Role of Sialidase in Pulmonary Host Defenses and Acute Lung Injury

描述（由申请方提供）：多形性白细胞（PMN）保护肺部免受微生物感染;然而，PMN的过度或长期流入可能导致组织损伤。急性肺损伤的特征在于广泛的炎性变化，包括弥漫性PMN浸润，肺上皮和内皮均受损。我们建立了从中性粒细胞和内皮细胞（EC）的表面去除唾液酸残基的内源性中性粒细胞唾液酸酶是至关重要的正常的中性粒细胞运输。此外，我们发现Toll样受体（TLR）4复合物的去唾液酸化增强LPS诱导的细胞因子产生，而唾液酸转移酶（ST）的唾液酸残基的恢复可能会恢复基础状态。待测试的总体假设是唾液酸酶和ST调节肺中的先天免疫应答，并且所实现的平衡将决定先天免疫应答是否发展成急性肺损伤。为了解决这一中心假设，我们提出以下建议：在具体目标1中，我们将定义哪种形式的PMN唾液酸酶（neu 1和neu 3）通过过表达和敲低特定neu蛋白来调节粘附和迁移穿过人肺微血管EC。在具体目标2中，我们将定义PMN和EC中PMN唾液酸酶的底物，并评估其唾液酸化状态如何影响体外PMN粘附和体内运输。由于中性粒细胞在内皮细胞中诱导信号级联反应，在具体目标3中，我们将研究是否“适当”水平的中性粒细胞唾液酸酶诱导的EC蛋白酪氨酸磷酸化打开EC-EC旁细胞途径足以跨内皮迁移而没有蛋白质泄漏，而在超过此阈值的水平，如果将有EC屏障功能的损失。已经提出了急性肺损伤的“两次打击”模型，其中LPS和趋化肽的组合促进肺损伤。在具体目标4中，我们将研究唾液酸酶在肺血管系统对循环LPS和肺损伤的敏感性中的作用。将检查去唾液酸化通过TLR 4/MD 2/CD 14复合物促进信号传导的分子机制，以及ST下调该应答的能力。将在肺白细胞停滞的鼠模型中体内研究唾液酸酶抑制剂逆转急性肺损伤的能力。由于唾液酸酶抑制剂是市售的，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.