367 Molecular Regulation of Il23a Gene Expression in Murine Macrophages and Experimental Colitis

Abstract

Background: Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. Aim: The aim of this study is to characterize the molecular regulation of Il23a gene expression and promoter activation in murine macrophages and experimental colitis. Results: Interferon-γ (IFN-γ) inhibits LPS-induced Il23a mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence across multiple species was identified in the promoter of the Il23a gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine Il23a luciferase reporter plasmid, LPS induces and IFN-γ inhibits LPSinduced p19 promoter activity in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter activity. To understand the contribution of specific IRFs, IRF-1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/BMMs demonstrate increased LPS-induced IL-23 expression. Primary response genes have promoters that either exist in an open chromatin structure or undergo rapid nucleosomal remodeling. In contrast, secondary response genes with delayed induction kinetics require new protein synthesis prior to transcription initiation. Il23a expression in LPS-activated BMMs demonstrates rapid induction kinetics and unlike Il12b (established secondary response gene) is not dependent on new protein synthesis. Interestingly, LPS plus IFN-γ activated BMMs demonstrate loss of the IFN-γ inhibitory effect, suggesting IFN-γ mediated regulation of Il23a requires new protein synthesis. Il23a regulation was then studied in murine colitis models. Following intrarectal administration of TNBS, IRF-1-/mice exhibit increased inflammation and increased colonic IL-23 expression compared to wild type (WT) mice. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free and conventionalized WT and colitis-prone IL-10-/mice. Enteric bacteria induce significantly higher intestinal IL-23 expression in IL-10-/-mice compared withWTmice. Conclusions: IFN-γ and IRF-1 negatively regulate Il23a in murine macrophages and experimental colitis. Regulation of intestinal Il23a by the enteric microflora is a significant event in the initiation of chronic intestinal inflammation.