TLR4 Mutations: Molecular Mechanisms of Impaired LPS Sensitivity

描述（由申请人提供）：TLR 4是LPS的主要信号受体，对于宿主防御革兰氏阴性细菌感染至关重要。TLR 4胞外域的两个点突变D299 G和T399 I与对吸入LPS的反应迟钝、革兰氏阴性细菌感染和脓毒症的发生率增加有关。这些结果表明，这些突变改变了TLR 4的信号能力，导致宿主对细菌病原体的免疫应答受损。TLR 4多态性影响受体介导的信号传导的分子机制目前尚不清楚。待检验的中心假设是单核苷酸多态性改变了TLR 4对革兰氏阴性菌和LPS的识别、其与共受体的相互作用以及衔接子和激酶的募集和激活，导致TLR 4信号传导受损和宿主对病原体的免疫应答缺陷。该假设将通过追求以下特定目的来检验：（1）确定TLR 4 SNP是否改变革兰氏阴性菌和LPS的识别、受体信号体组装以及激酶和转录因子的活化;（2）确定TLR 4 SNP对炎症介质的表达和革兰氏阴性菌的吞噬作用的影响。将使用补充方法进行我们的研究，包括转染细胞系、具有通过核转染引入的“敲入”人野生型或突变型TLR 4的原代TLR 4-/-小鼠巨噬细胞，以及从携带野生型或突变型TLR 4等位基因的人受试者获得的人单核细胞。在这些研究完成后，我们希望确定TLR 4突变影响革兰氏阴性菌和LPS传感的分子机制。这项研究将推进我们对TLR 4信号传导的总体理解，并为开发新的治疗方法提供理论基础，以纠正表达突变型TLR 4变体的个体中TLR信号传导途径组分的功能受损。公共卫生相关性：细菌性脓毒症是全球范围内对人类健康的主要威胁，在美国的年发病率为约750，000例患者，其中超过约210，000例（> 28%）死亡。TLR 4是革兰氏阴性菌和脂多糖的主要受体，其突变与许多感染性疾病和败血症有关。然而，这些突变影响受体信号传导和巨噬细胞对微生物反应的分子机制尚不清楚。我们提出的研究将确定这些TLR 4突变如何在分子水平上改变受体功能。我们的研究将确定由TLR 4突变引起的受体和信号分子功能的最早障碍，并可能有助于开发新的药物来纠正这些缺陷。

DESCRIPTION (provided by applicant):TLR4, the predominant signaling receptor for LPS, is critical for host defense against Gram negative bacterial infections. Two point mutations, D299G and T399I, in the ectodomain of TLR4 have been associated with a blunted response to inhaled LPS, increased incidence of Gram negative bacterial infection and sepsis. These results suggest that these mutations alter the capacity of TLR4 to signal, leading to impaired host immune response against bacterial pathogens. The molecular mechanisms by which TLR4 polymorphisms affect receptor-mediated signaling are currently unknown. The central hypothesis to be tested is that the single nucleotide polymorphisms alter TLR4 recognition of Gram negative bacteria and LPS, its interactions with co-receptors and recruitment and activation of adapters and kinases, resulting in impaired TLR4 signaling and deficient host immune response against pathogens. This hypothesis will be examined by pursuit of the following Specific Aims: (1) Define whether the TLR4 SNPs alter recognition of Gram negative bacteria and LPS, receptor signalosome assembly, and activation of kinases and transcription factors; (2) Determine the effect of the TLR4 SNPs on expression of inflammatory mediators and phagocytosis of Gram negative bacteria. Complementary approaches will be used to conduct our studies, including transfected cell lines, primary TLR4-/- mouse macrophages with "knocked-in" human wild-type or mutant TLR4s introduced by nucleofection, and human monocytes obtained from human subjects who carry wild-type or mutant TLR4 alleles. At the completion of these studies, we expect to identify molecular mechanisms by which TLR4 mutations affect sensing of Gram negative bacteria and LPS. This research will advance our general understanding of TLR4 signaling, and provide a rationale for the development of new therapeutic approaches to correct functions of components of the TLR signaling pathway compromised in individuals expressing mutant TLR4 variants. PUBLIC HEALTH RELEVANCE: Bacterial sepsis is a major threat to human health worldwide, with the annual incidence in the U.S.A. is ~750,000 patients, with more than ~210,000 (> 28%) deaths. Mutations in TLR4, the main receptor for Gram negative bacteria and lipopolysaccharide, have been associated with many infectious diseases and sepsis. However, molecular mechanisms by which these mutations affect receptor signaling and macrophage reactions to microbes are unknown. Our proposed studies will determine how these TLR4 mutations change receptor functions at the molecular level. Our research will identify earliest disturbances in functions of receptor and signaling molecules that are caused by TLR4 mutations, and may aid to development of new pharmaceutical drugs to correct such deficiencies.