Identification of age-related mediators of pro-inflammatory signaling

描述（由申请人提供）：先天免疫系统是依赖于模式识别受体（PRR）通过识别病原体相关分子模式（PAMP）来检测外源病原体的第一线防御机制。Toll样受体（TLR）包括响应多种PAMP的膜靶向PRR的一个家族。这些受体之一，TLR 4，识别存在于革兰氏阴性细菌细胞壁上的脂多糖（LPS）。TLR 4参与启动直接但受调节的信号传导级联，导致核因子KB（NF-κ B）和干扰素调节因子（IRF）蛋白的活化和炎性细胞因子以及干扰素的诱导。减弱的免疫功能，包括失调或慢性炎症状态，往往与年龄的增长相一致。有趣的是，老化的腹腔巨噬细胞对炎症刺激物（包括LPS）敏感。然而，分子介质，可能有助于年龄相关的免疫疾病仍然是模糊的，在很大程度上不确定。在这里，我们建议采用一种基于系统的方法，它将整合一系列的功能基因组学分析，以确定新的分子因子，有助于增加LPS的敏感性，在老年人的腹腔巨噬细胞。具体来说，我们将使用稳定转染NF-κ B-荧光素酶报告基因的巨噬细胞系优化和执行全基因组RNAi筛选。将用LPS处理细胞系以诱导TLR 4依赖性促炎级联反应，并通过评估荧光素酶活性来监测途径的激活。从高通量筛选产生的推定命中随后将被验证，并且将代表影响巨噬细胞中促炎或抗炎状态的基因组的子集。为了确定调节TLR 4信号传导的基因是否也在衰老中被交替调节，将使用微阵列分析来生成年轻和年老的腹膜巨噬细胞在不存在和存在LPS刺激的情况下的转录谱。将在机制水平上进一步表征被鉴定为既调节TLR 4信号传导又在老化巨噬细胞中差异表达的基因子集。这些研究将使翻译系统水平的分析对巨噬细胞的反应，脂多糖和年龄相关的慢性炎症的机制和生理的理解。 公共卫生相关性：本文提出的研究将促进对先天免疫反应分子基础的全面了解，并为解决年龄相关的免疫和炎症疾病提供新的治疗策略。

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.