Neutrophil interactions with apical ICAM-1 regulate intestinal epithelial homeost

中性粒细胞与顶端 ICAM-1 的相互作用调节肠上皮稳态

• 批准号: 8679593
• 负责人: Ronen Sumagin
• 金额: $ 1.83万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8679593
• 关键词: Actins; Actomyosin; Acute; Adhesions; Adhesives; Apical; Apoptosis; Bacterial Infections; Binding; Biochemical; Biological Assay; Biopsy; Blood; CD59 Antigen; Cell Adhesion Molecules; Cell Communication; Cell Line; Cell membrane; Cell surface; Cells; Chemotactic Factors; Complex; Crohn' s disease; Data; Development; Dextrans; Disease; E-Cadherin; Endothelial Cells; Endothelium; Epithelial; Epithelial Cell Junction; Epithelial Cell Proliferation; Epithelial Cells; Epithelium; Event; Fluorescence; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Focal Adhesion Kinase 1; Functional disorder; Gastrointestinal tract structure; Goals; Healed; Homeostasis; Host Defense; Image; Immigration; Immune; In Vitro; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Injury; Integrins; Intercellular adhesion molecule 1; Interferons; Intestines; Lateral; Lead; Ligands; Ligation; Light; Link; Lipoxins; Longevity; Luciferases; Mediating; Membrane; Modeling; Molecular; Mucositis; Mus; Patients; Permeability; Play; Process; Proteins; Receptor Signaling; Regulation; Reporter; Resistance; Resolution; Role; Salmonella; Signal Transduction; Staging; Surface; Testing; Therapeutic; Time; Tissues; Ulcerative Colitis; Up-Regulation; Wound Healing; cell motility; crosslink; dextran; healing; improved; in vivo; in vivo Model; injury and repair; intravital imaging; intravital microscopy; lipid mediator; migration; multi-photon; neutrophil; novel; occludin; overexpression; prevent; public health relevance; repaired; research study; response; two-photon; wound

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 β-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.

描述（由申请人提供）：众所周知，中性粒细胞（PMN）在促进组织损伤和愈合方面可充当“双刃剑”。中性粒细胞经上皮迁移（TEM）和在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相关。然而，也有大量证据表明PMN在炎症消退中的功能，包括释放脂质介质，如脂氧素、消退素和保护素，促进愈合。PMN TEM还显示诱导β-连环蛋白依赖性增殖反应，促进粘膜伤口修复。对活动性IBD患者的发炎肠组织的组织学分析显示，在完成TEM后，保持与顶端IEC表面紧密接触的PMN数量增加，从而持续接合肠上皮细胞（IEC）顶端配体。人们付出了大量努力来确定介导中性粒细胞TEM的关键参与者，并建立中性粒细胞TEM与上皮屏障功能之间的联系，然而，我们对这一过程的了解仍然有限。甚至更少的是已知的PMN与顶端IEC配体的相互作用，以及这些相互作用对上皮内环境稳定的影响，特别是上皮伤口修复。一种这样的配体是细胞内粘附分子1（ICAM-1）。ICAM-1在健康组织中以低水平表达，并且在炎症期间高度上调。与内皮细胞不同，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充分证实，而在上皮细胞中ICAM-1的作用仍不清楚。在炎症组织中，其表达仅限于顶端上皮表面，表明腔表面上的ICAM-1可能在介导PMN-上皮细胞相互作用、促进PMN滞留和触发信号事件以改变上皮功能中起作用。我们的初步数据表明，在完成TEM后，PMN通过结合ICAM-1而保留在管腔上皮膜上。PMN与ICAM-1的结合可显著抑制PMN凋亡，从而延长粘附于顶端上皮膜的PMN的寿命。此外，我们发现ICAM-1的参与导致上皮完整性降低，并诱导促进上皮伤口修复的增殖信号。因此，本提案的总体目标是确定顶上皮表面上的ICAM-1的PMN接合如何触发信号传导事件以增加上皮渗透性、促进PMN迁移和刺激上皮修复。在目标1中，我们将使用PMN TEM的体外和体内模型，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连接下游的特异性信号传导事件，其增加上皮通透性以促进增强的PMN TEM。在目标2中，我们将使用体外划痕试验和急性损伤和炎症的新型体内模型（包括结肠镜活检伤口和DSS诱导的结肠粘膜损伤）来确定ICAM-1在调节上皮稳态和伤口修复中的作用。在目前的建议中概述的实验将揭示新的光调节PMN TEM和保留在粘膜表面的机制，并有助于识别连接PMN上皮细胞与上皮屏障功能和伤口修复的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，并重建IEC稳态是必要的。