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, and is characterized by mucosal disruption and the translocation of lipopolysaccharide (LPS) across the inflamed intestine. We have recently demonstrated that the LPS receptor - Toll 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 also shown that mice with mutations in TLR4 are protected from the development of NEC, and accordingly demonstrate reduced apoptosis and enhanced intestinal healing. These findings suggest the possibility that TLR4 may be a useful therapeutic target in NEC. In the current proposal, we seek to explore a potential role for bacterial-derived DNA (CpG-DNA) as a novel therapeutic agent for NEC, through its inhibitory effects on TLR4 signaling in enterocytes. To do so, we hypothesize that: TLR9 activation with CpG-DNA leads to an inhibition of TLR4-mediated signaling in enterocytes resulting in attenuation in the extent of LPS-mediated enterocyte apoptosis and a reversal in the inhibition of enterocyte proliferation and migration. We further hypothesize that the mechanism by which TLR9 activation inhibits TLR4 signaling involves a clathrin-mediated disruption in TLR4 trafficking. Finally, we hypothesize that by inhibiting TLR4 signaling in enterocytes, activation of TLR9 with CpG-DNA will prevent and treat NEC by reducing the extent of intestinal injury and enhancing intestinal repair. To test these hypotheses, we propose the following specific aims, which will be tested using a variety of primary and cultured enterocyte lines, and in wild-type mice and those with mutations in TLR4 and TLR9:
AIM 1. To assess the protective effects of TLR9 activation with CpG-DNA on TLR4-mediated intestinal injury and repair.
AIM 2. To determine the mechanisms by which TLR9 activation with CpG-DNA inhibits TLR4 signaling in enterocytes through modifying the cellular distribution of TLR4.
AIM 3. To understand the role of TLR9 activation in the prevention and treatment of necrotizing enterocolitis through the inhibition of TLR4 activation.

Public Health Relevance

The relevance of this research to public health is found in 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 activating another receptor - called """"""""Toll like receptor 9 (TLR9)"""""""", which leads to the inhibition of TLR4, can serve as a novel therapeutic approach for infants with this devastating disorder.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZHD1-DSR-A (18))
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Hamilton, Frank A
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University of Pittsburgh
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