Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling.
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.
Lacaze
Paul
P
Division of Pathway Medicine, The University of Edinburgh, The Chancellor's Building, College of Medicine, Edinburgh, UK. Paul.Lacaze@ed.ac.uk
Raza
Sobia
S
Sing
Garwin
G
Page
David
D
Forster
Thorsten
T
Storm
Petter
P
Craigon
Marie
M
Awad
Tarif
T
Ghazal
Peter
P
Freeman
Tom C
TC
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
D034741:RNA, Small Interfering; D014157:Transcription Factors; D007371:Interferon-gamma