Role of Sialidase in Pulmonary Host Defenses and Acute Lung Injury

描述（由申请人提供）：多形核白细胞（PMN）保护肺部免受微生物感染；然而，过度或长时间的PMNs流入可能导致组织损伤。急性肺损伤的特征是广泛的炎症改变，包括弥漫性PMN浸润，肺上皮和内皮均受到损伤。我们证实了内源性PMN唾液酸酶从PMN和内皮细胞（EC）表面去除唾液酸残留物对正常的PMN运输至关重要。此外，我们发现toll样受体（TLR） 4复合物的脱氮化可以增强lps诱导的细胞因子产生，而通过唾液基转移酶（ST）恢复唾液基残基可能会恢复基础状态。需要验证的总体假设是唾液酸酶和STs调节肺部的先天免疫反应，其达到的平衡将决定先天免疫反应是否发展为急性肺损伤。为了解决这一中心假设，我们提出以下建议：在Specific Aim 1中，我们将定义哪种形式的PMN唾液酸酶（neul和neu3）通过过度表达和敲低特定的新蛋白来调节对人肺微血管EC的粘附和迁移。在Specific Aim 2中，我们将定义PMN唾液酸酶在PMN和ECs中的底物，并评估它们的唾液酸化状态如何影响PMN在体外的粘附和体内的转运。由于PMN在EC中诱导信号级联，在Specific Aim 3中，我们将研究是否“适当”水平的PMN唾液酸酶诱导EC蛋白的酪氨酸磷酸化足以打开EC-EC细胞旁通路，以进行跨内皮迁移而不发生蛋白泄漏，而当水平超过这个阈值时，EC屏障功能是否会丧失。提出了一种急性肺损伤的“双击”模型，即LPS和趋化肽的结合促进肺损伤。在特异性目标4中，我们将研究唾液酸酶在肺血管对循环LPS和肺损伤的敏感性中的作用。我们将研究脱氮化促进TLR4/MD2/CD14复合物信号传导的分子机制，以及STs下调这一反应的能力。唾液酸酶抑制剂逆转急性肺损伤的能力将在小鼠肺白斑模型中进行体内研究。由于唾液酸酶抑制剂在商业上是可用的，这些研究将为测试治疗肺损伤的新治疗方法提供实验依据。

DESCRIPTION (provided by applicant): Polymorphonuclear leukocytes (PMN) protect the lung against microbial infection; however, an excessive or prolonged influx of PMNs may cause tissue damage. Acute lung injury is characterized by widespread inflammatory changes, including diffuse PMN infiltration, with injury to both lung epithelium and endothelium. We established that the removal of sialyl residues from the surface of PMNs and endothelial cells (EC) by an endogenous PMN sialidase is critical to normal PMN trafficking. Further, we find that desialylation of the Toll-like receptor (TLR) 4 complex enhances LPS-induced cytokine production, while restoration of sialyl residues by sialyltransferases (ST) may restore the basal state. The overall hypothesis to be tested is that sialidases and STs regulate innate immune responses in the lung, and the balance achieved will dictate whether or not the innate immune response develops into acute lung injury. To address this central hypothesis, we propose the following: In Specific Aim 1 we will define which form(s) of PMN sialidase(s) (neul and neu3) regulates adherence to and migration across human lung microvascular EC by overexpression and knockdown of specific neu proteins. In Specific Aim 2 we will define the substrates for PMN sialidase in PMNs and ECs and assess how their sialylation status affects PMN adhesion in vitro and trafficking in vivo. Since PMNs induce signaling cascades in ECs, in Specific Aim 3 we will examine whether an "appropriate" level of PMN sialidase-induced tyrosine phosphorylation of EC proteins opens the EC-EC paracellular pathway sufficiently for transendothelial migration without protein leak, while at levels that exceed this threshold, if there will be loss of EC barrier function. A "two-hit" model of acute lung injury has been proposed whereby a combination of LPS and chemotactic peptide promotes lung injury. In Specific Aim 4, we will examine the role of sialidase in sensitizing the pulmonary vasculature to circulating LPS and lung injury. The molecular mechanism(s) by which desialylation promotes signaling through the TLR4/MD2/CD14 complex will be examined, as well as the ability of STs to downregulate this response. The ability of sialidase inhibitors to reverse acute lung injury will be studied in vivo in a murine model of pulmonary leukostasis. Since inhibitors of sialidase are commercially available, these studies will provide the experimental rationale for testing a new therapeutic approach to the treatment of lung injury.