Experimental colonization with Blastocystis ST4 is associated with protective immune responses and modulation of gut microbiome in a DSS-induced colitis mouse model

Abstract

Background: Blastocystis is a common gut protistan parasite in humans and animals worldwide, but its interrelationship with the host gut microbiota and mucosal immune responses remains poorly understood. Different murine models of Blastocystis colonization were used to examine the effect of a common Blastocystis subtype (ST4) on host gut microbial community and adaptive immune system.

Results: Blastocystis ST4-colonized normal healthy mice and Rag1^-/- mice asymptomatically and was able to alter the microbial community composition, mainly leading to increases in the proportion of Clostridia vadinBB60 group and Lachnospiraceae NK4A136 group, respectively. Blastocystis ST4 colonization promoted T helper 2 (Th2) response defined by interleukin (IL)-5 and IL-13 cytokine production, and T regulatory (Treg) induction from colonic lamina propria in normal healthy mice. Additionally, we observed that Blastocystis ST4 colonization can maintain the stability of bacterial community composition and induce Th2 and Treg immune responses to promote faster recovery from experimentally induced colitis. Furthermore, fecal microbiota transplantation of Blastocystis ST4-altered gut microbiome to colitis mice reduced the severity of colitis, which was associated with increased production of short-chain fat acids (SCFAs) and anti-inflammatory cytokine IL-10.

Conclusions: The data confirm our hypothesis that Blastocystis ST4 is a beneficial commensal, and the beneficial effects of Blastocystis ST4 colonization is mediated through modulating of the host gut bacterial composition, SCFAs production, and Th2 and Treg responses in different murine colonization models.

Keywords: Blastocystis; Colitis; Gut microbiota; Th2; Treg.

Fig. 1

Blastocystis ST4 colonization did not induce any abnormal effects on normal healthy mice. a Experimental design. b Weight changes and disease activity index (DAI) between control and ST4-colonized mice. c Blastocystis ST4 cells per milligram of stool in ST4-clonized mice. d Scanning electron microscopy (SEM) of colonic and cecum tissues from control and ST4-clonized mice, Blastocystis are indicated with red asterisk (*). Scale bar = 1 μm. e Colon length at endpoint. f Representative micrographs of H&E-stained colon sections from control and ST4-colonized mice, and colonic histological scores at day 7. Scale bar = 100 μm

Fig. 2

Blastocystis ST4 colonization alters the murine fecal bacterial community compositions. a Alpha diversity was measured by Shannon, Simpson, and Richness indexes in the fecal samples of control and ST4-colonized mice (n = 4 mice per group). b PCoA of fecal gut microbiota in control and ST4-clonized mice at day 0 and day 7. c Heatmap of ST4 colonization-associated taxonomic markers at day 7. d Bacterial genera (relative abundance in the top 35) showing significant differences in their relative abundance between control and ST4-colonized mice

Fig. 3

Colonization with Blastocystis ST4 induces accumulation of Th2 and Treg cells in the colonic lamina propria (LP). a Colored contour plots show staining for IL-4, IL-5, and IL-13 within CD4⁺ cells. b Bar charts show the percentage of IL-4, IL-5, and IL-13 expressing CD4⁺ T cells. c Colored contour plots show staining for IL-10, IL-17, TNF-α, and IFN-γ within CD4⁺ cells. d Bar charts show the percentage of IL-10, IL-17, TNF-α, and IFN-γ expressing CD4⁺ T cells. Statistical significance is indicated by *p < 0.05, and ***p < 0.001, unpaired Student’s t test

Fig. 4

Blastocystis ST4 colonizes asymptomatically in Rag1^−/− mice. a Experimental design. b Scanning electron microscopy (SEM) of colonic and cecum tissues from control and ST4-clonized mice, Blastocystis are indicated with red asterisk (*). Scale bar = 1 μm. c Representative micrographs of H&E-stained colon sections from ST4-colonized and control mice at day 14. Scale bar = 100 μm. d Colon length and colonic histological scores at endpoint. Blastocystis ST4 cells per milligram of stool in ST4-clonized mice

