Effects of intergenerational transmission of small intestinal bacteria cultured from stunted Bangladeshi children with enteropathy

Abstract

Environmental enteric dysfunction (EED), a small intestinal disorder found at a high prevalence in stunted children, is associated with gut mucosal barrier disruption and decreased absorptive capacity^1-4. To test the hypothesis that intergenerational transmission of a perturbed small intestinal microbiota contributes to undernutrition by inducing EED⁵, we characterized two consortia of bacterial strains cultured from duodenal aspirates from stunted Bangladeshi children with EED - one of which induced local and systemic inflammation in female gnotobiotic mice. Offspring of dams colonized with the inflammatory consortium exhibited impaired prenatal and postnatal growth, as well as immunologic changes phenocopying features of EED in children. Dam-to-pup transmission of the inflammatory consortium produced, in recently weaned offspring, alterations in (i) inter-cellular signaling pathways related to intestinal epithelial cell renewal, barrier integrity and immune function plus (ii) glial- and endothelial-neuronal signaling pathways that regulate neural growth, angiogenesis and inflammation in the cerebral cortex. Cohousing of mice harboring the inflammatory or non-inflammatory consortia and subsequent screening of candidate disease-promoting bacterial isolates identified Campylobacter concisus, an organism typically found in the oral microbiota, as a contributor to enteropathy. The C. concisus strain induced, in a host nitric oxide synthase (NOS)-dependent manner, pro-inflammatory cytokine signaling. Moreover, host-derived nutrients generated by NOS augmented C. concisus growth. This preclinical model should facilitate identification of small intestinal microbiota-targeted therapeutics for (intergenerational) undernutrition.

Extended Data Fig. 1.. Initial test of the host response to colonization with the cSI-N and cSI-I consortia.

a, Weight gain of mice colonized with all 184 isolates cultured from duodenal aspirates obtained from children with EED (cSI-I), or a species-representative set of isolates (cSI-N). A control group of CONV-D mice was colonized with cecal microbiota harvested from conventionally raised animals. All mice were fed Mirpur-18 diet ad libitum. Each small point represents the weight gain for an individual mouse; larger points denote the mean, error bars, standard deviation. n=5–10 mice/group, linear mixed effects model (Weight ~ Group*Day + (1|MouseID), P-values shown are the result of Tukey’s post-hoc tests). b, Serum IGF-1 correlates with mouse weight measured 9 (left) or 28 (right) days after colonization (n=5 mice per group per timepoint, linear regression). c, Serum leptin levels determined nine (left) or 28 days (right) days post-colonization (n=5 mice per group per timepoint, bars denote mean ± s.d.). d, Number of Gene Ontology (GO) categories related to the immune system that were significantly enriched (GSEA q value < 0.05) based on differential gene expression in the small intestine of mice colonized with all isolates (red) versus the species representative subset (blue). Bulk RNA-seq was performed in duodenal, jejunal, and ileal tissue 28 days following gavage (n=5 mice/group). e, Expression of genes (columns, un-labeled) that comprise the leading edge of the eight GO categories most significantly enriched in animals colonized with the full consortium (red) compared to the species-representative subset (blue). Bulk RNA-seq of duodenal tissue, 28 days post colonization. f, Serum lipocalin-2 (LCN2) levels 9 (left) or 28 days (right) days post-gavage of the consortia (n=5 mice per treatment group per timepoint.). g, Design of experiment to test whether systemic inflammation induced by cSI-I consortium is influenced by diet. h, Serum levels of LCN2 after mice were fed breeder chow for one week (day 7), Adult Mirpur the following seven days (day 14), and a final week of breeder chow (day 21) (Wilcoxon rank-sum tests, n=8–17 mice/group). For a,c,f,h, bars denote mean ± s.d.

