ABCG2 is an itaconate exporter that limits TFEB-mediated lysosomal biogenesis to impair antibacterial innate immunity

Itaconate is a metabolite that synthesized from cis-aconitate in mitochondria and transported into cytosol to exert multiple regulatory effects in macrophages. However, the mechanism by which itaconate exits from the cytosol of activated macrophages remains unknown. Using a genetic screen, we reveal that cytosolic itaconate is exported by ABCG2, an ATP-binding cassette (ABC) transporter, in an ATPase-dependent manner in human and mouse macrophages. As the human membrane transporter CRISPRi library (customized by Corus Biotechnology Inc., Nanjing in China) contains 7441 sgRNAs targeting 1241 membrane transporter genes, Cytosolic BioITA (cBioITA)-expressing THP-1 cells (5×107) were transduced with lentivirus from the library at a transduction effificiency of 30% and an average of 400-fold coverage of the library. The cells were stimulated with LPS (100 ng ml–1) for 8 hours. The cells were sorted into two populations by GFP expression: the top 25% of cBioITA’s intensity cells and bottom 25% of cBioITA’s intensity cells. Cells were washed in PBS and stored at -80°C for isolation of genomic DNA (gDNA). PCR were performed for identification of sgRNA inserts. The resulting amplicons were pooled and extracted from 2% agarose gels using the Gel Extraction Kit (Omega), and sequenced by Novogene Bioinformatics Institute (Beijing, China).