Rationally designed bacterial consortia to treat chronic immune-mediated colitis and restore intestinal homeostasis

Abstract

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.

Fig. 1. Effect of gavage with GUT-103 on the gut microbiome community composition, lipocalin-2 levels, histology of the gut epithelium, SCFA synthesis and tissue cytokine expression of Il10^−/− mice where experimental colitis was induced by inoculation with Escherichia coli LF82 (E), Enterococcus faecalis OG1RF (E) and Ruminococcus gnavus ATCC29149 (R).

a Schematic overview of experimental design. Gnotobiotic Il10^−/− mice were inoculated with GUT-103 (N = 5), EER (N = 9), GUT-103 plus EER (preventive protocol) (N-7), and EER plus GUT-103 (therapeutic protocol) (N = 7). After 5 or 6 weeks, animals were killed and tissue samples were collected for analysis; b Composition of the microbiome in fecal pellets of gnotobiotic 129 Il10^−/− mice inoculated with EER, GUT-103 plus EER (preventive protocol), and EER plus GUT-103 (therapeutic protocol) determined by qPCR. Il10^−/− mice were inoculated via gavage with GUT-103 (2 × 10⁺⁷ cfu/strain) or EER (2 × 10⁺⁷ cfu/strain). The composition of the gut microbiome was determined on fecal pellets or after sacrifice on cecal contents using qPCR with species-specific primers. The average community composition (pie diagrams) for seven animals per treatment are presented. Strain legend: A1: Megamonas hypermegale DSM1672; A2 Bacteroides stercoris DSM19555; A3: Anaerostipes hadrus DSM3319; A4: Clostridium symbiosum ATCC14940; A5: Clostridium boltea ATCC BAA-613; A6: Blautia producta DSM2950; A7: Clostridium scindens ATCC35704; A8: Akkermansia muciniphila ATCC BAA-835; A9: Megamonas funiformis DSM19343; A10: Acidaminococcus intestini DSM21505; A11: Bacteroides massiliensis DSM17679; A12: Barnesiella intestinihominis DSM21032; A13: Faecalibacterium prausnitzii DSM17677; A14: Subdoligranulum variabile DSM15176; A15: Anaerostipes caccae DSM14662; A16: Blautia hydrogenotrophica DSM10507; A17: Marvinbryantia formatexigens DSM14469. c Cecal content lipocalin-2 levels at time of sacrifice from GUT-103 group (N = 5), EER group (N = 9), GUT-103 plus EER group (N = 9), and EER plus GUT-103 group (N = 8), Mann–Whitney unpaired two-tailed t-test. Mean ± SE, *P < 0.05. d Representative histology pictures of H&E-stained cecum tissue (×100). The bar (upper right corner) in the pictures indicates 100 µm from GUT-103 group (experiment was repeated five times independently with similar results), EER group (experiment was repeated nine times independently with similar results), GUT-103 plus EER group (experiment was repeated eight times independently with similar results), and EER plus GUT-103 group (experiment was repeated eight times independently with similar results). e Histology scores of GUT-103 group (N = 5), EER group (N = 9), GUT-103 plus EER group (N = 8), and EER plus GUT-103 group (N = 8). Combined score of cecum, proximal colon, and distal colon/rectum. 1-way ANOVA, Mean ± SE, *P < 0.05, **P < 0.01. f Fecal metabolite profiles for butyrate and propionate in Il10^−/− mice inoculated with GUT-103 (N = 5), GUT-103 plus EER (preventive protocol, N = 8), and EER plus GUT-103 (therapeutic protocol, N = 8) compared to EER (N = 8). Mann–Whitney unpaired two-tailed t-test. ±SE, **P < 0.01, ****P < 0.001. g Cecal tissue levels of cytokine mRNA expression of GUT-103 (N = 5), EER (N = 8), GUT-103 plus EER (preventive protocol, N = 8), and EER plus GUT-103 (therapeutic protocol, N = 8). Samples were collected at necropsy. One-way ANOVA, Mean ± SE, *P < 0.05, **P < 0.01. Source data and calculated P-values are provided as a Source Data file.