Extended Data Fig. 2. Intergenerational transmission of duodenal bacteria.

a, Design of the intergenerational transmission experiment. b, Summary of animals collected during intergenerational experiment. Litter sizes and the age of offspring at the time of sample collection are shown for each dam (n=4 dams/group). t₁-t₃ denotes the number of days between first mating and birth of the litter. c, MAGs present at >0.001% relative abundance in gavage mixes, dams, and their P14 and P37 offspring. d,e, Principal component analysis of MAGs in feces of dams ordinated by their log₁₀ absolute abundance and colored by bacterial consortium (d) or timepoint (e). The composition of community membership differs significantly between cSI-I and cSI-N dams (i, P=0.01, PERMANOVA) but is stable after the first week of colonization (j, P=0.09, PERMANOVA weeks 4 through 17). Each small point represents a fecal sample from one animal. Larger filled circles are the centroids for the ellipses shown, which denote the 95% confidence interval. f, Detectable MAGs (log₁₀ count > 0) in mice from the ‘Diet oscillation experiment’ (Extended Data Fig. 1g) are included in the heatmap; MAGs that match ‘core taxa’ described in children with EED are highlighted in blue, as in Fig. 1b, MAGs whose absolute abundance differs significantly between cSI-I and cSI-N mice are indicated (•, P-adj < 0.05, FDR-corrected Wilcoxon rank-sum). g, Principal component analysis of MAGs in feces of mice during the transition from breeder chow (the larger shape denotes the centroid of points for each diet and timepoint, circle) to Adult Mirpur (triangle) and back to breeder chow (square).

Extended Data Fig. 3.. Growth and inflammatory phenotypes of P14 offspring and fetuses.

a, MAGs whose absolute abundance differs significantly between P14 cSI-I and cSI-N mice are indicated (•, P-adj < 0.05, ••, P-adj < 0.01, •••, P-adj < 0.001, FDR-corrected Wilcoxon rank-sum). All 51 MAGs are included in the heatmap. b, Average masses of fetuses per dam at E11.5, n=9–10 dams/group; n=4–8 fetuses/litter. c,d, Average masses of placentas within each dam at E11.5 (c) and E17.5 (d); n=7–10 dams/group; n=2–10 conceptuses/litter. For b-d, P-values determined with unpaired t-tests. e, Body mass of P14 offspring of dams colonized with the cSI-I consortium (red), cSI-N consortium (blue), or cecal contents from a conventional mouse (CONV-D). Each point represents an individual animal. f, Serum levels of IGF-1 in P14 offspring. g-j, Serum levels of the inflammation-associated proteins, LCN2 (g), S100A9 (h), MMP8 (i), and CXCL1 (j), in P14 offspring. k-m, Levels of the inflammation-associated proteins LCN2 (k), S100A9 (l), and CHI3L1 (m) in duodenal, ileal, and colonic tissue of P14 animals. For a,e-m, n=11–19 mice/group, 3–8 pups/litter, P-values were determined using Wilcoxon rank-sum with Tukey’s post-hoc tests. For b-m, all panels, mean values ± s.d. are shown.

Extended Data Fig. 4.. Assessment of EED biomarkers and immune profiling of P37 animals.

a, Ratio of villus length to crypt depth in the ileums of cSI-I and cSI-N mice (n=5 mice/group, Wilcoxon rank-sum test; boxes denote interquartile range). b,c, Levels of serum MMP8 (b), and CXCL1 (c), in the P37 offspring of C57Bl/6J dams colonized with the cSI-I or cSI-N consortia, or with mouse microbiota harvested from the cecal contents of conventionally raised (CONV-D) C57Bl/6J mice (n=21–25 mice/group). d,e, Levels of S100A9 (d), and CHI3L1 (e), in the duodenal, ileal, and colonic tissue of P37 animals (n=7–10 animals/group). f, Concentration of acylcarnitines in small intestinal tissue of P37 animals. Acylcarnitines shown exhibited a statistically significant difference in levels between cSI-I versus cSI-N groups (n=7–16 mice/group, Mann-Whitney tests with Benjamini, Krieger, Yekutieli two-stage step up FDR correction, q-values shown). g,h, Frequency of CD3⁺ (g), and CD4⁺ (h) immune cells in duodenal, ileal and colonic lamina propria (n=4–7 mice/group, except for colonic tissue collected from CONV-D animals for which n=2 and a statistical comparison was excluded). For panels b-e and g-h, bars denote mean ± s.d. and P-values were determined by Wilcoxon rank-sum tests.

