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

Cytokine xxx (2013) xxx–xxx

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IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells Eva Latorre, Carmen Mendoza 1, Nyurky Matheus 1, Marta Castro, Laura Grasa, José E. Mesonero ⇑, Ana I. Alcalde Department of Pharmacology and Physiology, Faculty of Veterinary Sciences, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain

a r t i c l e

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Article history: Received 21 May 2012 Received in revised form 16 November 2012 Accepted 13 January 2013 Available online xxxx Keywords: IL-10 5-HT Serotonin transporter Intestine Caco-2 cells

a b s t r a c t 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. Ó 2013 Elsevier Ltd. All rights reserved.

1. 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 [1–4], and its effects depend on 5-HT availability, which is mainly regulated by the serotonin transporter (SERT) located in the enterocytes [5]. Abbreviations: GI, gastrointestinal; 5-HT, 5-hydroxytryptamine, serotonin; SERT, serotonin transporter; IBS, Irritable Bowel Syndrome; IBD, Inflammatory Bowel Diseases; IL-10, Interleukin-10; IL-10R, Interleukin-10 receptor; IL10RA, Interleukin-10 receptor subunit alpha; IL10RB, Interleukin-10 receptor subunit beta; NF-jB, Nuclear Factor-jB; p38 MAPK, p38 Mitogen activated protein kinase; PI3K, Phosphatidylinositol 3 kinase; SB 220025, 5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(4-piperidinyl) imidazole trihydrochloride; sulfasalazine, 5-[4-(2-Pyridylsulfamoyl)phenylazo] salicylic acid; PDTC, pyrrolidinedithiocarbamate. ⇑ Corresponding author. Address: Departamento de Farmacología y Fisiología, Unidad de Fisiología, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain. Tel.: +34 976 762832; fax: +34 976 761612. E-mail address: [email protected] (J.E. Mesonero). 1 Present address: Department of Basic Sciences, Faculty of Veterinary Sciences, University Centroccidental Lisandro Alvarado, Núcleo Hector Ochoa Zuleta, Tarabana, Estado Lara, Venezuela.

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 [9–11], 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 [16–18] 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].

1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.01.012

Please cite this article in press as: Latorre E et al. IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells. Cytokine (2013), http://dx.doi.org/10.1016/j.cyto.2013.01.012

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The effect of pro-inflammatory factors on SERT has been described in previous research. However, the role of antiinflammatory factors on the regulation of SERT remains unknown. Interleukin-10 (IL-10) has been demonstrated to be an antiinflammatory 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.

2.3. 5-HT uptake studies

2. Materials and methods

2.4. RNA extraction and reverse transcription PCR

2.1. Reagents and antibodies

RNA extractions were carried out with the RNeasy mini kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions as described in a previous paper [28]. Total RNA was extracted from Caco-2 cells cultured in 25 cm2 flasks 14 days after seeding. The extracted RNA (1 lg) was used as a template for first-strand cDNA synthesis using oligo(dT) primers and a reverse transcriptase (Life Technologies). Negative amplification control was performed in the absence of reverse transcriptase. Expression of human IL10R subunit alpha (IL10RA) and subunit beta (IL10RB) was examined by RT-PCR using the following specific primers [29]: IL10RA sense (50 -ATTGCCATTCGCAAGGTG-30 ); IL10RA antisense (50 -TCTCC CACTTCTCCAGAGGTTA-30 ); IL10RB sense (50 -GCTGTGGTGCGTTTACAAGA-30 ); and IL10RB antisense (50 -TGAGGATGGCCCAAAAACT30 ). 35 cycles were carried out for PCR IL-10Rs amplification under the following conditions: 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. PCR products were electrophoresed on 1% agarose gel and visualized under UV light after ethidium bromide staining. The images from gels were captured using the Biodoc-It Imaging System (UPV Inc., USA). The fragment sizes were 90 and 96 bp for IL10RA and IL10RB, respectively.

