Rainey, John F.

The human gastrointestinal (GI) tract is home to trillions of microorganisms. There is dynamic interaction between the gut microbiota and the human host, but how gut microbiota modulates GI physiology is largely unknown. A recent study from our laboratory showed that human gut microbiota increases host serotonin (5‐hydroxytryptamine, 5‐HT) biosynthesis. 5‐HT is an important neurotransmitter and a paracrine messenger in the GI tract that increases intestinal secretion primarily through 5‐HT 3 and 5‐HT 4 receptors. In this study we investigated whether gut microbiota modulate 5‐HT 3 R and 5‐HT 4 R expression and host secretory response to 5‐HT. Methods We used proximal colon mucosa‐submucosa preparations from germ free (GF) and humanized (ex‐GF colonized with human bacteria for 4–5 weeks; HM) male mice. The change in short circuit current (ΔIsc) was determined in response to increasing concentrations of 5‐HT (0.003–300μM) on the submucosal side either alone or in the presence of 5‐HT 3 R antagonist ondansetron (100nM) in an Ussing chamber. Receptor expression was quantified using rt‐PCR (normalized to L32) and western blot (normalized to GAPDH). Immunohistochemistry (IHC) was used to determine localization of 5‐HT 3 R in the paraformaldehyde‐fixed 10μm sections from proximal colonic epithelium. Differences between groups were tested using Mann‐Whitney U (Mdn; IQ range) with P ‐value lower than 0.05 considered significant. Results 5‐HT induced ΔIsc was significantly higher in GF than HM mice (218; 184–274 vs 163; 132–182 μA/cm 2 , n=6, P = 0.04). To determine if this change was due to an alteration in 5‐HTR, we quantified receptor expression. We found significantly higher 5‐HT 3 R mRNA expression (1.82; 1.08–2.80 vs 1.00; 0.33–1.38, n=8–9, P = 0.046) in GF compared to HM mice, but no change in 5‐HT 4 R expression. 5‐HT 3 R protein expression was also significantly higher in GF mice compared to HM (4.62; 1.06–6.14 vs 1.00; 1–1.3, n= 11–12, P = 0.01) mice. There was a significantly higher ΔIsc in GF compared to HM (87.1; 51.7–149 vs 37.0; 19.5–44 μA/cm 2 , n=5, P = 0.02) mice, following application of 2‐methyl 5‐hydroxytryptamine hydrochloride (selective 5‐HT 3 R agonist) suggesting increased receptor expression augments secretory response. Furthermore, ondansetron significantly blocked 5‐HT induced maximum ΔIsc in GF mice (201; 189–260 vs 117.6; 92–149 μA/cm 2 , n=6–8, P = 0.001) but not in HM mice (163; 132–182 vs 120; 107–150 μA/cm 2 , n=6, P = 0.24). To determine the expression of 5‐HT 3 R in mouse colon mucosa‐submucosa we used 5‐HT 3 –tagged GFP mice. IHC identified 5‐HT 3 R‐GFP positive cells in the mouse colonic epithelium. To ascertain if the significant difference in 5‐HT‐induced ΔIsc between GF and HM mice (188; 152–200 vs 125; 99–139 μA/cm 2 , n=5, P = 0.008) is driven by epithelial 5‐HT 3 R, we measured the response in the presence of TTX (block neuronal transmission). TTX (500 nM) had no effect of ondansetron inhibition of 5‐HT‐induced ΔIsc, suggesting a role for epithelial 5HT 3 R. Conclusion Gut microbial colonization decreases host secretory response to 5‐HT by downregulating 5‐HT 3 R expression. Epithelial 5‐HT 3 R may be important in gut microbiota modulation of intestinal secretion Support or Funding Information NIH K08 DK

Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5‐hydroxytryptamine (5‐HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ‐free (GF) or humanized (HM; ex‐GF colonized with human gut microbiota). 5‐HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5‐HT synthesis; P < 0.01] and chromogranin A (neuroendocrine secretion; P < 0.01), with no effect on monoamine oxidase A (serotonin catabolism), serotonin receptor 5‐HT 4 , or mouse serotonin transporter. HM and CR mice also had increased colonic Tph1 protein ( P < 0.05) and 5‐HT concentrations (GF, 17 ± 3 ng/mg; HM, 25 ± 2 ng/mg; and CR, 35 ± 3 ng/mg; P < 0.05). Enterochromaffin (EC) cell numbers (cells producing 5‐HT) were unchanged. Short‐chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5‐HT production and homeostasis.—Reigstad, C. S., Salmonson, C. E., Rainey, III, J. F., Szurszewski, J. H., Linden, D. R., Sonnenburg, J. L., Farrugia, G., Kashyap, P. C. Gut microbes promote colonic serotonin production through an effect of short‐chain fatty acids on enterochromaffin cells. FASEB J. 29, 1395‐1403 (2015). www.fasebj.org