The Role of Lumican in the cornea

描述(申请人提供)：无血管角膜是一种保护性屏障，具有完整的天然免疫，但限制了适应性免疫反应，以实现最佳视力。手术、角膜损伤和感染会破坏先天性免疫反应和获得性免疫反应之间的平衡，导致严重的炎症和视力丧失。我们发现LUMICAN，一种丰富的细胞外基质(ECM)基质蛋白多糖，可以调节角膜免疫功能。Lumcan本身结合的内毒素可以被CD14竞争性清除，CD14是一种细胞表面蛋白，将内毒素转移到Toll样受体4，即跨膜IPS受体。缺乏鲁米肯(Lum-/-)的小鼠对细菌脂多糖内毒素(LPS)反应迟钝，产生的促炎细胞因子数量较少。更重要的是，在脂多糖诱导的角膜角膜炎中，Lum-/-小鼠早期表现出中性粒细胞流入减少，但总体上延迟愈合。因此，我们的Lum-/-小鼠的先天免疫反应受损。Lumcan还结合FasL和TGFbeta，在Lum-/-小鼠中，这些途径被破坏。Fas-FasL介导的角膜细胞凋亡和转化生长因子β信号有助于维持免疫抑制环境以获得免疫豁免。我们的主要假设是，在角膜中，鲁米肯促进了早期的先天性免疫反应，但提供了一个免疫抑制的微环境，以限制适应性免疫产生机制，以维持免疫豁免。以下目标将检验这一假设。1)检测Lumcan是否调节角膜炎症和免疫豁免，从而使Lum/-角膜移植物的存活率降低。通过评估Lum+/+和Lum-/-BMC嵌合体宿主的同种异体移植成功，测试Lumcan是否直接调节骨髓细胞(BMC)功能。2)检测Lumcan是否通过内毒素特异性的CD14-TLR4途径促进角膜天然免疫反应，a)在细胞培养中，通过评估重组Lumcan在同样缺乏CD14的Lum-/-巨噬细胞中拯救LPS诱导的TNFa的能力，以及b)通过比较Lum+/+和Lum-/-BMC CD14-零嵌合体和亲本株的内毒素诱导的角膜炎。3)研究LUMICAN与TLR4信号通路的关系及其治疗意义。在巨噬细胞培养中测试鲁米肯来源的合成肽与脂多糖的结合及其调节天然免疫反应的能力，以及它们对脂多糖诱导的角膜炎的影响。这项研究将阐明细胞外基质和角膜免疫功能之间的新联系，并导致治疗性流明产品的开发，以减轻角膜中的有害炎症反应，这是国家眼科研究所的主要重点。

DESCRIPTION (provided by applicant): The avascular cornea is a protective barrier with intact innate immune, but restricted adaptive immune response for optimal vision. Surgery, injury and infection of the cornea can disrupt this balance between innate and adaptive immune response leading to intense inflammation and loss of vision. We found lumican, an abundant extracellular matrix (ECM) stromal proteoglycan, to regulate corneal immune functions. Lumican itself binds LPS that can be removed competitively by CD14, the cell surface protein that transfers LPS to toll-like receptor 4, the trans-membrane IPS receptor. Mice, deficient in lumican (Lum-/-), are hypo-responsive to bacterial lipopolysacharide endotoxin (LPS), and produce lower amounts of proinflammatory cytokines. More importantly, in LPS-induced corneal keratitis, the Lum-/- mice show reduced neutrophil influx early, but overall delayed healing. Thus, innate immune response is impaired in our Lum-/- mice. Lumican also binds FasL and TGFbeta, and in the Lum-/- mice these pathways are disrupted. The Fas-FasL mediated apoptosis and TGFbeta signaling in the cornea help to maintain an immunosuppressive environment for immune privilege. Our overarching hypothesis is that in the cornea lumican promotes early innate immune response, but provides an immunosuppressive microenvironment to restrict adaptive immunogenic mechanisms to maintain immune privilege. The following aims will test this hypothesis. 1) Test if lumican regulates corneal inflammation and immune privilege such that Lum-/-corneal allografts have decreased survival. Test if lumican regulates bone marrow cell (BMC) functions directly by evaluating allograft success in Lum+/+ and Lum-/- BMC chimera hosts. 2) Test if lumican promotes corneal innate immune response via the LPS-specific CD14-TLR4 pathway, a) in cell culture by assessing the ability of recombinant lumican to rescue LPS-induction of TNFa in Lum-/- macrophages also lacking CD14, and b) by comparing LPS-induced keratitis in Lum+/+ and Lum-/- BMC CD14-null chimeras and parental strains. 3) Investigate connections between lumican and the TLR4-signaling pathway and its therapeutic implications. Test binding of lumican-derived synthetic peptides to LPS and their ability to regulate innate immune response in macrophage culture and their effects on LPS-induced keratitis. This study will elucidate a novel link between the ECM and corneal immune functions and lead to the development of therapeutic lumican-products to alleviate detrimental inflammatory responses in the cornea, a major focus of the National Eye Institute.