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 polyclonal antibody anti-human SERT (Abcam, ab36127, Cambridge, UK), goat polyclonal anti-human actin antibody (Santa Cruz Biotechnology, sc-1616r, Santa Cruz, CA), rat monoclonal antibody anti-human IL-10 receptor (IL-10R; Santa Cruz Biotechnology, sc-53654), and secondary antibodies coupled with horseradish peroxidase (Santa Cruz Biotechnology) were also used. All generic reagents were purchased from Sigma–Aldrich and Roche Applied Sciences (Sant Cugat del Vallés, Barcelona, Spain). 2.2. Cell culture This study was carried out in the human enterocyte-like cell line Caco-2/TC7 [27]. These cells were cultured at 37 °C in an atmosphere of 5% CO2 and maintained in high glucose DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin, 1% non-essential amino acids, and 20% heat-inactivated FBS (Life Technologies, Carlsbad, CA). The cells were passaged enzymatically (0.25% trypsin–1 mM EDTA) and sub-cultured in 25 cm2 plastic culture flasks (Sarstedt, Nuembrecht, Germany). The medium was changed 48 h after seeding and daily thereafter. For 5-HT uptake assays, cells were seeded in 24-well plates at a density of 4  104 cells/well, and uptake measurements were carried out 14 days after seeding (9 days after confluence). Previous results have shown that Caco-2 cells express SERT and that its activity reaches a plateau on the fifth day after confluence [26]. In the experiments, the cell medium was free of FBS 24 h before using the cells. IL-10 and the different modifiers were added to the culture medium at different concentrations and periods, depending on the experiment.

Uptake measurements were performed on cells attached to 24well plates either under control conditions or after treatment with IL-10. The transport medium composition in mM was as follows: 137 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 10 HEPES pH 7.4, 4 glutamine, 0.1% BSA, and both 5-HT 0.2 lM and [3H]-5-HT (1.5 lCi/ml). Before measuring uptake, cells were pre-incubated at 37 °C in an atmosphere of 5% CO2 with substrate-free transport medium for 30 min. The cells were immediately washed with substrate-free transport medium at 37 °C and then incubated with transport medium at 37 °C for 6 min. Transport was stopped by removing the transport medium and washing the cells twice with ice-cold, substrate-free transport medium containing 20 lM 5-HT. The cells were solubilized in 0.1 N NaOH, and samples were taken for radioactivity counting (Wallac Liquid Scintillation Counter, Perkin–Elmer). Protein was measured using the Bradford method (Bio-Rad, Hercules, CA), with BSA as standard. Results were calculated in pmol 5-HT/mg protein and were expressed as a percentage of control value (100%). In the kinetic study, 5-HT uptake was measured at different 5-HT concentrations (0.05, 0.1, 0.2, 1, 2, 5, and 10 lM), and the kinetic constants Vmax and Kt were calculated.

2.5. Quantitative RT-PCR The relative abundance of SERT mRNA in control and IL-10 treated cells was quantified by quantitative RT-PCR. cDNAs obtained by reverse transcription were used to determine SERT mRNA expression levels. Reactions were run using the StepOne Plus Real-Time PCR System (Life Technologies) and the hSERT Gene Expression Assay from Applied Biosystems (Life Technologies, Assay number Hs00169010_m1 [SLC6A4]) was used, following the manufacturer’s recommendations. Each sample was run in triplicate, and the mean Ct was determined from the three runs. Relative SERT mRNA expression under each experimental condition (control or treatment) was expressed as DCt = CtSERT  Ctcalibrator, with hGAPDH (Assay number Hs99999905_m1) and hHPRT1 (Assay number Hs99999909_m1) housekeeping gene expression used as calibrators. Then, relative SERT mRNA expression was calculated as DDCt = DCtcontrol  DCttreatment. Finally, the relative gene expression levels were converted and expressed as fold difference (=2DDCt).

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2.6. Brush border-enriched fraction from Caco-2 cells and western blotting

nevertheless, no significant effect was obtained at a high concentration (Fig. 1).

