Macrophages undergo functionally significant reprograming of nucleotide metabolism upon classical activation

During an immune response, macrophages specifically rewire their metabolism to support functional changes. Using a multi-omics approach, we identified nucleotide metabolism as one of the most significantly rewired pathways across the metabolic network in classically activated macrophages. Further isotopic tracing studies revealed the substantial changes in nucleotide de novo synthesis, degradation, and salvage fluxes in stimulated macrophages, as well as the key reactions where metabolic regulation occurs: 1) de novo synthesis of purine nucleotides is shut down and particularly blocked at the last step of IMP synthesis catalyzed by ATIC; 2) de novo synthesis of pyrimidines is maintained up to UMP, but further synthesis of CTP (catalyzed by CTPS) and dTMP (catalyzed by TYMS) is greatly reduced; 3) Nucleotide degradation to nitrogenous bases is increased, but further oxidation of purine bases (catalyzed by XOR) is inhibited, causing a great accumulation of nucleosides and bases; and 4) cells switch to salvaging the nucleosides and bases as the primary means to maintain purine nucleotides. Mechanistically, we found these changes are driven by a combination of transcriptional regulation and enzyme inhibition. Nitric oxide (NO) was identified as a major regulator, driving the strong inhibition of ATIC and XOR, and the transcriptional downregulation of Tyms. To understand the functional impact of the activation-induced switch from purine de novo synthesis to salvage, we knocked out the purine salvage enzyme Hprt. Hprt knockout significantly alters functional gene expression in activated macrophages, suppresses macrophage migration, and increases pyroptosis. Furthermore, knocking out Hprt or Xor increases the proliferation of the intracellular parasite Toxoplasma gondii in macrophages. Together, these results comprehensively uncovered the dynamic rewiring of nucleotide metabolism in classically activated macrophages, elucidated the key regulatory mechanisms, and identified the functional significance of such rewiring.