Collectin-Mediated Defense Against Influenza

描述（由申请方提供）：甲型流感病毒（IAV）是一种主要威胁，因为它通过遗传变异逃避适应性免疫。包括表面活性蛋白D（SP-D）在内的先天性防御在IAV感染的早期阶段至关重要。我们的核心假设是SP-D直接抑制IAV感染性，并通过对呼吸道上皮细胞和多形核中性粒细胞（PMNs）的影响减少IAV感染期间的炎症反应。我们的目标是确定这些影响是如何联系在一起的，以及哪些是最重要的。目的1将研究SP-D如何在体外和体内调节呼吸道上皮的感染。我们将利用SP-D耐药和敏感的IAV株和条件性SP-D基因缺失（SP-D-/-）小鼠来阐明SP-D如何抑制IAV复制以及这与SP-D减少炎症反应的关系。IAV感染呼吸道上皮细胞的体外研究将详细确定SP-D如何调节病毒生命周期和细胞信号传导。目的2将利用一组重组修饰形式的SP-D来确定SP-D的哪些分子特征对于抗病毒和抗炎活性是关键的。这些重组SP-D变体将在体外（在人呼吸道细胞培养物中）和体内使用滴注和遗传拯救进行检测，以纠正条件性SP-D -/-小鼠抗病毒应答的异常。我们的假设是SP-D的多聚化和糖结合特性在确定其抗病毒和抗炎作用方面都很重要，并且构建体将有助于分离这些作用。目的3将确定在IAV感染过程中SP-D如何下调PMN内流以及PMN对肺损伤或体内病毒复制控制的贡献。用人PMN进行的体外研究将确定SP-D如何调节PMN对IAV的摄取以及SP-D如何调节IAV感染的PMN的呼吸爆发反应。SP-D可以增加或减少IAV处理的PMN在体外的呼吸爆发反应，这取决于SP-D和IAV添加到细胞中的顺序。将评估IAV和SP-D的特异性PMN受体在这些效应中的作用。目的4将评估另外两种先天免疫蛋白，它们本身具有抗病毒活性，但也结合并修饰SP-D的功能。这些是富含清道夫受体的糖蛋白340（gp 340）和人嗜中性粒细胞防御素（HNP）。这些研究应该阐明防御IAV的重要方面，并与治疗和预防策略相关。

DESCRIPTION (provided by applicant): Influenza A virus (IAV) is a major threat due to evasion of adaptive immunity through genetic variation. Innate defenses, including surfactant protein D (SP-D) are critical in the early phase IAV infection. Our core hypothesis is that SP-D inhibits IAV infectivity directly and also reduces inflammatory responses during IAV infection through effects on respiratory epithelium and polymorphonuclear neutrophils (PMNs). We aim to determine how these effects relate and which is most important. Aim 1 will examine how SP-D modulates infection of respiratory epithelium in vitro and in vivo. We will make use of SP-D-resistant and -sensitive IAV strains and conditionally SP-D gene-deleted (SP-D-/-) mice to clarify how SP-D inhibits IAV replication and how this relates to reduction of inflammatory responses by SP-D. In vitro studies of IAV infection in respiratory epithelial cells will determine in detail how SP-D modulates the viral life cycle and cell signaling. Aim 2 will make use of a panel of recombinantly modified forms of SP-D to determine which molecular features of SP-D are critical for antiviral and anti-inflammatory activities. These recombinant SP-D variants will be tested both in vitro (in human respiratory cell culture) and in vivo using instillation and genetic rescue to correct abnormalities in the antiviral response of conditional SP-D -/- mice. Our hypothesis is that multimerzation and saccharide binding properties of SP-D are both important in determining its antiviral and anti-inflammatory effects, and that the constructs will help separate out these effects. Aim 3 will determine how SP-D downregulates PMN influx during IAV infection and the contribution of PMNs to lung injury or control of viral replication in vivo. In vitro studies with human PMNs will determine how SP-D modulates the uptake of IAV by PMNs and how SP-D modulates respiratory burst responses of lAV-infected PMNs. SP-D can either increase or reduce respiratory burst responses of lAV-treated PMNs in vitro depending on sequence of addition of SP-D and IAV to the cells. The role of specific PMN receptors for IAV and SP-D in these effects will be evaluated. Aim 4 will evaluate two other innate immune proteins that have antiviral activity in their own right but also bind to, and modify function of SP-D. These are scavenger receptor rich glycoprotein 340 (gp340) and human neutrophil defensins (HNPs). These studies should elucidate important aspects of defense against IAV and be relevant to treatment and prevention strategies.