Caco-2/TC7 cells were cultured in 75 cm2 flasks and used 14 days after seeding. The preparation of the intestinal brush border membrane-enriched fraction followed divalent cation precipitation and the differential centrifugation procedure described in a previous paper [30]. Briefly, cells were washed twice with PBS and immediately re-suspended with a cold Tris–manitol buffer (Tris 2 mM, Manitol 50 mM, pH 7.1) containing protease inhibitors and 0.02% sodium azide. The suspension was homogenized and the cells were disrupted by sonication (fifteen 1-s bursts, 60 W). One sample was taken from the lysate for total protein analysis and protein quantification. CaCl2 (final concentration 20 mM) was added to the cell lysate and, after standing for 10 min in ice, the mixture was centrifuged for 10 min at 950g. The supernatant was taken and centrifuged at 33,500g for 30 min. The pellet (brush border-enriched fraction) was re-suspended in phosphate buffer (KH2PO4/K2HPO4 10 mM pH 6.8), and a sample was taken for protein analysis. Protein was measured using the Bradford method. Brush border-enriched fraction and cell lysate (60 lg of total protein) from Caco-2 cells were electrophoresed in 8% SDS–PAGE gels and then transferred to PVDF membranes by electroblotting. The membranes were blocked with 5% non-fat dried milk plus 1% BSA and probed either with a goat polyclonal anti-human SERT (1:500) or with a rat monoclonal anti-human IL-10R (1:100). The primary antibody was detected using secondary antibodies coupled with horseradish peroxidase and the ECL Plus detection kit (GE Healthcare, Buckinghamshire, UK) and visualized with X-ray films. The blots were re-probed, after stripping, with goat polyclonal anti-human b-actin to determine differences in the sample load. The SERT/b-actin protein ratio was calculated in densitometric units from the film using Quantity One Analysis Software (BioRad).

3.2. Kinetic study of IL-10 effect on 5-HT uptake

2.7. Statistical analysis All results are expressed as means ± the standard error of the mean (SE). Statistical comparisons were performed using oneway ANOVA, followed by the Bonferroni post-test with a confidence interval of 95% (p < 0.05). Normal distribution was previously confirmed with the D’Agostino-Pearson test. Kinetic study of the 5-HT transport values was performed by non-linear regression, fitting the results to an equation containing a saturable (Michaelis–Menten) plus a non-saturable (diffusion) component. Statistical analysis was carried out with the computer-assisted Prism GraphPad Program (Prism version 4.0, GraphPad Software, San Diego, CA).

3. Results

In order to characterize the IL-10 dual effect on SERT activity, a kinetic study was carried out and Vmax and Kt, which inform the capacity and affinity of SERT respectively, were calculated. The kinetic constants were analyzed in cells treated for 6 h with IL-10 (0.01 ng/ml or 25 ng/ml). 5-HT concentration ranged between 5  108 and 105 M, and the period of uptake incubation was 6 min. The results showed that the treatment with IL-10, at both 0.01 and 25 ng/ml concentrations, significantly affected Vmax by reducing or increasing, respectively, its value; however, Kt did not seem to be altered by any IL-10 treatment (Table 1). 3.3. Effect of IL-10 treatment on SERT mRNA level and protein expression The levels of SERT mRNA and protein were measured by quantitative RT-PCR and western blotting, respectively, in Caco-2 cells treated for 6 h with IL-10 (0.01 or 25 ng/ml). The results showed that none of the IL-10 treatments altered SERT mRNA levels (Fig. 2A). In relation to SERT protein, the expression in cell lysate was not significantly affected by any IL-10 treatment; however, the level of SERT protein in the brush border-enriched membrane fraction significantly increased after IL-10 25 ng/ml treatment (Fig. 2B and C), in accordance with the increase obtained in the 5-HT uptake experiments. 3.4. Expression of IL-10R subunits in Caco-2 cells and analysis of its mediation in IL-10 effects on SERT Most of the IL-10 activity in the cells has been described to be mediated through IL-10R, which includes two subunits, A and B [31]. The expression of IL-10R in Caco-2 cells was assessed by RT-PCR and western blotting, under conditions described in Sections 2.4 and 2.6, respectively. Fig. 3A shows two bands of 90 and 96 bp amplified by PCR, which correspond to the expected size of both the subunits IL10RA and IL10RB, and Fig. 3B shows a band of 63 kDa, which corresponds to IL-10R protein. The involvement of IL-10R in IL-10 effects on SERT activity was analyzed by blocking the receptor with a specific antibody. To do so, SERT activity was measured in cells pretreated with IL-10R antibody at 1 lg/ml 30 min prior to treatment, and then with IL-10 at either 0.01 or 25 ng/ml, plus the antibody for 6 h. The results showed that the effect of IL-10 25 ng/ml on SERT activity was reversed in cells treated with IL-10R antibody; however, antibody receptor blocking did not modify the inhibitory effect of IL-10 at a concentration of 0.01 ng/ml (Fig. 3C). 3.5. Analysis of signaling pathways involved in the IL-10 effect on hSERT

