Elsevier

Cytokine

Volume 61, Issue 3, March 2013, Pages 778-784
Cytokine

IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells

https://doi.org/10.1016/j.cyto.2013.01.012Get rights and content

Abstract

Serotonin is a neuromodulator mainly synthesized by intestinal enterochromaffin cells that regulate overall intestinal physiology. The serotonin transporter (SERT) determines the final serotonin availability and has been described as altered in inflammatory bowel diseases. IL-10 is an anti-inflammatory cytokine that is involved in intestinal inflammatory processes and also contributes to intestinal mucosa homeostasis. The regulation of SERT by pro-inflammatory factors is well known; however, the effect of IL-10 on the intestinal serotoninergic system mediated by SERT remains unknown. Therefore, the aim of the present study is to determine whether IL-10 affects SERT activity and expression in enterocyte-like Caco-2 cells. Treatment with IL-10 was assessed and SERT activity was determined by 5-HT uptake. SERT mRNA and protein expression was analyzed using quantitative RT-PCR and western blotting. The results showed that IL-10 induced a dual effect on SERT after 6 h of treatment. On one hand, IL-10, at a low concentration, inhibited SERT activity, and this effect might be explained by a non-competitive inhibition of SERT. On the other hand, IL-10, at a high concentration, increased SERT activity and molecular expression in the membrane of the cells. This effect was mediated by the IL-10 receptor and triggered by the PI3K intracellular pathway. Our results demonstrate that IL-10 modulates SERT activity and expression, depending on its extracellular conditions. This study may contribute to understand serotoninergic responses in intestinal pathophysiology.

Highlights

► Serotonin and IL-10 are involved in intestinal homeostasis and pathophysiology. ► Serotonin transporter regulation by IL-10 was studied in Caco-2 cells. ► IL-10 at a low concentration inhibited SERT activity. ► IL-10 at a high concentration increased SERT activity and membrane expression. ► IL-10 modulates SERT and serotonin availability depending on intestinal conditions.

Introduction

The gastrointestinal (GI) tract is the main source of endogenous serotonin (5-hydroxytryptamine, 5-HT). 5-HT plays a critical role in the regulation of overall intestinal physiology [1], [2], [3], [4], and its effects depend on 5-HT availability, which is mainly regulated by the serotonin transporter (SERT) located in the enterocytes [5].

The alteration of the serotoninergic system has been described to be involved in the origin and/or prevalence of chronic GI pathologies such as Irritable Bowel Syndrome (IBS) [6], [7] and Inflammatory Bowel Diseases (IBD) [8]. Actually, numerous studies have demonstrated that 5-HT levels are altered in experimental intestinal inflammation and in IBD [9], [10], [11], and high levels of 5-HT have also been described to be involved in several inflammatory and diarrheal conditions [12].

Down-regulation of SERT has been implicated in the pathophysiology of various functional gut disorders. Thus, SERT expression has shown to be reduced in experimentally induced colitis [13] and in the gut of patients with ulcerative colitis and IBS [7], [14]. Accordingly, transgenic mice with targeted deletion of SERT frequently exhibit diarrhea interspersed with transient constipation [15]. In this context, recent results have demonstrated that pro-inflammatory factors affect the GI serotoninergic system, leading to a decrease in SERT activity [16], [17], [18] and, consequently, to an increase of the 5-HT extracellular level in the intestine. In addition, many results support the concept that 5-HT is a potent immunoregulator [19], [20].

The effect of pro-inflammatory factors on SERT has been described in previous research. However, the role of anti-inflammatory factors on the regulation of SERT remains unknown.

Interleukin-10 (IL-10) has been demonstrated to be an anti-inflammatory factor, in part through the inhibition of the microbially induced production of pro-inflammatory cytokines, which are known to play a role in the pathogenesis of IBD [21]. Supporting evidence for the role of IL-10 in inflammation is derived from studies of enterocolitis in mice deficient in IL-10 or harboring mutated IL-10, [22], [23] as well as in rats with experimental colitis [24].

In relation to 5-HT activity, a recent study has concluded that colitis associated with IL-10 deficiency is enhanced when it is combined with a SERT deficiency [25]. However, the effect of IL-10 on SERT activity has not been analyzed.

Consequently, the aim of the present work is to determine whether IL-10 affects SERT activity and expression in intestinal epithelial cells. The present study has been carried out in human enterocyte-like Caco-2 cells, which express SERT endogenously [26]. These cells were treated with IL-10, and different experimental conditions were assayed.

Section snippets

Reagents and antibodies

The following drugs and substances were used (abbreviations and suppliers in parentheses): serotonin (5-HT), 5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(4-piperidinyl) imidazole trihydrochloride (SB 220025), 5-[4-(2-Pyridylsulfamoyl)phenylazo] salicylic acid (sulfasalazine), pyrrolidinedithiocarbamate (PDTC), and specific primers (Sigma–Aldrich, St. Louis, MO). In addition, wortmannin (Jena Bioscience, Jena, Germany), [3H]-5-HT (specific activity 28 Ci/mmol; Perkin–Elmer, Boston, MA), goat

Effect of IL-10 on 5-HT uptake

Caco-2 cells were treated with IL-10 at different concentrations, ranging between 0.01 and 25 ng/ml, for different periods—30 min, 6 h, or 1 day—and 5-HT uptake (SERT activity) was measured. The results showed that IL-10 affected 5-HT transport, depending on the duration of treatment and the concentration. Thus, at 30 min of treatment, IL-10 reduced 5-HT uptake. In contrast, after 6 h of treatment, IL-10 induced a dual effect on SERT activity such that, at a low concentration (0.01 ng/ml), IL-10

Discussion

The results of the present study showed that IL-10 induced a dual effect on SERT activity in Caco-2 cells. Thus, the 6 h treatment with IL-10 at a low concentration (0.01 ng/ml) inhibited 5-HT uptake, whereas at a high concentration (25 ng/ml), it stimulated this process. Previous studies have shown that IL-10 may induce a dual effect in enteric cell proliferation, depending on the IL-10 concentration [32], and in the immune response, depending on the environmental context [33]. Moreover,

Conclusion

This study has analyzed the role of IL-10 in SERT activity and expression in enterocyte-like Caco-2 cells. In summary, IL-10 showed a dual effect on SERT. First, at a high concentration, IL-10 induced an increase of SERT activity and expression in the cell membrane that seemed to be mediated by IL-10R and the intracellular PI3K signaling pathway. Secondly, IL-10 at a low concentration inhibited SERT activity by affecting the capacity of the transporter in an IL-10R independent way. Therefore,

Acknowledgments

This work was funded by grants from the Spanish Ministry of Science and Innovation and the European Regional Development Fund (ERDF/FEDER) (BFU2009-08149; BFU2010-18971), European Social Found (ESF), the Aragon Regional Government (B61) and the Foundation for the Study of Inflammatory Bowel Diseases in Aragón (ARAINF 012/2008). Eva Latorre is a PhD student under a fellowship from Aragon Regional Government (B105/11). The authors would like to thank Dr. Brot-Laroche (INSERM, UMR S 872, Centre de

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    Present address: Department of Basic Sciences, Faculty of Veterinary Sciences, University Centroccidental Lisandro Alvarado, Núcleo Hector Ochoa Zuleta, Tarabana, Estado Lara, Venezuela.

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