title: Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling.
abstract: BACKGROUND
  Interferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs). Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNgamma.
  
  
  RESULTS
  Transfection of murine bone-marrow derived macrophages (BMDMs) with a non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNgamma. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNgamma. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response.
  
  
  CONCLUSIONS
  Our results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated with type I and type II IFN signalling and a suppression of macrophage M1 polarization.
authors: Division of Pathway Medicine, The University of Edinburgh, The Chancellor's Building, College of Medicine, Edinburgh, UK. Paul.Lacaze@ed.ac.uk
authors: Paul
authors: P
authors: Lacaze
authors: Sobia
authors: S
authors: Raza
authors: Garwin
authors: G
authors: Sing
authors: David
authors: D
authors: Page
authors: Thorsten
authors: T
authors: Forster
authors: Petter
authors: P
authors: Storm
authors: Marie
authors: M
authors: Craigon
authors: Tarif
authors: T
authors: Awad
authors: Peter
authors: P
authors: Ghazal
authors: Tom C
authors: TC
authors: Freeman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D055106:Genome-Wide Association Study / Q000379:methods; D007371:Interferon-gamma / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D014157:Transcription Factors; D007371:Interferon-gamma