3.1. 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 yielded a significant reduction of 5-HT uptake but, at a high concentration (25 ng/ml), IL-10 significantly increased 5-HT uptake. After 1 day of treatment, the effect of IL-10 on SERT activity at a low concentration persisted

To further study the IL-10 effects on SERT, the involvement of the intracellular signaling pathways NF-jB, p38 MAPK and PI3K, which have been described to mediate IL-10 activity, was assessed. Caco-2 cells were pretreated for 30 min with PDTC, sulfasalazine, SB 220025 or wortmannin, specific inhibitors of NF-jB, p38 MAPK, and PI3K intracellular signaling pathways, respectively, and then with IL-10 at either 0.01 or 25 ng/ml for 6 h. The results showed that PDTC, sulfasalazine, and SB220025 inhibited SERT activity under control conditions (data not shown). However, wortmannin 10 nM, a specific PI3K inhibitor, reversed the effect of IL-10 25 ng/ml on SERT activity, whereas the effect of IL-10 0.01 ng/ml persisted (Fig. 4A). This result was corroborated by measuring SERT

Please cite this article in press as: Latorre E et al. IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells. Cytokine (2013), http://dx.doi.org/10.1016/j.cyto.2013.01.012

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Fig. 1. Effect of the dose and period of treatment of IL-10 on 5-HT uptake. Uptake was measured after a 6 min incubation with 0.2 lM 5-HT. The IL-10 concentrations tested were 0.01, 0.1, 1, and 25 ng/ml. The treatment periods were 30 min, 6 h, and 1 day. The results are expressed as the percentage of the uptake control (100%) and are the mean ± SE of five independent experiments. Absolute control values were 10.94 ± 0.39, 9.98 ± 0.61, and 11.16 ± 0.34 pmol 5-HT/mg prot at 30 min, 6 h, or 1 day, respectively.  p < 0.05, p < 0.01, and p < 0.001 compared with the control (untreated cells).

Table 1 Kinetic constants calculated from kinetic analysis of 5-HT transport in Caco-2 cells treated with IL-10 at 0.01 or 25 ng/ml concentration during 6 h. Conditions

Vmax (pmol 5-HT/mg prot)

Kt (lM)

Control IL-10 (0.01 ng/ml) IL-10 (25 ng/ml)

25.72 ± 1.12 18.30 ± 1.14* 32.97 ± 1.84*

0.45 ± 0.07 0.46 ± 0.04 0.48 ± 0.06

The values are the means ± SE of eight independent experiments. p < 0.05 compared with control value.

*

protein expression in the brush border-enriched membrane fraction of cells treated with wortmannin and/or IL-10 at 0.01 or 25 ng/ml (Fig. 4B). In fact, wortmannin was shown to reverse the increase of the SERT protein expression yielded by IL-10 25 ng/ ml treatment.

4. 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, anti-inflammatory [34,35] and pro-inflammatory IL-10 effects [36,37] have been described, depending on the tissue and the pathological conditions. The IL-10 effect at a high concentration (25 ng/ml) seemed to be due to an increase of SERT in the membrane of the cells, since both Vmax, which indicates capacity, and SERT protein content in enriched cell membrane fraction were augmented. However, SERT mRNA or SERT total protein levels were not affected by IL-10, which might suggest a post-translational effect. To further study whether IL-10 at 25 ng/ml yielded a direct effect on Caco-2 cells, the mediation of IL-10R and the signaling pathway involved were analyzed. Previous results have shown that IL-10R is expressed in intestinal epithelial cells [38], and the present study demonstrates that Caco-2 cells expressed IL-10R. The results showed that IL-10R blocking with a specific antibody reversed the IL-10 effect on SERT, suggesting IL-10R mediation. Human IL-10R has been shown to bind ligands with high affinity (Kd = 0.035–0.200 nM) [39] therefore, IL-10 at 25 ng/ml (1.34 nM) may induce the activation of IL-10R and, as a consequence, the effect on SERT.

