial taxa ( Figures S2 B–S2D). We observed decreased relative detection of Bacteroides, Allobaculum, Clostridium, Desulfovibrio, and Lactobacillus spp., while relative detection of Anaerostipes, Coprococcus, and Streptococcus was increased. The data suggest that fungal and bacterial communities in the gut are codependent and that disruption of one community affects the other.

Candida albicans and bacterial microbiota interactions in the cecum during recolonization following broad-spectrum antibiotic therapy.

The effect of antifungal drug treatment on intestinal and lung disease was initially predicted to be due to depletion of commensal fungal populations; however, we consistently observed only a 3- to 5-fold reduction in the total amount of fungal 18 s rDNA in the feces after several weeks of fluconazole treatment ( Figure 3 A), indicating that antifungals might only target certain populations of fungi in the gut. To better understand the changes that occur within the intestinal microbiota following fluconazole treatment, we utilized high-throughput rDNA sequencing to determine if there was a general reduction of all fungal species or if the fungal community was restructured in a way that could potentially contribute to the effects observed in the lung and gut models. We isolated DNA from stool samples, amplified and sequenced fungal ITS1 (internal transcribed spacer 1) rDNA regions, and assigned operational taxonomic units using a custom-curated ITS database (). The approach mapped an average of 80% of all sequences at the fungal genera and species level, identifying 57 genera of fungi. While fluconazole treatment consistently led to a relative decrease in detection of Candida spp., other fungal genera, such as Aspergillus, Wallemia, and Epicoccum, expanded upon this treatment ( Figures 3 B and 3C). We next asked whether amphotericin-B affected gut fungal communities in a similar way. Principle coordinates analysis confirmed that fluconazole and amphotericin treatment led to changes in gut fungal communities that were distinct from nontreated controls ( Figure 3 D). Furthermore, using an algorithm for high-dimensional biomarker discovery, LEfSe (), we observed that relative detection of specific fungi including Penicillium brevicompactum and Candida tropicalis was significantly decreased upon antifungal treatment ( Figures 3 E and 3F and S2 A), while this treatment led to relative expansion of Aspergillus amstelodami, Epicoccum nigrum, and Wallemia sebi ( Figures 3 E, 3G, and S2 A).

(F and G) 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 and 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 five mice/group for each condition. See also Figure S2

(E) Taxonomic distribution of most abundant fungal species in control mice and mice treated with fluconazole or amphotericin.

(D) PCoA analysis of fungal communities in control mice and mice treated with fluconazole or amphotericin.

(C) PCR quantification of Candida and Aspergillus DNA, relative to total 18S fungal rDNA, in the feces of mice before and after fluconazole treatment.

(B) Bar graphs show relative abundance of specific fungal genera sequences before and after treatment with fluconazole assessed by ITS1 amplicon sequencing.

(A) Quantitative PCR for fungal 18S rDNA in feces of mice before and after 3 weeks of treatment with fluconazole.

Treatment of Mice with Antifungal Drugs Results in Intestinal Dysbiosis of Commensal Fungal and Bacterial Populations

To determine if fluconazole might have nonspecific effects that could influence the response, we monitored weight change, intestinal permeability, and levels of the liver enzyme alanine transaminase (ALT) in mice treated for 3 weeks with fluconazole and did not observe differences ( Figures S1 C–S1E). We also assessed the induction of allergic airway disease in mice treated with two additional antifungal agents, amphotericin-B and 5-fluorocytosine (5-FC), which target fungi through different mechanisms compared to fluconazole (). Consistent with the fluconazole observations, mice treated with amphotericin-B ( Figures 2 F–2H and S1 G) or 5-FC ( Figures S1 F and S1G) displayed exacerbated disease following HDM immunization characterized by severe eosinophilia in the lungs and increased Th2 polarization of CD4T cells from restimulated mediastinal lymph node cultures compared to control mice. Elevation in Th2-associated antibodies was also observed in serum from amphotericin-B-treated mice immunized with HDM ( Figure 2 H). Amphotericin-B is poorly absorbed through the gastrointestinal tract (), suggesting that the observed effect on lung inflammatory disease is likely mediated by disruption of fungal populations in the gut rather than an extraintestinal site such as the lungs or skin. Together, these results suggest that the enhanced response observed with fluconazole was not due to an off-target effect of the drug but was likely a specific effect of disrupting the fungal microbiota in the intestine.

It is well-established that bacterial populations in the gut can have systemic effects on the immune system, and this has been largely investigated in the context of allergic diseases such as asthma (). We therefore wondered if disruption of the fungal community in the gut might also impact immune responses at distant sites in the body. To test this, wild-type mice were treated with fluconazole in the drinking water followed by induction of allergic airway disease by intratracheal HDM immunization ( Figure 2 A). Several fungi are known to exacerbate symptoms in a subset of severe asthma patients (), so we anticipated that antifungal treatment might be protective in this model. Surprisingly, fluconazole-treated mice displayed exacerbated disease, characterized by increased inflammatory cellular infiltration (mainly eosinophils) into the lungs ( Figures 2 B and 2C). Consistent with this observation, mRNA levels of the eosinophil chemoattractants CCL11 and CCL21 were elevated in lung tissue of fluconazole-treated mice immunized with HDM (see Figure S1 A available online). Fluconazole treatment also resulted in elevation of serum Th2-associated antibodies, IgE and HDM-specific IgG1, in response to HDM immunization ( Figure 2 D), as well as increases in Th2 cytokines (IL-4, IL-5, IL-10) measured in the supernatants of HDM-restimulated mediastinal lymph node cultures ( Figure S1 B). Furthermore, the percentage of CD4T cells producing IL-4 and IL-13 from restimulated lymph node cultures was elevated in fluconazole-treated mice, whereas IL-17- and IFN-γ-producing CD4T cells were unchanged compared to control mice immunized with HDM ( Figure 2 E). These findings indicate that the exacerbating effect of fluconazole treatment is restricted to Th2-mediated inflammation in this model.

