TLR4 signaling negative modulators – clathrin and caveolin as potential therapeutics in endotoxin-related inflammation in immature enterocytes
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Microbe-induced TLR4 trafficking is needed to promote innate immunity and an inflammatory reaction. We showed that increased TLR4 expression in immature enterocytes is associated with severe inflammation and the development of necrotizing enterocolitis (NEC). Thus, we need to determine the intracellular regulatory mechanisms for TLR4 trafficking in fetal enteric inflammation. Here, we show that both clathrin- and caveolin-dependent endocytosis are necessary for the prevention of the fetal colonic (FHC) IL-1β response to endotoxin (LPS, a TLR4 agonist). In response to LPS stimulation, the inhibitor of clathrin, chlorpromazine (CPZ), and caveolin 1, Methyl-β- cyclodextrin (MβCD), significantly increased TLR4 mRNA and surface protein expression by retaining TLR4 in the early endosome. An increased TLR4 resulted in increased proinflammatory cytokine IL-1β mRNA and protein induction in a time dependent manner. TICAM2, elicited by TLR4, mediates down-stream TLR4 signaling. MβCD did not increase TICAM2 mRNA expression indicating that CPZ and MβCD affect TLR4 signaling differently. In addition, both CPZ or MβCD alone and CPZ or MβCD plus LPS reduced IL-10 mRNA expression, which functions to reduce LPS IL-1β. Collectively, these data identify an additional role for intestinal expression of clathrin and caveolin in anti-inflammation via gram negative pathogen LPS through regulation of TLR4 trafficking and cytokine expression in immature enterocytes. Study of the endocytosis pathway is likely to lead to successful strategies for prevention, treatment and improved outcomes of immature inflammatory diseases such as NEC.