Fig. 2. Effect of IL-10 on SERT mRNA and protein expression levels. (A) Quantitative RT-PCR analysis of SERT mRNA expression level in cells treated for 6 h with IL-10 (0.01 or 25 ng/ml). Relative quantification was performed using comparative Ct (2DDCt). (B) Immunodetection of SERT by western blot in cell lysate and apical membrane from Caco-2 cells treated with IL-10 (0.01 or 25 ng/ml) for 6 h. (C) Quantitation of SERT protein in both cell lysate and apical membrane using b-actin as an internal control of the protein load (SERT/b-actin ratio). The results are expressed in arbitrary densitometric units and are the mean ± SE of four independent experiments. p < 0.001 compared with the control value.

In order to determine the intracellular signaling triggered by IL10 at 25 ng/ml, NF-jB, p38 MAPK and PI3K pathways, which have been shown to be activated by IL-10 [34,35,40,41], were analyzed. The results showed that the inhibition of PI3K with wortmannin blocked the effect of IL-10 on both SERT activity and membrane protein expression, and the treatment with wortmannin itself was not shown to affect then. This result suggests the mediation

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Fig. 3. Study of IL-10R expression in Caco-2/TC7 cells and its role in the IL-10 effect on SERT activity. (A) IL10RA and IL10RB mRNA expression was determined by RTPCR using primers and conditions detailed in Section 2.4. Two bands of 90 and 96 bp corresponding to IL10RA and IL10RB, respectively, are shown. (B) IL-10R expression by western blotting in cell lysate (CL) and brush-border (BB) enriched membrane fraction of Caco-2 cells. The size of protein band was about 63 kDa for IL-10R. (C) Involvement of IL-10R in IL-10 effect on SERT activity. Cells were treated with IL-10 receptor antibody (1 lg/ml) 30 min before the experiment and then with IL-10 (0.01 or 25 ng/ml) with or without the antibody for 6 h. 5-HT uptake was measured after a 6 min incubation with 5-HT 0.2 lM. The results are expressed as the percentage of the uptake control and are the mean ± SE of four independent experiments. Absolute control value was 10.39 ± 0.95 pmol 5-HT/mg prot.  p < 0.001 compared with control; ####p < 0.001 compared with IL-10 25 ng/ml treated cells.

of the PI3K signaling pathway in the high concentration IL-10 effect on SERT. The role of NF-jB and p38 MAPK pathways in the IL-10 effect on SERT was also assessed. However, treatment with sulfasalazine or PDTC, inhibitors of NF-jB, or SB220025, a specific inhibitor of p38 MAPK, inhibited 5-HT uptake under control conditions (without treatment with IL-10) (data not shown). Therefore, based on our results, the involvement of either NF-jB or p38 MAPK in IL-10 effects cannot be disregarded. In relation to the effect of IL-10 at a low concentration (0.01 ng/ ml) on SERT activity, the results showed that IL-10 induced a reduction of Vmax, which indicated a diminution of SERT capacity. However, levels of SERT mRNA and SERT protein in the cell lysate or in the cell membrane were not altered by IL-10, suggesting a non-competitive inhibition on SERT. This kind of inhibition has been previously described with regard to SERT activity in intestinal epithelial cells, and it seemed to be due to the effect on the allosteric site of the protein in the membrane [30,42]. The allosteric site has been described in SERT as a low-affinity binding site that regulates the binding of ligands at the site that mediates 5-HT reuptake, named as primary site [43]. In addition, the results show that IL-10 inhibition of SERT activity did not seem to be mediated by IL-10R, because the blockade with the specific IL-10R antibody did not alter the effect of IL-10 on 5-HT uptake, and it did not appear to be mediated by any of the signaling pathways assessed (p38 MAPK, NF-jB or PI3K). The results of the present work have also shown that the dual effect of IL-10 on SERT activity appears gradually as IL-10 concentration increases and after 6 h of treatment. In relation to the IL-10 effects on SERT at 30 min or 1 day of treatment, the results obtained show that treatment with low IL10 concentration (0.01 ng/ml) inhibited SERT activity in any period of treatment, which may suggest that the effect of low concentra-

