The goals of the proposed research are to determine the mechanisms that lead to the development of necrotizing enterocolitis (NEC), which is the leading cause of death and disability from gastrointestinal disease in premature infants, and to determine novel therapeutic strategies for this devastating disorder. To do so, we will explore the role of the bacterial endotoxin receptor Toll like receptor 4 (TLR4) in the pathogenesis of NEC through its previously unrecognized effects on the induction of endoplasmic reticulum (ER) stress in the intestinal mucosa of the premature infant. ER stress reflects a cellular state of accumulated mis-folded proteins within the lumen of the ER, which leads to rapid apoptosis. In the previous funding period, we discovered that TLR4 signaling in the intestinal mucosa is required for NEC development and that NEC is characterized by a TLR4-dependent induction of enterocyte apoptosis leading to mucosal injury. We also showed that the homologous receptor for TLR4, namely TLR9 - which recognizes bacterial DNA ("CpG-DNA") - attenuated NEC through the inhibition of TLR4 signaling and a reduction in enterocyte apoptosis. Importantly however, the mechanisms by which TLR9 inhibited the TLR4 signaling pathways that lead to NEC remained unexplained. We now provide evidence that TLR4 activation leads to NEC through an increase in ER stress within the newborn intestinal epithelium, and that TLR9 activation protects against NEC by inhibiting TLR4- mediated ER stress via the intracellular chaperone heat shock protein 70 (Hsp70). Strikingly, the premature mouse was characterized by a TLR4-dependent increase in ER stress in the intestinal epithelium, a finding also seen in human infants. We now hypothesize that TLR4 activation within the newborn intestinal mucosa leads to the development of NEC by inducing ER stress in the intestinal epithelium leading to enterocyte apoptosis, which can be reversed by TLR9 activation through the intracellular chaperone Hsp70. We further hypothesize that the in-utero regulation of intestinal TLR4 and TLR9 in the developing fetus can reduce ER stress and prevent the development of NEC. We will test this hypothesis in three specific aims:
AIM 1. To investigate the role of TLR4 activation in regulating ER stress in the newborn intestinal epithelium in the pathogenesis of necrotizing enterocolitis.
AIM 2. To determine the mechanisms by which TLR9 activation reduces TLR4-induced ER stress and NEC severity.
AIM 3. To evaluate whether the in utero regulation of TLR4 and TLR9 can inhibit ER stress and prevent the development of NEC. These studies will make a significant conceptual advance by defining how TLR4 signaling leads to enterocyte apoptosis and mucosal injury in NEC, and by explaining the susceptibility of the premature infant to NEC based on increased TLR4-induced mucosal ER stress, and through the evaluation of novel anti-NEC therapies based upon the attenuation of ER stress within the premature small intestine.

Public Health Relevance

Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in premature infants, and for which there exists no effective cure. The current proposal seeks to understand the causes of necrotizing enterocolitis by focusing on how activation of the immune system causes destruction of the lining of the intestine, and seeks to reverse this damage in order to discover novel treatments for this devastating disease.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01DK083752-08
Application #
8691794
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hamilton, Frank A
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Surgery
Type
Schools of Medicine
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Lu, Peng; Sodhi, Chhinder P; Jia, Hongpeng et al. (2014) Animal models of gastrointestinal and liver diseases. Animal models of necrotizing enterocolitis: pathophysiology, translational relevance, and challenges. Am J Physiol Gastrointest Liver Physiol 306:G917-28
Good, Misty; Sodhi, Chhinder P; Ozolek, John A et al. (2014) Lactobacillus rhamnosus HN001 decreases the severity of necrotizing enterocolitis in neonatal mice and preterm piglets: evidence in mice for a role of TLR9. Am J Physiol Gastrointest Liver Physiol 306:G1021-32
Costello, Cait M; Hongpeng, Jia; Shaffiey, Shahab et al. (2014) Synthetic small intestinal scaffolds for improved studies of intestinal differentiation. Biotechnol Bioeng 111:1222-32
Bauer, Eileen M; Chanthaphavong, R Savanh; Sodhi, Chhinder P et al. (2014) Genetic deletion of toll-like receptor 4 on platelets attenuates experimental pulmonary hypertension. Circ Res 114:1596-600
Ding, Ning; Chen, Guoqiang; Hoffman, Rosemary et al. (2014) Toll-like receptor 4 regulates platelet function and contributes to coagulation abnormality and organ injury in hemorrhagic shock and resuscitation. Circ Cardiovasc Genet 7:615-24
Good, Misty; Sodhi, Chhinder P; Hackam, David J (2014) Evidence-based feeding strategies before and after the development of necrotizing enterocolitis. Expert Rev Clin Immunol 10:875-84
Lu, Peng; Sodhi, Chhinder P; Hackam, David J (2014) Toll-like receptor regulation of intestinal development and inflammation in the pathogenesis of necrotizing enterocolitis. Pathophysiology 21:81-93
Hackam, David J; Good, Misty; Sodhi, Chhinder P (2013) Mechanisms of gut barrier failure in the pathogenesis of necrotizing enterocolitis: Toll-like receptors throw the switch. Semin Pediatr Surg 22:76-82
Ford, Henri R; Hackam, David J (2013) Management of premature infants. Preface. Semin Pediatr Surg 22:67-8
Neal, Matthew D; Sodhi, Chhinder P; Dyer, Mitchell et al. (2013) A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis. J Immunol 190:3541-51

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