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.

Intestinal health depends on maintaining a balanced microbial community within the highly dynamic environment of the intestine. Every few minutes, this environment is rocked by peristaltic waves of muscular contraction and relaxation through a process regulated by the enteric nervous system (ENS). We hypothesized that normal, healthy intestinal microbial communities are adapted to this dynamic environment, and that their composition would become perturbed without a functional ENS. To test this idea, we used a model organism, the zebrafish, with a genetic mutation that prevents formation of the ENS. We found that some mutant individuals without an ENS develop high levels of inflammation, whereas other mutants have normal intestines. We profiled the intestinal bacteria of inflamed and healthy mutants and found that the intestines of inflamed individuals have an overabundance of pro-inflammatory bacterial lineages, lack anti-inflammatory bacterial lineages, and are able to transmit inflammation to individuals with a normally functioning ENS. Conversely, we were able to prevent inflammation in the ENS mutants by either administering a representative anti-inflammatory bacterial strain or restoring ENS function. From these experiments, we conclude that the ENS modulates intestinal microbiota community membership to maintain intestinal health.

Funding: NIGMS (grant number P50GM098911). Received by KJG. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIDDK (grant number 1F32DK098884-01A1). Received by ASR. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Broad Medical Research Program (grant number IBD-0209). Received by KJG. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. John Simon Guggenheim Memorial Foundation Fellowship. Received by JSE. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NICHD (grant number P01HD22486). Received by JSE. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2017 Rolig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we demonstrate that the ENS constrains the abundance and composition of the microbiota. We find that loss of the ENS in sox10 t3 mutants results in assembly of a dysbiotic community leading to a microbe-driven intestinal inflammation that varies among individuals and resembles HAEC. Microbiota profiling across the spectrum of inflammatory states revealed that extreme intestinal inflammation is linked to an outgrowth of pro-inflammatory bacterial lineages and a reduction of anti-inflammatory bacterial lineages. Moreover, administering representative anti-inflammatory bacterial strains or transplanting wild-type (WT) ENS precursors to restore a WT ENS corrects the pathology in sox10 t3 mutant hosts. Our analysis reveals that ENS function is a key feature of intestinal health that constrains the composition of the resident microbiota and prevents overgrowth of bacterial lineages that can drive disease.

To explore how the ENS may prevent dysbiosis by constraining microbial populations, we turned to a zebrafish model of HSCR. Multiple well-described zebrafish lines carry mutations in HSCR loci [ 19 – 22 ]. The most extreme ENS loss is seen in mutants homozygous for a null mutation in the HSCR gene sox10 [ 23 , 24 ]; these mutants entirely lack an ENS [ 24 ]. The mutant allele t3 (sox10 t3 ) homozygotes have diminished rhythmic peristaltic activity [ 21 ], making this an ideal model for dissecting the role of the ENS in host–microbe interactions. Zebrafish are well suited for examining ENS contributions to microbiota composition because we can monitor ENS development, absolute bacterial abundance, and disease phenotypes, such as neutrophil accumulation, across the entire intestine of individual larvae. Thus, we can assess system-level functional readouts that describe properties of the associated microbiota. Furthermore, the high fecundity and ease of working with zebrafish provide us with large sample sizes to increase the power of our experiments such that we can monitor how natural microbiota variation at the species level drives phenotypic variation.

The most severe example of ENS dysfunction in humans is Hirschsprung disease (HSCR), an enteric neuropathy that results from a failure of neural crest–derived cells to form the distal ENS [ 3 ]. Approximately 30% of HSCR patients develop a severe form of intestinal dysbiosis, known as Hirschsprung-associated enterocolitis (HAEC) [ 9 – 11 ], which is distinguished by diarrhea, distension, fever, and, in extreme cases, sepsis and death [ 12 ]. Studies suggest that the etiology of HAEC has a microbial component, as both pathogenic bacteria [ 13 ] and alterations in commensal communities [ 9 , 10 ] have been linked to HAEC. Interestingly, patients with a broad range of human diseases, such as inflammatory bowel disease (IBD), cystic fibrosis [ 14 ], diabetes [ 15 ], malnutrition [ 16 ], and myotonic muscular dystrophy [ 17 , 18 ], also experience debilitating gastrointestinal (GI) symptoms. Although cause and effect are difficult to determine, these diseases are associated with both small intestinal bacterial overgrowth, a clinical syndrome often seen with impaired intestinal motility, and an altered microbiota, suggesting that impaired ENS function could be a driver of dysbiosis.

