Colonization with two different Blastocystis subtypes in DSS-induced colitis mice is associated with strikingly different microbiome and pathological features

Abstract

Rationale: The gut microbiota plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). However, the role of Blastocystis infection and Blastocystis-altered gut microbiota in the development of inflammatory diseases and their underlying mechanisms are not well understood. Methods: We investigated the effect of Blastocystis ST4 and ST7 infection on the intestinal microbiota, metabolism, and host immune responses, and then explored the role of Blastocystis-altered gut microbiome in the development of dextran sulfate sodium (DSS)-induced colitis in mice. Results: This study showed that prior colonization with ST4 conferred protection from DSS-induced colitis through elevating the abundance of beneficial bacteria, short-chain fatty acid (SCFA) production and the proportion of Foxp3⁺ and IL-10-producing CD4⁺ T cells. Conversely, prior ST7 infection exacerbated the severity of colitis by increasing the proportion of pathogenic bacteria and inducing pro-inflammatory IL-17A and TNF-α-producing CD4⁺ T cells. Furthermore, transplantation of ST4- and ST7-altered microbiota resulted in similar phenotypes. Conclusions: Our data showed that ST4 and ST7 infection exert strikingly differential effects on the gut microbiota, and these could influence the susceptibility to colitis. ST4 colonization prevented DSS-induced colitis in mice and may be considered as a novel therapeutic strategy against immunological diseases in the future, while ST7 infection is a potential risk factor for the development of experimentally induced colitis that warrants attention.

Keywords: Blastocystis; DSS-induced colitis; Gut microbiota; IBD; Short-chain fatty acids.

Figure 1

Blastocystis ST4 and ST7 infection caused differential gut microbial signatures. (A) Alpha diversity was measured by Shannon and Chao1 indices (n = 6). (B) Principal co-ordinates analysis (PCoA) based on Bray-Curtis dissimilarities of fecal gut microbiota derived from control (green dots), ST4 (blue dots), and ST7 (red dots) mice at day 0 and day 14. (C) Relative abundance of the different taxa at the class level. (D) Heatmap showing Blastocystis ST4 and ST7-associated taxonomic markers at day 14. (E) LefSe analysis showed the differentially abundant bacterial taxa between ST4 and ST7 groups relative to the baseline. Data are representative of two independent experiments and shown as the mean ± SEM.

Figure 2

Blastocystis ST4 and ST7 infection influence SCFAs production and colonic T helper cells. (A) The concentration of acetic, propionic, butyric, and isobutyric in control, ST4, and ST7 infected mice. (B) The concentration of valeric acid, isovaleric, 2-methylbutyric, and caproic acid, and 4-methylvaleric acid. (C) Zebra plots show staining for IL-4, IL-17A, TNF-α, IFN-γ and IL-10 within CD4⁺ T cells. (D) Bar charts show the percentage of IL-4, IL-17A, TNF-α, IFN-γ and IL-10 expressing CD4⁺ T cells. One-way ANOVA p-values adjusted for Tukey's multiple comparisons are shown. Data are representative of two independent experiments and shown as the mean ± SEM.

Figure 3

The effects of Blastocystis ST4 and ST7 colonization on subsequent DSS challenge. (A) Study design. (B) SEM of colonic and cecum tissues from control, ST4, and ST7 infected mice (upper panel), and Blastocystis are indicated with red asterisk (Scale bar = 1 μm). H&E-stained colon sections (lower panel) from the three groups (Scale bar = 100 μm). Weight changes (C), and DAI (D) in control, ST4, and ST7 infected mice. (E) The number of Blastocystis cells in ST4 and ST7 infected mice. Colon length (F) and histological scores (G) in the three groups. Data are representative of two independent experiments and shown as the mean ± SEM. Two-way ANOVA with Dunnett's multiple comparison testing were used for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Gene expression in ST4 and ST7-infected colon tissues after DSS treatment. (A) Principal component analysis (PCA) plot comparing mouse transcriptomes among control, ST4, and ST7 infected mice. (B) Volcano plots showing log2 fold change plotted against log of mean normalized expression counts. (C) The top 10 most enriched GO terms found in the analysis of DEGs in ST4 vs. Control group, and ST7 vs. Control group. GO terms were ranked by their significance. (D) GSEA analysis showed the gene sets of inflammatory bowel disease, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, and Th17 cell differentiation. Graphs depict the enrichment score (y axis) with negative values where gene sets are inhibited, and positive values where they are induced. Each vertical bar on the x axis represents an individual gene within the gene set, and its relative ranking against all genes analyzed. P and Padj value is indicated on the graph.

Figure 5

Colonic immune profiles in control, ST4, and ST7 infected mice after treatment with DSS. (A) Zebra plots and bar charts show staining and the percentage of IL-4, and IL-17A within CD4⁺ T cells. (B) Zebra plots and bar charts show staining and the percentage of TNF-α, and IFN-γ within CD4⁺ T cells. Zebra plots and bar charts show staining and the percentage of IL-10 (C) and Foxp3 (D) within CD4⁺ T cells. Data are representative of two independent experiments and shown as the mean ± SEM. One-way ANOVA p-values adjusted for Tukey's multiple comparisons are shown.

Figure 6

Effects of Blastocystis-altered gut microbiota on the severity of experimental colitis. (A) Study design. Weight changes (B) and DAI (C) in control, ST4, and ST7 altered microbiota recipient mice. (D) H&E-stained colon sections from the three groups (Scale bar = 100 μm). (E) colon length and histological scores in the three groups. (F) The concentration of acetic, propionic, butyric, and isobutyric, valeric acid, isovaleric, 2-methylbutyric, and caproic acid, and 4-methylvaleric acid in the recipient mice. Data are representative of two independent experiments and shown as the mean ± SEM. One-way ANOVA p-values adjusted for Tukey's multiple comparisons are shown (E, F). Two-way ANOVA with Dunnett's multiple comparison testing were used for multiple comparisons (B, C). *p < 0.05; **p < 0.01.

Figure 7

Colonic immune profiles from recipient mice. (A) Zebra plots and bar charts show staining and the percentage of IL-4, and IL-17A within CD4⁺ T cells. (B) Zebra plots and bar charts show staining and the percentage of TNF-α, and IFN-γ within CD4⁺ T cells. Zebra plots and bar charts show staining and the percentage of IL-10 (C) and Foxp3 (D) within CD4⁺ T cells. Data are representative of two independent experiments and shown as the mean ± SEM. One-way ANOVA p-values adjusted for Tukey's multiple comparisons are shown.