Dysbiosis of gut fungal microbiota is associated with mucosal inflammation in Crohn's disease

Abstract

Goals: We aim to characterize the fungal microbiota in the intestinal mucosa and feces in patients with Crohn's disease (CD).

Background: Fungi represent a diverse microbial community in the human intestine and might play a role in the pathogenesis of CD; however, little is known about the structure and composition of the fungal microbiota especially adhering to the intestinal mucosa in CD patient.

Study: Nineteen patients with active CD and 7 healthy individuals were recruited in this study. The mucosa-associated and fecal fungal microbiotas in CD patients were analyzed using culture-independent community fingerprint techniques.

Results: The fungal richness and diversity were significantly elevated in the inflamed mucosa compared with the noninflamed mucosa. The predominant fungal composition in the inflamed mucosa was strikingly altered, mainly characterized by expansion in the proportions of Candida spp., Gibberella moniliformis, Alternaria brassicicola, and Cryptococcus neoformans. The fecal fungal community was perturbed in CD patients as accompanied by increased fungal diversity and prevalence in Candida albicans, Aspergillus clavatus, and C. neoformans. The species richness and diversity of the mucosal fungal community were associated with the expression of TNF-α, IFN-γ, or IL-10 (P<0.05). The diversity of the fecal fungal microbiota positively correlated with serum C-reactive protein and CD activity index (P<0.05).

Conclusions: This study first demonstrates that the fungal microbiota in the inflamed mucosa is distinguishable from that of the noninflamed area. Shifts of gut fungal microbiota composition may be associated with mucosal inflammation and disease activity of CD. Our data would provide novel insights into understanding the potential of gut fungal microbiota in the pathogenesis of CD.

FIGURE 1

Comparison of the fungal communities in the inflamed and noninflamed mucosa. A, Molecular fingerprinting of the mucosa-associated fungal populations. I indicates inflamed ileal mucosa; N, noninflamed specimen. The figures represent the number of the patients. The similarity coefficient of the fungal microbiota between the inflamed and noninflamed mucosa is shown in the down regions of the panel. B, Clustering analysis of mucosal DGGE profiles from CD patients. The similarities between mucosal specimens are shown in the dendrogram. C, PCA plot of the mucosal fungal flora in CD patients. The plot demonstrated the difference of fungal community composition in the inflamed and noninflamed regions. The percentage of variation explained by each principal component is shown in brackets. D, Biodiversity of mucosal fungal populations from the inflamed and noninflamed ileum. The mean values and SD (bars) are shown, *P<0.05. E, Pie charts showing proportion of the predominant fungi in each class (top) and species (bottom) for the noninflamed and inflamed mucosa. F, The frequencies of fungal species presented in the noninflamed and inflamed regions.

FIGURE 2

The variability of intestinal fungal communities as determined by DGGE analysis of samples from the feces. A, Representative DGGE profiles of the fecal samples from CD patients and healthy controls. B, Dendrogram generated from the fungal community fingerprints of the feces, which was conducted with cluster analysis by the unweighted pair group method using arithmetic averages. C, Principal component analysis of the DGGE data for the fecal fungal community compositions from CD patients and controls. Each circle is representative of a single sample and shaded according to the relative abundance of DGGE bands. The plot shows different fungal community composition in the feces from the patients and controls. The percentage of variation explained by each principal component is shown in brackets. D, The diversity of fungal communities revealed by the numbers of DGGE bands and Shannon-Wiener diversity indices. For each group, the mean value and SD (bars) are shown, **P<0.01. E, Visualization of taxonomic levels. Pie charts showing proportion of fecal predominant fungi in each class (top) and species (bottom) for the controls and the CD. F, The incidences of fungal species presented in the fecal samples from the controls and CD patients. CD indicates Crohn’s disease; H, healthy control.

FIGURE 3

Analysis of T-cell subsets in the intestinal mucosal tissues. A, Representative histograms of flow cytometry showing dynamics of percentage of CD4⁺CD3⁺ and CD8⁺CD3⁺. B, The proportions of CD4⁺CD3⁺ and CD8⁺CD3⁺ in the inflamed and noninflamed mucosa. Data are presented as mean±SD.

FIGURE 4

Expression of cytokines in ileal mucosa by immunofluorescent staining. A, Representative images of cytokine expression in the inflamed and noninflamed mucosa. Longitudinal visualization of TNF-α, IFN-γ, and IL-10 production (red) was performed by confocal laser scanning microscopy. Nuclei were stained with DAPI (blue). B, The intensities of TNF-α, IFN-γ, and IL-10 expression per HPF (magnification, ×200) were measured by Image-Pro Plus 6.0. Data are presented as mean±SD.