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

坏死性小肠结肠炎(NEC)是一种破坏性的肠炎性疾病，主要影响 早产儿和极低出生体重儿。通常观察到的NEC风险因素有 早产、配方奶喂养、肠道生态失调和感染。以前的研究有力地表明了 在NEC发育过程中不适当的微生物定植和激活新生儿免疫系统。 然而，NEC的发病机制尚不清楚。特别是，目前还不清楚NEC相关的风险是如何 导致这种紊乱的因素有很多。在初步研究中，我们描述了配方奶喂养对 新生小鼠肠道菌群、基因表达和免疫生物学。我们还检查了肠道病理。 用配方奶粉喂养小鼠幼鼠，然后诱导特定的抗微生物免疫反应。 我们发现，仅配方奶喂养会导致一种不同类型的肠道微生物失调和NEC前期肠道分子 使新生儿肠道容易受到不适当的微生物定植/感染并使肠道 粘膜成为细胞毒炎性细胞的靶点。我们进一步揭示了配方奶喂养但不是水坝喂养 小鼠幼鼠在激活细胞毒性炎症细胞相关的抗微生物免疫后发生NEC 回应。因此，NEC似乎是在早产儿不适当的微生物定植后发展起来的。 婴儿是“多发的病理生理事件”的结果。在这个项目中，我们将研究新的机械 对这些事件的洞察，并确定多个命中事件的相互作用如何促进发展 NEC的两个相辅相成的目标：(1)我们将首先描述一系列病理生理事件 导致NEC的发展，使用一种新的和病理相关的NEC小鼠模型和最新的 体内实验病理学和免疫学方法。然后，我们将使用RNAseq和尖端 用生物信息学分析方法研究小鼠小肠对多效唑的转录反应 Hit挑战，并揭示这种新型的NEC小鼠模型与人类NEC的相关性。此外，我们 将通过重点研究主要NEC风险因素引发的“多发事件”如何促进NEC的发展 炎症细胞，粘膜炎症相关的炎症介质，以及一个独特的信号轴 保护肠道上皮细胞免受炎性细胞攻击。我们将通过在体内实现这一目标 利用分子生物学和小鼠基因工程技术的实验方法。(2)我们会 阐明配方奶喂养如何引起早产儿小肠NEC前期分子变化 通过采取多学科的体内和体外方法，结合有机培养技术， 分子和细胞生物学、微生物学、小鼠基因工程和生理学。共同努力，我们的工作将 提供了一种与人类NEC相关的新型小鼠模型，加深了对NEC之间相互作用的了解 风险因素和新生儿不成熟免疫系统的激活触发NEC的发展，并获得 将为制定预防和治疗NEC的新战略提供信息的机械性见解。

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.