Extended Data Fig. 5.. snRNA-Seq differential gene expression in the duodenum and ileum of P37 cSI-I versus cSI-N animals.

a, Normalized enrichment scores (NES) for all GO categories (rows) that were significantly enriched (q < 0.05, GSEA) in at least two of the indicated cell populations (columns) in either the duodenum (left) or ileum (right). Immune-related GO terms are labeled. If a GO category was non-significant, it was assigned an NES value of 0. b, Significantly differentially expressed genes comprising the ‘leading edge’ of the efferocytosis gene set in duodenal mid-villus enterocytes (cSI-I versus cSI-N; P-adj < 0.05, GSEA). See Supplementary Table 3c for annotations.

Extended Data Fig. 6.. Expression of genes encoding orthologs of proteins quantified in the duodenal mucosa of children with EED and proliferative inter-cellular signaling in P37 animals.

a, NicheNet analysis of the expression of receptors (right) in stem and transit-amplifying (TA) cells for ligands (left) expressed by various cell types in the ileum of P37 animals. Upper panel – ligands expressed more highly in cSI-I animals; lower panel – ligands expressed more highly in cSI-N animals. Boxes are sized proportionally to the weighted ligand/receptor score, related to Fig. 2d. b, Representative sections of crypts from the duodenum of cSI-N (left) and cSI-I (right) P37 mice. Sections were stained with antibodies to E-cadherin (red) and Ki67 (green). Scale bar, 50 μm. c, Expression of transcripts (rows) in cell populations (columns) in the duodenum of P37 animals that match proteins, quantified in biopsies of the duodenal mucosa of Bangladeshi children with EED, that were (i) negatively correlated with their LAZ and (ii) positively correlated with the absolute abundances of ‘core’ EED bacterial taxa; the abundances of these taxa were positively correlated with their degree of stunting); related to Fig. 2c (see Supplementary Table 3c for a list of the mouse homologs of LAZ-associated human proteins).

Extended Data Fig. 7.. Abundance of Actinomyces in isolate ‘add-in’ experiments and.

a,b, Mice were initially colonized with either the cSI-N or cSI-I consortium (‘gavage 1’). Gavages 2–4 were performed on experimental days 9 through 11, as in Fig. 4a. The absolute abundances of A. odontolyticus isolate Bg044 (corresponding to MAG044, panel a) and A. naeslundii isolate Bg041 (corresponding to MAG041, panel b), shown relative to their absolute abundance in the cSI-N/sham-gavaged controls (a ratio of 1 is indicated by dotted line; mixed-effects analysis with Dunnett’s multiple comparisons to cSI-N/sham control; mean values ± s.d. are shown.) c, Changes in body mass relative to time of colonization in mice in ‘add-in’ experiments (points and error bars denote mean values ± s.e.m.; shaded, thicker line represents results of linear regression. *P<0.05, **P<0.01, ***P<0.001 for Tukey’s multiple comparisons demonstrating significant differences between C. concisus-gavaged animals compared to cSI-N/mock, cSI-N/A.naeslundii, A. odontolyticus, or Actinomyces from two-way repeated measures ANOVA; all stats are reported in Supplementary Table 2b). d, Relative weight gain in isolate-gavaged and cSI-I/mock-gavaged animals relative to weight gain in cSI-N mice (one-way ANOVA with Tukey’s post-hoc, mean ± s.e.m.). e, Levels of LCN2 protein in duodenal tissue on experimental day 18 (one-way ANOVA with Tukey’s multiple comparisons; mean values ± s.e.m. are shown). The inset shows comparisons between individual isolates. f, Tissue levels of CHI3L1 (Chitinase-3-like protein 1) in the duodenum and colon (one-way ANOVA with Tukey’s multiple comparisons, n=5–6 mice/group, mean ± s.d. shown). For a-e, n=5–11 mice/group combined across two independent experiments.