Fig. 4. Role of PI3K in IL-10 effect on SERT activity. Cells were treated with wortmannin 10 nM for 30 min prior to the experiment and then with IL-10 (0.01 and 25 ng/ml) and/or wortmannin 10 nM for 6 h. (A) Uptake of 5-HT was measured after a 6 min incubation with 5-HT 0.2 lM. Control condition correspond to untreated cells. The results are expressed as the percentage of the uptake control and are the mean ± SE of eight independent experiments. Control absolute value was 10.26 ± 0.68 pmol 5-HT/mg prot. p < 0.001 compared with the control; ### p < 0.001 compared with IL-10 25 ng/ml treatment. (B) Immunodetection of SERT by western blot in apical membrane from cells treated for 6 h with IL-10 (0.01 or 25 ng/ml) and/or wortmannin 10 nM. (C) Quantitation of SERT protein apical membrane using b-actin as an internal control of the protein load (SERT/b-actin ratio). The results are expressed in arbitrary densitometric units and are the mean ± SE of three independent experiments. p < 0.05 compared with the control value; #p < 0.05 compared with IL-10 25 ng/ml treatment.

tions of IL-10 on SERT is independent of the treatment duration. However, IL-10 at short-term of treatment acts as an inhibitor independently on the concentration. In cases of high concentration and long-term period treatment (1 day), IL-10 did not affect SERT. This result might be due to IL-10 receptor down-regulation induced by the persistent treatment of the cells with elevated doses of IL-10. Previous results support the concept that 5-HT is a potent immunoregulator [19,20] and a pro-inflammatory mediator in the gut [25,44]. The dual effect of IL-10 on SERT activity suggests that IL-10 may induce either an increase or a decrease in 5-HT extracellular availability, depending on IL-10 concentration. Therefore, the results of the current study suggest that IL-10 at a low concentration might contribute to inflammation (extracellular 5HT is increased) and at a high concentration may contribute to anti-inflammatory activity (extracellular 5-HT is decreased). Moreover, our results also suggest that IL-10 may contribute to intestinal mucosa homeostasis [45] by regulating intestinal 5-HT levels.

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5. 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, in our model, IL-10 may have a pro-inflammatory or an anti-inflammatory effect acting on 5-HT availability, depending on IL-10 concentration and/ or the mechanisms involved. The results of the current study may be useful for clarifying the serotoninergic response in intestinal inflammation and may also contribute to our knowledge and understanding of the regulation of the intestinal serotoninergic system under physiological and pathological conditions.

[16]

[17]

[18]

[19]

[20]

[21]

[22]

Acknowledgments [23]

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 Recherche des Cordeliers, Paris) for providing Caco-2/TC7 cells and Alba De Martino for technical assistance.

[24]

[25]

[26]

[27]

