title: Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages.
abstract: Macrophages play a critical role in the innate immune response to pathogen infection, but few tools exist for systematic dissection of these responses using modern genome-wide perturbation methods. To develop an assay platform for high-throughput analysis of macrophage activation by pathogenic stimuli, we generated reporter systems in human and mouse macrophages with dynamic readouts for NF-κB and/or TNF-α responses. These reporter cells show responsiveness to a broad range of TLR ligands and to gram-negative bacterial infection. There are significant challenges to the use of RNAi in innate immune cells, including efficient small RNA delivery and non-specific immune responses to dsRNA. To permit the interrogation of the macrophage pathogen response pathways with RNAi, we employed the stably expressed reporter genes to develop efficient siRNA delivery protocols for maximal target gene silencing with minimal activation of the innate macrophage response to nucleic acids. We demonstrate the utility of these macrophage cell systems for siRNA screening of pathogen responses by targeting components of the human and mouse TLR pathways, and observe species-specific perturbation of signaling and cytokine responses. Our approach to reporter cell development and siRNA delivery optimization provides an experimental paradigm with significant potential for developing genetic screening platforms in mammalian cells.
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Zachary L
authors.initials: ZL
authors.lastname: Benet
authors.affiliation: Lymphocyte Biology Section, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ze
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Souhaila
authors.initials: S
authors.lastname: Al-Khodor
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Sinu P
authors.initials: SP
authors.lastname: John
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Lin
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Myong-Hee
authors.initials: MH
authors.lastname: Sung
authors.affiliation: Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Iain D C
authors.initials: ID
authors.lastname: Fraser
mesh_terms: D000818:Animals; D002460:Cell Line; D016000:Cluster Analysis; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D016905:Gram-Negative Bacterial Infections / Q000235:genetics / Q000276:immunology; D054884:Host-Pathogen Interactions / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D008024:Ligands; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D015203:Reproducibility of Results; D051193:Toll-Like Receptors / Q000378:metabolism
chemical_list: D008024:Ligands; D034741:RNA, Small Interfering; D051193:Toll-Like Receptors
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