S928 Enteropathogenic Infections Modulate Intestinal Serotonin Transporter (SERT) Function by Activating Toll-Like Receptor 2 (TLR-2) in Crohn’s Disease

Abstract

Introduction: Availability of intestinal serotonin (5-HT) is dependent on serotonin transporter (SERT), which uptakes 5-HT and facilitates its degradation. Interestingly, Toll-like receptor 2 (TLR-2) is co-localized with 5-HT, which suggests a possible impact of neuroendocrine cells in the inflammatory response through TLR-2 activation. Whether modulating SERT activity and intestinal 5-HT accumulation contributes to tissue injury caused by enteropathogenic infections is unknown. Methods: Serum 5-HT levels were measured in 80 CD patients and 40 healthy control subjects. Additionally, fully differentiated Caco-2 monolayers were infected with Mycobacteria paratuberculosis (MAP), L. monocytogenes or M. smegmatis in the presence of exogenous 5-HT at different concentrations. Cells were harvested and used for measuring SERT activity, RNA isolation followed by RT-PCR, protein quantification, and tissue damage markers (DHE, LDH, GSH and MDA). TLR-2 intracellular signaling pathways were assessed by pre-incubating Caco-2 monolayers with selective blockers of ERK, cAMP/PKA, p38 MAPK, and 5-HT3 receptors. Results: Serum 5-HT levels were significantly higher among CD patients in comparison to healthy subjects [424.14 ± 9.42 ng/mL vs 211.69 ± 8.11 ng/mL, respectively]. TLR-2 activation by enteropathogenic bacteria inhibited SERT activity in the presence of exogenous 5-HT by up to 50%. These effects were increasing gradually over 72 hours, and MAP infection had the greatest effect on SERT inhibition when cells were exposed to 5-HT in a concentration dependent manner. Additionally, inhibition of SERT activity was accompanied with higher level of pro-inflammatory cytokines (TNF-α, IL-6, IL-8) and oxidative stress markers (DHE, LDH and MDA), whereas SERT expression and protein level were downregulated. Consequently, inhibition of TLR-2 and p38 MAPK pathways or blocking 5-HT3 restored SERT activity and reduced the production of pro-inflammatory cytokines by about 2 folds, which was reflected on downregulating oxidative stress and tissue damage markers. Conclusion: The involvement of TLR-2 in intestinal pathology might be concluded not only from its innate immune role, but also from its effect on modulating the intestinal serotonergic response. Ultimately, regulating the intestinal serotonergic system could provide therapeutic approaches in enteropathogenic infections, which will help in understanding the gut-microbiome-brain connection.Figure 1.: Proposed mechanism for increasing intestinal and serum 5-HT among CD patients. Enterochromaffin cells release 5-HT into the intestinal lumen. Under normal conditions, SERT activity is able to reuptake 5-HT back into the intracellular compartment, which regulates intestinal 5-HT availability. However, enteropathogenic infections cause inflammation by activating TLR-2, which reduces SERT expression and function, leading to accumulation of 5-HT and disruption of the intestinal tight junctions. This results in paracellular movement of 5-HT across the intestinal epithelium to the blood vessels, where it can cause further systemic complications.