Transferable Immunoglobulin A-Coated Odoribacter splanchnicus in Responders to Fecal Microbiota Transplantation for Ulcerative Colitis Limits Colonic Inflammation

Abstract

Background & aims: Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC.

Methods: Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A-coated microbiota (called IgA-seq) was used to define immune-reactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity.

Results: Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3+/RORγt+ regulatory T cells, induction of interleukin (IL) 10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models.

Conclusions: This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the efficacy of microbial therapy for UC. Clinicaltrials.gov ID NCT02516384.

Keywords: Fecal Microbiota Transplant; IgA-Seq; Odoribacter; Ulcerative Colitis.

Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Clinical response to FMT is associated with a core transferable microbiota.

(A) Venn diagram depicting the number of bacterial species unique to a single variable (DON, PRE or WK4) or shared by two, or all three variables. Microbial cut off for prevalence was defined as ≥ 50% and relative abundance as ≥ 0.1%. Heatmap depicting the relative abundance of the 17 species defined by the Venn diagram as being part of the CTM. (B) Boxplot comparing the median relative abundance of the CTM in DON, PRE and WK4 post FMT. **p unique (n = 12) and therefore absent in the NRTM. (F) Proportion of bacterial strains detected on WK4 belonging to the RTMunique (n = 12) shown in figure 1F. Strains detected on WK4 were categorized as derived from DON, PRE, DON and PRE, and WK4. Each dot represents a subject (strain analysis for two patients is missing due to insufficient RTMunique genome coverage) and error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, t test (Tukey adjusted). (G) Paramsothy et al, 2017 cohort is shown (FMT patients n = 54; Δ based on final follow up time point). Boxplots present the median, 25th and 75th percentiles; **p < 0.01, Mann-Whitney test. (H) Receiver operating characteristics (ROC) curve of the Δ relative abundance shown in G. Prediction on Paramsothy cohort yielded an AUC of 0.8 (80% accuracy, sensitivity = 60%, specificity = 80%). Mann-Whitney test P-value is shown.

Figure 2.. IgA-seq identifies donor-derived Odoribacter as an immune reactive bacteria associated with clinical response to FMT.

(A) Boxplot comparing the median percentage of IgA-coated bacteria in fecal homogenate of DON, PRE and WK4 groups. Boxplots present the median, 25th and 75th percentiles. (B) Venn diagram depicting the number of IgA-coated bacterial taxa unique to a single variable (DON, PRE or WK4) or shared by two, or all three variables. Heatmap depicting the relative abundance of the 29 taxa defined by the Venn diagram as being part of the IgA-coated transferable microbiota. (C) Gnotobiotic mouse model experimental design and IgA-sequencing strategy are shown. Germ-free mice, 6–8 weeks old, received fecal transplant from a 2-donor FMP. Fecal homogenate was filtered, blocked, and stained with cell-permeable DNA dye Syto BC and anti-IgA antibody. Samples were gated on the basis of forward scatter (FSC) and side scatter (SSC). (D) IgA coating index (ICI) was calculated for genera found to be differentially abundant between IgA+ and IgA− microbial communities of the humanized mice. (E) Correlation between IgA-coated Odoribacter and Δ Mayo. Linear regression analysis revealed a significant correlation for Δ relative abundance of IgA-coated Odoribacter genus and Δ Mayo.

Figure 3.. O. splanchnicus induces iTreg and limits DSS colitis.

(A) Germ-free C57BL/6 WT mice were colonized with patient-derived A.finegoldii (Alistipes), B. producta (Blautia) and O. splanchnicus (Odori) for seven days and exposed to 2% DSS ad libitum for 6 days. Weight loss and levels of lipocalin in cecal contents are shown. Graphs show data from two independent experiments. Error bars represent SEM; ****p < 0.0001, ANOVA test. Boxplots present the median, 25th and 75th percentiles; *p < 0.05, **p < 0.01, Mann–Whitney test (Tukey adjusted). (B) Flow cytometry of live CD4+ T cells was used to evaluate RORγt and Foxp3 expression in colonic lamina propria 21 days post-colonization. Mean percentages of RORγt, RORγt+Foxp3+, or RORγt+Foxp3+/ RORγt+ CD4+ T cells per colon is shown in germ-free or mice mono-colonized with patient-derived bacteria. Data are from one representative out of four independent experiments. Error bars represent SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t test (Tukey adjusted). (C) Correlation between IgA-coated Odoribacter and Δ Foxp3. Linear regression analysis revealed a significant correlation for Δ IgA-coated Odoribacter genus and Δ Foxp3. Lamina propria mononuclear cells (LPMCs) were isolated from rectal endoscopic biopsies taken pre and 4 weeks after FMT. Flow cytometry of live, CD4+ T cells was used to evaluate Foxp3. (D) Germ-free C57BL/6 WT or RAG1−/− mice were colonized with patient-derived O. splanchnicus for seven days and exposed to 2% DSS ad libitum for 6 days. Weight loss and levels of lipocalin in cecal contents are shown. Graphs show data from two out of three independent experiments. Error bars represent SEM; ****p < 0.0001, anova test. Boxplots present the median, 25th and 75th percentiles; *p < 0.05, **p < 0.01, Mann–Whitney test (Tukey adjusted).

Figure 4.. O. splanchnicus induces IL-10 to limit T cell inflammation and colitis.

(A) Germ-free C57BL/6 mice were colonized with patient-derived O. splanchnicus and Il10 expression in the colonic lamina propria were measured 21 days after colonization. Graph shows data from one out of three independent experiments. Error bars represent the SEM. **p ≤ 0.01, t test. (B - E) Germ-free C57BL/6 WT or IL10−/− mice were colonized with patient-derived O. splanchnicus. Seven days post-colonization, mice were exposed to 2% DSS ad libitum for 6 days. Weight loss (B), and fecal levels of lipocalin (C) are shown. Flow cytometry of live CD4+ T cells was used to evaluate RORγt, Foxp3, RORγt+Foxp3+ (D) and IL17 as well as IFNγ (E) expression. Graphs show data from two independent experiments (WT, n = 8; IL10−/−, n =7). Error bars represent SEM; ****p < 0.0001, ANOVA test. Boxplots present the median, 25th and 75th percentiles; *p < 0.05, **p < 0.01, Mann–Whitney test. Bar plot error bars represent the SEM. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 t test.

Figure 5.. SCFAs production by O. splanchnicus limits colitis.

(A) Germ-free C57BL/6 WT mice were colonized with patient-derived A.finegoldii (Alistipes), B. producta (Blautia) and O. splanchnicus (Odori), respectively. Acetate, butyrate and propionate levels in cecal contents were assessed 10 days post-colonization. Error bars represent SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001, t test (Tukey adjusted). (B-E) SPF GPR43−/−, GPR109a−/− and het littermate controls were colonized with patient-derived O. splanchnicus and then exposed to 2% DSS ad libitum for 6 or 3 (GPR43−/−) days. Weight loss (B-D), percent survival (B-D) and lipocalin in the cecal contents (E) are shown. Graph shows data from one representative experiment (Het control, n = 10; Het Odori n = 7; GPR109a−/− control, n = 6; GPR109a−/− Odori, n = 6; GPR43−/− Odori, n = 8; GPR43−/− Odori, n = 7). Error bars in weight loss graphs represent SEM; ****p < 0.0001, repeated measures ANOVA test. Survival analysis; *p < 0.05, **p < 0.01, log-rank (Mantel-Cox) test. Boxplots present the median, 25th and 75th percentiles, Kruskal-Wallis test.