Intestinal Serotonin Transporter Inhibition by Toll-Like Receptor 2 Activation. A Feedback Modulation

Abstract

TLR2 is a microbiota recognition receptor that has been described to contribute to intestinal homeostasis and to ameliorate inflammatory intestinal injury. In this context, serotonin (5-HT) has shown to be an essential intestinal physiological neuromodulator that is also involved in intestinal inflammatory diseases. Since the interaction between TLR2 activation and the intestinal serotoninergic system remains non-investigated, our main aim was to analyze the effect of TLR2 on intestinal serotonin transporter (SERT) activity and expression and the intracellular pathways involved. Caco-2/TC7 cells were used to analyze SERT and TLR2 molecular expression and SERT activity by measuring 5-HT uptake. The results showed that apical TLR2 activation inhibits SERT activity in Caco-2/TC7 cells mainly by reducing SERT protein level either in the plasma membrane, after short-term TLR2 activation or in both the plasma membrane and cell lysate, after long-term activation. cAMP/PKA pathway appears to mediate short-term inhibitory effect of TLR2 on SERT; however, p38 MAPK pathway has been shown to be involved in both short- and long-term TLR2 effect. Reciprocally, 5-HT long-term treatment yielded TLR2 down regulation in Caco-2/TC7 cells. Finally, results from in vivo showed an augmented intestinal SERT expression in mice Tlr2-/-, thus confirming our inhibitory effect of TLR2 on intestinal SERT in vitro. The present work infers that TLR2 may act in intestinal pathophysiology, not only by its inherent innate immune role, but also by regulating the intestinal serotoninergic system.

NOD2 Modulates Serotonin Transporter and Interacts with TLR2 and TLR4 in Intestinal Epithelial Cells

“…This suggests that long-term NOD2 activation may decrease SERT expression via transcriptional and/or post-transcriptional mechanisms, offering an explanation for the 5-HT uptake reduction. In agreement with our results, other PRRs have also demonstrated inhibition of SERT activity with [21,22] or without [30] alteration of SERT expression. The activation of NOD2, with low concentrations of MPD, induces an inhibitory effect observed only in the short (30 minutes) and medium terms (6 hours), suggesting that a brief stimulation of NOD2 induces a rapid inhibition of SERT activity, increasing 5-HT extracellular availability to promote a fast inflammatory response.…”

“…Similarly, previous results have concluded that TLR2 (either TLR2/1 or TLR2/6) and TLR4 activation decrease significantly 5-HT uptake in Caco-2/TC7 cells [21,22]. Moreover, the over-expression of both receptors has been implicated in IBD [14].…”

“…However, when we compared high 5-HT concentration (10 -4 M), which occurs in inflammatory pathologies [2] with lower (5-HT, 10 -8 M) physiological conditions no significant differences were observed in NOD2 expression, suggesting that a NOD2 induced extracellular 5-HT increase would not modify the observed effect. Previous studies, however, have demonstrated feedback regulation between TLR2 and 5-HT [21], where a high 5-HT concentration decreases TLR2 expression, modulating TLR2 responses. Our results demonstrate that 5-HT, an intestinal non-immunological modulator, could regulate the expression of innate immune receptors in different ways and may contribute to modulation of the intestinal response to inflammation.…”

“…After this time, samples were centrifuged for 10 minutes at 950 g. The supernatant was centrifuged (30 minutes, 40, 000 g) and the pellet was re-suspended in a phosphate buffer (10 mM KH 2 PO 4 /K 2 HPO 4 pH 6.8) to obtain the brush border membrane-enriched fraction. Mice intestinal samples (ileum and colon) were cleaned and homogenized using ultra-turrax in Tris-mannitol buffer pH 7.1 [21]. Then, all of the samples were disrupted using a PotterElvehjem homogenizer with a PTFE pestle and the obtained suspension was homogenized and disrupted by sonication (fifteen 1-s bursts, 60W).…”

“…Recent works have shown that both TLR2 and TLR4 activation downregulates intestinal serotonin transporter activity and expression [21,22]. However, the implication of other PRRs, such as NOD2, in the intestinal serotonergic system remains unknown.…”