IRF1 Is Required for MDA5 (IFIH1) Induction by IFN-α, LPS, and poly(I:C) in Murine Macrophages

Abstract

Melanoma differentiation-associated protein 5 (MDA5) induces type I interferons (IFNs) after the recognition of viral RNA. In addition, gain-of-function mutations in the interferon induced with helicase C domain 1 (<i>IFIH1</i>) gene, which encodes MDA5, lead to type I interferonopathies. Here, we show that <i>Mda5</i> is highly expressed in murine macrophages and is regulated by pro-inflammatory stimuli such as the cytokines IFN-α and IFN-γ, the TLR ligand LPS, and a mimic of dsRNA, poly(I:C). <i>Mda5</i> induction is mediated through the production of reactive oxygen species. The induction by IFN-α or LPS occurs at the transcriptional level since the <i>Mda5</i> mRNA half-life before and after induction is very stable. Interestingly, STAT1 is required for <i>Mda5</i> induction by IFN-α, LPS, or poly(I:C). The time course of induction of at least 3 h and the need for protein synthesis indicate that <i>Mda5</i> requires an intermediate protein for transcription. In transient transfection experiments, we found that a 105-bp fragment of this gene, between −1153 and −1258 bp relative to the transcription start site, is required for transcription. In this specific region, we observed a sequence containing an IRF-binding motif, which, when mutated, abolishes the induction of <i>Mda5</i>. This sequence is strongly conserved in the <i>IFIH1</i> promoters of eutherian mammals and in other distant species. Kinetic experiments, chromatin immunoprecipitation assays, and gene-silencing experiments revealed that IRF1 is required for induction of <i>Mda5</i> expression.

Lipopolysaccharide Enhances Microglia Antiviral Control of Betacoronavirus Infection through TLR4-Dependent Induction of IFNβ with Concomitant Modulation of dsRNA Sensors MDA5 and TLR3

“…TLR4 signaling, induced by LPS, has been previously shown to increase the expression of MDA5 in U373MG human astrocytoma cells [91]. Moreover, LPS induced MDA5 expression in primary murine macrophages [92]. Therefore, our study expands previous knowledge on other cell types and demonstrates that LPS:EB induces upregulation of the dsRNA sensor MDA5 in murine microglia.…”

“…Type I IFNs induce hundreds of genes with antiviral functions, including the interferon induced with helicase C domain 1 (IFIH1) gene encoding MDA5, a cytosolic dsRNA sensor with critical roles in some viral infections, including coronaviruses [87][88][89][90]. Furthermore, in vitro LPS stimulation of murine macrophages and U373MG human astrocytoma cells induces upregulation of MDA5 expression [91,92]. Although LPS is the most potent stimuli that induces microglial activation, the effects of TLR4 stimulation on dsRNA sensors including MDA5 remain to be elucidated.…”