The Role of TLR4 and RSV F Protein in Immunity to RSV

描述（由申请人提供）：呼吸道是病原体的主要入口。位于粘膜表面的支气管肺泡巨噬细胞通过称为“模式识别受体（PRRs）”的结构识别“病原体相关分子模式（PAMPs）”。一个密切相关的哺乳动物PRRs家族，“toll样受体（TLRs）”，是跨膜信号分子，对多种PAMPs作出反应。革兰氏阴性菌的脂多糖（LPS）通过TLR4刺激巨噬细胞，引发强烈促炎的基因表达模式，如TNF-a、IL-1¿、IL-12和IFN-？导致“th1型”细胞因子环境。呼吸道合胞病毒（RSV）是全世界婴幼儿肺炎和细支气管炎的主要原因，最近被认为是老年人和免疫抑制人群发病率和死亡率增加的原因。在一项失败的临床试验中，福尔马林灭活RSV （FI-RSV）疫苗导致RSV疾病加重。通过组织病理学和气道高反应性证明，疫苗增强的RSV疾病在棉花大鼠（S. hispidus）中得到了真实再现，并且与th2型细胞因子基因表达模式相关。接种FI-RSV和TLR4激动剂可减轻疾病加重。最近，RSV融合(F)蛋白被证明是一种TLR4激动剂。在体外实验中，棉花大鼠巨噬细胞对LPS和F蛋白产生反应，引发相同的炎症基因谱，尽管F蛋白的作用较弱。需要验证的总体假设是，F蛋白与TLR4的相互作用对于thl型环境的建立至关重要，而RSV的福尔马林固定破坏了F蛋白与TLR4相互作用的能力，导致th2型反应。该提案详细介绍了新的实验方法：(i)评估通过TLR4的F蛋白信号传导对th1型细胞因子谱发展的贡献和调控，（ii）确定TLR4在体内RSV介导的疾病发展中的作用，（iii）确定先前与气道对LPS低反应性相关的TLR4多态性形式是否表现出对F蛋白的敏感性降低，以及它们是否在RSV高风险人群中过度代表。预计在这笔拨款完成后，我们将确定原发性和疫苗增强型RSV疾病的分子基础，并确定开发人类RSV疫苗的策略。

DESCRIPTION (provided by applicant): The respiratory tract is a major portal for pathogens. The bronchoalveolar macrophage, positioned at the mucosal surface, recognizes "pathogen associated molecular patterns (PAMPs)" through structures called "pattern recognition receptors (PRRs)." A family of closely related mammalian PRRs, the "Toll-like receptors (TLRs)," are transmembrane signaling molecules that respond to diverse PAMPs. The lipopolysaccharide (LPS) of Gram negative bacteria stimulates macrophages through TLR4 to elicit a pattern of gene expression that is strongly proinflammatory, e.g., TNF-a, IL-1¿, IL-12, and IFN-? resulting in a "Th1-type" cytokine milieu. Respiratory syncytial virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and young children worldwide, and has recently been attributed to increased morbidity and mortality in the elderly and immunosuppressed. In a failed clinical trial, a formalin-inactivated RSV (FI-RSV) vaccine led to exacerbated RSV disease. Vaccine-enhanced RSV disease, demonstrated by histopathology and airway hyperreactivity, are faithfully recapitulated in the cotton rat (S. hispidus), and is associated with a Th2-type pattern of cytokine gene expression. Vaccination with FI-RSV and a TLR4 agonist mitigates enhanced disease. Recently, the RSV fusion (F) protein was shown to be a TLR4 agonist. In vitro, cotton rat macrophages respond to LPS and F protein to elicit the same spectrum of inflammatory genes, although the F protein is less potent. The overall hypothesis to be tested is that the interaction of F protein with TLR4 is essential for establishment of a Thl-type milieu and that formalin fixation of RSV destroys the capacity of F to interact with TLR4, resulting in a Th2-type response. This proposal details novel experimental approaches: (i) to evaluate the contribution and regulation of F protein signaling through TLR4 to the development of a Th1-type cytokine profile, (ii) to determine the role of TLR4 in the development of RSV-mediated disease in vivo, and (iii) to determine if polymorphic forms of TLR4 previously associated with airway hyporesponsiveness to LPS exhibit diminished sensitivity to F protein and if they are overrepresented in a population at high risk for RSV. It is expected that at the completion of this grant, we will have determined the molecular basis for primary and vaccine-enhanced RSV disease, and have identified strategies for the development of a human RSV vaccine.