The intestinal tract serves to harvest nutrients and energy, protect against harmful toxins and pathogens, and clear out waste. These functions can be modulated by both the enteric nervous system (ENS) and the trillions of symbiotic bacteria that reside within the gut [ 1 – 3 ]. Importantly, the influence of microbiota on intestinal functions and health depends on the constituent microbes. Alterations in microbial composition from those observed in “healthy” subjects are often defined as “dysbiotic,” which refers to communities that become perturbed in their composition such that they acquire pathogenic properties [ 4 – 6 ]. Given that the composition of the microbiota is critical for host health, it is significant that the intestinal microbial community is generally stable despite the highly dynamic internal environment of the intestinal tract [ 7 , 8 ], which experiences disruptions such as influxes of ingested matter, host secretion and epithelial cell turnover, and coordinated outward flow of material. How microbial community stability is achieved amid these constant perturbations is unknown. Hosts with impaired intestinal motility can develop dysbiosis and intestinal pathology [ 9 , 10 ], which suggests a profound role for the ENS in constraining microbiota composition. Here, we explore how the ENS shapes the ecology of the intestine, and we address key questions about the assembly of dysbiotic microbial communities, their functional properties, and strategies for their treatment—three aspects of dysbiosis that have been challenging to address from observational studies in humans. Our analysis reveals how, without ENS constraint, imbalances in pro- and anti-inflammatory members of the microbiota can drive intestinal pathology.

Results

Loss of sox10 results in intestinal bacterial overgrowth The complete loss of ENS in sox10t3 mutants (S1 Fig) results in defective intestinal motility [21]. Given the connection between altered intestinal motility and small intestinal bacterial overgrowth, we hypothesized that functional consequences of these mutants would include changes to intestinal ecology and alterations in resident microbial populations. To visualize the abundance and distribution of bacteria along the length of the intestine, we used fluorescent in situ hybridization (FISH). In sox10t3 mutants, we noted large populations of bacteria throughout the intestine, with marked accumulations of bacteria at the esophageal-intestinal junction (Fig 1A and 1B), a location not typically heavily colonized with bacteria. We also quantified the total number of colony-forming units (CFU) per intestine and found that sox10t3 mutants had a significantly higher bacterial load (Fig 1C). These results suggest that sox10t3 mutants experience bacterial overgrowth, which is consistent with defective intestinal transit. Defective intestinal transit has been observed in mutants in another allele, sox10m241, which have intestinal peristalsis but do not clear ingested fluorescent beads as well as WTs [25]. To demonstrate delayed intestinal transit in sox10t3 mutants, we adapted a previous single color assay [26] into a two-color intestinal transit assay (S1 Fig). The delayed transit and impaired clearance we observed in sox10 mutants likely contribute to bacterial overgrowth within their intestines. For the work described in this manuscript, we use sox10t3 mutants, hereafter referred to as sox10 mutant or sox10-. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.2000689.g001

The sox10- intestine exhibits increased neutrophil response and epithelial cell proliferation We next asked whether the bacterial overgrowth phenotype in sox10- resulted in signs of intestinal inflammation. Thus, we quantified intestinal neutrophil populations, a marker of inflammation, in cohoused WTs and sox10 mutants by staining for the neutrophil-specific enzyme myeloid peroxidase. At 6 d post fertilization (dpf), intestinal neutrophil accumulation in sox10 mutants was significantly increased compared to WTs (Fig 1D and 1E). Notably, sox10 mutants exhibited a much greater variation in intestinal neutrophil accumulation (0–18; n = 30) compared to WT siblings (0–7; n = 31); some sox10 mutants had intestinal neutrophil levels similar to WTs, whereas others had significantly elevated neutrophil populations. Intestinal neutrophil accumulation under homeostatic conditions in WT fish requires the pro-inflammatory tumor necrosis factor (TNF) pathway [27,28]. The increased neutrophil response in sox10 mutants also depends on this pathway, as inhibiting expression of the TNF receptor using an antisense morpholino [27,28] abolished the increased neutrophil response (Fig 1D and 1E). Another indicator of intestinal pathology is epithelial cell proliferation. At 6 dpf, sox10 mutants had markedly increased intestinal cell proliferation relative to cohoused WT animals. Unlike the normal intestinal epithelial cell proliferation response to microbiota, which is TNF independent [29], we found that elevated cell proliferation in the sox10 mutant intestine was TNF dependent (Fig 1F), suggesting that this was an inflammation-dependent pathological response.

