Gene Regulatory Network Inference of Immunoresponsive Gene 1 (IRG1) Identifies Interferon Regulatory Factor 1 (IRF1) as Its Transcriptional Regulator in Mammalian Macrophages.

Tallam A, Perumal TM, Antony PM, Jäger C, Fritz JV, Vallar L, Balling R, Del Sol A, Michelucci A.
Source: PLoS ONE
Publication Date: (2016)
Issue: 11(2): 1-28
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Macrophage, human
Species: human
Tissue Origin: blood
RAW 264.7
Species: mouse
Tissue Origin: blood
4D-Nucleofector® X-Unit
4D-Nucleofector® Y-Unit
The ON-TARGETplus SMARTpool, containing four different siRNA sequences specifically targeting each of murine Irf1 (siRNA Irf1, L-046743-01-0005), murine Cebpb (siRNA Cebpb, L-043110-00-0005), human IRF1 (siRNA IRF1, L-011704-00-0005) and the corresponding non-targeting control (siRNA NEG, D-001810-10-05), were designed and synthesized by Thermo Scientific Dharmacon. Murine RAW264.7 macrophages were transfected with Amaxa 4D Nucleofector device, Xunit (Lonza) using the Amaxa SG cell line 4D Nucleofector Kit for THP-1 cells according to the manufacturer’s instructions. Briefly, transfection with siRNA complexes was carried out from pelleted and resuspended cells (1x106 cells per condition). Transfection reagent and siRNA were prepared according to the manufacturer’s instructions (Amaxa). Specific siRNAs were added at a final concentration of 100nM. After nucleofection using the program “RAW264.7 (ATCC)”, the cells were seeded at a density of 1x106 cells per well in 12-well plates in DMEM supplemented with 10% FBS and incubated for 24h. Human PBMCs-derived macrophages were transfected using the Amaxa 4D Nucleofector device, Y-unit, which was specifically designed for transfection of adherent cells. For these experiments, cells were seeded at 1x106 cells in 24-well plates for 11 days and the medium was then replaced with nucleofection reagent and specific siRNAs (2µM) according to the manufacturer’s protocol. The reagent solution was removed and the medium was added to the cells which were then incubated for 24h and stimulated with LPS.
Immunoresponsive gene 1 (IRG1) is one of the highest induced genes in macrophages under pro-inflammatory conditions. Its function has been recently described: it codes for immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (IRG1/CAD), an enzyme catalysing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (TCA) cycle intermediate. Itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the TCA cycle and enables bacteria to survive on limited carbon conditions. To elucidate the mechanisms underlying itaconic acid production through IRG1 induction in macrophages, we examined the transcriptional regulation of IRG1. To this end, we studied IRG1 expression in human immune cells under different inflammatory stimuli, such as TNFa and IFN?, in addition to lipopolysaccharides. Under these conditions, as previously shown in mouse macrophages, IRG1/CAD accumulates in mitochondria. Furthermore, using literature information and transcription factor prediction models, we re-constructed raw gene regulatory networks (GRNs) for IRG1 in mouse and human macrophages. We further implemented a contextualization algorithm that relies on genome-wide gene expression data to infer putative cell type-specific gene regulatory interactions in mouse and human macrophages, which allowed us to predict potential transcriptional regulators of IRG1. Among the computationally identified regulators, siRNA-mediated gene silencing of interferon regulatory factor 1 (IRF1) in macrophages significantly decreased the expression of IRG1/CAD at the gene and protein level, which correlated with a reduced production of itaconic acid. Using a synergistic approach of both computational and experimental methods, we here shed more light on the transcriptional machinery of IRG1 expression and could pave the way to therapeutic approaches targeting itaconic acid levels.