Double-stranded RNA induces cathelicidin expression in the intestinal epithelial cells through phosphatidylinositol 3-kinase-protein kinase Cζ-Sp1 pathway and ameliorates shigellosis in mice

Abstract

Cathelicidin antimicrobial peptide is a key component of the host innate immune system. It is constitutively expressed by the intestinal epithelial cells, but induced at further higher levels by different host-derived and microbial stimuli, including the ligands for Toll-like receptors (TLRs). While the underlying mechanisms of cathelicidin expression remain incompletely understood, altered expression may be associated with gastro-intestinal infections and inflammatory diseases. We demonstrate here that viral double-stranded RNA and its synthetic analog poly(I:C) are potent and tissue-specific inducers of cathelicidin mRNA and protein expression in the mouse as well as human intestinal epithelial cells. Reporter assays showed that poly(I:C) transcriptionally regulates murine cathelicidin-related antimicrobial peptide (mCRAMP) by recruiting Sp1 transcription factor to the GC-box cis-regulatory element at -71bp of the mCRAMP putative promoter. Sp1 recruitment to the endogenous mCRAMP promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Immunoblotting, qPCR, ChIP and siRNA-mediated gene knockdown studies revealed that the activation of phosphatidylinositol 3-kinase/protein kinase Cζ pathways in poly(I:C)-stimulated cells underlies Sp1 phosphorylation and recruitment to the mCRAMP promoter, leading to enhanced transcription. We further showed that intra-rectal poly(I:C) administration in mice reduces intestinal bacterial load and mucosal inflammation following Shigella flexneri 2a infection by inducing mCRAMP expression in the colonic epithelial cells. This study reports novel regulatory mechanisms of cathelicidin expression that may be targeted to treat gastro-intestinal infections.