Extended Data Fig. 8.. Comparative genomic analysis of C. concisus isolates.

120 whole C. concisus genomes are included in the circle phylogram that were isolated from intestinal biopsies, feces, or saliva of healthy or diseased individuals. Genes that are present are shown in black; if genes are absent, in gray. The Bangladeshi isolate from children with EED (Bg048) is highlighted in green. The nitrate-inducible formate dehydrogenase (fdnG) unique to this Bangladeshi isolate and a selenate di-kinase (selD) unique to the Bangladeshi isolate and one other genome are highlighted. Heatmap shows ANI comparisons between all isolates.

Extended Data Fig. 9.. C. concisus growth in spent medium collected from colonic epithelial cells.

a, C. concisus gene expression in the gut (Concisus, Concisus+Actinomyces) from the isolate ‘add-in’ experiment (Fig. 5) compared to growth in rich medium (Bolton broth, or Bolton broth supplemented with 1% mucin). Green, P-adj < 0.05 and higher expression in the gut; brown, P-adj < 0.05 and higher expression in vitro (DESeq2 Wald test, n=4 biological replicates per condition). Genes involved in formate metabolism and anaerobic respiration are labelled. b, Abbreviations of cell lines and treatments used to generate conditioned medium from mouse and human colonic epithelial cells that were live or apoptotic. C. concisus growth in conditioned medium acquired from live cells are colored in green; apoptotic cells, in red. QVD, Quinoline-Val-Asp-Difluorophenoxymethylketone (caspase inhibitor); L-NIO, N⁵-(1-Iminoethyl)-L-ornithine, dihydrochloride (nitric oxide synthase inhibitor). c,d, Microaerophilic growth of C. concisus after 8 (c) or 24 hours (d) in conditioned medium from mouse CT26 colonic epithelial cells that were live (CT26:FADD, z-VAD, z-VAD+BB) or treated with an inducer of apoptosis (BB). Points shown with an open rather than filled circle denote abundances that were below the limit of detection at the time of quantification and are presented at the limit of detection of the assay. e-g, Microaerophilic growth of C. concisus after 6 (e), 12 (f), or 24 (g) hours in spent medium from HTC116 human colonic epithelial cells that were live (QVD, QVD+STS) or that had been treated with an inducer of apoptosis (STS). h-k, Anaerobic growth of C. concisus after 8 (h, j) or 24 hours (i, k) in conditioned medium from live CT26 mouse colonic epithelial cells (CT26:FADD, z-VAD, z-VAD+BB) or from cells treated with an inducer of apoptosis (BB). Panels j and k show growth relative to (as a ratio to) growth in tissue culture medium. l, Levels of nitrite in cecal contents 9 days post-isolate gavage (n=5–11 mice/condition, combined across two independent experiments). m,n, Microaerophilic growth of C. concisus after 8 (m) or 24 (n) hours growth in conditioned medium from HTC116 human colonic epithelial cells acquired from live cells (QVD, QVD+STS) or cells treated with an inducer of apoptosis (STS). All cells were treated with an inhibitor of nitric oxide synthase (L-NIO). Growth shown as a ratio to growth in cell culture medium alone. o,p, Anaerobic growth of C. concisus after 24 hours of incubation in conditioned medium from HTC116 cells collected from live cells (QVD, QVD+STS) or cells that had been treated with an inducer of apoptosis (STS). All cells were treated with an inhibitor of nitric oxide synthase (L-NIO). Panel o shows growth as a ratio to growth in tissue culture medium alone; p, total bacterial growth. For c-p, bars denote mean ± s.d., P-values determined with one-way ANOVA and Tukey’s multiple comparisons. For c,d, g-k, m-p, n=4 biological replicates/condition; for e,f, n=3 biological replicates/condition.

