Extracellular citrate modulates glutamine metabolism in human macrophages during infection

Abstract Citrate is a central metabolite linking tricarboxylic acid (TCA) cycle activity to energy and lipid metabolism and supports the synthesis of inflammatory mediators, including itaconate, in macrophages. While citrate is primarily generated endogenously, extracellular citrate levels are elevated under pathological conditions such as citrate transporter disorder. Cells import extracellular citrate through SLC13 transporters, including the sodium-dependent citrate transporter NaCT (encoded by SLC13A5). However, whether macrophages take up extracellular citrate and how this affects metabolism and function remains unclear. Here, we combined mass spectrometry and tracing approaches to investigate the metabolic fate of citrate in human macrophage cell lines, primary, and iPSC-derived macrophages. We demonstrate that cells take up extracellular citrate, which was enhanced under metabolic stress conditions. Exogenous citrate was not substantially utilized as a carbon source but selectively altered glutamine metabolism and responses to bacterial infection with Salmonella enterica Typhimurium and Legionella pneumophila Corby. Our work identifies extracellular citrate as a context-dependent regulator in macrophages that decouples uptake from metabolic utilization. Highlights Macrophages import extracellular citrate via SLC13 transporters Extracellular citrate accumulates under hypoxia and inflammatory activation Extracellular citrate does not fuel central carbon metabolism in human macrophages Citrate modulates glutamine immunometabolism and modulates immune responses eTOC blurb Voß-Willenbockel et al. demonstrate that human macrophages accumulate extracellular citrate without using it as a major carbon source. Instead, citrate modulates glutamine utilization, inflammatory responses, and host-pathogen interactions revealing a context-dependent regulatory role for extracellular metabolites in immune cell function. Competing Interest Statement The authors have declared no competing interest. List of Abbreviations - [2,4-13C2]citrate - 2,4 labeled citrate - [U-13C5]glutamine - Uniformly labeled glutamine - [U-13C6]glucose - Uniformly labeled glucose - 12C - Carbon 12 - 13C - Carbon 13 - ACES - N-(2-acetamido)-2-aminoethanesulfonic acid - ACLY - ATP-citrate-lyase - ATCC - American Type Culture Collection - BCA - Bicinchoninic Acid Assay - BCYE - Buffered charcoal yeast extract - BPTES - Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide - BSA - Bovine serum albumin - CFU - Colony-forming units - CoA - Coenzyme A - cRPMI - Complete Roswell Park Memorial Institute medium - CSF - Cerebrospinal fluid - CT - Cycling threshold - DAMP - Damage-associated molecular pattern - DMEM - Dulbecco’s Modified Eagle Medium - ELISA - Enzyme-linked immunosorbent assay - FAMEs - Fatty acid methyl esters - FBS - Fetal bovine serum - FCCP - Trifluoromethoxy carbonyl cyanide phenylhydrazone - GC - Gas chromatography - GLS - Glutaminase - hMDMs - Human monocyte-derived macrophages - HRP - Horse radish peroxidase - IFNγ - Interferon γ - IL-6 - Interleukin 6 - IPSC - Induced pluripotent stem cell - L. pneumophila - Legionella pneumophila Corby - LPS - Lipopolysaccharide - LRS - Leucocyte reduction system - MASLD - Metabolically-dysfunction-associated steatotic liver disease - MOI - Multiplicity of Infection - MS - Mass spectrometry - MTBSTFA - N-tert-Butyldimethylsilyl-N-methyltrifluoracetamide - NaCT - Sodium-dependent citrate transporter (SLC13A5) - NaDC1 - Sodium-dependent dicarboxylate transporter 1 (SLC13A2) - NaDC3 - Sodium-dependent dicarboxylate transporter 3 (SLC13A3) - NAFLD - Non-alcoholic fatty liver disease - OCR - Oxygen consumption rate - PMA - Phorbol 12-myristate 13-acetate - P/S - Penicillin/Streptomycin - PBMC - Peripheral Blood Mononuclear Cell - PBS - Phosphate-buffered saline - Rh M-CSF - Recombinant human macrophage colony-stimulating factor - RIPA - Radioimmunoprecipitation Assay buffer - RPMI - Roswell Park Memorial Institute medium - RPL27 - Ribosomal protein L27 (human gene) - RT - Room temperature - s.e.m. - Standard error of mean - S. Typhimurium - Salmonella enterica Typhimurium - SDH - Succinate dehydrogenase - SLC7A5 - Solute carrier family 7 member 5 (human gene) - SLC13A3 - Solute carrier family 13 member 3 (human gene) - SLC13A5 - Solute carrier family 13 member 5 (human gene) - tBDMS - Tert-Butyldimethylsilylchloride - TCA - Tricarboxylic acid - TNFα - Tumor necrosis factor alpha - UHPLC - Ultra-high performance liquid chromatography - WT - Wild type - YEB - Yeast extract beef