Human derived Gut Microbiota Modulates Colonic Secretion in Mice by Regulating 5‐HT3 Receptor Expression

Abstract

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‐HT3 and 5‐HT4 receptors. In this study we investigated whether gut microbiota modulate 5‐HT3R and 5‐HT4R expression and host secretory response to 5‐HT.MethodsWe 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‐HT3R 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‐HT3R 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.Results5‐HT induced ΔIsc was significantly higher in GF than HM mice (218; 184–274 vs 163; 132–182 μA/cm2, 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‐HT3R 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‐HT4R expression. 5‐HT3R 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/cm2, n=5, P = 0.02) mice, following application of 2‐methyl 5‐hydroxytryptamine hydrochloride (selective 5‐HT3R 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/cm2, n=6–8, P = 0.001) but not in HM mice (163; 132–182 vs 120; 107–150 μA/cm2, n=6, P = 0.24). To determine the expression of 5‐HT3R in mouse colon mucosa‐submucosa we used 5‐HT3–tagged GFP mice. IHC identified 5‐HT3R‐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/cm2, n=5, P = 0.008) is driven by epithelial 5‐HT3R, 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 5HT3R.ConclusionGut microbial colonization decreases host secretory response to 5‐HT by downregulating 5‐HT3R expression. Epithelial 5‐HT3R may be important in gut microbiota modulation of intestinal secretionSupport or Funding InformationNIH K08 DK