Extended Data Fig. 10.. C. concisus induces iNOS and immune signaling.

a,b, Bone marrow cells from wild-type (WT) mice were incubated for 48-hours with fresh (Live) or heat-killed (HK) C. concisus culture, or bacterial medium as a negative control. b, Bone marrow cells were co-cultured for 48-hours with increasing amounts of live C. concisus culture. Proteins were extracted from bone marrow cell pellets and iNOS levels were quantified. c-j, Bone marrow cells collected from wild-type (WT) mice were incubated for 48 hours with fresh C. concisus culture (Live, CFU), heat-killed C. concisus culture (HK), or bacterial medium as a negative control (BHI). IL-1ß (c,d), TNFa (e), IL-5 (f), IL-17 (g,h), and IL-22 (i,j) were quantified in cell supernatant. k-o, Bone marrow cells collected from Nos2^−/− mice were incubated for 48 hours with fresh (Live) or heat-killed (HK) C. concisus culture. Again, IL-1ß (k), TNFa (l), IL-22 (m), IL-5 (n) and IL-17 (o) were quantified from cell culture supernatants. p, p38 MAPK activation was assessed in bone marrow cell lysates after 48 hours incubation bacterial medium (BHI), fresh C. concisus culture (Live), or heat-killed (HK) C. concisus culture (multiple unpaired t-tests with two-stage step-up FDR correction). q, Expression of the C. concisus nitrate-inducible formate dehydrogenase (fdnG) and formate transporter (focA) when cultured in supernatants collected from HTC116 cells that were or were not treated with the NOS inhibitor L-NIO or cell culture medium alone (statistics shown are unadjusted P values from the DESeq2 Wald test, transcripts per million). r, C. concisus was cultured in Bolton broth supplemented with various levels of formate; growth was quantified after 24 hours. s, Formate was quantified in supernatants from bone marrow cells incubated for 48 hours with bacterial culture medium (BHI), fresh C. concisus culture (Live), or heat-killed C. concisus culture (HK). For all panels, n=4–5 biological replicates/condition, bars denote mean ± s.d.. For a-o, r,s, P-values shown are the result of one-way ANOVA followed with Tukey’s multiple comparisons.

Fig. 1.. Dam-to-offspring transmission of bacteria cultured from duodenal aspirates collected from Bangladeshi children with EED.

a, Design of intergenerational transmission experiment. b, Differential abundances of bacterial MAGs (rows) along the length of the intestine (columns) of P37 pups born to dams harboring cSI-N or cSI-I bacterial consortia (n=21–25 mice/group). MAGs were included in the heatmap if they demonstrated significant differential log₁₀ absolute abundance in at least one of the intestinal locations. MAG taxonomy colored in blue indicates correspondence to ASVs representing ‘core taxa’ identified in the small intestines of stunted children with EED in the BEED study. •, P-adj < 0.05; ••, P-adj < 0.01; •••, P-adj < 0.001, FDR-corrected Wilcoxon rank-sum tests. c, Average fetal mass for a given dam at E17.5. Dams were colonized with the cSI-I or cSI-N consortia for two weeks prior to mating (n=7–9 dams/group; n=2–10 fetuses/litter, unpaired t-test). d,e, Villus length (d), and ratio of villus length to crypt depth (e) in the duodenum of P37 cSI-I compared to cSI-N mice (n=5 animals/group, boxes denote interquartile range). f, Levels of lipocalin-2 (LCN2) protein in intestinal tissue from P37 mice (n=7–10 mice/group). g,h, Serum levels of LCN2 (g), and S100A9 protein (h), in P37 pups (n=21–25 mice/group). For c-e, n=4–6 litters/group, 3–7 pups/litter. i,j, Frequency of neutrophils (i), and Th17 cells (j), along the length of the gut (n=5–7 mice/group, each point represents an individual animal). For d-j, statistics shown are the result of Wilcoxon-rank sum tests. For c-j, mean values ± s.d. are shown.

