Mucosal Serotonin Reuptake Transporter Expression in Irritable Bowel Syndrome Is Modulated by Gut Microbiota Via Mast Cell-Prostaglandin E2

Abstract

Background & aims: Increased colonic serotonin (5-HT) level and decreased serotonin reuptake transporter (SERT) expression in irritable bowel syndrome (IBS) may contribute to diarrhea and visceral hypersensitivity. We investigated whether mucosal SERT is modulated by gut microbiota via a mast cell-prostaglandin E2 (PGE2) pathway.

Methods: C57Bl/6 mice received intracolonic infusion of fecal supernatant (FS) from healthy controls or patients with diarrhea-predominant irritable bowel syndrome (IBS-D). The role of mast cells was studied in mast cell-deficient mice. Colonic organoids and/or mast cells were used for in vitro experiments. SERT expression was measured by quantitative polymerase chain reaction and Western blot. Visceromotor responses to colorectal distension and colonic transit were assessed.

Results: Intracolonic infusion of IBS-D FS in mice caused an increase in mucosal 5-HT compared with healthy control FS, accompanied by ∼50% reduction in SERT expression. Mast cell stabilizers, cyclooxygenase-2 inhibitors, and PGE2 receptor antagonist prevented SERT downregulation. Intracolonic infusion of IBS-D FS failed to reduce SERT expression in mast cell-deficient (W/Wv) mice. This response was restored by mast cell reconstitution. The downregulation of SERT expression evoked by IBS FS was prevented by lipopolysaccharide (LPS) antagonist LPS from Rhodobacter sphaeroides and a bacterial trypsin inhibitor. In vitro LPS treatment caused increased cyclooxygenase-2 expression and PGE2 release from cultured mouse mast cells. Intracolonic infusion of IBS-D FS in mice reduced colonic transit, increased fecal water content, and increased visceromotor responses to colorectal distension. Ondansetron prevented these changes.

Conclusions: Fecal LPS acting in concert with trypsin in patients with IBS-D stimulates mucosal mast cells to release PGE2, which downregulates mucosal SERT, resulting in increased mucosal 5-HT. This may contribute to diarrhea and abdominal pain common in IBS.

Keywords: Colonic Mucosa; IBS; Mast Cells; SERT.

Figure 1.. Increased 5-HT level and decreased SERT expression in mouse colon induced by IBS-D patient fecal supernatant.

(A) 5-HT release from distal colonic mucosa in mice 18 h after intracolonic infusion of fecal supernatant (FS) from healthy controls (HC) or IBS-D patients. Results are expressed as mean ± SEM, *P < 0.01 from HC, Student t test, n = 6–9 in each group with duplicated measurements. (B) RT–PCR of SERT in distal colon from mice treated with intracolonic infusion of HC or IBS-D FS. Results are expressed as mean ± SEM, *P < 0.01 from HC, Student t test, n = 6–7 in each group. (C) Western blot of SERT in distal colon from mice treated with HC or IBS-D FS, quantified by densitometry analysis. Results are expressed as mean ± SEM, *P = 0.02 from HC, Student t test, n = 4 in each group. (D,E) Immunofluorescent staining for SERT was performed on sections of normal mouse colon. SERT staining is in green contrasting with blue counterstained nuclei. SERT is principally expressed on the surface area of colon mucosa, but not in the lamina propria. (F,G) Immunofluorescent staining for SERT and E-cadherin was performed on sections of normal mouse colon. SERT staining is in green and E-cadherin in red, contrasting with blue counterstained nuclei. SERT is principally expressed in the mature differentiated epithelial cells where E-cadherin is primarily localized. Immunofluorescent staining for SERT in normal (H) and IBS-D FS injected mouse colon (I). The average fluorescence intensity was lower in mouse colon after IBS-D FS treatment. (J) RT–PCR of TPH1 in distal colon from mice treated with HC or IBS-D FS. Results are expressed as mean ± SEM, *P = 0.29 from HC, Student t test, n = 10 in each group. (K) Western blot of TPH1 in distal colon from mice treated with HC or IBS-D FS, quantified by densitometry analysis. Results are expressed as mean ± SEM, *P = 0.26 from HC, Student t test, n = 4 in each group. (L and M) RT-PCR of Chag and MAOA in distal colon from mice treated with HC or IBS-D FS. Results are expressed as mean ± SEM. *P = 0.11 from HC for Chag, *P > 0.99 from HC for MAOA, Student t test, n = 6 in each group.

Figure 2.. Decreased colonic SERT expression induced by IBS-D fecal supernatant is mast cell–dependent.

(A) RT–PCR of SERT in murine colonic organoids alone or co-cultured with mast cells (MC), treated with fecal supernatant (FS) from HC or IBS-D patients. Results are expressed as mean ± SEM, two-way ANOVA, n = 6 in each group. (B) colonic SERT expression in response to intracolonic infusion of IBS-D FS in wild-type (WT), W/Wv, W/Wv mice reconstituted with bone marrow–derived MC from wild-type mice, and W/Wv mice reconstituted with bone marrow–derived MC from TLR deficient mice. Results are expressed as mean ± SEM, one-way ANOVA, n = 4–6 in each group. (C) Decreased colonic SERT expression evoked by intracolonic IBS-D FS infusion was abolished by ketotifen or cromolyn sodium pretreatment (30 min before infusion, IP). Results are expressed as mean ± SEM, one-way ANOVA, n = 4–7 in each group.

