Itaconate drives mtRNA-mediated Type I interferon production via inhibition of succinate dehydrogenase

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.

Methods
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.

衣康酸（itaconate）是炎症巨噬细胞中上调水平最高的代谢物之一。现有研究表明其具有免疫调节活性，但具体分子机制尚未完全阐明。本研究探究了衣康酸对I型干扰素（type I interferon）产生的调控机制。通过药理学与遗传学实验手段，我们发现该调控过程依赖琥珀酸脱氢酶（succinate dehydrogenase, SDH）的抑制。衣康酸对SDH的抑制可诱导线粒体RNA（mitochondrial RNA, mtRNA）释放，该过程依赖VDAC1（voltage-dependent anion channel 1, VDAC1）介导形成的线粒体孔道。后续实验证实，MDA5（melanoma differentiation-associated gene 5, MDA5）与RIG-I（retinoic acid-inducible gene I, RIG-I）这两种双链RNA感受器，是衣康酸通过抑制SDH诱导干扰素β（interferon β, IFNβ）产生的必需元件。综上，衣康酸对SDH的抑制通过线粒体RNA通路，将三羧酸循环（tricarboxylic acid cycle, TCA cycle）的调控与I型干扰素产生关联起来。

实验方法
将骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs，来源于3只小鼠）按指示条件处理，按照前述步骤提取总RNA。利用偶联poly-T寡核苷酸的磁珠从总RNA中富集mRNA。经片段化处理后，使用随机六聚体引物合成第一链cDNA，随后完成第二链cDNA的合成。采用Qubit定量仪与实时荧光定量PCR对构建好的文库进行定量，并通过生物分析仪检测文库的片段大小分布。将定量合格的文库混合后，在NovaSeq 6000 S4测序平台（Illumina）上进行高通量测序。采用计数读段与DESeq2 R包67完成两组处理条件间的差异表达分析。通路富集分析按照后续“定量与统计分析”章节所述方法开展。