Enhancing Collectin-Mediated Defense Against Influenza

集合是存在于血清和肺分泌物中的胶原凝集素，与先天免疫有关。肺表面活性蛋白A和D （SP-A和SP-D）在防御甲型流感病毒（IAV）中发挥重要作用。与对照组相比，由于基因缺失而缺乏SP-D （SP-D -/-）的小鼠在IAV感染后肺部病毒滴度和炎症明显增加。本文将对这些小鼠的IAV感染进行更详细的描述。更好地了解SP-D抑制IAV复制的机制可以解释为什么某些人更容易受到IAV感染并发症的影响。本研究将在体外研究SP-D在宿主防御IAV中的重要功能，包括其结合、聚集和中和IAV感染的能力，以及调节吞噬细胞与IAV的相互作用。此外，还将测试SP-D多态变体重组制剂的抗病毒活性。SP-D -/-有肺脂质沉积。由于磷脂积累可能在SP-D -/-小鼠对IAV感染的易感性增加中起作用，因此SP-D或IAV与表面活性剂磷脂的相关相互作用也将被研究。另一种被称为gp340的呼吸道蛋白与SP-D结合，当与SP-D结合时具有协同抗病毒作用。本研究将进一步研究gp340的抗病毒活性及其增强SP-D抗病毒作用的机制。由于SP-D通过其碳水化合物识别结构域（CRD）与IAV、吞噬细胞、磷脂和gp340结合，因此CRD的特定部分对这些功能活动的贡献将被深入研究。一系列重组结构将被制成，其中SP-D CRD的部分被血清收集的类似部分所取代。由于一些血清收集物比SP-D具有更强的中和IAV传染性的能力，并且在与吞噬细胞、磷脂和gp340的结合方面可能与SP-D不同，因此该方法应该更准确地确定CRD上哪些氨基酸群对这些功能至关重要。根据先前的实验，本提案产生的SP-D的修饰形式可能比野生型SP-D具有更强的抑制IAV感染的能力。这将在体外和体内通过鼻内滴注或在SP-D -/-小鼠中转基因表达一些新的重组SP-D进行测试。这一建议不仅增加了我们对SP-D参与宿主防御IAV的复杂机制的理解，而且还提供了通过使用重组修饰的集合恢复或增强收集素介导的防御的可行性的证据。

The collectins are collagenous lectins present in serum and lung secretions involved in innate immunity. Pulmonary surfactant proteins A and D (SP-A and SP-D), play important roles in defense against influenza A virus (IAV). Compared to controls, mice lacking SP-D due to gene-deletion (SP-D -/-) have markedly increased lung viral titers and inflammation after IAV infection. IAV infection of these mice will be characterized in more detail for this proposal. A better understanding of the mechanisms through which SP-D inhibits replication of IAV could elucidate why certain people are more susceptible to complications of IAV infection. This proposal will examine in vitro important functional aspects of SP-D relevant to its role in host defense against IAV, including its ability to bind to, aggregate and neutralize infectivity of IAV, and to modulate the interactions of phagocytic cells with IAV. In addition, the antiviral activities of recombinant preparations of polymorphic variants of SP-D will be tested. SP-D -/- have pulmonary lipidosis. Since phospholipid accumulation could play a role in the increased susceptibility SP-D -/- mice to IAV infection, relevant interactions of SP-D or IAV with surfactant phospholipids will also be examined. Another respiratory tract protein called gp340 binds to SP-D, and has cooperative antiviral effects when combined with SP-D. This proposal will examine further the antiviral activity of gp340 and the mechanisms through which it potentiates the antiviral effects of SP-D. Since SP-D binds to IAV, phagocytes, phospholipids and gp340, through its carbohydrate recognition domain (CRD), the contribution of specific parts of the CRD to these functional activities will be intensively examined. A series of recombinant constructs will be made in which parts of the SP-D CRD are replaced with analogous parts of serum collectins. Since several of the serum collectins have stronger ability to neutralize infectivity of IAV than SP-D, and probably differ from SP-D in terms of binding to phagocytes, phospholipids and gp340, this approach should identify more precisely which groups of amino acids on the CRD are critical for these functions. Based on prior experiments, it is likely that the modified forms of SP-D generated for this proposal will have greater ability to inhibit infectivity of IAV than wild type SP-D. This will be tested in vitro and in vivo by intranasal instillation or transgenic expression of some of these novel, recombinant SP-Ds in SP-D -/- mice. This proposal should not only increase our understanding of the complex mechanisms through which SP-D participates in host defense against IAV, but also provide evidence of the feasibility of restoring or enhancing collectin-mediated defense through use of recombinantly modified collectins.