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Abstract
BackgroundInterferons (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 IFNγ.ResultsTransfection 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 IFNγ. 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 IFNγ. 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.ConclusionOur 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.
Highlights
Interferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs)
We observed significantly increased levels of replication of mouse cytomegalorvirus (mCMV) in bone-marrow derived macrophages (BMDMs) following treatment of the cells with a number siRNAs the mechanisms by which the genes targeted contribute to the antiviral phenotype are largely unknown
In order to examine this affect in BMDMs we performed a series of mock transfections, treating cells for 24 hours with the cationic lipid reagent Lipofectamine2000 alone or combined with a non-targeting control siRNA (RISCFree siRNA, Thermo Fisher)
Summary
Interferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs). Interferons (IFNs) are cytokines capable of causing a major shift in cellular gene expression through engagement of signal transduction pathways and subsequent activation of transcriptional networks. IFNs have proven useful clinically as potent agents against both RNA and DNA viruses, and as response modifiers for oncology and as therapeutic agents for autoimmune diseases such as multiple sclerosis [1]. In spite of this our understanding of the signalling pathways and transcriptional networks associated with IFN signalling remains incomplete. Macrophages activated by IFNγ are sometimes referred to as being polarized into an M1 phenotype [8,9]
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