Fig. 2. Levels of unconjugated and conjugated bile acids in the cecal content of Il10^−/− mice at the time of sacrifice where experimental colitis was induced by inoculation with Escherichia coli LF82 (E), Enterococcus faecalis OG1RF (E), and Ruminococcus gnavus ATCC29149 (R).

Gnotobiotic Il10^−/− mice were inoculated with GUT-103 (N = 5), EER (N = 9), GUT-103 plus EER (preventive protocol) (N = 7), and EER plus GUT-103 (therapeutic protocol) (N = 7). Il10^−/− mice were inoculated via gavage with GUT-103 (2 × 10⁺⁷ cfu/strain) or EER (2 × 10⁺⁷ cfu/strain). a Unconjugated bile acids: αMCA α-muricholic acid, βMCA β-muricholic acid, CA cholic acid, CDCA chenodeoxycholic acid, LCA lithocholic acid, UDCA ursodeoxycholic acid, DCA deoxycholic acid. b Conjugated bile acids: TβMCA taurine-conjugated β-muricholic acid, TCA taurocholic acid, TCDCA taurochenodeoxycholic acid, TUDCA tauroursodeoxycholic acid, GUDCA glycoursodeoxycholic acid, GCA glycine-conjugated cholic acid, GLCA glycine-conjugated lithocholic acid. 1e-way ANOVA, Mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. Source data and calculated P-values are provided as a Source Data file.

Fig. 3. Effect on the gut microbiome community composition, colonic histology, and fecal metabolomics of gnotobiotic mice inoculated with GUT-108 or treated with PBS for 2 weeks.

Il10⁺/^eGFP VertX reporter mice (a) or Il10^−/− mice (b) were inoculated via gavage with GUT-108 (2 × 10⁺⁷ cfu/strain), and after 2 weeks community composition of the gut microbiome was determined on cecal contents using qPCR with species-specific primers. The average community composition (circle diagram) as well as the individual community composition for six Il10⁺/^eGFP VertX reporter mice and five Il10^−/− mice are presented. The lower detection limit for qPCR was >0.0001%. c Representative distal colonic photomicrographs of H&E-stained tissue showing the lack of colonic inflammation two weeks after inoculation of gnotobiotic Il10^−/− mice with GUT-108 or PBS. The experiment was repeated independently with similar results for each animal in the GUT-108 (N = 11) or PBS (N = 8) group. The bar (upper right corner) in the pictures indicates 100 µm. d Histological scoring showing the lack of colonic inflammation 2 weeks after inoculation of gnotobiotic Il10⁺/^eGFP VertX reporter mice or Il10^−/− mice with GUT-108: combined score of cecum, proximal colon, and distal colon/rectum. Two-Way ANOVA with Sidak’s multiple comparison test. NS not significant. N = 5–6/group. e Metabolite analysis of fecal material from Il10^−/− mice inoculated with GUT-108 to confirm the successful restoration of bacterial synthesis of acetate, propionate, butyrate and IAA. Two-Way ANOVA with Sidak’s multiple comparison test. Bar indicates mean ± SE. ***P < 0.001, ****P < 0.0005, N.S. not significant. N = 8/group or N = 11/group, for PBS and GUT-108 treatment, respectively. f Colonization of gnotobiotic Il10⁺/^eGFP reporter mice with GUT-108 for 2 weeks induced IL-10-producing T and B cells, dendritic cells (DC) and macrophages (Mf) (top panel) and different types of IL-10⁺ regulatory T cells (lower panel⁾. GUT-108 refers to Il10⁺/^eGFP mice treated with GUT-108 (N = 11); PBS refers to Il10⁺/^eGFP mice treated with PBS buffer (N = 8). Dots represent individual mouse results. Bars indicate mean. Mann–Whitney unpaired two-tailed t-test. Mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. N = 8 for PBS, N = 11 for GUT-108. Source data and calculated P-values are provided as a Source Data file.