References [1] Arruebo MP, Mesonero JE, Murillo MD, Alcalde AI. Effect of serotonin on Dgalactose transport across the rabbit jejunum. Reprod Nutr Dev 1989;29:441–8. [2] Gershon MD. Serotonin receptors and transporters – roles in normal and abnormal gastrointestinal motility. Aliment Pharmacol Ther 2004;20:3–14. [3] Hansen MB, Witte AB. The role of serotonin in intestinal luminal sensing and secretion. Acta Physiol (Oxford) 2008;193:311–23. [4] Salvador MT, Murillo MD, Rodriguez-Yoldi MC, Alcalde AI, Mesonero JE, Rodriguez-Yoldi MJ. Effects of serotonin on the physiology of the rabbit small intestine. Can J Physiol Pharmacol 2000;78:359–66. [5] Chen JX, Pan H, Rothman TP, Wade PR, Gershon MD. Guinea pig 5-HT transporter: cloning, expression, distribution, and function in intestinal sensory reception. Am J Physiol 1998;275:48–G433. [6] Cremon C, Carini G, Wang B, Vasina V, Cogliandro RF, De Giorgio R, et al. Intestinal serotonin release, sensory neuron activation, and abdominal pain in irritable bowel syndrome. Am J Gastroenterol 2011;106:1290–8. [7] Faure C, Patey N, Gauthier C, Brooks EM, Mawe GM. Serotonin signaling is altered in irritable bowel syndrome with diarrhea but not in functional dyspepsia in pediatric age patients. Gastroenterology 2010;139:249–58. [8] Margolis KG, Gershon MD. Neuropeptides and inflammatory bowel disease. Curr Opin Gastroenterol 2009;25:503–11. [9] Bertrand PP, Barajas-Espinosa A, Neshat S, Bertrand RL, Lomax AE. Analysis of real-time serotonin (5-HT) availability during experimental colitis in mouse. Am J Physiol Gastrointest Liver Physiol 2010;298:55–G446. [10] O’Hara JR, Ho W, Linden DR, Mawe GM, Sharkey KA. Enteroendocrine cells and 5-HT availability are altered in mucosa of guinea pigs with TNBS ileitis. Am J Physiol Gastrointest Liver Physiol 2004;287:G998–G1007. [11] Spiller R. Recent advances in understanding the role of serotonin in gastrointestinal motility in functional bowel disorders: alterations in 5-HT signalling and metabolism in human disease. Neurogastroenterol Motil 2007;19:25–31. [12] Spiller R. Serotonin and GI clinical disorders. Neuropharmacology 2008;55:1072–80. [13] Coates MD, Johnson AC, Greenwood-Van Meerveld B, Mawe GM. Effects of serotonin transporter inhibition on gastrointestinal motility and colonic sensitivity in the mouse. Neurogastroenterol Motil 2006;18:464–71. [14] Coates MD, Mahoney CR, Linden DR, Sampson JE, Chen J, Blaszyk H, et al. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology 2004;126:1657–64. [15] Chen JJ, Li Z, Pan H, Murphy DL, Tamir H, Koepsell H, et al. Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-

[28] [29]

[30]

