Transcriptional Regulation of IL-10 Expression by Peroxisome Proliferator-Activated Receptor (Pparγ) in Human Intestinal Epithelial Cells

Abstract

IL-10 is an important immunomodulatory cytokine in the regulation of intestinal inflammation in inflammatory bowel disease (IBD). PPARγ is a nuclear receptor known to modulate transcriptional activities of many cytokines and is highly expressed in intestinal epithelial cells (IECs). Human IECs have been shown to express IL-10, but how its expression is regulated remains obscure especially in the setting of inflammation. We have demonstrated that intestinal 15d-PGJ2 (an endogenous PPARγ ligand) is predominantly induced by infiltrated macrophages (Mφs) through toll-like receptor 4 (TLR4)-mediated Cox-2 induction during colitis. In this study, we addressed whether PPARγ activation regulates expression of IL-10 in IECs. Human IEC cell line SW480-APC expressing TLR4 (top) and mouse peritoneal Mφs (bottom) were co-cultured in transwells. 15d-PGJ2 production and IL-10 mRNA expression were measured by enzyme linked assay and real-time PCR in the presence or absence of lipopolysaccharide (LPS). In order to examine whether the IL-10 promoter has PPARγ responsive elements, a 2,000 bp of IL-10 promoter was cloned from SW480-APC genomic DNA into the luciferase reporter vector. Expression and activity of PPARγ were blocked by siRNA and a specific inhibitor SR202, respectively. The IL-10 promoter activity was analyzed by luciferase reporter assay. PPARγ activity was confirmed using a reporter vector PPRE X3-TK-luc. Co-culturing SW480-APC with Mφs increased IL-10 expression in IECs within 24 hours, which was significantly enhanced by LPS stimulation in later time point (72 hours). LPS stimulation of SW480-APC resulted in PPARγ activation peaked at 48 hours. During SW480-APC/Mφ co-culture, LPS stimulation induced 15d-PGJ2 production, which is abolished when TLR4-deficient Mφs were used. Knocking down the expression of PPARγ suppressed IL-10 promoter activity and mRNA expression in SW480-APC in baseline as well as after LPS stimulation. These results suggest that PPARγ positively regulates IL-10 expression in IECs at the transcriptional level. During inflammation, mucosal Mφs induce PPARγ ligand (15d-PGJ2) in a TLR4-dependent manner that contributes to PPARγ activation in IECs and thus IL-10 expression. This may be an intrinsic immunoregulatory program that is mediated through host-bacterial interactions, which dampens on-going inflammation during intestinal inflammation and thus can be a novel therapeutic target for IBD.