The long term goal of the current proposal is to understand the pathogenesis and to design novel therapeutic approaches for necrotizing enterocolitis (NEC). NEC is the leading cause of death from gastrointestinal disease in preterm infants, characterized by mucosal disruption and lipopolysaccharide (LPS) translocation across the inflamed intestine. We have recently demonstrated that the LPS receptor,ToII-like receptor 4 (TLR4) plays a critical role in the pathogenesis of NEC. Activation of TLR4 on enterocytes by LPS leads to an increase in enterocyte apoptosis and villus loss, as well as reduced intestinal repair by blocking intestinal restitution and proliferation, leading to mucosal disruption. We have shown that TLR4 mutant mice are protected from the development of NEC, and demonstrating reduced apoptosis and enhanced intestinal healing, suggesting that TLR4 is useful therapeutic target in NEC. We recently discovered a novel TLR4 inhibitory pathway, through treatment with a TLR9 ligand, conferring similar protection, and are awaiting clinical trials, demonstrating that approaches aimed at blocking exaggerated TLR4 signaling will yield vital treatments for this disease. Here we aim to demonstrate that (1) Heat shock protein 70 (Hsp70), a molecular chaperone, is an endogenous inhibitor of TLR4 signaling and that (2) Hsp70 induction plays a cytoprotective role in vivo via inhibition of TLR4 in settings of exaggerated TLR4 signaling, such as NEC. We will utilize an enteroctye cell line for in vitro studies including RT-PCR, SDS-PAGE and immunofluorence microscopy, where upstream markers of TLR4 signaling (pMAPKs) and downstream markers (inflammatory cytokine production, enterocyte migration and apoptosis) will be assessed with or without Hsp70 induction or inhibition. Recently developed FRET constructs and site- directed mutagenesis of Hsp70 will be used to further characterize the inhibitory mechanism. We will utilize genetic strains of mice we have created, including Hsp70 knockout and enteroctye specific knock-in mice for all in vivo model studies. Hsp70 induction experiments in wild-type mice subjected to the NEC model are planned to model practical avenues for human infants.
The relevance of this research to public health relates to the fact that this work seeks to understand the causes and to identify novel therapies for necrotizing enterocolitis, which is a major cause of death and disability in newborn infants. We have shown that NEC develops when an immune receptor - called toll like receptor 4 (TLR4) - becomes activated within the intestine. The current proposal seeks to test whether increasing levels of Heat Shock Protein 70 can serve as a novel therapeutic approach for infants with this devastating disorder.
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