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. 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).

衣康酸盐（itaconate）是一种免疫调节代谢物，由炎症巨噬细胞中的线粒体酶免疫反应基因1（IRG1）产生。我们近期明确了衣康酸盐从炎症巨噬细胞释放的关键机制，但目前仍不清楚胞外衣康酸盐是否会被非髓系细胞摄取并发挥免疫调节功能。
本研究采用定制化CRISPR筛选（CRISPR screen）技术，将二羧酸转运蛋白溶质载体家族13成员3（SLC13A3）鉴定为衣康酸盐摄入转运体，并表征了SLC13A3在衣康酸盐改善肝脏抗菌天然免疫中的作用。功能实验结果显示，Slc13a3的肝脏特异性敲除会在体内及体外均削弱肝脏的抗菌天然免疫能力。从机制上来说，通过SLC13A3摄取的衣康酸盐可诱导转录因子EB（TFEB）依赖的溶酶体生物发生，进而增强小鼠肝细胞的抗菌天然免疫功能。本研究鉴定出SLC13A3是小鼠肝细胞中关键的衣康酸盐摄入转运体，将有助于开发基于衣康酸盐的强效抗菌治疗手段。
由中国南京科勒斯生物技术有限公司（Corus Biotechnology Inc.）定制的人类膜转运蛋白CRISPR干扰（CRISPRi）文库包含靶向1241个膜转运蛋白基因的7441条单引导RNA（sgRNA）。我们以感染复数（MOI）为0.3将慢病毒文库感染表达cBioITA的293T细胞，以实现该文库平均400倍的覆盖度。感染后48小时，使用2 μg·mL⁻¹的嘌呤霉素对细胞进行为期5天的筛选。将经3 mM衣康酸盐处理1小时的携带gRNA文库的感染细胞，根据cBioITA荧光强度分为cBioITA低表达群（最低25%的细胞）与cBioITA高表达群（最高25%的细胞）并进行分选。收集分选后的细胞（每个群体约3×10⁶个细胞），采用血液与细胞培养DNA大提试剂盒（Qiagen）提取基因组DNA（gDNA），随后使用Phanta超保真DNA聚合酶试剂盒（Vazyme）扩增包含gRNA序列的DNA片段。最后，由中国苏州吉因生生物信息学研究所（Genewiz Bioinformatics Institute）对PCR扩增产物进行测序。