TLR4 Mutations: Molecular Mechanisms of Impaired LPS Sensitivity

描述(申请人提供)：TLR4是脂多糖的主要信号受体，对宿主抵御革兰氏阴性细菌感染至关重要。TLR4胞外区的两个点突变D299G和T399I与吸入内毒素反应迟钝、革兰氏阴性细菌感染和败血症的发生率增加有关。这些结果表明，这些突变改变了TLR4的信号传递能力，导致宿主对细菌病原体的免疫反应减弱。TLR4基因多态影响受体介导的信号转导的分子机制目前尚不清楚。需要检验的中心假设是，单核苷酸多态改变了TLR4对革兰氏阴性细菌和内毒素的识别，它与辅助受体的相互作用，以及适配器和激酶的招募和激活，导致TLR4信号受损和宿主对病原体的免疫反应不足。这一假说将通过追求以下具体目标来验证：(1)确定TLR4 SNPs是否改变了对革兰氏阴性细菌和内毒素的识别、受体信号体的组装以及激酶和转录因子的激活；(2)确定TLR4 SNPs对革兰氏阴性细菌的炎症介质表达和吞噬功能的影响。我们的研究将使用互补的方法进行，包括转基因细胞系，通过核重组导入野生型或突变型TLR4的原代TLR4小鼠巨噬细胞，以及从携带野生型或突变型TLR4等位基因的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TLR4突变影响革兰氏阴性细菌和内毒素感知的分子机制。这项研究将促进我们对TLR4信号的总体理解，并为开发新的治疗方法以纠正表达TLR4突变个体中受损的TLR信号通路组件的功能提供理论基础。公共卫生相关性：细菌性败血症是全球人类健康的主要威胁，美国每年的发病率约为75万名患者，其中超过21万人(&gt；28%)死亡。TLR4是革兰氏阴性细菌和脂多糖的主要受体，其突变与许多感染性疾病和脓毒症有关。然而，这些突变影响受体信号和巨噬细胞对微生物反应的分子机制尚不清楚。我们拟议的研究将在分子水平上确定这些TLR4突变如何改变受体功能。我们的研究将确定由TLR4突变引起的受体和信号分子功能的早期障碍，并可能有助于开发新的药物来纠正这种缺陷。

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.