Fig. 4. GUT-108 strains decreased colitis in Il10^−/− mice colonized with human fecal microbiota.

a Schematic overview of experimental design. On day 1, mice were inoculated with a human stool previously verified to induce aggressive colitis in gnotobiotic Il10^−/− mice. Therapeutic application of GUT-108 or PBS control started after 2 weeks. After 4 weeks necropsies were performed, and tissue samples were collected for analysis. b Bacterial species whose abundance significantly changed in the gut microbiome of mice that received therapeutic application of GUT-108 compared to PBS control mice. Differences in relative species abundance between the two treatments is expressed on a Log10 scale. Strains with decreased (red) or increased (blue) relative abundance are indicated. GUT-108 strains are indicated in black. *P < 0.05 value using the two-sided Benjamini–Hochberg corrected Wilcoxon test. c Serial fecal lipocalin-2 levels were measured using ELISA after 2 (before start of GUT-108 or PBS therapy), 3 and 4 weeks. Dots indicate individual mice data. Mann–Whitney unpaired two-tailed t-test. Bars indicate Mean ± SE, *P < 0.05, ***P < 0.001, N.S. indicates not significant. N = 11/group. d Blinded histologic scoring assessed the level of inflammation for different parts of the intestine, including the distal ileum (Ile), cecum (Ce), proximal- (Pc) and distal- (Dc) colon, rectum (Re), and combined (Ce + Pc + Dc + Re). Dots indicate individual mice data. Mann–Whitney unpaired two-tailed t-test. Bars indicate Mean ± SE. Unpaired two-tailed t-test was performed on the data. *P < 0.05, ***P < 0.001, NS not significant. N = 11/group. e Representative distal colonic photomicrographs of H&E-stained tissue showing colonic tissues 2 weeks after starting therapeutic treatment of humanized Il10^−/− mice with GUT-108 or PBS. The experiment was repeated independently with similar results for each animal in the GUT-108 (N = 11) or PBS (N = 11) group. The bar in the picture indicates 100 µm; Hu + GUT-108 refers to humanized Il10^−/− mice treated with GUT-108; Hu + PBS refers to humanized Il10^−/− mice that received PBS as a placebo control. f Induction of protective fecal metabolites by therapeutic GUT-108 treatment beginning at 2 weeks after the induction of inflammation by human stool. Week 4 represents 2 weeks of therapeutic treatment. Hu+GUT-108 refers to humanized Il10^−/− mice treated with GUT-108; Hu + PBS refers to humanized Il10^−/− mice that received PBS buffer. DCA deoxycholic acid, LCA lithocholic acid, IPA indole-3-propionic acid. Two-way ANOVA and adjusted P-values were calculated by the multiple comparisons test. Bar indicates Mean ± SE. N.S. not significant; *: adjusted P < 0.05; **: adjusted P < 0.01; ***: adjusted P < 0.001. Source data and calculated P-values are provided as a Source Data file.

Fig. 5. Effects of treatment with GUT-108 on the levels of inflammatory biomarkers.

a Colonic lamina propria effector CD4 + T cells, including Th1 and Th17 cells that produce IFNγ and/or IL-17α. The mRNA expression levels of b inflammatory cytokines and c functions important for mucosal homeostasis in the distal colon. Expression levels were determined by RT-Q-PCR and presented as fold change (FC) of expression after therapeutic application of GUT-108 compared to expression after PBS treatment. Hu + GUT-108 refers to humanized Il10^−/− mice treated with GUT-108 2 weeks after fecal colonization; Hu+PBS refers to humanized Il10^−/− mice that received PBS as a placebo control. AhR aryl hydrocarbon receptor gene, AhrR aryl hydrocarbon receptor repressor gene, Cyp1A1 Cytochrome P450 Family 1 Subfamily A Member 1gene, DefCR1 and DefA genes defensins, Aldh1A1 and Aldh1A2 genes aldehyde dehydrogenases. Bar indicates Mean ± SE. Two-way ANOVA and adjusted P-value were calculated by the multiple comparisons test. *: adjusted P < 0.05. N = 11/group. Source data and calculated P-values are provided as a Source Data file.