Identification of age-related mediators of pro-inflammatory signaling

描述(申请人提供)：先天免疫系统是一种一线防御机制，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。Toll样受体(Toll-like Receptor，TLRs)是一类膜靶向的PRR家族，可对多种PAMP产生反应。其中一个受体，TLR4，识别存在于革兰氏阴性细菌细胞壁上的脂多糖(LPS)。TLR4参与启动即时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，并诱导炎性细胞因子和干扰素。免疫功能减弱，包括失控或慢性炎症状态，通常与年龄的增长一致。有趣的是，老化的腹膜巨噬细胞对包括内毒素在内的炎性刺激敏感。然而，可能导致年龄相关免疫疾病的分子介质仍然含糊不清，在很大程度上还没有定义。在这里，我们建议采用一种基于系统的方法，整合一系列功能基因组学分析，以确定导致老龄腹膜巨噬细胞对内毒素敏感性增加的新分子因素。具体地说，我们将优化和执行全基因组RNAi筛选，使用稳定转染了NF-KB-荧光素酶报告的巨噬细胞系。该细胞系将被内毒素处理以诱导依赖于TLR4的促炎级联反应，并将通过评估荧光素酶的活性来监测该途径的激活。高通量筛选产生的假定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的一个子集。为了确定调控TLR4信号的基因在衰老过程中是否也被交替调节，将使用微阵列分析在没有和存在内毒素刺激的情况下，建立年轻和老年腹膜巨噬细胞的转录图谱。被发现既调节TLR4信号又在衰老巨噬细胞中差异表达的基因子集将在机制水平上进一步表征。这些研究将使系统水平的分析能够转化为对巨噬细胞对内毒素和年龄相关慢性炎症的反应的机制和生理方面的理解。 公共卫生相关性：这里提出的研究将促进对先天免疫反应分子基础的全球洞察，并为解决与年龄相关的免疫和炎症性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.