167 : Investigating the mechanism of TLR signaling crosstalk in mouse macrophages

Abstract

Toll-like receptors (TLRs) are important pathogen-recognition receptors in innate immune sensing and are also important for initiating adaptive immunity. While the signaling of individual TLR pathways has been intensively studied, less is known about how innate immune cells integrate the combined TLR inputs presented by a typical microorganism. Based on the observation that certain TLR ligand combinations can induce synergistic production of cytokine mediators, we are using a combination of gene expression analysis and siRNA screening to identify the cellular components that regulate the signaling crosstalk between TLR pathways. We carried out gene expression profiling in bone marrow-derived macrophages stimulated with either poly (I:C) (TLR3 ligand), R848 (TLR7 ligand), or both ligands combined. We identified genes that were synergistically induced under dual TLR stimulation, including IL-6, IL-10, IL-1 α , IL-1 β , haptoglobin, lipocalin 2, and SOCS3. Synergy at the mRNA level was observed as early as 2 h. Based on these data, over 200 synergy factor candidates were picked and subjected to RNAi screening. In a pilot screening of known TLR pathway genes, knock-down of TLR3 pathway-specific components led to a loss of synergy in the secretion of IL-6 and IL-12p40, which validated the effectiveness of the screening protocol. We predict that the described approach will identify important mediators in TLR signaling crosstalk, and shed light on the regulation of late cytokines such as IL-6 and IL-12p40. This study was supported by the Intramural Research Program of NIAID, NIH.