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 from gastrointestinal disease in premature infants, and to determine novel therapeutic strategies for this devastating disease. To do so, we will explore the role of th bacterial endotoxin receptor Toll-like receptor 4 (TLR4) in the pathogenesis of NEC through its previously unrecognized effects on the adaptive immune system of the premature intestine. In the last funding period, we discovered that TLR4 activation on the intestinal epithelium plays a critical role in NEC development, as mice lacking TLR4 on the intestinal epithelium are protected from NEC. We also determined that TLR4 expression on the intestinal epithelium is higher in the premature human and mouse intestine, which in mice reflects a novel role played by TLR4 in the regulation of intestinal differentiation. However, the mechanisms by which TLR4 signaling in the premature intestine causes NEC are unknown. We now show that TLR4 activation leads to NEC through a STAT3-dependent increase in pro-inflammatory Th-17 lymphocytes in the intestinal mucosa, as lymphocyte deficient mice were protected from NEC, the adoptive transfer of intestinal lymphocytes obtained from mice with NEC induced intestinal inflammation in nave mice, and STAT3 inhibition prevented TLR4-mediated Th-17 induction. Th-17 cells caused intestinal injury via the release of IL-17, which induced enterocyte apoptosis and reduced enterocyte proliferation. Further, using in silico screening, we identified a novel class of TLR4 inhibitors that treat NEC in mice and piglets, and reduced pro-inflammatory Th-17 lymphocytes while increasing anti-inflammatory Treg lymphocytes. Based upon these findings, we now hypothesize that exaggerated TLR4 signaling within the mucosa of the newborn intestine leads to NEC through its novel role in the induction of pro-inflammatory Th-17 cells and reduction of anti- inflammatory Treg lymphocytes, that the induction of Th-17 cells leads to NEC by increasing enterocyte apoptosis and reducing mucosal repair, and that a novel TLR4 inhibitor protects against NEC by reducing Th- 17 cells and increasing Tregs. We will test these hypotheses in three aims:
Aim 1 : To understand how TLR4 signaling in the premature gut increases in pro-inflammatory Th-17 cells and reduces anti-inflammatory Tregs within the intestinal mucosa in the pathogenesis of NEC;
Aim 2 : To understand how IL-17 release leads to NEC via increased apoptosis and reduced proliferation in the premature gut;
Aim 3. To evaluate experimental strategies of TLR4 inhibition to prevent NEC in mice and piglets by inhibiting Th-17 cells and increasing Tregs. These studies will make a significant conceptual advance by defining how TLR4 signaling leads to mucosal injury in NEC through previously unrecognized effects on the balance between pro-inflammatory Th-17 and anti-inflammatory Tregs, and through the evaluation of novel anti-NEC therapies based TLR4 inhibition in the premature 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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Zhao, Xiaoli
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Johns Hopkins University
Schools of Medicine
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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
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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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