The Role of Lumican in the Cornea

项目摘要 这一竞争性的更新集中在由LUMICAN调节眼表炎症，一种丰富的 眼表面的细胞外基质(ECM)蛋白。全球有800万人遭受感染， 过敏和自身免疫状况导致角膜结疤和失明。ECM具有重要的作用 在调节局部炎症环境中，虽然其分解可以呈现内源性免疫细胞 危险信号会导致过度的免疫反应。然而，ECM的确切分子机制-- 免疫相互作用还不是很清楚。在其最丰富的形式中，Lumcan是一种结构蛋白， 调节胶原蛋白结构，形成透明的角膜。最近，我们鉴定了它的N-末端 与白细胞表面相互作用的结构域促进Toll样受体4(TLR4)介导的宿主反应 革兰氏阴性细菌脂多糖内毒素。在感染和感染期间重塑的ECM中 炎症，鲁米肯的免疫相互作用可能是一把双刃剑-发展所需的 保护性的先天免疫，但不受限制会导致慢性炎症。我们目前的目标是获得 对LUMICAN-先天免疫细胞相互作用的分子洞察，以开发加强保护的策略 免疫和减少免疫失调使用基于鲁米肯的多肽和抗体。因为我们在体内 炎症环境我们将使用脂多糖和铜绿假单胞菌(PA)角膜炎模型在野生型和 缺乏流明基因的小鼠。其他重组鲁米肯变异体、多肽、抗鲁米肯抗体和细胞 为了这项研究，所有的培养模型都在实验室里开发出来了。以下假设将是 在三个目标上进行了测试。目的1：假设-Lumcan通过增加细胞周内毒素促进免疫反应 或调节脂筏以增强TLR4对细胞表面的募集。脂筏在心脏疾病中起重要作用 作为治疗靶点的先天免疫信号转导和临床相关。我们将使用主服务器 巨噬细胞，细胞系，重组鲁米肯，生化，共聚焦和FRET分析，以解决鲁米肯 细胞表面的受累情况。目标2：假设-免疫活性N-末端结构域是 在胶原蛋白相关的LUMICAN中不能用于免疫细胞的相互作用。我们将使用重组鲁米肯和 三个具有N-末端结构域或中央胶原结合结构域的变体来测试这一点 假设。胶原蛋白处理过的LUMICAN变异体将被测试其诱导内毒素反应的能力 文化。其次，变异体将在野生型和光明型的眼表瞬时表达 缺陷小鼠，角膜炎挑战，以解决它们在调节细胞因子环境中的作用，先天和 适应性免疫细胞与眼表炎症。目标3：假说-眼表炎症可以 被基于鲁米肯的多肽或抗卢米肯抗体调节。我们将测试一种针对N-的抗体- 末端结构域或模拟多肽可以调节发光蛋白-免疫细胞的相互作用以改善 在角膜炎模型中发展炎症，为局部治疗炎症提供前提。

Project Summary This competitive renewal focuses on the regulation of ocular surface inflammation by lumican, an abundant extracellular matrix (ECM) protein at the ocular surface. Eight million people worldwide suffer from infections, allergies and autoimmune conditions leading to corneal scarring and blindness. The ECM has a significant role in modulating the local inflammatory milieu, while its breakdown can present immune cells with endogenous danger signals to drive excessive immune responses. However, the precise molecular mechanisms of ECM- immune interactions are not understood. In its most abundant form lumican is a structural protein that regulates collagen architecture to generate a transparent cornea. Recently, we identified its N-terminal domain to interact with leukocyte surfaces to promote toll-like receptor 4 (TLR4) mediated host response to gram-negative bacterial lipopolysaccharide (LPS) endotoxins. In a remodeling ECM during infections and inflammation, the immune-interactions of lumican can be a double-edged sword - needed for development of protective innate immunity, but unrestricted can drive chronic inflammation. Our current goals are to gain molecular insights into lumican - innate immune cell interactions, to develop strategies to strengthen protective immunity and diminish immune dysregulation using lumican-based peptides and antibodies. As our in vivo inflammatory setting we will use LPS and Pseudomonas aeruginosa (PA) keratitis models in wild type and lumican-deficient mice. Additional recombinant lumican variants, peptides, anti-lumican antibodies and cell cultures models have all been developed in the laboratory for this study. The following hypotheses will be tested in three aims. Aim 1: Hypothesis - Lumican promotes immune response by increasing pericellular LPS or modulates lipid rafts to enhance TLR4 recruitment to the cell surface. Lipid rafts are centrally important in innate immune signal transduction and clinically relevant as targets of therapy. We will use primary macrophages, cell lines, recombinant lumican, biochemical, confocal and FRET analyses to address lumican involvements at the cell surface. Aim 2: Hypothesis - The immunologically active N-terminal domain is unavailable for immune cell interactions in collagen-associated lumican. We will use recombinant lumican and three variants that either have the N-terminal domain or the central collagen binding domain to test this hypothesis. Collagen-pretreated lumican variants will be tested for their abilities to induced LPS response in culture. Secondly, the variants will be transiently expressed at the ocular surface of wild type and lumican deficient mice, and challenged with keratitis to address their role in modulating the cytokine milieu, innate and adaptive immune cells and ocular surface inflammation. Aim 3: Hypothesis - Ocular surface inflammation can be modulated by lumican-based peptides or anti-lumican antibodies. We will test if an antibody against the N- terminal domain or mimetic peptides can modulate lumican-immune cell interactions to ameliorate inflammation in the keratitis models to develop the premises for localized treatments of inflammation.