Substrate availability and citrate alter TCA cycle metabolism and SLC13A3 in macrophage immune responses

Abstract Cell culture media are commonly formulated to enhance cell growth and often lack the physiological nutrient composition found in human blood plasma. The impact of substrate availability on immune cell metabolism and function remains incompletely understood. Here, we demonstrate that changes in culture medium composition affect mitochondrial metabolic pathways, immune responses, and transport in macrophages. Using mass spectrometry and stable isotope tracing, we identify citrate as a mediator linking extracellular substrate availability to intracellular metabolism. We also observe increased IL-6 secretion and elevated expression of plasma membrane transporter NaDC3 (SLC13A3) under physiological carbon source conditions that are reversed when citrate is excluded from the medium. Our findings demonstrate that extracellular substrate composition shapes macrophage immunometabolism and identify citrate as an extracellular signal that modulates immune responses. This work highlights the importance of physiologically relevant nutrient availability in studying and targeting immunometabolic pathways.