Insights into a multi-hit process in the development of necrotizing enterocolitis

坏死性小肠结肠炎（NEC）是一种毁灭性的肠道炎症性疾病，主要影响 早产儿和极低出生体重儿。NEC的常见风险因素包括 早产、配方奶粉喂养、肠道生态失调和感染。先前的研究强烈表明， 不适当的微生物组定植和激活新生儿免疫系统在NEC的发展。 然而，NEC的发病机制是难以捉摸的。特别是，目前尚不清楚NEC相关风险如何 这些因素导致了这种疾病。在初步研究中，我们描述了配方奶喂养对 新生小鼠肠道植物群、基因表达和免疫生物学。我们还检查了肠道病理学 用配方奶喂养小鼠幼崽，然后诱导特定的抗微生物免疫应答。 我们发现，配方奶粉喂养单独导致一种独特的肠道生态失调和前NEC肠道分子 使新生儿肠道易受不适当的微生物组定植/感染的变化， 粘膜是细胞毒性炎性细胞的靶点。我们进一步发现，配方奶喂养的，但不是母鼠喂养的， 在激活细胞毒性炎症细胞相关的抗微生物免疫后，小鼠幼崽发生NEC 反应因此，NEC似乎是在早产儿中不适当的微生物组定植后发展的。 婴儿是“多次发病的病理生理事件”的结果。在这个项目中，我们将研究新的机制， 深入了解这些事件，并确定多重打击事件的相互作用如何有助于发展 NEC的两个互补的目标：（1）我们将首先描述一系列的病理生理事件， 导致NEC的发展，使用一种新的和病理相关的小鼠幼仔模型NEC和最新的 体内实验病理学和免疫学方法。然后，我们将使用RNAseq和尖端的 生物信息学分析来描绘小鼠幼仔小肠对多种 挑战和解开这种新的NEC小鼠模型与人类NEC的相关性。而且我们 将研究主要NEC风险因素引发的“多击事件”如何通过关注以下方面促进NEC发展 炎性细胞、粘膜炎症相关的炎症介质和独特的信号轴， 保护肠上皮细胞免受炎性细胞攻击。我们将在体内使用 利用分子生物学和小鼠基因工程技术的实验方法。(2)我们将 阐明配方奶喂养如何导致早产儿小肠中NEC前分子的变化 通过采用结合类器官培养技术的多学科体内和体外方法， 分子和细胞生物学、微生物学、小鼠基因工程和基因工程学。我们一起努力， 提供了一种新的与人类NEC相关的小鼠模型， 危险因素和新生儿不成熟免疫系统的激活触发NEC的发展， 机械的见解，将告知新的战略，为预防和治疗NEC的发展。

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease that primarily affects premature infants and extremely low birth weight babies. Commonly observed risk factors for NEC are prematurity, formula feeding, intestinal dysbiosis, and infection. Previous studies strongly suggest a critical role of the inappropriate microbiome colonization and activation neonatal immune system in NEC development. However, the pathogenesis of NEC is elusive. Particularly, it remains unclear how the NEC-associated risk factors contribute to the disorder. In preliminary studies, we characterized the effect of formula-feeding on intestinal flora, gene expression, and immunobiology in neonatal mice. We also examined intestinal pathology of mouse pups which were fed with formula followed by induction of a particular antimicrobial immune response. We found that formula-feeding alone resulted in a distinct type of gut dysbiosis and pre-NEC intestinal molecular changes that predispose the neonatal gut to inappropriate microbiome colonization/infection and render intestinal mucosa to be a target of cytotoxic inflammatory cells. We further revealed that formula-fed but not dam-fed mouse pups developed NEC upon activation of a cytotoxic inflammatory cell-associated antimicrobial immune response. Thus, it appears that NEC develops following inappropriate microbiome colonization in premature infants as a consequence of “multi-hit pathophysiological events”. In this project, we will study new mechanistic insights into these events and determine how the interaction of multiple-hit events contributes to the development of NEC in two complementary aims: (1) We will first characterize the series of pathophysiological events that leads to NEC development, using a novel and pathologically relevant mouse pup model of NEC and up-to-dated in vivo experimental pathological and immunological approaches. Then, we will use RNAseq and cutting-edge bioinformatic analysis to delineate the transcriptomic response of the small intestine of mouse pups to multiple- hit challenges and to unravel the relevance of this novel mouse model of NEC for human NEC. Furthermore, we will study how major NEC risk factor-induced “multi-hit events” contribute to NEC development by focusing on inflammatory cells, mucosal inflammation-associated inflammatory mediators, and a unique signal axis that protects intestinal epithelial cells against inflammatory cell attack. We will achieve this aim using in vivo experimental approaches that draw on molecular biology and mouse genetic engineering techniques. (2) We will elucidate how formula feeding causes pre-NEC molecular changes in the small intestine of premature neonates by taking a multidisciplinary in vivo and in vitro approaches that incorporate techniques of organoid culture, molecular and cellular biology, microbiology, mouse genetic engineering and gnotobiogy. Together, our work will provide a novel mouse model relevant for human NEC, advance knowledge of how the interaction between NEC risk factors and activation of the neonatal immature immune system triggers NEC development, and gain mechanistic insights that will inform the development of new strategies for the prevention and treatment of NEC.