Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii served as key components of fecal microbiota transplantation to alleviate colitis

Abstract

Fecal microbiota transplantation (FMT) is a promising therapy for inflammatory bowel disease (IBD) via rectifying gut microbiota. The aim of this study was to identify a mechanism of how specific bacteria-associated immune response contributes to alleviated colitis. Forty donors were divided into high (donor H) and low (donor L) groups according to the diversity and the abundance of Bacteroides and Faecalibacterium by 16S rRNA sequencing. FMT was performed on dextran sulfate sodium (DSS)-induced colitis in mice. Mice with colitis showed significant improvement in intestinal injury and immune imbalance after FMT with group donor H (P < 0.05). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii were identified as targeted strains in donor feces by real-time PCR and droplet digital PCR. Mice with colitis were treated with mono- or dual-bacterial gavage therapy. Dual-bacterial therapy significantly ameliorated intestinal injury compared with mono-bacterial therapy (P < 0.05). Dual-bacterial therapy increased the M2/M1 macrophage polarization and improved the Th17/Treg imbalance and elevated IL-10 production by Tregs compared with the DSS group (P < 0.05). Metabolomics showed increased abundance of lecithin in the glycerophospholipid metabolism pathway. In conclusion, B. thetaiotaomicron and F. prausnitzii, as the key bacteria in donor feces, alleviate colitis in mice. The mechanism may involve increasing lecithin and regulating IL-10 production of intestinal Tregs.NEW & NOTEWORTHY We demonstrate that donors with high abundance of Bacteroides and Faecalibacterium ameliorate dextran sulfate sodium (DSS)-induced colitis in mice by fecal microbiota transplantation (FMT). The combination therapy of Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii is superior to mono-bacterial therapy in ameliorating colitis in mice, of which mechanism may involve promoting lecithin and inducing IL-10 production of intestinal Tregs.

Keywords: Bacteroides thetaiotaomicron; Faecalibacterium prausnitzii; fecal microbiota transplantation; gut microbiota; inflammatory bowel disease.

Graphical abstract

Figure 1.

Donors with different bacterial abundance ameliorate colitis differently by FMT. A: taxonomic composition distribution on genus-level of donor fecal microbiota in group donor H and group donor L. B: α-diversity (Shannon index and Simpson index) of donor fecal microbiota in group donor H and group donor L. C: principal coordination analysis (PCoA) based on OTU abundance of donor fecal microbiota in group donor H and group donor L. D: mice (n = 6–8/group) were administered 2.5% DSS in drinking water for 7 days and then were transplanted daily with fresh feces (0.1 mL/10 g/day) from different donors for 5 days. Daily changes in body weight (E) and disease activity index (DAI) (F) in different groups. G: macroscopic observation of colons. H: histopathological changes of colonic samples observed by H&E staining. Photomicrographs of Claudin-1 (I) and ZO-1 immunofluorescence (J) in the colon (×200 magnification). Data are provided as means ± SE. In B, *P < 0.05 and ***P < 0.001. In E–J, *means compared with the CON group; #means compared with the DSS group. When comparing between group H and group L, $means compared with L1; &means compared with L2; † means compared with L3. *, #, and †P < 0.05; **, ##, and $$P < 0.01; ***, ###, &&&, and †††P < 0.001. DSS, dextran sulfate sodium; FMT, fecal microbiota transplantation; H&E, hematoxylin-eosin.

Figure 2.

Changes of colonic cytokines and immune cells in colitis with FMT therapy from donors after different bacterial abundance. mRNA expression levels of colonic TNF-α (A), IL-1β (B), and IL-6 (C). mRNA expression levels of colonic RORγ-t (D), iNOS (E), Ly6G (F), T-bet (G), Foxp3 (H), GATA3 (I). Data are provided as means ± SE (n = 6–8/group). *Means compared with the CON group; #means compared with the DSS group. When comparing between group H and group L, $means compared with L1; & means compared with L2; †means compared with L3. *, #, &P < 0.05; **, ##, &&, and ††P < 0.01; ***, ###, $$$, &&&, and †††P < 0.001. DSS, dextran sulfate sodium; FMT, fecal microbiota transplantation; Foxp3, forkhead box protein P3; GATA3, GATA binding protein 3; iNOS, inducible nitric oxide synthase; Ly6G, lymphocyte antigen 6 G; RORγ-t, retinoid-related orphan nuclear receptor γ-t; T-bet, T-box expressed in T cell; TNF-α, tumor necrosis factor-α.