Data are representative of four (A–E) and two (F–H) independent experiments. Each dot represents an individual mouse.p < 0.05,p < 0.01, Student’s t test. See also Figure S1

(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.

(E) Representative intracellular cytokine staining (left panel) and quantification of cytokine producing CD4 + T cells (right panel) from HDM restimulated mediastinal lymph nodes from control and fluconazole treated mice.

(A–D) (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).

The link between fungi and severe asthma: a summary of the evidence.

To determine if disruption of the healthy intestinal fungal community impacts the response to immune challenge, mice were treated for 3 weeks with the commonly used antifungal agent fluconazole and were subsequently given DSS in their drinking water to induce acute colitis. We have previously shown that fluconazole is protective in situations where antifungal immunity is genetically compromised and pathogenic fungi have expanded (); however, we were surprised to find that prolonged treatment of healthy wild-type mice with fluconazole promoted more severe colitis. This was measured by failure to recover weights and histology ( Figures 1 A and 1B ) and correlated with higher frequencies of inflammatory CD4T cells in the intestinal lamina propria ( Figure 1 C). These results are consistent with a recent study showing similar effects of fluconazole treatment on the severity of DSS colitis (). We next examined whether disruption of gut fungal communities would also affect intestinal disease in the T cell transfer-mediated model of chronic colitis. Rag1-deficient mice were adoptively transferred with naive CD4CD45RBT cells and treated or not with fluconazole for the duration of the experiment. Consistent with our results in the DSS-induced model of colitis, prolonged fluconazole treatment led to exacerbated disease characterized by increased mucosal erosion, crypt destruction, and infiltration of inflammatory cells in the colons of fluconazole-treated mice ( Figure 1 D). Although fluconazole treatment did not affect the total number of CD4T cells infiltrating the colon and MLNs of adoptively transferred mice ( Figure 1 E), the frequencies of inflammatory Th1 and Th17 cells significantly increased during fluconazole-induced gut fungal community dysbiosis ( Figures 1 F and 1G). Together, these data suggest that a healthy gut fungal community is important in resistance to intestinal inflammatory disease.

(F and G) 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 naive T cell transfer. Each symbol represents a different mouse. One of several independent experiments with similar outcome is shown. Error bars, SD; ∗ p < 0.05, ∗∗ p < 0.01, Student’s t test.

(D) H&E-stained colonic sections from Rag1 −/− mice adoptively transferred with CD4 + CD45RB high T cells and treated with fluconazole for 6 weeks.

(A–C) 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.

Changes in the composition of intestinal fungi and their role in mice with dextran sulfate sodium-induced colitis.

Discussion

In this study we utilized oral antifungal treatment to determine if disruption of the “healthy mycobiota” in the intestines alters local and peripheral immune homeostasis. Treatment of mice with antifungal drugs increased disease severity in chemically induced and T cell transfer-mediated models of experimental colitis and exacerbated development of HDM-induced allergic airway disease. High-throughput ITS1 and 16s sequencing of fungal and bacterial rDNA showed that antifungal treatment restructured intestinal fungal and bacterial communities. We specifically noted expansion of three commensal fungi (A. amstelodami, E. nigrum, and W. sebi) that, when orally delivered to mice, recapitulated the effects of antifungal treatment on allergic airway disease. These findings provide compelling evidence to support a functional role of the fungal “mycobiota” in modulating immune function and development of inflammatory disease.

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et al. Inhibition of Dectin-1 Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine. The finding that antifungal treatment exacerbated disease during both intestinal and allergic lung inflammation was surprising. We have previously reported that acute treatment with fluconazole is protective during DSS colitis in situations where opportunistic fungi have expanded and invaded the intestinal mucosa (); however, it was not known if commensal fungi play a protective or detrimental role under steady-state conditions. The observation that extended pretreatment with antifungals exacerbated disease in both chemically induced and T cell transfer-mediated models of experimental colitis suggests that steady-state fungal populations of the gut directly or indirectly help to maintain healthy intestinal homeostasis. There is a substantial body of literature showing that intestinal bacterial populations can both positively and negatively influence the development and severity of inflammatory bowel disease (). Whether or not the effects we observed following antifungal drug treatment were due to primary alterations in the fungal community or to secondary effects on bacterial populations is unclear. It is interesting to note, however, that antifungal treatment resulted in substantial reduction in lactobacillus populations ( Figure S2 ), which are thought to be protective in the context of both intestinal and allergic airway inflammation ().