Age-associated Toll-like receptor dysfunction in the lungs

描述（由申请人提供）：社区获得性肺炎（CAP）是老年人（65岁以下）死亡的主要原因；尽管可以获得抗微生物药物和支持性重症监护，但患有CAP的老年人的病死率为15-30%。Toll样受体（TLR）是一种模式识别分子，可识别与入侵微生物相关的分子模式，并启动先天免疫反应（如NFkB激活、细胞因子产生）。现在很明显，衰老与TLR功能障碍有关；从老年动物中分离的吞噬细胞表现出TLR表达减少，TLR信号受损，TLR介导的细胞因子产生减少。目前，年龄依赖性TLR功能障碍（ADTD）对肺部的影响尚不清楚。然而，adhd可能有助于解释：1)为什么CAP在老年人中更严重；2)为什么10%的老年肺炎患者没有明显的临床感染症状，如发热、咳嗽或呼吸困难；3)为什么不发烧是临床预后不良的预后指标。我们假设多动症发生在老年动物的肺部，它增加了对肺炎的易感性。支持这一假设的实验室观察包括：1)老年小鼠肺中TLR-1、-2和-4蛋白水平低于年轻小鼠；2)老年小鼠感染肺炎链球菌后会发展为暴发性肺炎并在36小时内死亡，而年轻小鼠则相对健康；3)感染期间老龄小鼠肺部NFkB活性降低；4)肺部感染肺炎链球菌成分后，老龄小鼠肺中TNF1和IL-6水平降低。该提案的重点是肺炎期间肺部的多动症。我们将：目标1：确定衰老对TLR和TLR信号蛋白水平的影响。使用实时(RT)- PCR，定量Western blot和FACs分析，我们将确定从老年小鼠分离的肺泡巨噬细胞和II型肺细胞（肺粘膜上皮细胞）中TLRs 1-9的mRNA和蛋白水平以及TLR细胞信号分子Myd88， IRAK-1和IRAK-4是否降低。目的2：确定年龄对肺内肺炎链球菌成分TLR反应的影响。使用原代肺细胞和活小鼠，我们将分别测定衰老对TLR细胞信号蛋白IRAK-1磷酸化、NFkB激活、细胞因子产生和白细胞粘附分子表达的影响，以响应肽聚糖和溶气酶、TLR-2和TLR-4配体。-该提案的完成将扩大我们对ADTD的理解，包括肺泡巨噬细胞和粘膜上皮细胞，这是肺部呼吸道病原体的主要接触点。拟议的实验可能会确定adhd是老年人肺炎易感性增加的主要机制。该项目符合NIA既定的目标，即检查与年龄相关的变化对功能性疾病结果的影响。公共卫生相关性：toll样受体（TLR）识别入侵微生物并启动宿主先天免疫反应。我们实验室收集的证据表明，年龄依赖性TLR功能障碍（ADTD）发生在肺部。这项研究将确定肺泡巨噬细胞和粘膜上皮细胞（肺部呼吸道病原体的主要接触点）是否会经历ADTD。

DESCRIPTION (provided by applicant): Community-acquired pneumonia (CAP) is a leading cause of death among the elderly (>65 years); despite access to antimicrobials and supportive critical care, the case-fatality rate for elderly with CAP is 15-30%. Toll- like receptors (TLR) are pattern recognition molecules that identify molecular patterns associated with invading microorganisms and initiate the innate immune response (e.g. NFkB activation, cytokine production). It is now evident that aging is associated with TLR dysfunction; phagocytes isolated from aged animals exhibit decreased TLR expression, impaired TLR signaling, and a reduction in TLR-mediated cytokine production. Currently the impact of age-dependent TLR dysfunction (ADTD) in the lungs is unknown. However, ADTD may help explain: 1) why CAP is more severe in the elderly; 2) why 10% of the elderly with pneumonia show no overt clinical signs of infection such as fever, cough, or dyspnea; and 3) why lack of fever is a prognostic indicator of poor clinical outcome. We hypothesize that ADTD occurs in lungs of aged animals and that it increases susceptibility to pneumonia. Laboratory observations that support this hypothesis include: 1) decreased TLR-1, -2, and -4 protein levels in the lungs of aged mice versus young mice; 2) aged mice infected with Streptococcus pneumoniae develop fulminate pneumonia and die within 36 hours whereas young mice remain relatively healthy; 3) diminished NFkB activation in the lungs of aged mice during infection; and 4) reduced TNF1 and IL-6 levels in the lungs of aged mice following tracheal challenge with S. pneumoniae components. The focus of this proposal is ADTD in the lungs during pneumonia. We will: Aim 1: Determine the effects of aging on TLRs and TLR signaling protein levels. Using Real Time (RT)- PCR, quantitative Western blot, and FACs analysis we will determine if mRNA and protein levels of TLRs 1-9, and the TLR cell signaling molecules Myd88, IRAK-1, and IRAK-4 are decreased in alveolar macrophages and type II pneumocytes (lung mucosal epithelial cells) isolated from aged versus young and mature mice. Aim 2: Determine the effects of aging on the TLR response to S. pneumoniae components in the lung. Using primary lung cells and live mice, we will determine the effects of aging on phosphorylation of the TLR cell signaling protein IRAK-1, NFkB activation, cytokine production, and expression of leukocyte adhesion molecules in response to peptidoglycan and pneumolysin, TLR-2 and TLR-4 ligands, respectively. -Completion of this proposal will expand our understanding of ADTD to include alveolar macrophages and mucosal epithelial cells, the primary point of contact for respiratory tract pathogens in the lungs. Proposed experiments may determine that ADTD is a principal mechanism responsible for the increased susceptibility of the elderly to pneumonia. This project meets established NIA goals of examining the effects of age-related changes on functional disease outcomes. PUBLIC HEALTH RELEVANCE: Toll-like receptors (TLR) recognize invading microorganisms and initiate the host innate immune response. Evidence collected in our laboratory indicates that age-dependent TLR dysfunction (ADTD) occurs in the lungs. This proposal will determine if alveolar macrophages and mucosal epithelial cells, the primary point of contact for respiratory tract pathogens in the lungs, experience ADTD.