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.