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: 8850858
• 负责人: Ronen Sumagin
• 金额: $ 15.07万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-19 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8850858
• 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; 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）可以充当促进组织损伤和愈合的“双刃剑”。 PMN 跨上皮迁移（TEM）和结肠上皮隐窝内的积聚是粘膜炎症，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，也有大量证据表明中性粒细胞在消退炎症方面具有功能，包括释放脂质介质，例如促进愈合的脂氧素、分解素和保护素。 PMN TEM 还被证明可以诱导 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 患者发炎肠道组织的组织学分析显示，在完成 TEM 后，与顶端 IEC 表面保持密切接触的 PMN 数量增加，从而持续与肠上皮细胞 (IEC) 顶端配体结合。人们付出了大量的努力来确定介导 PMN TEM 的关键参与者，并建立 PMN TEM 和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。关于 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后续影响，人们知之甚少。一种这样的配体是细胞内粘附分子1 (ICAM-1)。 ICAM-1 在健康组织中表达水平较低，在炎症过程中表达高度上调。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确立，而在上皮细胞中，ICAM-1 的作用仍不清楚。在发炎组织中，其表达仅限于顶端上皮表面，表明管腔表面上的 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，完成 TEM 后，PMN 通过与 ICAM-1 结合而保留在管腔上皮膜上。 PMN 与 ICAM-1 结合显着抑制 PMN 凋亡，从而延长粘附于根尖上皮膜的 PMN 寿命。此外，我们发现 ICAM-1 的参与会导致上皮完整性降低，并诱导增殖信号传导促进上皮伤口修复。因此，该提案的总体目标是确定 PMN 与根尖上皮表面 ICAM-1 的结合如何触发信号事件，从而增加上皮通透性、促进 PMN 迁移并刺激上皮修复。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 连接下游的特定信号事件，这些事件增加上皮通透性，以促进增强的 PMN TEM。在目标 2 中，我们将使用体外划痕伤口测定和新型体内急性损伤和炎症模型（包括结肠镜活检伤口和 DSS 诱导的结肠粘膜损伤）来定义 ICAM-1 在调节上皮稳态和伤口修复中的特定作用。当前提案中概述的实验将为调节 PMN TEM 和粘膜表面保留的机制提供新的线索，并有助于识别将 PMN-上皮细胞相互作用与上皮屏障功能和伤口修复联系起来的特定分子。这对于开发新的和改进的治疗方法至关重要，以促进增强宿主防御功能和解决粘膜炎症，并重建 IEC 稳态。