Abstract
The mammalian innate immune system senses many bacterial stimuli through the toll-like receptor (TLR) family. Activation of the TLR4 receptor by bacterial lipopolysaccharide (LPS) is the most widely studied TLR pathway due to its central role in host responses to gram-negative bacterial infection and its contribution to endotoxemia and sepsis. Here we describe a genome-wide siRNA screen to identify genes regulating the human macrophage TNF-α response to LPS. We include a secondary validation screen conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits. We also provide microarray data from the same LPS-treated macrophage cells to facilitate downstream data analysis. Tertiary screening with multiple TLR ligands and a microbial extract demonstrate that novel screen hits have broad effects on the innate inflammatory response to microbial stimuli. These data provide a resource for analyzing gene function in the predominant pathway driving inflammatory cytokine expression in human macrophages.
Highlights
Background & SummaryToll-like receptors (TLR) are a major class of pattern recognition receptors in innate immune cells, sensing a wide range of bacterial, viral, and microbial stimuli[1,2]
TLR4, a toll-like receptor (TLR) conserved in human and mouse whose activation is central to the immune response to gram-negative bacteria[3,4], is the most widely studied TLR with established roles in clinical cases such as sepsis and endotoxemia[5,6]
Due to its critical and initiating role in the inflammatory cytokine response, the TLR pathway has been the target of screening studies to identify novel regulators from across the genome[7,8,9,10,11]
Summary
Toll-like receptors (TLR) are a major class of pattern recognition receptors in innate immune cells, sensing a wide range of bacterial, viral, and microbial stimuli[1,2]. Given the intricacy of the signaling pathways shared across multiple TLR receptors, more remains to be understood of how the candidate regulators identified by these studies are shared across multiple TLRs and which are specific to the TLR4 response To address these challenge we have conducted a genome-wide siRNA screen in the human THP-1 macrophage-like cell line, modified to express a dual luciferase reporter for an inflammatory readout and corrective for perturbation effects on cell viability[12]. Our screen provides a comprehensive genome-wide study of the global regulation of the TLR4 response in a human cell line, identifying multiple novel regulators It can serve as a complimentary data set to genome-wide studies in mouse cell lines for comparative analysis of conserved versus species-specific regulators, and whether the pathways downstream of multiple TLR receptors share these regulators. Data from our tertiary screen utilizing diverse TLR ligands can be used for further analysis of shared and specific regulators across the TLR receptors and their intricate downstream signaling cascades
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