Defined microbiota transplant restores Th17/RORγt+ regulatory T cell balance in mice colonized with inflammatory bowel disease microbiotas

Significance Composition of gut microbiota is altered in many human diseases, including inflammatory bowel disease. Some hope that restoring microbiota to a healthy state could help treat such diseases. We have used mice colonized with microbiotas from humans with inflammatory bowel disease to study what happens when these mice receive a microbiota transplant from a set of healthy humans. We find that the mouse gut immune system is changed by microbiota transplants, becoming broadly less inflammatory and protecting mice from colitis. By culturing bacteria from these microbiotas, we identify one strain that induces inflammatory responses in mice and show that it is modified by microbiota transplant. We also show that increases in the density of microbiota following transplant may be antiinflammatory.


Fig S1. Modulation of mucosal Th17 cells by defined microbiota transplant. (A)
The proportion of RORgt + Th17 cells (of live, CD4 + FoxP3 -cells) in the ileum lamina propria of gnotobiotic mice colonized with the five HD-derived microbiotas used as DMT donors. Red lines indicate the proportion of Th17 cells induced by each HD alone. Plots show the mean and standard error of each indicated group of mice. *p<0.05, **p<0.01, ****p<0.0001 by ANOVA with Tukey correction.

Fig S2. Identification of a Th17-inducing strain from a donor with Crohn's disease. (A)
FDR-adjusted one-tailed p values for the positive association of each strain from defined microbiota IBD A with the proportion of colon IL-17A + CD4 T cells derived from the in vivo gnotobiotic screen described in Figure 3. Data is combined from 2 independent experiments. (B) The proportion of RORgt + Treg cells in colon lamina proporia of mice colonized with communities in which E. coli strain A6 was present (+) or absent (-). (C) The proportion of IL-17A + CD4 T cells induced in the colon lamina propria of mice colonized with a synthetic community of seven commensals (see methods for details) with or without the addition of E. coli A6. Each point represents data from one mouse, bars indicate the mean +/-SEM of each group. Data in A and B combined from 2 independent experiments. (D) The ability of E. coli A6 and E2 to adhere to and invade Caco-2 cells, persist in J774 macrophage cells and migrate on soft agar relative to a negative control strain (DH5a) or positive control strain (LF82 or 541-15). (E) Vb chain usage by CD4 + T cells from mLN and colon and small intestine lamina propria of mice colonized with IBD A or an SFB-containing SPF microbiota. Each bar shows data from one mouse. P value in (B) and (C) calculated by unpaired t-test and in (D) by ANOVA with Tukey correction. The correlation between the absolute abundance of E. coli strain A6 and the proportion of RORgt + Th17 cells in mice colonized with IBD A alone, or following transplant with each of the five HD microbiotas. *p<0.05, **p<0.01, ***p<0.001 as calculated by ANOVA with Tukey correction comparing the relative abundance of the strain before transplant and at each timepoint after transplant in (A) and by f-test in (B).

Fig S4. Modulation of FoxP3 + Treg cells and mucosal dendritic cell phenotype following microbiota transplant. (A-B)
The proportion of colon and ileum lamina propria total FoxP3 + Treg cells (of live, CD4 + cells) in groups of mice colonized with each IBD donor alone or three weeks following DMT with one of the five HD microbiotas. Red lines indicate the proportion of FoxP3 + Treg cells induced by each HD alone. (C) The expression of CD86 on total dendritic cells (MHC-II + , CD64 -, CD11c + ) and double-positive dendritic cells (MHC-II + , CD64 -, CD11c +, CD11b + , CD103 + ) in colon of mice colonized with donor IBD A or HD 1 alone, or following microbiota transplant with HD 1. (D-E) The association between the proportion of the indicated cell types in mice colonized with a HD microbiota alone, or in mice receiving the HD microbiota as a transplant following with one of three IBD microbiotas. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by ANOVA with Tukey correction (A-C) or f-test (E and E), ns -not significant.

Fig S5. The association of microbiota diversity and density with mucosal RORgt + Treg cells. (A)
The association between fecal microbiota density and the proportion of mucosal FoxP3 + Treg cells in mice colonized with HD and IBD donor microbiotas alone or following DMT.
(B-C) The association between fecal microbiota alpha diversity and the proportion of mucosal RORgt + Treg and FoxP3 + Treg cells in mice colonized with HD and IBD donor microbiotas alone or following DMT. (D) The association between fecal microbiota density and the proportion of mucosal RORgt + Th17 cells in mice colonized with HD and IBD donor microbiotas alone or following DMT. (E) The association between fecal microbiota density and the proportion of mucosal RORgt + Treg cells in specific pathogen free mice treated with the indicated antibiotic in drinking water. P values calculated by by f-test.