Neutrophil interactions with apical ICAM-1 regulate intestinal epithelial homeost

描述（由申请人提供）：中性粒细胞（PMN）可以作为促进组织损伤和愈合的“双刃剑”。PMN经上皮迁移（TEM）和结肠上皮隐窝内积聚是粘膜炎症，特别是胃肠道疾病的标志。这些疾病包括炎症性肠病、溃疡性结肠炎和克罗恩病，其中PMN TEM通常与上皮损伤和屏障功能障碍有关。然而，也有大量证据表明PMN在炎症消退中起作用，包括释放脂质介质，如脂质毒素、溶解蛋白和促进愈合的保护蛋白。PMN TEM也被证明可以诱导-catenin依赖性增殖反应，促进粘膜伤口修复。活动期IBD患者炎症肠组织的组织学分析显示，TEM完成后，与肠上皮细胞（IEC）根尖表面密切接触的PMN数量增加，从而持续与肠上皮细胞（IEC）根尖配体结合。广泛的努力致力于确定介导PMN TEM的关键参与者，并建立PMN TEM与上皮屏障功能之间的联系，然而，我们对这一过程的理解仍然有限。PMN与顶端IEC配体的相互作用以及这些相互作用对上皮稳态，特别是上皮伤口修复的影响，我们所知的就更少了。其中一种配体是细胞内粘附分子1 （ICAM-1）。ICAM-1在健康组织中表达水平较低，在炎症期间高度上调。在内皮细胞中，ICAM-1在介导PMN跨内皮迁移和调节屏障功能中的作用已得到证实，但在上皮细胞中，ICAM-1的作用尚不清楚。在炎症组织中，ICAM-1的表达仅限于根尖上皮表面，提示管腔表面的ICAM-1可能在介导PMN-上皮细胞相互作用、促进PMN保留和触发信号事件以改变上皮功能方面发挥作用。我们的初步数据表明，TEM完成后PMN通过与ICAM-1的结合保留在管腔上皮膜上。PMN与ICAM-1结合可显著抑制PMN的凋亡，从而延长附着在根尖上皮膜上的PMN的寿命。此外，我们发现ICAM-1的参与导致上皮完整性降低，并诱导增殖信号，促进上皮伤口修复。因此，本研究的总体目标是确定ICAM-1在顶端上皮表面的PMN参与如何触发信号事件，从而增加上皮通透性，促进PMN转运并刺激上皮修复。在Aim 1中，我们将使用体外和体内PMN TEM模型，辅以分子和蛋白质方法，以及先进的多光子活体成像，定义ICAM-1连接下游增加上皮通透性以促进PMN TEM增强的特定信号事件。在Aim 2中，我们将定义ICAM-1在调节上皮稳态和伤口修复中的特定作用，使用体外抓伤试验和新型急性损伤和炎症的体内模型，包括结肠镜活检伤口和dss诱导的结肠粘膜损伤。当前提案中概述的实验将揭示PMN TEM和粘膜表面保留的调节机制，并有助于鉴定PMN-上皮细胞与上皮屏障功能和伤口修复相互作用的特定分子。这对于开发新的和改进的治疗方法促进增强宿主防御功能和解决粘膜炎症以及重建IEC稳态是必要的。

DESCRIPTION (provided by applicant): It is well appreciated that neutrophils (PMN) can act as a "double edged sword" in promoting both tissue injury and healing. PMN transepithelial migration (TEM), and accumulation within colonic epithelial crypts, is a hallmark of mucosal inflammation, and particularly, diseases of the gastrointestinal tract. These include inflammatory bowel diseases, ulcerative colitis and Crohn's disease, where PMN TEM is often associated with epithelial injury and barrier dysfunction. However, there is also abundant evidence for PMN function in resolution of inflammation, including release of lipid mediators, such as lipoxins, resolvins and protectins that facilitate healing. PMN TEM has also been shown to induce �-catenin dependent proliferative responses promoting mucosal wound repair. Histological analysis of inflamed intestinal tissue from patients with active IBD show increased numbers of PMN that remain in intimate contact with apical IEC surface after completing TEM, thus continuously engaging intestinal epithelial cell (IEC) apical ligands. Extensive efforts were dedicated to identifying key players that mediate PMN TEM, and establishing the link between PMN TEM and epithelial barrier function, however, our understanding of this process is still limited. Even less is known about PMN interactions with apical IEC ligands and the consequent effects of these interactions on epithelial homeostasis, and specifically epithelial wound repair. One such ligand is intracellular adhesion molecule 1 (ICAM-1). ICAM-1 is expressed at low levels in healthy tissue, and is highly upregulated during inflammation. Unlike in endothelium, where the role of ICAM-1 in mediating PMN transendothelial migration and in regulating barrier function is well established, in epithelium the role for ICAM-1 is still not clear. In inflamed tisue its expression is restricted to apical epithelial surface, suggesting that ICAM-1 on the luminal surface may play a role in mediating PMN-epithelial cell interactions, promoting PMN retention and triggering signaling events to alter epithelial function. Our preliminary data suggest that PMN after completion of TEM are retained on the luminal epithelial membrane through binging to ICAM-1. PMN binding to ICAM-1 significantly inhibits PMN apoptosis, resulting in prolonged life span of PMN adherent to the apical epithelial membrane. Moreover, we found that engagement of ICAM-1 leads to decreased epithelial integrity and an induction of proliferative signaling facilitating epithelial wound repair. Thus the overall goal of this proposal is to determine how PMN engagement of ICAM-1 on the apical epithelial surface triggers signaling events to increase epithelial permeability, promote PMN transmigration and stimulate epithelial repair. In Aim 1 we will define the specific signaling events downstream of ICAM-1 ligation that increase epithelial permeability to facilitate enhanced PMN TEM, using in- vitro and in-vivo models of PMN TEM, supplemented with molecular and protein approaches, and advanced multi-photon intravital imaging. In Aim 2 we will define the role for specific engagement of ICAM-1 in regulation of epithelial homeostasis and wound repair using in-vitro scratch wound assays and novel in-vivo models of acute injury and inflammation, including colonoscopic biopsy-wound and DSS-induced colonic mucosal injury. Experiments outlined in the current proposal will shed new light on mechanisms regulating PMN TEM and retention at the mucosal surfaces, and aid in identification of specific molecules that link PMN-epithelial cell interactions with epithelial barrier function and wound repair. This is imperative for the development of new and improved therapeutic approaches facilitating enhanced host defense function and resolution of mucosal inflammation, and reestablishing IEC homeostasis.