Fig. 2.. Increased proliferative signaling in the small intestine of P37 offspring of cSI-I dams.

a,b, Uniform manifold and projection (UMAP) plot of single nuclei isolated from the duodenum (a), and ileum (b) of P37 offspring (n=3 mice/group). c, Intercellular signaling from cell populations (columns) to stem/transit amplifying (TA) cells in the ileum. A subset of ligands identified by NicheNet are shown that were significantly differentially expressed between cSI-I and cSI-N animals. Annotations for these ligands (left) are based on literature findings. Their paired receptors are shown in Extended Data Fig. 6a. Ligands that were more highly expressed in cSI-I mice are shown in red (top of panel); those more highly expressed in cSI-N animals are shown in blue (bottom of panel). d, Percent of all cells in a crypt that were Ki67⁺ in the duodenum (left) or ileum (right) (n=3–5 mice/group; each dot represents a single crypt-villus unit; 10 crypts analyzed per intestinal segment per mouse). P-values were determined by Tukey’s post-hoc tests; mean values ± s.d. are shown. e, Gene set enrichment analysis (GSEA) along the crypt-villus axis, focusing on genes encoding duodenal mucosal proteins whose levels were quantified in children with EED in the BEED study.

Fig. 3.. Identification of bacteria associated with pathology after cohousing.

a, Experimental design of cohousing experiment. b, Serum levels of LCN2 protein on experimental day 18, 9 days after initiation of cohousing (n=14 mice/control group, n=7 mice/cohoused group). c, Intestinal tissue levels LCN2 measured on experimental day 18, 9 days after cohousing (n=8 mice/control group, n=4 mice/cohoused group). d,e, Frequency of neutrophils (d), and Th17 cells (e), in the small intestinal lamina propria (n=4 mice/group; duodenal, jejunal and ileal segments were combined prior to analysis). f,g, Frequency of neutrophils in the spleen (f), and meninges (g) (n=4 mice/group). For panels d-f, each point represents an individual animal; the color code matches that used in panel a. h, Differences in absolute abundances of MAGs along the length of the gut (n=6 mice/group). MAGs were included if they demonstrated statistically significant differences in their absolute abundance for any comparison between groups in at least one segment of the intestine. MAG taxonomy colored in blue indicates correspondence to ASVs representing ‘core taxa’ in the duodenal microbiota of stunted Bangladeshi children with EED in the BEED study. The three red arrows point to MAGs defined as ‘pathology-associated’. • P-adj < 0.05, FDR-corrected Wilcoxon rank-sum. i,j, Log₁₀ absolute abundances (AA) of the pathology-associated MAGs (MAG041, MAG044 and MAG048) along the length of the intestine in either the cohousing experiment (i) or P37 animals from the intergenerational experiment (j). Values are expressed relative to cSI-N non-cohoused controls (n=6 mice/control group and n=3/cohoused groups, i) or P37 cSI-N offspring in the intergenerational transmission experiment (n=22–25 mice/group, j). For b-j, P-values were determined by Wilcoxon rank-sum test; mean values ± s.d. are shown in b-g, i-j.

Fig. 4.. Direct test of candidate pathology-inducing small intestinal bacterial strains in cSI-N mice.

a, Experimental design of ‘add-in’ experiment. Individual cultured isolates, alone or in combinations, were gavaged once daily on days 9–11 into mice previously colonized with the cSI-N bacterial consortium. b, Ratio of the absolute abundance of C. concisus strain Bg048 (corresponding to MAG048) shown relative to its absolute abundance in cSI-N/sham controls (a ratio of 1 is indicated by the dotted line; two-way ANOVA with Dunnett’s multiple comparisons to cSI-N/sham control; mean values ± s.d. are shown). Colors correspond to the groups shown in panel a. c, Levels of LCN2 protein in colonic tissue measured on experimental day 18, 9 days after isolate gavage. The inset shows colonic LCN2 levels after addition of the individual isolates alone (one-way ANOVAs with Tukey’s multiple comparisons; mean values ± s.e.m. are shown). d,e, Serum LCN2 at 9 (c), or 35 days (d), following isolate gavage (mean ± s.d. shown, one-way ANOVAs with Tukey’s multiple comparisons). f, Results of bulk-RNA-Seq analysis: normalized enrichment scores (NES) for all Reactome pathways that were significantly enriched in the colon (GSEA q-value <0.05) of the indicated treatment groups compared to their respective cSI-N counterparts (non-cohoused cSI-N controls, or cSI-N/sham). Pathways related to the immune system are labeled. Pathways were included in the heatmap if they were significantly enriched with the addition of C. concisus and in cohoused animals. If a pathway was not significantly enriched, it was assigned a NES score of zero. For panels b, c, f, n=5–11 mice/group combined across two independent experiments. For d and e, n=5 mice/group in a third independent experiment.

