Pulmonary Collectins, Hyaluronan and Macrophages

描述（由申请人提供）：氧化应激和硝化应激被认为是肺损伤和随后诱导炎症反应的关键介质。蛋白质是超氧化物(O)和一氧化氮（NO）反应的主要靶标。暴露于这些反应性应激下，蛋白质修饰如s -亚硝基化（SNO）、羰基化和3-硝基酪氨酸（3NT）发生，并已被证明通过改变蛋白质功能在生理和病理上起作用。肺集合素、表面活性蛋白(SP)-A和SP- d调节肺的免疫功能，SP-A的硝化作用使其丧失功能。初步数据表明，博莱霉素诱导的啮齿动物肺损伤后，SNO和3NT的定位和表达存在差异。此外，我们已经记录了损伤肺中灌洗透明质酸（HA）的急剧增加，HA结合肽治疗限制了损伤的炎症和纤维化反应。有趣的是，活性氧和活性氮（RONS）将高分子量（HMVV） HA裂解成低分子量（LMW）形式，促进巨噬细胞活化、细胞因子基因表达和趋化。在体内和体外，HA受体CD44和RHAMM都参与了这些炎症反应。初步数据表明，抗rhamm抗体阻断SP- a介导的巨噬细胞趋化性，并且SP和HA信号通路需要膜脂筏。利用啮齿类动物气管内博来霉素肺损伤模型，我们将验证肺损伤引起的ron同时产生GAG加合物和post的假设。翻译修饰特异性蛋白靶点，共同调节巨噬细胞活化、炎症细胞因子基因表达和趋化性，从而促进肺部炎症。利用药物（化学阻滞剂）和转基因（SP-ND敲除）方法，Aim 1将确定RONS对博莱霉素损伤后SPND翻译后修饰、LMW HA形成和炎症的贡献。此外，Aim 2将使用特异性抗体和肽阻断剂，重点确定HA、CD44和RHAMM在体外天然和硝化SP-ND对巨噬细胞功能的调节中的作用。在Aim 3中，将通过确定脂筏相关信号单元的形成来进一步研究巨噬细胞功能改变的机制，该信号单元调节SP-ND和LMW ha介导的巨噬细胞行为。使用脂筏特异性阻滞剂进行治疗干预，然后测试其在肺损伤后阻断炎症的功效。本方案所述的实验将明确肺收集和LMW HA调节巨噬细胞功能的分子机制，并将探索新的治疗干预靶点，以限制肺损伤的炎症反应。

DESCRIPTION (provided by applicant): Oxidative and nitrative stresses are thought to be critical mediators of lung injury and subsequent induction of inflammatory responses. Proteins constitute a major target of superoxide (O) and nitric oxide (NO) reactivity. Under exposure to these reactive stresses, protein modifications such as S-nitrosylation (SNO), carbonylation and 3-nitrotyrosine (3NT) occur and have been shown to act both physiologically and pathologically by altering protein function. The pulmonary collectins, Surfactant Proteins (SP)-A and SP-D modulate immune functions in the lung and nitration of SP-A abrogates its functions. Preliminary data indicate differential localization and expression of SNO and 3NT after bleomycin-induced lung injury in rodents. In addition, we have documented dramatic increases in lavage hyaluronic acid (HA) in injured lungs and HA-binding peptide treatment limits the inflammatory and fibrotic response to injury. Interestingly, reactive oxygen and nitrogen species (RONS) fragment high molecular weight (HMVV) HA into low molecular weight (LMW) forms that promote macrophage activation, cytokine gene expression and chemotaxis. The HA receptors CD44 and RHAMM have been implicated in these inflammatory responses both in vitro and in vivo. Preliminary data indicate that anti-RHAMM antibody blocks SP-A-mediated macrophage chemotaxis and that membrane lipid rafts are required for SP and HA signaling. Using the rodent intratracheal bleomycin model of lung injury, we will test the hypothesis that RONS, occurring as a result of lung injury, generate both GAG adducts and the post.translational modification of specific protein targets that collectively regulate macrophage activation, inflammatory cytokine gene expression and chemotaxis so as to promote pulmonary inflammation. Using both pharmacologic (chemical blockers) and transgenic (SP-ND knockouts) approaches, Aim 1 will determine the contribution of RONS to post-translational modifications of SPND, formation of LMW HA and inflammation after bleomycin injury. In addition, using specific antibody and peptide blockers, Aim 2 will focus on defining the roles of HA, CD44 and RHAMM in the regulation of macrophage functions by native and nitrated SP-ND in vitro. In Aim 3, the mechanisms of altered macrophage function will be further examined by determining the formation of a lipid raft-associated signaling unit that regulates SP-ND and LMW HA-mediated macrophage behavior. Therapeutic intervention with specific blockers of lipid rafts will then be tested for their efficacy in blocking inflammation after lung injury. The experiments described in this proposal will define the molecular mechanisms of pulmonary collectin and LMW HA regulation of macrophage function and will explore novel targets for therapeutic intervention to limit the inflammatory response to lung injury.