Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice

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 rRNA 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) andd 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 nNOS 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 ENPCs expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from ENPCs to approximately 10%, compared to 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.

[1]  Anthony M. Haag,et al.  Neonatal Antibiotics Disrupt Motility and Enteric Neural Circuits in Mouse Colon , 2019, Cellular and molecular gastroenterology and hepatology.

[2]  Danielle Hunt,et al.  Murine macrophage TLR2-FcγR synergy via FcγR licensing of IL-6 cytokine mRNA ribosome binding and translation , 2018, PloS one.

[3]  P. Trosvik,et al.  Individuality and convergence of the infant gut microbiota during the first year of life , 2018, Nature Communications.

[4]  F. Bäckhed,et al.  Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks , 2018, Proceedings of the National Academy of Sciences.

[5]  Hongjun Song,et al.  Adult enteric nervous system in health is maintained by a dynamic balance between neuronal apoptosis and neurogenesis , 2017, Proceedings of the National Academy of Sciences of the United States of America.

[6]  P. Pasricha,et al.  Age-dependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system , 2017, Gut.

[7]  D. Chaussabel,et al.  Inherited human IRAK-1 deficiency selectively impairs TLR signaling in fibroblasts , 2017, Proceedings of the National Academy of Sciences.

[8]  C. Mittal,et al.  Remnant lipoprotein size distribution profiling via dynamic light scattering analysis. , 2016, Clinica chimica acta; international journal of clinical chemistry.

[9]  M. Neunlist,et al.  TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide , 2016, Journal of Neuroinflammation.

[10]  Paul J. McMurdie,et al.  DADA2: High resolution sample inference from Illumina amplicon data , 2016, Nature Methods.

[11]  S. Srinivasan,et al.  Intestinal Dysbiosis Contributes to the Delayed Gastrointestinal Transit in High-Fat Diet Fed Mice , 2016, Cellular and molecular gastroenterology and hepatology.

[12]  Eric S. Lander,et al.  Natural history of the infant gut microbiome and impact of antibiotic treatment on bacterial strain diversity and stability , 2015, Science Translational Medicine.

[13]  Paul J. McMurdie,et al.  Bioconductor Workflow for Microbiome Data Analysis: from raw reads to community analyses , 2016, F1000Research.

[14]  Ulrich Bodenhofer,et al.  msa: an R package for multiple sequence alignment , 2015, Bioinform..

[15]  M. Schemann,et al.  Mechanical stress activates neurites and somata of myenteric neurons , 2015, Front. Cell. Neurosci..

[16]  N. Bódi,et al.  Gut region-dependent alterations of nitrergic myenteric neurons after chronic alcohol consumption. , 2015, World journal of gastrointestinal pathophysiology.

[17]  J. Bienenstock,et al.  The gut microbiome restores intrinsic and extrinsic nerve function in germ‐free mice accompanied by changes in calbindin , 2015, Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society.

[18]  M. Neunlist,et al.  Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system , 2014, Journal of Neuroinflammation.

[19]  James J Collins,et al.  Antibiotics and the gut microbiota. , 2014, The Journal of clinical investigation.

[20]  M. Merad,et al.  Crosstalk between Muscularis Macrophages and Enteric Neurons Regulates Gastrointestinal Motility , 2014, Cell.

[21]  M. Neunlist,et al.  Nutrient‐induced changes in the phenotype and function of the enteric nervous system , 2014, The Journal of physiology.

[22]  Jean M. Macklaim,et al.  Unifying the analysis of high-throughput sequencing datasets: characterizing RNA-seq, 16S rRNA gene sequencing and selective growth experiments by compositional data analysis , 2014, Microbiome.

[23]  J. Huizinga,et al.  Intestinal microbiota influence the early postnatal development of the enteric nervous system , 2014, Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society.

[24]  James R. Cole,et al.  Ribosomal Database Project: data and tools for high throughput rRNA analysis , 2013, Nucleic Acids Res..

[25]  A. Rosato,et al.  Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system. , 2013, Gastroenterology.