Fig. 5.. Host epithelial cell nitrate metabolism boosts C. concisus growth in vitro.

a, Absolute abundance of the Campylobacter ASV identified in duodenal aspirates from children in the BEED study. b, Heatmap of significantly differentially expressed genes (DESeq2 Wald test) in the colon between cSI-I and C. concisus-gavaged animals (‘inflamed’) compared to sham-gavaged cSI-N controls. The gene products for the most significantly differentially expressed genes across all three pairwise conditions are labeled. c, Expression of C. concisus nitrate reductase (napA) and nitrate transporter (ntrD) 9 days post-isolate gavage, or in cSI-I and cSI-N sham-gavaged controls (tpm, transcripts per million). d, Abbreviations of cell lines and treatments used to collect conditioned medium from mouse and human colonic epithelial cells that were live or apoptotic. QVD, Quinoline-Val-Asp-Difluorophenoxymethylketone (caspase inhibitor); L-NIO, N⁵-(1-Iminoethyl)-L-ornithine, dihydrochloride (nitric oxide synthase inhibitor). Conditioned medium acquired from live epithelial cells is colored in green; apoptotic cells, in red. e,f, Growth of C. concisus after 8 (e) or 24 hours (f) in conditioned medium collected from CT26 mouse colonic epithelial cells that were live (CT26:FADD, z-VAD, z-VAD+BB) or were treated with an inducer of apoptosis (BB). Growth is presented relative to (as a ratio to) growth in cell culture medium (n=4 biological replicates per condition). g-i, Growth of C. concisus after 6 (g), 12 (h), or 24 (i), hours in spent medium collected from HTC116 human colonic epithelial cells that were live (QVD, QVD+STS), or that had been treated with an inducer of apoptosis (STS) relative to growth in cell culture medium (n=3 biological replicates per condition, growth is presented as a ratio to growth in cell culture medium). j, Levels of nitrate in homogenized ileal tissue 9 days post-isolate gavage. k, Comparison of C. concisus growth after 24 hours in spent medium collected from live (QVD, QVD+STS) or apoptotic (STS) HTC116 cells that were or were not treated with L-NIO (n=3–4 biological replicates per condition, pairwise t-tests for each ± L-NIO comparison). l, C. concisus genes that were significantly differentially expressed when cultured in conditioned medium collected from HTC116 cells compared to medium alone (DESeq2 Wald test). For c-j, bars denote mean ± s.d.. For b-c, j, n=5–11 mice/group combined across two independent experiments. For e-j, P-values were determined with one-way ANOVA and Tukey’s multiple comparisons.

Fig. 6.. C. concisus elicits cytokine signaling in a Nos2-dependent manner.

a-e, Bone marrow cells harvested from conventionally-raised wild-type (WT) or iNOS^−/− (Nos2^−/−) mice were incubated with live or heat-killed (HK) C. concisus for 48 hours and cytokines were quantified in bone marrow cell supernatants (n=4–5 biological replicates per condition, mean ± s.d., multiple unpaired t-tests with two-stage step-up FDR correction). f, IL-1ß quantified in bone marrow supernatants after 24 hours incubation with C. concisus pre-treated (grown for the 24 hours prior to co-culture) with various concentrations of formate (n=5 biological replicates per condition, mean ± s.d., one-way ANOVA: F_(3,16)=527.4, P<0.0001, Tukey’s multiple comparisons shown).