Itaconate uptake via SLC13A3 improves hepatic antibacterial innate immunity

Itaconate is an immunoregulatory metabolite produced by the mitochondrial enzyme immune-responsive gene 1 (IRG1) in inflammatory macrophages. We recently identified an important mechanism by which itaconate is released from inflammatory macrophages. However, it remains unknown whether extracellular itaconate is taken up by non-myeloid cells to exert immunoregulatory functions. Here, we used a custom-designed CRISPR screen to identify the dicarboxylate transporter solute carrier family 13 member 3 (SLC13A3) as an itaconate importer and to characterize the role of SLC13A3 in itaconate-improved hepatic antibacterial innate immunity. Functionally, liver-specific deletion of Slc13a3 impairs hepatic antibacterial innate immunity in vivo and in vitro. Mechanistically, itaconate uptake via SLC13A3 induces transcription factor EB (TFEB)-dependent lysosomal biogenesis and subsequently improves antibacterial innate immunity in murine hepatocytes. These findings identify SLC13A3 as a key itaconate importer in murine hepatocytes and will aid in the development of potent itaconate-based antibacterial therapeutics. Overall design: As the human membrane transporter CRISPRi library (customized by Corus Biotechnology Inc., Nanjing in China) contains 7441 sgRNAs targeting 1241 membrane transporter genes, cBioITA-expressing 293T cells were infected with the lentivirus library at an MOI of 0.3 to obtain an average of 400-fold coverage of the library. At 48 hours post-infection, the cells were selected with 2 µg ml-1 of puromycin for 5 days. Infected cells expressing the gRNA library treated with 3 mM itaconate for 1 hour, and then sorted into a cBioITAbottom population (the lowest 25% of cells) and a cBioITAtop population (the highest 25% of cells) according to the cBioITA fluorescence intensity. The sorted cells (approximate 3 × 10^6 cells/population) were collected for genomic DNA (gDNA) extraction using a Blood & Cells Culture DNA Maxi kit (Qiagen), followed by PCR amplification of DNA fragments containing gRNA sequences using a Phanta Super-Fidelity DNA Polymerase kit (Vazyme). The PCR amplicons were sequenced by the Genewiz Bioinformatics Institute (Suzhou, China).