Collectin-Mediated Defense Against Influenza

描述(申请人提供)：甲型流感病毒(IAV)是一个主要威胁，因为通过遗传变异逃避适应性免疫。包括表面活性蛋白D(SP-D)在内的天然防御在IAV感染的早期阶段起着关键作用。我们的核心假设是，SP-D直接抑制IAV的感染性，并通过影响呼吸道上皮和中性粒细胞(PMN)来减轻IAV感染过程中的炎症反应。我们的目标是确定这些影响是如何关联的，以及哪些是最重要的。目的1研究SP-D在体外和体内对呼吸道上皮细胞感染的调节作用。我们将利用SP-D耐药和敏感的IAV毒株和SP-D基因条件性缺失(SP-D-/-)小鼠来阐明SP-D如何抑制IAV的复制，以及这与SP-D减少炎症反应的关系。对呼吸道上皮细胞中IAV感染的体外研究将详细确定SP-D如何调节病毒的生命周期和细胞信号。目的2将利用一组重组修饰形式的SP-D来确定SP-D的哪些分子特征对抗病毒和抗炎活性至关重要。这些重组的SP-D变异体将在体外(在人类呼吸道细胞培养中)和体内进行测试，使用滴注和遗传拯救来纠正条件SP-D-/-小鼠的抗病毒反应异常。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，并且这些结构将有助于分离这些影响。目的3将确定SP-D如何在IAV感染过程中下调PMN的流入，以及PMN在体内对肺损伤或控制病毒复制的作用。对人PMN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LAV感染PMN的呼吸爆发反应。在体外，SP-D可以增加或减少LAV处理的PMN的呼吸爆发反应，这取决于SP-D和IAV加入细胞的顺序。将评估IAV和SP-D的特异性PMN受体在这些效应中的作用。AIM 4将评估另外两种天然免疫蛋白，它们本身具有抗病毒活性，但也与SP-D结合并改变其功能。它们是清道夫受体富含糖蛋白340(Gp340)和人中性粒细胞防御素(HNPs)。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.