TLR4 Signaling in the Pathogenesis of Surgical Necrotizing Enterocolitis

描述(由申请人提供)：拟议研究的目标是确定导致坏死性小肠结肠炎(NEC)发展的机制，NEC是早产儿胃肠道疾病导致死亡和残疾的主要原因，并确定针对这种破坏性疾病的新治疗策略。为了实现这些目标，我们现在重点研究先天免疫受体Toll样受体-4(TLR4)，它是已知的脂多糖(LPS)受体，在第一个资助期，我们已经确定它在本病的发病机制中发挥关键作用。我们现在试图定义TLR4激活导致NEC的分子和细胞机制。为此，我们培育了肠道上皮细胞上缺乏TLR4的小鼠，与野生型小鼠相比，这些小鼠被发现受到保护，不受NEC发育的影响。在试图了解相关机制的过程中，我们现在证明了TLR4在调节正常的肠上皮细胞分化中发挥了一个以前未被认识到的重要作用。我们还确定TLR4表达在肠干细胞(ISC)上，并且ISC增殖-肠粘膜愈合能力的关键决定因素-在NEC中减少。为了确定ISC上的TLR4激活是否是失去ISC增殖所必需的，我们产生了ISC上缺乏TLR4的小鼠，与野生型小鼠相比，ISC上的TLR4激活后表现出完整的增殖。我们现在假设，早产儿中不适当的TLR4信号通过损害肠道干细胞的活性和功能而导致NEC的发展。为了验证这一假说，我们提出了以下三个目标：目的1.探讨TLR4激活在坏死性小肠结肠炎发病机制中的作用。目的2.探讨TLR4在发育过程中的异常激活对肠道炎症的影响。目的3.探讨TLR4介导的肠干细胞增殖丧失在坏死性小肠结肠炎发病机制中的作用，以及恢复肠干细胞增殖能否预防或减轻疾病的严重程度。为了实现这些研究目标，我们培育了在肠上皮中缺乏或过表达TLR4或在ISC上缺乏TLR4的小鼠品系，开发了肠道干细胞分离和培养技术，并成功地将TLR4激动剂引入到子宫发育中的小鼠肠道中。这些研究将在将先天性免疫系统与肠道发育联系起来方面取得重大的概念性进展，将通过解释基于TLR4在肠道干细胞中的作用来解释早产儿的易感性来促进我们对NEC的理解，并将评估试图逆转TLR4对肠道干细胞修复早产小肠受损黏膜能力的有害影响的新型抗NEC疗法。 公共卫生相关性：坏死性小肠结肠炎是早产儿胃肠道疾病死亡的主要原因，目前还没有有效的治疗方法。目前的提议试图通过关注免疫系统和新生儿肠道干细胞之间的相互作用来了解坏死性小肠结肠炎的原因，从而发现这种毁灭性疾病的新疗法。

DESCRIPTION (provided by applicant): 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 accomplish these goals, we now focus on the innate immune receptor Toll like receptor-4 (TLR4), which is known to be the receptor for lipopolysaccharide (LPS), and which in the first funding period, we have identified to play a critical role in the pathogenesis of this disease. We now seek to define the molecular and cellular mechanisms by which TLR4 activation leads to NEC. To do so, we generated mice that lack TLR4 specifically on the intestinal epithelium, which were found to be protected from NEC development as compared with wild-type mice. In seeking to understand the mechanisms involved, we now demonstrate that TLR4 exerts an important and previously unrecognized role in the regulation of normal intestinal epithelial differentiation. We also determined that TLR4 is expressed on the intestinal stem cells (ISC's), and that ISC proliferation - a key determinant of the ability of the intestinal mucosa to heal - is reduced in NEC. To define whether TLR4 activation on ISC's was required for the loss of ISC proliferation, we generated mice that lack TLR4 specifically on the ISC's, which showed intact proliferation after TLR4 activation compared with wild-type mice. We now hypothesize that inappropriate TLR4 signaling in the preterm infant leads to the development of NEC by impairing the activity and function of the intestinal stem cells. To test this hypothesize we propose the following three aims: Aim 1. To investigate the role of TLR4 activation in regulating intestinal epithelial differentiation in the pathogenesis of necrotizing enterocolitis. Aim 2. To determine the effects of inappropriate activation of TLR4 in the developing intestine on the induction of intestinal inflammation. Aim 3. To characterize the role of TLR4-mediated loss of intestinal stem cell proliferation in the pathogenesis of necrotizing enterocolitis, and to determine whether restoring intestinal stem cell proliferation can prevent or attenuate the severity of the disease. To accomplish these research goals, we have generated mice strains that lack or overexpress TLR4 in intestinal epithelium or that lack TLR4 on the ISC's, have developed techniques of intestinal stem cell isolation and culture and have successfully introduced TLR4 agonists into the developing mouse intestine in utero. These studies will make a significant conceptual advance in linking the innate immune system with intestinal development, will advance our understanding of NEC by explaining the susceptibility of the premature infant based on the role of TLR4 in the intestinal stem cells, and will evaluate novel anti-NEC therapies which seek to reverse the deleterious effects of TLR4 on the ability of the intestinal stem cells to repair the injured mucosa of 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 the interaction between the immune system and the stem cells of the newborn intestine, and therefore to discover novel treatments for this devastating disease.