Ocular surface functions of SLURP1

摘要 我们对眼表免疫调节认识的持续空白是开发新的免疫调节的障碍 炎症性疾病的治疗是眼科门诊就诊的主要原因。 本实验室以前发表的文献证实，分泌型Ly6/uPAR相关蛋白-1(SLURP1)， Ly6蛋白家族中的一个成员是眼表面的一种免疫调节分子，它：(I)高度 在角膜上皮细胞中表达，并分泌到泪液中；(Ii)作为尿激酶的可溶性清除剂- 型纤溶酶原激活剂；(Iii)抑制人脐静脉内皮细胞(HUVEC)管形成； (Iv)体外抑制中性粒细胞通过融合内皮细胞单层的趋化和迁移；以及 (V)稳定上皮细胞连接，抑制肿瘤坏死因子-α诱导的细胞因子产生，与 具有抗炎作用。总的来说，这些研究确定SLURP1为潜在的治疗靶点 眼表的炎症性疾病。在这里，我们建议在这些重要发现的基础上，通过测试 SLURP1抑制角膜新生血管炎症和中性粒细胞募集的中心假说 通过调节转化生长因子-β和尿激酶型纤溶酶原激活剂活性促进核因子κB介导的促炎反应 分子的。这一假设得到了我们之前发表的上述文章的支持，并从以下方面获得了令人兴奋的结果 我们未发表的初步研究中，Slurp1基因敲除(Slurp1X-/-)小鼠的角膜表现出致密 硝酸银灼伤后5天，角膜新生血管和过量中性粒细胞涌入。此外， 天真野生型和Slurp1X-/-小鼠角膜转录本的RNA-Seq比较 包括转化生长因子-β和核因子κB途径在内的血管生成炎症激活因子将在 Slurp1的缺失为这一假说提供了额外的支持。我们将通过使用以下方法来验证这一假设 小鼠模型和体外研究追求以下具体目标：目的1)。检验以下假设 Slurp1通过抑制未减轻的转化生长因子-β和 目的2)参与核转录因子κB途径的uPA活性。检验Slurp1抑制中性粒细胞的假设 通过促进中性粒细胞成熟和清除进入健康角膜，并干扰其 外渗；和目标3)。检验SLURP1与股骨头损伤严重程度负相关的假设 人类干眼病和眼表面炎症性疾病的有效治疗靶点。通过 阐明与SLURP1免疫调节功能相关的有希望的新信息，一个丰富的 表达但未被充分研究的蛋白质，这一提议的预期结果直接涉及NIH的任务 寻找有关生命系统的性质和行为的基本知识，并为 验证一种治疗眼表炎症性疾病的新靶点 给我们的医疗系统带来了沉重的负担。

Summary The persistent gap in our understanding of ocular surface immunomodulation is a barrier for developing new therapies for inflammatory disorders that are responsible for a bulk of outpatient visits to an ophthalmologist. Previous publications from our laboratory established that the secreted Ly6/uPAR related protein-1 (SLURP1), a member of the Ly6 family of proteins is an immunomodulatory molecule at the ocular surface that: (i) is highly expressed in the corneal epithelium and secreted to the tear fluid; (ii) acts as a soluble scavenger of urokinase- type plasminogen activator (uPA); (iii) inhibits human umbilical vein endothelial cell (HUVEC) tube formation; (iv) suppresses neutrophil chemotaxis and transmigration through confluent endothelial monolayer in vitro; and (v) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production consistent with an anti-inflammatory function. Collectively, these studies identified SLURP1 as a potential therapeutic target for inflammatory disorders of the ocular surface. Here we propose to build upon these salient findings by testing the central hypothesis that ‘SLURP1 suppresses corneal angiogenic inflammation and neutrophil recruitment by regulating the TGF-β- and uPA-activities that promote NFκB-mediated production of pro-inflammatory molecules’. This hypothesis is supported by our prior publications described above, and exciting results from our unpublished preliminary studies wherein Slurp1 knockout (Slurp1X-/-) mouse corneas displayed dense corneal neovascularization and excessive neutrophil influx five days after silver nitrate cautery. Furthermore, RNA-Seq comparison of the naïve wild type and Slurp1X-/- mouse corneal transcriptomes identified key activators of angiogenic inflammation including TGF-β and NFκB pathway components to be upregulated in the absence of Slurp1, lending additional support for this hypothesis. We will test this hypothesis by employing mouse models and in vitro studies to pursue the following Specific Aims: Aim 1). Test the hypothesis that Slurp1 protects the cornea from undesirable angiogenic inflammation by suppressing unmitigated TGF-β and uPA activities that feed into NFκB pathway; Aim 2). Test the hypothesis that Slurp1 suppresses neutrophil influx into healthy corneas by promoting neutrophil maturation and clearance, and interfering with their extravasation; and Aim 3). Test the hypothesis that SLURP1 is negatively correlated with the severity of human dry eye disease and a useful therapeutic target for ocular surface inflammatory disorders. By elucidating promising new information related to the immunomodulatory functions of SLURP1, an abundantly expressed yet understudied protein, anticipated outcomes of this proposal directly address the NIH mission of ‘seeking fundamental knowledge about the nature and behavior of living systems’ and offer the potential for validating a novel therapeutic target for inflammatory disorders of the ocular surface that account for a significant burden on our healthcare system.