Interferon-g and Rip3 Play an Critical Role in Apoptosis-Induced Necrotizing Enterocolitis-Like Intestinal Injury in Neonatal Mice

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of preterm infants characterized by intestinal inflammation and intestinal epithelial cells (IEC) death. Evidence suggests that scattered crypt IEC apoptosis is a critical pathogenic event that subsequently leads to excessive villus necrosis in human NEC and rodent pups with experimental NEC. Necroptosis, programmed necrosis, is a proinflammatory cell death process that has been linked to several intestinal diseases. However, the critical role of necroptosis and its upstream pathways in the pathogenesis of NEC remains largely unexplored. Here, we aimed to study the linkage between scattered crypt IEC apoptosis and necroptosis during NEC development using a mouse genetic engineering approach. First, a unique triple-transgenic mouse model, 3xTg-iEAP (inducible Enterocyte specific Apoptosis Phenotype), was developed to induce IECs-specific over-expression of FasL using LoxP-Villin-Cre-mediated recombination strategy in combination with the rtTA-based regulatory system. Second, we created 3xTg-iEAP Ifng-/- and 3xTg-iEAP Rip3-/- mice by breeding 3xTg-iEAP with either Ifnγ knockout (KO) mice or Rip3 KO mice. The three days old neonatal pups of 3xTg-iEAP, 3xTg-iEAP Ifng-/- , 3xTg-iEAP Rip3-/- and littermate controls were challenged with a single dose doxycycline (Dox; 20 μg/gm, subcutaneously). Their growth was monitored on daily basis. The intestinal tissue samples were collected at different time points (8 h, 24 h, 48 h, and 72 h) and subjected to histological and qRT-PCR analysis. We confirmed that 3xTg-iEAP, 3xTg-iEAP Ifng-/- , and 3xTg-iEAP Rip3-/- mice developed normal, viable, and fertile, and indistinguishable from littermate controls without exposure to Dox. Treatment with Dox resulted in scattered IEC apoptosis in crypts of 3xTg-iEAP, 3xTg-iEAP Ifng-/- , and 3xTg-iEAP Rip3-/- mice within 8 h. Compared to littermate controls, we observed NEC-like damage with profound intestinal villus epithelial necrosis at 48 h after Dox treatment in 3xTg-iEAP mice that mimics histological features of human NEC. qRT-PCR analysis showed that the development of villus necrosis was associated with the increased expression of pro-inflammatory mediator genes and necroptosis-associated genes including Ifnγ and Rip3 in 3xTg-iEAP mice during the phase of NEC development. Interestingly, compared to 3xTg-iEAP mice, 3xTg-iEAP Ifng-/- and 3xTg-iEAP Rip3-/- mice displayed a lack of NEC-like incidence at 48h after Dox treatment, suggesting the crucial role of IFN-γ and RIP3 in scattered IEC apoptosis-induced NEC in our model. Taken together, the inhibition of necroptosis in IECs prevents intestinal injury during NEC. Our findings may advance knowledge in preventing or treating this devastating disorder in humans. Grants from NIH and the U.S. Department of Veterans Affairs support this work. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.