Modulation of TLR4 and TLR9 Signaling in NEC

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。为此，我们将通过细菌内毒素受体Toll样受体4(TLR4)在早产儿肠粘膜内质网(ER)应激诱导中的作用来探讨其在NEC发病机制中的作用。内质网应激反映了内质网管腔内累积的错误折叠蛋白的细胞状态，这导致了快速的细胞凋亡。在之前的资助期间，我们发现肠粘膜中的TLR4信号是NEC发生所必需的，并且NEC的特征是依赖TLR4诱导肠细胞凋亡，导致粘膜损伤。我们还发现，TLR4的同源受体，即识别细菌DNA(“CpG-DNA”)的TLR9，通过抑制TLR4信号和减少肠细胞凋亡来减弱NEC。然而，重要的是，TLR9抑制导致NEC的TLR4信号通路的机制仍然不清楚。我们现在提供的证据表明，TLR4激活通过增加新生儿肠上皮内ER应激而导致NEC，TLR9激活通过细胞内伴侣热休克蛋白70(Hsp70)抑制TLR4介导的ER应激来保护NEC。值得注意的是，早产小鼠的特点是肠道上皮细胞内质网应激增加依赖TLR4，这一发现也见于人类婴儿。我们现在假设，TLR4在新生儿肠粘膜中的激活通过诱导肠上皮细胞内ER应激导致肠细胞凋亡而导致NEC的发生，而TLR9通过细胞内伴侣Hsp70激活可以逆转这一过程。我们进一步假设，发育中胎儿肠道TLR4和TLR9的宫内调节可以减轻内质网应激，阻止NEC的发生。我们将在三个具体目标中检验这一假说：目的1.研究TLR4激活的作用 在坏死性小肠结肠炎发病机制中调节新生儿肠道上皮细胞的内质网应激。目的2.探讨TLR9激活减轻TLR4诱导的内质网应激和NEC严重程度的机制。目的3.探讨TLR4和TLR9的宫内调节能否抑制内质网应激，预防NEC的发生。这些研究将通过确定TLR4信号如何导致NEC中的肠细胞凋亡和粘膜损伤，通过解释基于TLR4诱导的粘膜ER应激增加的早产儿对NEC的易感性，以及通过评估基于早期小肠内ER应激的减弱的新型抗NEC治疗方法，取得重大的概念性进展。

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 do so, we will explore the role of the bacterial endotoxin receptor Toll like receptor 4 (TLR4) in the pathogenesis of NEC through its previously unrecognized effects on the induction of endoplasmic reticulum (ER) stress in the intestinal mucosa of the premature infant. ER stress reflects a cellular state of accumulated mis-folded proteins within the lumen of the ER, which leads to rapid apoptosis. In the previous funding period, we discovered that TLR4 signaling in the intestinal mucosa is required for NEC development and that NEC is characterized by a TLR4-dependent induction of enterocyte apoptosis leading to mucosal injury. We also showed that the homologous receptor for TLR4, namely TLR9 - which recognizes bacterial DNA ("CpG-DNA") - attenuated NEC through the inhibition of TLR4 signaling and a reduction in enterocyte apoptosis. Importantly however, the mechanisms by which TLR9 inhibited the TLR4 signaling pathways that lead to NEC remained unexplained. We now provide evidence that TLR4 activation leads to NEC through an increase in ER stress within the newborn intestinal epithelium, and that TLR9 activation protects against NEC by inhibiting TLR4- mediated ER stress via the intracellular chaperone heat shock protein 70 (Hsp70). Strikingly, the premature mouse was characterized by a TLR4-dependent increase in ER stress in the intestinal epithelium, a finding also seen in human infants. We now hypothesize that TLR4 activation within the newborn intestinal mucosa leads to the development of NEC by inducing ER stress in the intestinal epithelium leading to enterocyte apoptosis, which can be reversed by TLR9 activation through the intracellular chaperone Hsp70. We further hypothesize that the in-utero regulation of intestinal TLR4 and TLR9 in the developing fetus can reduce ER stress and prevent the development of NEC. We will test this hypothesis in three specific aims: AIM 1. To investigate the role of TLR4 activation in regulating ER stress in the newborn intestinal epithelium in the pathogenesis of necrotizing enterocolitis. AIM 2. To determine the mechanisms by which TLR9 activation reduces TLR4-induced ER stress and NEC severity. AIM 3. To evaluate whether the in utero regulation of TLR4 and TLR9 can inhibit ER stress and prevent the development of NEC. These studies will make a significant conceptual advance by defining how TLR4 signaling leads to enterocyte apoptosis and mucosal injury in NEC, and by explaining the susceptibility of the premature infant to NEC based on increased TLR4-induced mucosal ER stress, and through the evaluation of novel anti-NEC therapies based upon the attenuation of ER stress within the premature small intestine.