The intestinal microbiota of sox10 mutants is necessary and sufficient to induce a hyper-inflammatory state To determine whether the intestinal microbiota of sox10- hosts is necessary to induce the increased intestinal neutrophil response, we derived sox10 mutants and their WT siblings germ free (GF). We found that GF sox10 mutants have a low neutrophil population, indistinguishable from their WT siblings (Fig 2A). To determine if the microbial community established in sox10 mutants is sufficient to induce inflammation, we performed an experiment in which we transferred microbiota from sox10 mutants into WTs. As donors, we used microbial communities from conventionally raised (CV) WT, sox10 mutant, or WT intestinal alkaline phosphatase morpholino (iap MO)-injected larvae. iap MO-injected fish are hypersensitive to lipopolysaccharide and thus develop elevated intestinal inflammation without evidence of dysbiosis [27]. These fish serve as control for the possibility that nonbacterial factors such as host pro-inflammatory cytokines rather than microbial derived factors cause transmissible intestinal inflammation (Fig 2B) [30]. At 6 dpf, for each separate group (WT, sox10-, and iap MO), we dissected, pooled, and homogenized the donor intestines. As a negative control, we included transplantation from homogenized intestines of GF fish. The homogenate from each group was inoculated into flasks housing GF 4 dpf WT fish (Fig 2C). We found that inoculation with microbes from sox10 mutants was sufficient to induce elevated intestinal inflammation in WTs as compared to inocula from GF, CV WT, or CV iap MO fish, none of which induced intestinal inflammation (Fig 2D). To test whether the capacity of sox10 mutant microbiota to induce elevated neutrophils was due to increased bacterial load, we transplanted 5× CV WT microbes, which corresponded to the bacterial load of sox10 mutant transplants. This larger inoculum did not induce more intestinal inflammation (S2 Fig), which indicates that the microbial community assembled in sox10- hosts is functionally distinct from WT microbiota and is sufficient to induce inflammation in fish with a normal, functional ENS. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.2000689.g002

Bacterial overgrowth does not explain increased intestinal neutrophil response sox10 mutants exhibit a wide range of intestinal neutrophil populations (Figs 1D and 2A) as well as variation in bacterial load (Fig 1B). Therefore, we asked whether intestinal neutrophil abundance corresponded to increased bacterial abundance. We used transgenic sox10 mutant hosts expressing green fluorescent protein (GFP) under control of the neutrophil-specific mpx promoter to quantify both neutrophil population and intestinal bacterial load in individual fish (Fig 3A). When we compared sox10 mutants that fell in the bottom half of neutrophil response (“sox10- low”) or in the top half of neutrophil response (“sox10- high”) to WTs, we found that all sox10 mutants, regardless of neutrophil level, carried significantly higher bacterial loads than WTs (Fig 3B). Thus, impaired intestinal clearance (S1 Fig) leads to an increased bacterial load; however, the bacterial overgrowth per se in sox10- does not drive an increased intestinal neutrophil response. We further characterized the pro-inflammatory signature of the sox10- high- and low-neutrophil subsets by monitoring expression of a panel of immune genes in the intestine (Fig 3C). These results aligned with our observations of the neutrophil population, as the sox10- high-neutrophil subset had elevated levels of mpx, saa, and tnfα expression compared to WT and the sox10- low-neutrophil subset (Fig 3C); however, the increase in saa transcription was the only one to reach statistical significance. Consistent with the significantly elevated intestinal neutrophil response in these samples, saa is known to mediate intestinal neutrophil behavior stimulated by microbes [31]. Collectively, our results suggest that a pro-inflammatory compositional change occurs in the microbial community of a subset of sox10 mutants. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.2000689.g003