Figure 3.. PGE2 is the mast cell mediator regulating colonic SERT expression in response to IBS-D supernatant.

(A) SERT expression in colonic mucosa of mice treated with HC or IBS-D FS with or without pretreatment (30 min before infusion, ip) with nafamostat mesylate, ebastine, or PF-04418948. Results are expressed as mean ± SEM, one-way ANOVA, n = 3–7 in each group. (B) SERT expression in colonic mucosa of mice treated with intracolonic infusion of PBS, SLIGKV-NH2, histamine, or PGE2 for 18 h. Results are expressed as mean ± SEM, one-way ANOVA, n = 4–7 in each group. (C) Western blot of SERT in distal colon from mice treated with intracolonic infusion of HC FS, IBS-D FS, or PGE2. Results are expressed as mean ± SEM, one-way ANOVA, n = 5 in each group. (D) SERT expression in murine colonic organoids after treatment with PBS, SLIGKV-NH2, histamine, or PGE2 for 18 h. Results are expressed as mean ± SEM, one-way ANOVA, n = 4–9 in each group. (E) IBS-D FS–induced SERT downregulation in the ex vivo murine colonoid–mast cell co-culture system was reversed by PF-04418948 (administered simultaneously with IBS-D FS to culture media). Results are expressed as mean ± SEM, one-way ANOVA, n = 3–7 in each group. (F) IBS-D FS–induced SERT downregulation in the ex vivo murine colonoid–mast cell co-culture system was reversed by indomethacin or celecoxib (administered simultaneously with IBS-D FS to culture media). Results are expressed as mean ± SEM, one-way ANOVA, n = 4 in each group. (G) Pretreatment with specific antagonists of EP1 (ONO-8130, ip., 10 mg/kg), EP3 (L-798106, ip., 10 mg/kg), and EP4 (ONO-AE3–208, ip., 10 mg/kg) failed to prevent downregulation of SERT induced by IBS-D FS (2way ANOVA with Dunnett’s Post Hoc test with vehicle group, n=3, P values as marked). In contrast, blocking PGE2 receptor EP2 by the EP2-specific antagonist PF-04418948 (10 mg/kg, ip) significantly diminished the inhibitory effect of IBS-D FS on colonic SERT expression (2way ANOVA with Dunnett’s Post Hoc test with vehicle group, P = 0.01, n = 3).

Figure 4.. LPS is a required component in the IBS-D fecal supernatant for mast cell activation and SERT downregulation.

(A) Intracolonic infusion of FS from IBS-D patients who responded to 4-wk low-FODMAP treatment resulted in increased SERT expression in mouse colon compared to infusion of FS from the same patients before low-FODMAP. Results are expressed as individual values, Wilcoxon signed-rank test, n = 8 with duplicated measurements. (B) SERT expression in colonic mucosa of mice treated with HC FS, IBS-D FS, pretreatment with LPS-RS (30-min intracolonic infusion) followed by IBS-D FS or IBS-D FS pretreated with endotoxin removal. Results are expressed as mean ± SEM, one-way ANOVA, n = 4–7 in each group. (C) SERT expression in murine colonic organoids co-cultured with mast cells, treated with PBS, IBS-D FS, or IBS-D FS pretreated with endotoxin removal. Results are expressed as mean ± SEM, one-way ANOVA, n = 6 in each group. (D) RT–PCR of COX-2 in cultured bone marrow–derived mast cells treated with LPS for 4 h (0, 3, 10 μg/mL). Results are expressed as mean ± SEM. one-way ANOVA, n = 6–8 in each group. (E) Release of PGE2 from mast cells treated with LPS for 4 h (0, 3, 10 μg/mL). Results are expressed as mean ± SEM. one-way ANOVA, n = 5–6 in each group.

Figure 5.. IBS-D fecal supernatant causes diarrhea and visceral hypersensitivity in a mast cell–, PGE2-, and 5-HT–dependent manner.

(A) Colonic transit time in mice treated with intracolonic infusion of HC or IBS-D FS, with or without pretreatment (30 min before infusion, IP) with ondansetron, cromolyn sodium, or PF-0441894. Results are expressed as mean ± SEM, one-way repeated measures ANOVA, n = 4. (B) Stool water weight (left) and percentage (right) of mice treated with intracolonic infusion of HC or IBS-D FS, with or without pretreatment (30 min before infusion, IP) with ondansetron, cromolyn sodium, or PF-0441894. Results are expressed as mean ± SEM, one-way repeated measures ANOVA, n = 4. (C) VMR to CRD in mice treated with intracolonic administration of HC or IBS-D FS, with or without pretreatment (30 min before infusion, IP) with ondansetron, cromolyn sodium, or PF-0441894. Results are expressed as mean ± SEM, *P < 0.05 from HC, ^#P < 0.05 from IBS-D, two-way repeated measures ANOVA, n = 6.

Figure 6.. Mucosal SERT is modulated by gut microbiota via mast cell–COX–PGE2 pathway in IBS.

Proinflammatory bacterial products such as LPS and trypsin, among others, are increased in the gut lumen of IBS patients. On entry to the lamina propria, these mediators activate the mucosal mast cells to increase COX-dependent PEG2 synthesis and release, which acts on EP2 receptors on the epithelial cells to downregulate SERT expression, leading to increased mucosal 5-HT availability, which stimulates motility, epithelial secretion, and visceral sensitivity via intrinsic and extrinsic sensory neuronal networks.