The enteric nervous system promotes intestinal health by constraining microbiota composition

Abstract

Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that zebrafish lacking an ENS due to a mutation in the Hirschsprung disease gene, sox10, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health.

Fig 1. sox10 mutants experience bacterial overgrowth and physiological indications of dysbiosis.

(A) Schematic representation of the location and orientation of images in B and D. (B) Representative images of the panbacterial population by FISH on the esophageal-intestinal junction of WT (left) and sox10^- (right) fish. Blue, DNA; red, eubacteria. (C) Quantification of bacterial colonization level in sox10 mutants and WT siblings. (D) Representative images of WT, sox10 mutant, and tumor necrosis factor receptor (tnfr) morpholino (MO) injected larvae of both genotypes. Arrowhead indicates neutrophil. (E) Quantification of intestinal neutrophil number per 140 μm of distal intestine. (F) Total numbers of proliferating cells over 30 serial sections beginning at the esophageal-intestinal junction and proceeding into the bulb in 6-d-post-fertilization (dpf) fish. Box plots represent the median and interquartile range; whiskers represent the 5–95 percentile. n > 15 per group, *p < 0.05, ***p < 0.001, ****p < 0.0001, ANOVA with Tukey’s range test. Also see S1 Fig. Scale bars = 50 μm.

Fig 2. Intestinal microbiota are necessary and sufficient to induce increased intestinal neutrophil accumulation in sox10 mutants.

(A) Quantification of intestinal neutrophil number per 140 μm of distal intestine. Neutrophil accumulation was inhibited when sox10 mutants were raised GF compared to CV controls. n > 21 per condition. (B) Schematic of fish used as donors in the transmission experiment. Intensity of red indicates level of intestinal inflammation. (C) Schematic of the experimental protocol. Intestines of GF, CV WT, sox10 mutants, or iap MO were dissected for use as inoculum for 4 dpf GF WT recipients. Recipient fish were colonized for 2 d before examination of intestinal neutrophil number. (D) Transfer of intestinal microbes from inflamed intestines of sox10 mutants causes increased intestinal neutrophil number in WTs. n ≥ 10, *p < 0.05, ***p < 0.001, ****p < 0.0001, ANOVA with Tukey’s range test. See also S2 Fig.

Fig 3. Increased bacterial colonization level does not drive increased intestinal neutrophil accumulation or pro-inflammatory gene expression.

Quantification of intestinal neutrophil number (A) and bacterial colonization level (B) in the sox10^-, Tg(mpx:GFP) line. sox10^- fish were split into two groups, “sox10^- low” (bottom half) and “sox10^- high” (top half) based on intestinal neutrophil number. Ten representative fish from each group were plated to determine total CFU/intestine. n ≥ 9 per group. *p < 0.05, **p < 0.01, ****p < 0.0001, ANOVA with Tukey’s range test. (C) Relative expression calculated by the 2^-ΔΔCt method of immune genes from dissected intestines. For mpx, saa, il1b, and c3, n = 5 pools of 5 dissected intestines; for tnfα and mmp9, n = 3 pools of 18 dissected intestines. Graph displays average ± standard deviation (SD); **p < 0.01, t test corrected for multiple comparisons using Holm–Šidák method.

Fig 4. Changes in microbial lineages define low and high neutrophil accumulation.

(A) Intestinal neutrophil accumulation from samples used for 16S rRNA gene sequencing. Each dot represents an individual fish; black circles indicate sequenced samples, WT, n = 32; sox10^- high, n = 30; sox10^- low, n = 31. Gray circles indicate samples that were not sequenced. Line indicates median. **p < 0.01 Student’s t test. (B) Spearman’s rank correlation between intestinal neutrophil number and each operational taxonomic unit (OTU) present in at least 20 samples. After false discovery rate correction, two genera, Escherichia and Vibrio, stand out with correlations to neutrophil number. Asterisks represent significance of Spearman correlation, *p < 0.05, #p = 0.08, c: class, g: genus. The percent abundance of Escherichia (C) and Vibrio (D) across genotypes and intestinal neutrophil levels. (E) Phylogenetic tree of OTUs from the Vibrio genus and our Vibrio zebrafish isolate (Vibrio Z20). For D, E: *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA. See also S3 Fig.

Fig 5. An increase in Vibrio contributes dominantly to intestinal neutrophil number.

(A) Correlation between the log percent Vibrio abundance (left) and log percent Escherichia abundance (right) with log₁₀(intestinal neutrophil number +1). Linear regression analysis with 95% confidence intervals. Each point represents an individual fish: WT, squares, n = 32; sox10^- high, open circles, n = 30; sox10^- low, closed circles, n = 31. (B) Correlation between both log₁₀(percent Escherichia) (z-axis) and log₁₀(percent Vibrio) (x-axis) abundance with log₁₀(intestinal neutrophil number +1) (y-axis). Planar regression analysis, n = 93. WT, black squares; sox10^- low, red closed circles; sox10^- high, red open circles. (C) Addition of Vibrio Z20 increased intestinal neutrophil accumulation in both CV and GF sox10 mutants. n ≥ 49, from at least three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001, indicates difference from CV, ANOVA. (D) Correlation between the absolute abundance of Vibrio Z20 in CV sox10 mutants and log₁₀(intestinal neutrophil number + 1) in experiments with exogenously added Vibrio Z20. Linear regression analysis with 95% confidence intervals. For C-D, data from four to six independent experiments, n > 35. See also S4 Fig.

Fig 6. Inflamed intestines are rescued by anti-inflammatory bacterial isolates or transplantation of WT ENS into sox10 mutants.

(A) Addition of a representative Escherichia isolate, E. coli HS, to CV sox10 mutants reduces intestinal neutrophil accumulation. Monoassociation of sox10 mutants with E. coli HS does not increase neutrophil level over that observed in GF zebrafish. n > 20, from at least three independent experiments. (B) Correlation between absolute abundance of E. coli HS and log₁₀(intestinal neutrophil number + 1) in experiments with added E. coli HS. Linear regression analysis with 95% confidence intervals. For A, B: n > 35, from three to six independent experiments. (C) Representative images of distal intestine from WT, sox10^-, and sox10^- rescued by WT ENS precursor transplantation. Anti-ElavI1–labeled enteric neurons are white (white arrow); neutrophils are black (black arrow). Scale bar = 100 μm. (D) Quantification of intestinal neutrophil number per 140 μm of distal intestine. n > 6 for all conditions, *p < 0.05, **p < 0.01, ****p < 0.0001, ANOVA with Tukey’s range test. See also S4 Fig.

Fig 7. Proposed model of sox10 mutant intestinal pathology.

sox10 mutants have altered intestinal motility and an increased bacterial load. Given the role of the ENS in intestinal function, sox10 mutants likely also experience alterations in epithelial secretion and permeability, although these phenotypes are yet to be examined. sox10 mutants can assemble a microbiota that mirrors WT intestinal microbiota (host population 2) or is dysbiotic (host population 1), characterized by an expansion of the Vibrio lineage and reduction of the Escherichia lineage. We do not yet know what determines which bacterial community assembles in sox10 mutants (dashed lines) but hypothesize that it could be due to the timing or order of exposure to bacterial strains, differences in epithelial permeability or secretion, or differences in other host compensatory mechanisms.