[26]  Mathieu Almeida,et al.  Dietary intervention impact on gut microbial gene richness , 2013, Nature.

[27]  Sarah L. Westcott,et al.  Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform , 2013, Applied and Environmental Microbiology.

[28]  P. Wipf,et al.  Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors , 2013, PloS one.

[29]  Susan Holmes,et al.  phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data , 2013, PloS one.

[30]  P. Pasricha,et al.  Ex Vivo Neurogenesis within Enteric Ganglia Occurs in a PTEN Dependent Manner , 2013, PloS one.

[31]  J. Bornstein,et al.  Effects of oxaliplatin on mouse myenteric neurons and colonic motility , 2013, Front. Neurosci..

[32]  J. Garthwaite,et al.  On the selectivity of neuronal NOS inhibitors , 2013, British journal of pharmacology.

[33]  J. Foster,et al.  The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse , 2013, Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society.

[34]  S. Srinivasan,et al.  Gut microbial products regulate murine gastrointestinal motility via Toll-like receptor 4 signaling. , 2012, Gastroenterology.

[35]  D. Sinderen,et al.  Gut microbiota composition correlates with diet and health in the elderly , 2012, Nature.

[36]  S. Yooseph,et al.  Analyses of the Microbial Diversity across the Human Microbiome , 2012, PloS one.

[37]  John B. Furness,et al.  The enteric nervous system and neurogastroenterology , 2012, Nature Reviews Gastroenterology &Hepatology.

[38]  S. Morrison,et al.  Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. , 2011, The Journal of clinical investigation.

[39]  C. Weber,et al.  Tight junction pore and leak pathways: a dynamic duo. , 2011, Annual review of physiology.

[40]  Klaus Peter Schliep,et al.  phangorn: phylogenetic analysis in R , 2010, Bioinform..

[41]  K. Ravid,et al.  Adenosine 2B receptors (A2BAR) on enteric neurons regulate murine distal colonic motility , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[42]  Les Dethlefsen,et al.  The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing , 2008, PLoS biology.

[43]  R. Knight,et al.  Evolution of Mammals and Their Gut Microbes , 2008, Science.

[44]  Yaniv Ziv,et al.  Toll-like receptors modulate adult hippocampal neurogenesis , 2007, Nature Cell Biology.

[45]  S. Akira,et al.  Activation of smooth muscle and myenteric plexus cells of jejunum via toll‐like receptor 4 , 2006, Journal of cellular physiology.

[46]  G. Taglialatela,et al.  Suppression of nNOS expression in rat enteric neurones by the receptor for advanced glycation end‐products , 2006, Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society.

[47]  Elisabeth M Bik,et al.  Molecular analysis of the bacterial microbiota in the human stomach. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[48]  M. Camilleri,et al.  Human enteric neuropathies: morphology and molecular pathology , 2004, Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society.

[49]  T. Hartung,et al.  Lipoteichoic Acid and Toll-like Receptor 2 Internalization and Targeting to the Golgi Are Lipid Raft-dependent* , 2004, Journal of Biological Chemistry.

[50]  D. Steindler,et al.  Neural stem and progenitor cells in nestin‐GFP transgenic mice , 2004, The Journal of comparative neurology.

[51]  M. Blaser,et al.  Long-Term Persistence of Resistant Enterococcus Species after Antibiotics To Eradicate Helicobacter pylori , 2003, Annals of Internal Medicine.

[52]  C. Gariepy Intestinal Motility Disorders and Development of the Enteric Nervous System , 2001, Pediatric Research.

[53]  S. Akira,et al.  Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. , 1999, Immunity.

[54]  Toku Takahashi,et al.  Nitrergic regulation of colonic transit in rats. , 1999, American journal of physiology. Gastrointestinal and liver physiology.

[55]  S. Korsmeyer,et al.  Enx (Hox11L1)-deficient mice develop myenteric neuronal hyperplasia and megacolon , 1997, Nature Medicine.

[56]  S. Snyder,et al.  Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. , 1991, Biochemical and biophysical research communications.