Figure 3.

B. thetaiotaomicron and F. prausnitzii are the key differentiating bacteria in the two groups of fecal microbes. Relative quantitative levels of B. ovatus (A), B. thetaiotaomicron (B), B. stercoris (C), B. uniformis (D), B. eggerthii (E), B. caccae (F), and B. vulgatus (G) in donor feces. H: absolute quantitative levels of B. thetaiotaomicron in donor feces by droplet digital PCR (ddPCR). I: relative quantitative levels of F. prausnitzii. J: absolute quantitative levels of F. prausnitzii in donor feces by ddPCR. Data are provided as means ± SE (n = 12/group). ns, not significant; **P < 0.01 and ***P < 0.001.

Figure 4.

Dual-bacterial therapy is protective against DSS-induced colitis. A: schematic of therapeutic administration of bacteria in DSS-induced colitis model. Daily changes in body weight (B), disease activity index (DAI) (C), and survival in different groups (D). E: histopathological changes of colonic samples observed by H&E staining and pathological scores of colons. F: macroscopic observation of colons and the lengths of colon from each group. G and H: photomicrographs of claudin-1 (G) and ZO-1 immunofluorescence(H) in the colon (×200 magnification). Data are provided as means ± SE (n = 6–13/group). *Means compared with the CON group; #means compared with the DSS group; †means compared with the DSS+Bt+Fp group. # and †P < 0.05; ** and ##P < 0.01; ***, ###, and †††P < 0.001. DSS, dextran sulfate sodium; H&E, hematoxylin-eosin.

Figure 5.

Dual-bacterial therapy modulates the levels of inflammation in colitis. mRNA expression levels of colonic IL-1β (A), IL-6 (B), TNF-α (C), IL-10 (D), TGF-β (E), IL-19 (F), IL-20 (G), IL-22 (H), IL-24 (I), and IL-28 (J). Data are provided as means ± SE (n = 6–13/group). *Means compared with the CON group; #means compared with the DSS group; †means compared with the DSS+Bt+Fp group. *, #, and †P < 0.05; ** and ††P < 0.01; *** and ###P < 0.001. DSS, dextran sulfate sodium; TNF-α, tumor necrosis factor-α.

Figure 6.

Dual-bacterial therapy modulates immune cells in the lamina propria of the colon. A–G: flow cytometry analysis of immune cells in the lamina propria of the colon. Monocyte (A); macrophage (B); dendritic cell (C); regulatory T cell (D); T helper 17 cell (E); ratio of Treg to Th17 (F); M2-macrophages and M1-macrophages and their ratios (G). Data are provided as means ± SE (n = 6/group). *Means compared with the CON group; #means compared with the DSS group; †means compared with the DSS+Bt+Fp group. *, #, and †P < 0.05; ** and ##P < 0.01; *** and ###P < 0.001. DSS, dextran sulfate sodium.

Figure 7.

Dual-bacterial therapy promotes the release of IL-10 from Treg. A and B: flow cytometry analysis of immune cells in the lamina propria of the colon. IL-10 secreting Treg (A); IL-10 secreting macrophages (B). Data are provided as means ± SE (n = 6/group). *Means compared with the CON group; #means compared with the DSS group; †means compared with the DSS+Bt+Fp group. *, #, and †P < 0.05; **P < 0.01; *** and ###P < 0.001. DSS, dextran sulfate sodium.

Figure 8.

Metabolomic data profiles and pathway enrichment analysis. A: orthogonal partial least squares-discrimination analysis (OPLS-DA) for DSS+Bt+Fp vs. DSS, DSS+Bt vs. DSS, DSS+Fp vs. DSS. Volcano plot of changed metabolites for DSS+Bt+Fp vs. DSS (B), DSS+Bt vs. DSS (C), DSS+Fp vs. DSS (D). Kegg pathway enrichment analysis of changed metabolites for DSS+Bt+Fp vs. DSS (E), DSS+Bt vs. DSS (F), DSS+Fp vs. DSS (G). Altered metabolites in glycerophospholipid metabolic pathways in DSS+Bt+Fp vs. DSS (H) and DSS+Fp vs. DSS (I). Data are provided as means ± SE (n = 5/group). DSS, dextran sulfate sodium.