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