[31] [32] [33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

affinity serotonin transporter: abnormal intestinal motility and the expression of cation transporters. J Neurosci 2001;21:6348–61. Foley KF, Pantano C, Ciolino A, Mawe GM. IFN-cand TNF-a decrease serotonin transporter function and expression in Caco2 cells. Am J Physiol Gastrointest Liver Physiol 2007;292:84–G774. Matheus N, Mendoza C, Iceta R, Mesonero JE, Alcalde AI. Regulation of serotonin transporter activity by adenosine in intestinal epithelial cells. Biochem Pharmacol 2009;78:1198–204. Mendoza C, Matheus N, Iceta R, Mesonero JE, Alcalde AI. Lipopolysaccharide induces alteration of serotonin transporter in human intestinal epithelial cells. Innate Immun 2009;15:243–50. Dürk T, Panther E, Müller T, Sorichter S, Ferrari D, Pizzirani C, et al. 5Hydroxytryptamine modulates cytokine and chemokine production in LPSprimed human monocytes via stimulation of different 5-HTR subtypes. Int Immunol 2005;17:599–606. Müller T, Dürk T, Blumenthal B, Grimm M, Cicko S, Panther E, et al. 5Hydroxytryptamine modulates migration, cytokine and chemokine release and T-cell priming capacity of dendritic cells in vitro and in vivo. PLoS One 2009;4:e6453. Bolger AP, Sharma R, von Haehling S, Doehner W, Oliver B, Rauchhaus M, et al. Effect of interleukin-10 on the production of tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with chronic heart failure. Am J Cardiol 2002;90:384–9. Kang SS, Bloom SM, Norian LA, Geske MJ, Flavell RA, Stappenbeck TS, et al. An antibiotic-responsive mouse model of fulminant ulcerative colitis. PLoS Med 2008;5:e41. Rennick DM, Fort MM. Lessons from genetically engineered animal models. XII. IL-10-deficient (IL-10(/) mice and intestinal inflammation. Am J Physiol Gastrointest Liver Physiol 2000;278:33–G829. Barada KA, Mourad FH, Sawah SI, Khoury C, Safieh-Garabedian B, Nassar CF, et al. Up-regulation of nerve growth factor and interleukin-10 in inflamed and non-inflamed intestinal segments in rats with experimental colitis. Cytokine 2007;37:236–45. Haub S, Ritze Y, Bergheim I, Pabst O, Gershon MD, Bischoff SC. Enhancement of intestinal inflammation in mice lacking interleukin 10 by deletion of the serotonin reuptake transporter. Neurogastroenterol Motil 2010;22(826– 34):e229. Iceta R, Mesonero JE, Aramayona JJ, Alcalde AI. Molecular characterization and intracellular regulation of the human serotonin transporter in Caco-2 cells. J Physiol Pharmacol 2006;57:119–30. Chantret I, Rodolosse A, Barbat A, Dussaulx E, Brot-Laroche E, Zweibaum A, et al. Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation. J Cell Sci 1994;107:213–25. Iceta R, Mesonero JE, Alcalde AI. Effect of long-term fluoxetine treatment on the human serotonin transporter in Caco-2 cells. Life Sci 2007;80:1517–24. Kamizato M, Nishida K, Masuda K, Takeo K, Yamamoto Y, Kawai T, et al. Interleukin 10 inhibits interferon c- and tumor necrosis factor a-stimulated activation of NADPH oxidase 1 in human colonic epithelial cells and the mouse colon. J Gastroenterol 2009;44:1172–84. Iceta R, Aramayona JJ, Mesonero JE, Alcalde AI. Regulation of the human serotonin transporter mediated by long-term action of serotonin in Caco-2 cells. Acta Physiol (Oxford) 2008;193:57–65. Liu Y, Wei SH, Ho AS, De Waal Malefyt R, Moore KW. Expression cloning and characterization of a human IL-10 receptor. J Immunol 1994;152:1821–9. Rühl A, Franzke S, Stremmel W. IL-1b and IL-10 have dual effects on enteric glial cell proliferation. Neurogastroenterol Motil 2001;13:89–94. Wilke CM, Wei S, Wang L, Kryczek I, Kao J, Zou W. Dual biological effects of the cytokines interleukin-10 and interferon-c. Cancer Immunol Immunother 2011;60:1529–41. Al-Ashy R, Chakroun I, El-Sabban ME, Homaidan FR. The role of NF-jB in mediating the anti-inflammatory effects of IL-10 in intestinal epithelial cells. Cytokine 2006;36:1–8. Antoniv TT, Ivashkiv LB. Interleukin-10-induced gene expression and suppressive function are selectively modulated by the PI3K-Akt-GSK3 pathway. Immunology 2011;132:567–77. Lauw FN, Pajkrt D, Hack CE, Kurimoto M, van Deventer SJ, van der Poll T. Proinflammatory effects of IL-10 during human endotoxemia. J Immunol 2000;165:2783–9. Mitchell MD, Simpson KL, Keelan JA. Paradoxical proinflammatory actions of interleukin-10 in human amnion: potential roles in term and preterm labour. J Clin Endocrinol Metab 2004;89:4149–52. Denning TL, Campbell NA, Song F, Garofalo RP, Klimpel GR, Reyes VE, et al. Expression of IL-10 receptors on epithelial cells from the murine small and large intestine. Int Immunol 2000;12:133–9. Moore KW, De Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765. Patel S, Vetale S, Teli P, Mistry R, Chiplunkar S. IL-10 production in non-small cell lung carcinoma patients is regulated by ERK, P38 and COX-2. J Cell Mol Med 2012;16:531–44. Sharma S, Yang B, Xi X, Grotta JC, Aronowski J, Savitz SI. IL-10 directly protects cortical neurons by activating PI-3 kinase and STAT-3 pathways. Brain Res 2011;1373:189–94. Matheus N, Mendoza C, Iceta R, Mesonero JE, Alcalde AI. Melatonin inhibits serotonin transporter activity in intestinal epithelial cells. J Pineal Res 2010;48:332–9.

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E. Latorre et al. / Cytokine xxx (2013) xxx–xxx [43] Plenge P, Gether U, Rasmussen SG. Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites. Eur J Pharmacol 2007;567:1–9. [44] Khan WI, Ghia JE. Gut hormones: emerging role in immune activation and inflammation. Clin Exp Immunol 2010;161:19–27.

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[45] Jarry A, Bossard C, Bou-Hanna C, Masson D, Espaze E, Denis MG, et al. Mucosal IL-10 and TGF-b play crucial roles in preventing LPS-driven, IFN-c-mediated epithelial damage in human colon explants. J Clin Invest 2008;118:1132–42.

Please cite this article in press as: Latorre E et al. IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells. Cytokine (2013), http://dx.doi.org/10.1016/j.cyto.2013.01.012