Extended Data Fig. 1: MCT1-mediated L-lactate uptake inhibits innate immunity by suppressing cGAS activity.
From: AARS1 and AARS2 sense l-lactate to regulate cGAS as global lysine lactyltransferases
a, Upper: volcano plots (ggplot2) of the RNA-seq analysis of Peripheral Blood Mononuclear Cells (PBMCs) from Normal (Nor.), High-Lac (H-lac) and Lac-Acidosis (LA) patients. The significantly up- or down- regulated genes are reported as red or blue dots, respectively. Left: differential expression analysis in the LA versus the H-Lac groups (edgeR, P < 0.05 and log2 (FC)  > 1). Middle and right: differential expression analysis in the H-Lac and LA groups compared to the Nor. groups (edgeR, P < 0.0001 and log2 (FC)  >  2), respectively. Lower: Gene Ontology (GO) analysis of the top 10 enriched pathways are listed. Up- or down- regulated pathways are reported as red or blue strips, respectively. Pathways related to immune response are highlighted in brown. Functional enrichment analysis using DAVID functional annotation tool with the Gene Ontology biological process term annotations. P values were derived from DAVID v.6.8 database (https://david.ncifcrf.gov/), which uses a modified one-sided Fisher’s exact test with Benjamini correction. b, Peritoneal macrophages (PM) from wild-type mice pre-treated with control PBS, L-lactate (Lac, 25 mM) or sodium L-lactate (NaLac, 25 mM) were infected for 12 h with mock (PBS), HCMV, HSV-1 (MOI (multiplicity of infection), 10). respectively, or transfected with HT-DNA or cGAMP for 12 h. ELISA of cellular cGAMP (upper) and IFN-β (lower) were shown. c, Immunoblot (IB) analysis of monomeric (Mono) and dimeric (Dimer) IRF3 (top blot; native PAGE) and total lysates (bottom blot; denaturing gel) of primary peritoneal macrophages pre-treated with control PBS, L-lactate (25 mM) or NaLac (25 mM) for 12 h, followed by infection with HSV-1 (left) or HCMV (right) for indicated time points. d, Immunoblot (IB) analysis of monomeric (Mono) and dimeric (Dimer) IRF3 (top blot; native PAGE) and total lysate (bottom blot; denaturing gel) of primary peritoneal macrophages pre-treated for 12 h with PBS, L-lactate (25 mM) or NaLac (25 mM), followed by transfection with HT-DNA (2.5 ng/ul) or cGAMP (100 nM) for 12 h. e, Measurement of intracellular L-lactate in PBMCs, BMDMs, CT26 and HCT116 cells. PBMCs and BMDMs were pre-treated with control DMSO, AR-C155858 (100 nM), Oxamate (50 mM) or AZD3965 (100 nM) for 4 h, followed by incubation for 24 h with NaCl (25 mM) or NaLac (25 mM). Two-way ANOVA with Dunnett’s test. f, ELISA of cGAMP in PBMCs and BMDMs treated as in e followed by mock infection (PBS) or infection for 12 h with HSV-1(MOI, 10). g, qPCR analysis of Ifnb1 mRNA (left), HSV-1-specific genomic DNA (gDNA) (middle) and plaque assay of HSV-1 titers (right) in PBMCs and BMDMs treated as in f. Data are representative of at least three independent experiments (b-g). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (b, e-g). ND, not detected. Supplementary Fig. 1 shows full gels.
