Glutamine Is Required for M1-like Polarization of Macrophages in Response to Mycobacterium tuberculosis Infection

In response to Mycobacterium tuberculosis infection, macrophages mount proinflammatory and antimicrobial responses similar to those observed in M1 macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). A metabolic reprogramming to hypoxia-inducible-factor 1 (HIF-1)-mediated uptake of glucose and its metabolism by glycolysis is required for M1-like polarization, but little is known about other metabolic programs driving the M1-like polarization during infection. We report that glutamine serves as a carbon and nitrogen source for the metabolic reprogramming to M1-like macrophages. Widely targeted metabolite screening identified an association of glutamine and/or glutamate with highly affected metabolic pathways of M1-like macrophages. Moreover, stable isotope-assisted metabolomics of U¹³C glutamine and U¹³C glucose revealed that glutamine, rather than glucose, is catabolized in both the oxidative and reductive tricarboxylic acid (TCA) cycles of M1-like macrophages, thereby generating signaling molecules that include succinate, biosynthetic precursors such as aspartate, and itaconate. U¹⁵N glutamine-tracing metabolomics further revealed participation of glutamine nitrogen in synthesis of intermediates of purine and pyrimidine metabolism plus amino acids, including aspartate. These findings were corroborated by diminished M1 polarization from chemical inhibition of glutaminase (GLS), the key enzyme in the glutaminolysis pathway, and by genetic deletion of GLS in infected macrophages. Thus, the catabolism of glutamine is an integral component of metabolic reprogramming in activating macrophages and it coordinates with elevated cytosolic glycolysis to satisfy the cellular demand for bioenergetic and biosynthetic precursors of M1-like macrophages. Knowledge of these new immunometabolic features of M1-like macrophages should advance the development of host-directed therapies for tuberculosis. IMPORTANCE Macrophages play essential roles in determining the progression and final outcome of human infection by Mycobacterium tuberculosis. While upregulation of hypoxia-inducible-factor 1 (HIF-1) and a metabolic reprogramming to the Warburg Effect-like state are known to be critical for immune cell activation in response to M. tuberculosis infection, our overall knowledge about the immunometabolism of M1-like macrophages is poor. Using widely targeted small-metabolite screening, stable isotope tracing metabolomics, and pharmacological and genetic approaches, we report that, in addition to enhanced glucose catabolism by glycolysis, glutamine is utilized as an important carbon and nitrogen source for the generation of biosynthetic precursors, signaling molecules, and itaconate in M. tuberculosis-induced M1-like macrophages. Recognizing this novel contribution of glutamine to the immunometabolic properties of M. tuberculosis-infected macrophages may facilitate the development of treatments for tuberculosis and stimulate comparable studies with other pathogen-macrophage interactions.