Gastroenterology

Gastroenterology

Volume 159, Issue 1, July 2020, Pages 200-213.e8
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice

https://doi.org/10.1053/j.gastro.2020.03.050Get rights and content

Background & Aims

The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice.

Methods

We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creERT2:tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creERT2:tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry.

Results

Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice.

Conclusions

In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors.

Section snippets

Animals

All mice used for the experiments were between the ages of 8 and 16 weeks and are designated as adult mice. Experimental protocols were approved by the Johns Hopkins University’s Animal Care and Use Committee in accordance with the guidelines provided by the National Institutes of Health. Details of wild-type and transgenic mice used for the experiments are presented in Supplementary Material.

Antibiotic Treatment

Eight- to 16-week-old C57/BL6 male mice were treated either with ampicillin (0.5 g/L) through drinking

Neurogenesis in the Adult Colonic Myenteric Plexus Is Driven by Nestin+-neuronal Precursors

We have previously identified Nestin as a marker of the ENPCs in the small intestine and showed that a subset of Nestin+ cells give rise to neurons in vivo.21 Nestin+ cells also exist in the colon and surround the neurons within the colonic myenteric ganglion (Supplementary Figure 1A). In inducible Cre transgenic mice (Nestin-creERT2:tdTomato) the pan-neuronal marker HuC/D did not show overlap between tdTomato-expressing cells and neurons at 12 hours after tamoxifen induction (Supplementary

Discussion

Our understanding of the maintenance of the adult ENS is limited and until recently constrained by the dogma that it is a static system with a stable population of neurons through most of life.4 The recent demonstration that the ENS of the small intestine is in a state of constant turnover with active neurogenesis required to replace continually dying neurons21 has highlighted the vulnerability of this system and the importance of understanding the underlying mechanisms that maintain it. The

CRediT Authorship Contributions

Shadi Yarandi, MD, assistant professor (Formal analysis: Equal; Funding acquisition:

Equal; conceived the study, designed the research study, conducted the experiments:

Equal). Subhash Kulkarni, PhD, assistant professor (designed the research studies, drafted the manuscript: Equal). Monalee Saha, PhD, postdoctoral research fellow (conducted the experiments: Equal). Kristyn E. Sylvia, PhD (Formal analysis: Equal; Software: Equal). Cynthia L. Sears, MD, Professor (Funding acquisition: Supporting;

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    Conflict of interest The authors disclose no conflicts.

    Funding This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01DK080920 (to Pankaj Jay Pasricha); Bloomberg Philanthropies (to Cynthia L. Sears); Grant P30 DK089502 (to Shadi S. Yarandi); and a grant from the Amos family (to Pankaj Jay Pasricha).

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