Fig. 5

Effect of Blastocystis ST4 colonization on fecal bacterial community composition in Rag1^−/− mice. a Alpha diversity was measured by Shannon, Simpson, and Richness indexes (n = 6 in the control group and n = 7 in the ST4-colonized group). b PCoA of fecal gut microbiota in control and ST4-clonized mice at day 0 and day 14. c Heatmap of ST4 colonization-associated taxonomic markers at day 14. d Bacterial genera (relative abundance in the top 35) showing significant differences in their relative abundance between ST4-colonized and control mice

Fig. 6

Blastocystis ST4 colonization contributes to mice recovery from experimentally induced colitis. a Experimental design. b Blastocystis ST4 cells per milligram of stool in ST4-clonized mice. c Weight changes and DAI between control and ST4-colonized mice during treatment. d Representative micrographs of H&E-stained colon sections from control and ST4-colonized mice at day 21. Colon length and colonic histological at end point. Scale bar = 100 μm. *p < 0.05, unpaired Student’s t test

Fig. 7

Improvement of experimentally induced colitis is associated with gut microbiota. a Alpha diversity was measured by Shannon, Simpson, and Richness indexes (n = 4 per group). b PCoA of fecal gut microbiota in control and ST4-clonized mice at day 0 and day 21. c Heatmap of ST4 colonization-associated taxonomic markers. d Bacterial genera showing significant differences in their relative abundance between ST4-colonized and control mice

Fig. 8

Blastocystis ST4 colonization activates Th2 and Treg cells responses in colonic mucosa of DSS-induced colitis mice. a Colored contour plots show staining for IL-4, IL-5, and IL-13 within CD4⁺ cells. b Bar charts show the percentage of IL-4, IL-5, and IL-13 expressing CD4⁺ T cells. c Colored contour plots show staining for IL-10, IL-17, TNF-α, and IFN-γ within CD4⁺ cells. d Bar charts show the percentage of IL-10, IL-17, TNF-α, and IFN-γ expressing CD4⁺ T cells. Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001, unpaired Student’s t test

Fig. 9

Transfer of ST4-altered microbiome to colitis mice reduces colonic inflammation. a Experimental design. b Blastocystis ST4 cells per milligram of stool in DSS^{FMT + ST4−clonized} mice. c Weight changes and DAI between DSS^{FMT + Control} mice and DSS^{FMT + ST4−clonized} mice. d Scanning electron microscopy (SEM) of colonic and cecum tissues from DSS^{FMT + Control} mice and DSS^{FMT + ST4−clonized} mice, Blastocystis are indicated with red asterisk (*). Scale bar = 1 μm. e Representative micrographs of H&E-stained colon sections from DSS^{FMT + Control} mice and DSS^{FMT + ST4−clonized} mice at day 17. Scale bar = 100 μm. Colon length and colonic histological scores at end point

Fig. 10

Gut microbiota analysis upon different FMT conditions treatment in DSS-induced colitis mice. a Alpha diversity was measured by Shannon, Simpson, and Richness indexes (n = 4 in control group and n = 5 in ST4-colonized group). b PCoA of fecal gut microbiota in control and ST4-clonized mice at day 0 and day 17. c Heatmap of taxonomic markers among different groups. d Bacterial genera showing significant differences in their relative abundance among groups

Fig. 11

FMT influences SCFAs and Treg cells IL-10 production in DSS-induced colitis recipients. a Fold change of each SCFA relative to levels at day 0 from DSS^FMT mice (recipients). b Colored contour plots show staining for IL-10 within CD4⁺ cells, and bar chart shows the percentage of IL-10 expressing CD4⁺ T cells. c Colored contour plots show staining for TNF-α within CD4⁺ cells, and bar chart shows the percentage of TNF-α expressing CD4⁺ T cells. *p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA (a) and one-way ANOVA (b, c) analysis with Tukey multiple comparison test