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Genome-wide siRNA screen of genes regulating the Lipopolysaccharide-induced TNF-α response in human macrophages

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. These data provide a resource for analyzing gene function in the predominant pathway driving inflammatory cytokine expression in human macrophages.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83824 | GEO | 2017/02/23

SECONDARY ACCESSION(S): PRJNA327121

REPOSITORIES: GEO

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Project description:Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations. Keywords: time course For each treatment (Sigma LPS, LIST LPS, and media only), three biological replicates (separate macrophage cultures and RNA isolations) were profiled. Each sample was labeled with Cy3 and Cy5 and co-hybridized with Stratagene Universal Mouse Reference (dye flip techical replicates). Expression at 2 timepoints (6 and 24 hours post-treatment) was assessed.

Project description:Molecular Pathways and Transcriptional Networks Involved in the Macrophage Response to LPS, poly(I:C) and CpG DNA Stimulation Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations. For each treatment (LPS, polyIC, CpG DNA, media only), three biological replicates (separate macrophage cultures and RNA isolations) were profiled. Each sample was labeled with Cy3 and Cy5 and co-hybridized with Stratagene Universal Mouse Reference (dye flip techical replicates). Expression at 2 timepoints (6 and 24 hours post-treatment) was assessed.

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Genome-wide siRNA screen of genes regulating the Lipopolysaccharide-induced TNF-α response in human macrophages

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