Evidence for a core gut microbiota in the zebrafish

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status

Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations

Animal hosts must co-exist with beneficial microbes while simultaneously being able to mount rapid, non-specific, innate immune responses to pathogenic microbes. How this balance is achieved is not fully understood, and disruption of this relationship can lead to disease. Excessive inflammatory responses to resident microbes are characteristic of certain gastrointestinal pathologies such as inflammatory bowel disease (IBD). The immune dysregulation of IBD has complex genetic underpinnings that cannot be fully recapitulated with single-gene-knockout models. A deeper understanding of the genetic regulation of innate immune responses to resident microbes requires the ability to measure immune responses in the presence and absence of the microbiota using vertebrate models with complex genetic variation. Here, we describe a new gnotobiotic vertebrate model to explore the natural genetic variation that contributes to differences in innate immune responses to microbiota. Threespine stickleback, Gasterosteus aculeatus, has been used to study the developmental genetics of complex traits during the repeated evolution from ancestral oceanic to derived freshwater forms. We established methods to rear germ-free stickleback larvae and gnotobiotic animals monoassociated with single bacterial isolates. We characterized the innate immune response of these fish to resident gut microbes by quantifying the neutrophil cells in conventionally reared monoassociated or germ-free stickleback from both oceanic and freshwater populations grown in a common intermediate salinity environment. We found that oceanic and freshwater fish in the wild and in the laboratory share many intestinal microbial community members. However, oceanic fish mount a strong immune response to residential microbiota, whereas freshwater fish frequently do not. A strong innate immune response was uniformly observed across oceanic families, but this response varied among families of freshwater fish. The gnotobiotic stickleback model that we have developed therefore provides a platform for future studies mapping the natural genetic basis of the variation in immune response to microbes

The enteric nervous system promotes intestinal health by constraining microbiota composition

<div><p>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, <i>sox10</i>, 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.</p></div

Intestinal microbiota are necessary and sufficient to induce increased intestinal neutrophil accumulation in <i>sox10</i> mutants.

<p><b>(A)</b> Quantification of intestinal neutrophil number per 140 μm of distal intestine. Neutrophil accumulation was inhibited when <i>sox10</i> mutants were raised GF compared to CV controls. <i>n</i> > 21 per condition. <b>(B)</b> Schematic of fish used as donors in the transmission experiment. Intensity of red indicates level of intestinal inflammation. <b>(C)</b> Schematic of the experimental protocol. Intestines of GF, CV WT, <i>sox10</i> mutants, or <i>iap</i> 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. <b>(D)</b> Transfer of intestinal microbes from inflamed intestines of <i>sox10</i> mutants causes increased intestinal neutrophil number in WTs. <i>n</i> ≥ 10, *<i>p</i> < 0.05, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001, ANOVA with Tukey’s range test. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000689#pbio.2000689.s002" target="_blank">S2 Fig</a>.</p

Proposed model of <i>sox10</i> mutant intestinal pathology.

<p><i>sox10</i> mutants have altered intestinal motility and an increased bacterial load. Given the role of the ENS in intestinal function, <i>sox10</i> mutants likely also experience alterations in epithelial secretion and permeability, although these phenotypes are yet to be examined. <i>sox10</i> 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 <i>Vibrio</i> lineage and reduction of the <i>Escherichia</i> lineage. We do not yet know what determines which bacterial community assembles in <i>sox10</i> 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.</p

Coefficients of regression analysis of <i>Escherichia</i>, <i>Vibrio</i>, and intestinal neutrophil number.

<p>Coefficients of regression analysis of <i>Escherichia</i>, <i>Vibrio</i>, and intestinal neutrophil number.</p

Inflamed intestines are rescued by anti-inflammatory bacterial isolates or transplantation of WT ENS into <i>sox10</i> mutants.

<p><b>(A)</b> Addition of a representative <i>Escherichia</i> isolate, <i>E</i>. <i>coli</i> HS, to CV <i>sox10</i> mutants reduces intestinal neutrophil accumulation. Monoassociation of <i>sox10</i> mutants with <i>E</i>. <i>coli</i> HS does not increase neutrophil level over that observed in GF zebrafish. <i>n</i> > 20, from at least three independent experiments. <b>(B)</b> Correlation between absolute abundance of <i>E</i>. <i>coli</i> HS and log₁₀(intestinal neutrophil number + 1) in experiments with added <i>E</i>. <i>coli</i> HS. Linear regression analysis with 95% confidence intervals. For A, B: <i>n</i> > 35, from three to six independent experiments. <b>(C)</b> Representative images of distal intestine from WT, <i>sox10</i>^<i>-</i>, and <i>sox10</i>^<i>-</i> rescued by WT ENS precursor transplantation. Anti-ElavI1–labeled enteric neurons are white (white arrow); neutrophils are black (black arrow). Scale bar = 100 μm. <b>(D)</b> Quantification of intestinal neutrophil number per 140 μm of distal intestine. <i>n</i> > 6 for all conditions, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ****<i>p</i> < 0.0001, ANOVA with Tukey’s range test. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000689#pbio.2000689.s004" target="_blank">S4 Fig</a>.</p

<i>sox10</i> mutants experience bacterial overgrowth and physiological indications of dysbiosis.

<p><b>(A)</b> Schematic representation of the location and orientation of images in B and D. <b>(B)</b> Representative images of the panbacterial population by FISH on the esophageal-intestinal junction of WT (left) and <i>sox10</i>^<i>-</i> (right) fish. Blue, DNA; red, eubacteria. <b>(C)</b> Quantification of bacterial colonization level in <i>sox10</i> mutants and WT siblings. <b>(D)</b> Representative images of WT, <i>sox10</i> mutant, and tumor necrosis factor receptor (<i>tnfr</i>) morpholino (MO) injected larvae of both genotypes. Arrowhead indicates neutrophil. <b>(E)</b> Quantification of intestinal neutrophil number per 140 μm of distal intestine. <b>(F)</b> 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. <i>n</i> > 15 per group, *<i>p</i> < 0.05, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001, ANOVA with Tukey’s range test. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000689#pbio.2000689.s001" target="_blank">S1 Fig</a>. Scale bars = 50 μm.</p

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

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