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. 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, 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).