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)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK083752-06
Application #
8449869
Study Section
Special Emphasis Panel (ZRG1-SBIB-J (02))
Program Officer
Hamilton, Frank A
Project Start
2008-09-15
Project End
2017-06-30
Budget Start
2012-09-18
Budget End
2013-06-30
Support Year
6
Fiscal Year
2012
Total Cost
$321,421
Indirect Cost
$103,921
Name
University of Pittsburgh
Department
Surgery
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Hackam, David J; Sodhi, Chhinder P (2018) Toll-Like Receptor-Mediated Intestinal Inflammatory Imbalance in the Pathogenesis of Necrotizing Enterocolitis. Cell Mol Gastroenterol Hepatol 6:229-238.e1
Renz, Harald; Adkins, Becky D; Bartfeld, Sina et al. (2018) The neonatal window of opportunity-early priming for life. J Allergy Clin Immunol 141:1212-1214
Goldstein, Seth D; Beaulieu, Robert J; NiƱo, Diego F et al. (2018) Early detection of necrotizing enterocolitis using broadband optical spectroscopy. J Pediatr Surg 53:1192-1196
Sodhi, Chhinder P; Fulton, William B; Good, Misty et al. (2018) Fat composition in infant formula contributes to the severity of necrotising enterocolitis. Br J Nutr 120:665-680
Lu, P; Sodhi, C P; Yamaguchi, Y et al. (2018) Intestinal epithelial Toll-like receptor 4 prevents metabolic syndrome by regulating interactions between microbes and intestinal epithelial cells in mice. Mucosal Immunol 11:727-740
Chen, May W; Reyes, Michael; Kulikowicz, Ewa et al. (2018) Abdominal near-infrared spectroscopy in a piglet model of gastrointestinal hypoxia produced by graded hypoxia or superior mesenteric artery ligation. Pediatr Res 83:1172-1181
Sodhi, Chhinder P; Wohlford-Lenane, Christine; Yamaguchi, Yukihiro et al. (2018) Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration. Am J Physiol Lung Cell Mol Physiol 314:L17-L31
Martin, Laura Y; Ladd, Mitchell R; Werts, Adam et al. (2018) Tissue engineering for the treatment of short bowel syndrome in children. Pediatr Res 83:249-257
Hackam, David J; Sodhi, Chhinder P; Good, Misty (2018) New insights into necrotizing enterocolitis: From laboratory observation to personalized prevention and treatment. J Pediatr Surg :
Jia, Hongpeng; Sodhi, Chhinder P; Yamaguchi, Yukihiro et al. (2018) Toll Like Receptor 4 Mediated Lymphocyte Imbalance Induces Nec-Induced Lung Injury. Shock :

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