The Role of Lumican in the cornea

描述（由申请人提供）：无血管角膜是一种保护性屏障，具有完整的先天免疫，但限制了获得最佳视力的适应性免疫反应。角膜的手术、损伤和感染会破坏先天性和适应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，调节角膜免疫功能。Lumican本身结合LPS，其可以被CD 14竞争性地去除，CD 14是将LPS转移到跨膜IPS受体toll样受体4的细胞表面蛋白。缺乏Lumican（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量较低。更重要的是，在LPS诱导的角膜角膜炎中，Lum-/-小鼠早期显示出减少的中性粒细胞流入，但总体上延迟愈合。因此，先天免疫应答在我们的Lum-/-小鼠中受损。Lumican还结合FasL和TGF β，在Lum-/-小鼠中，这些途径被破坏。角膜中的Fas-FasL介导的细胞凋亡和TGF β信号传导有助于维持免疫赦免的免疫抑制环境。我们的总体假设是，在角膜中，lumican促进早期先天免疫应答，但提供免疫抑制微环境以限制适应性免疫原性机制以维持免疫赦免。以下目标将检验这一假设。1)测试Lumican是否调节角膜炎症和免疫豁免，使得Lum-/-角膜同种异体移植物的存活率降低。通过评估Lum+/+和Lum-/- BMC嵌合体宿主中的同种异体移植成功率，测试Lumican是否直接调节骨髓细胞（BMC）功能。2)测试Lumican是否通过LPS特异性CD 14-TLR 4途径促进角膜先天免疫应答，a）在细胞培养物中，通过评估重组Lumican在同样缺乏CD 14的Lum-/-巨噬细胞中拯救LPS诱导TNF α的能力，和B）通过比较Lum+/+和Lum-/- BMC CD 14缺失嵌合体和亲本菌株中LPS诱导的角膜炎。3)研究lumican和TLR 4信号通路之间的联系及其治疗意义。测试光蛋白聚糖衍生的合成肽与LPS的结合及其在巨噬细胞培养物中调节先天免疫应答的能力及其对LPS诱导的角膜炎的影响。这项研究将阐明ECM和角膜免疫功能之间的新联系，并导致治疗性Lumican产品的开发，以减轻角膜中有害的炎症反应，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.