Itaconate is one of the most highly upregulated metabolites in inflammatory macrophages. It has been shown to have immunomodulatory properties, although the underlying mechanisms are not fully elucidated. Here, we have investigated how itaconate regulates type I interferon production. Using pharmacological and genetic approaches, we have found that inhibition of succinate dehydrogenase (SDH) is required for this response. SDH inhibition by itaconate leads to double-stranded mitochondrial RNA (mtRNA) release, which is dependent on the mitochondrial pore formed by VDAC1. Following this, the dsRNA sensors MDA5 and RIG-I are required for IFNβ production in response to SDH inhibition by itaconate. Inhibition of SDH by itaconate links TCA cycle modulation to type I Interferon production via mtRNA.

BMDMs (three mice) were treated as indicated, and RNA was extracted as detailed above. mRNA was extracted from total RNA using poly-T-oligo-attached magnetic beads. After fragmentation, the first strand of cDNA was synthesised using random hexamer primers, followed by the second strand of cDNA synthesis. The library was checked using Qubit and real-time PCR for quantification and a bioanalyser for size distribution detection. Quantified libraries were pooled and sequenced on a NovaSeq 6000 S4 (Illumina). Differential expression analysis of two conditions per group was performed using counted reads and the DESeq2 R package67. Pathway enrichment analyses were performed as indicated below in the quantification 444 and statistical analysis section.