Identification of age-related mediators of pro-inflammatory signaling

描述（由申请人提供）：先天免疫系统是依靠模式识别受体（PRRs）通过识别病原体相关分子模式（PAMPs）来检测外来病原体的一线防御机制。toll样受体（TLRs）包括一个膜靶向PRRs家族，可响应多种PAMPs。其中一种受体TLR4识别存在于革兰氏阴性菌细胞壁上的脂多糖（LPS）。TLR4参与会立即启动但受调控的信号级联反应，导致核因子KB （NF-KB）和干扰素调节因子（IRF）蛋白的激活，并诱导炎症细胞因子和干扰素的产生。免疫功能的减弱，包括失控或慢性炎症状态，往往与年龄的增长同时发生。有趣的是，衰老的腹膜巨噬细胞对炎症刺激敏感，包括LPS。然而，可能导致年龄相关免疫疾病的分子介质仍然不明确，而且在很大程度上没有定义。在这里，我们建议采用一种基于系统的方法，该方法将整合一系列功能基因组学分析，以确定导致老年腹膜巨噬细胞LPS敏感性增加的新分子因素。具体来说，我们将使用稳定转染nf - kb荧光素酶报告基因的巨噬细胞系来优化和执行全基因组RNAi筛选。将对细胞系进行LPS处理以诱导tlr4依赖性促炎级联反应，并通过评估荧光素酶活性来监测该途径的激活情况。高通量筛选产生的假设命中将随后得到验证，并将代表影响巨噬细胞促炎或抗炎状态的基因组子集。为了确定调节TLR4信号的基因是否也在衰老过程中被交替调节，将使用微阵列分析来生成年轻和老年腹膜巨噬细胞的转录谱，无论是在缺乏还是存在LPS刺激的情况下。一组基因既调节TLR4信号，又在衰老巨噬细胞中差异表达，将在机制水平上进一步表征。这些研究将使巨噬细胞对脂多糖和年龄相关慢性炎症反应的机制和生理理解的系统水平分析的翻译成为可能。

DESCRIPTION (provided by applicant): The innate immune system is a first line defense mechanism that relies on pattern recognition receptors (PRRs) to detect foreign pathogens by recognizing pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) comprise one family of membrane-targeted PRRs that respond to a variety of PAMPs. One of these receptors, TLR4, recognizes lipopolysaccharide (LPS) present on cell walls of gram-negative bacteria. TLR4 engagement initiates immediate, but regulated, signaling cascades leading to activation of Nuclear factor KB (NF-KB) and Interferon Regulatory Factor (IRF) proteins and induction of inflammatory cytokines, as well as interferons. Attenuated immune functions, including deregulated or chronic inflammatory states, are often coincident with increasing age. Intriguingly, aged peritoneal macrophages are sensitized to inflammatory stimuli, including LPS. However, the molecular mediators that may contribute to age-related immune disorders remain ambiguous and largely undefined. Here, we propose to employ a systems-based approach, which will integrate a series of functional genomics analyses, to identify novel molecular factors that contribute to increased LPS sensitivity in aged peritoneal macrophages. Specifically, we will optimize and execute genome-wide RNAi screen using a macrophage cell line stably transfected with an NF-KB-luciferase reporter. The cell line will be treated LPS to induce TLR4-dependent pro-inflammatory cascades, and activation of the pathway will be monitored by assessing luciferase activity. Putative hits generated from high-throughput screening will be subsequently validated, and will represent a subset of the genome affecting pro- or anti- inflammatory states in macrophages. In order to determine if genes modulating TLR4 signaling are also alternatively regulated in aging, microarray analysis will be used to generate transcriptional profiles of young and aged peritoneal macrophages, both in the absence and presence of LPS stimulation. A subset of genes which are identified to both regulate TLR4 signaling and are also differentially expressed in aged macrophages will be further characterized at a mechanistic level. These studies will enable the translation of systems-level analyses towards mechanistic and physiological understanding of macrophage response to lipopolysaccharides and age-related chronic inflammation. PUBLIC HEALTH RELEVANCE: The studies proposed here will promote global insights into the molecular bases of innate immune response, and provide novel therapeutic strategies to address age-related immune and inflammatory diseases.