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Classically activated macrophages undergo functionally significant nucleotide metabolism remodelling driven by nitric oxide

Nature Metabolism volume 7, pages 1681–1702 (2025)Cite this article

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Abstract

During an immune response, macrophages specifically reprogramme their metabolism to support functional changes. Here, we revealed that nucleotide metabolism is one of the most significantly reprogrammed pathways upon classical activation. Specifically, de novo synthesis of pyrimidines is maintained up to uridine monophosphate, but blocked at cytidine triphosphate and deoxythymidine monophosphate synthesis; de novo synthesis of purines is shut off at the last step (catalysed by AICAR transformylase/IMP cyclohydrolase, ATIC), and cells switch to increased purine salvage. Nucleotide degradation to nitrogenous bases is upregulated but complete oxidation of purine bases (catalysed by xanthine oxidoreductase, XOR) is inhibited, diverting flux into salvage. Mechanistically, nitric oxide was identified as a major regulator of nucleotide metabolism, simultaneously driving multiple key changes, including the transcriptional downregulation of Tyms and profound inhibition of ATIC and XOR. Inhibiting purine salvage using Hgprt knockout or inhibition alters the expression of many stimulation-induced genes, suppresses macrophage migration and phagocytosis, and increases the proliferation of the intracellular parasite Toxoplasma gondii. Together, these results thoroughly uncover the dynamic reprogramming of macrophage nucleotide metabolism upon classical activation and elucidate the regulatory mechanisms and functional significance of such reprogramming.

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Fig. 1: Multi-omics analysis identifies nucleotide metabolism as a substantially reprogrammed metabolic pathway upon classical stimulation.
Fig. 2: Kinetic glutamine labelling reveals blockages in nucleotide de novo synthesis.
Fig. 3: Macrophages shift to purine salvage upon stimulation.
Fig. 4: Alterations in nucleotide degradation upon stimulation.
Fig. 5: NO has a key role in nucleotide metabolism reprogramming.
Fig. 6: NO causes the decrease of TYMS and inhibition of ATIC and XOR at the enzyme activity level.
Fig. 7: Alteration of nucleotide metabolism affects macrophage function and pathogen proliferation.

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Data availability

All data and uncropped scans for all immunoblots are included in the Source data files. The RNA-seq data reported in this article have been deposited in the Gene Expression Omnibus (GEO) under accessions GSE267544 and GSE267546. The unique reagents generated in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grant nos. R35 GM147014 (J.Fan), R01 AI172874 (L.J.K), and R01 AG078756 and U01 AG088679 (S.A.L.), and the Morgridge Institute for Research. G.L.S. was supported by National Research Service Award (NRSA) Individual Predoctoral Fellowship no. F31AI152280. B.J.E.-F. was supported by training grant T32 AI007414. N.L.A. was supported by NRSA Individual Predoctoral Fellowship no. F30AI183563. U.S.U. was supported by an UW–Madison Biotechnology Training Program NIH grant no. 5 T32 GM135066. I.R. was supported by the University of Wisconsin–Madison Post-Baccalaureate Research Education Program supported by grant no. R25 GM144251. Flow cytometry and single-cell sorting were performed with the instrument and assistance of the University of Wisconsin Carbone Cancer Center Flow Lab Core, supported by a Cancer Center Support Grant P30 CA014520. We thank M. Stefely for assistance in figure editing and A. Williams for text editing.

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Authors and Affiliations

  1. Morgridge Institute for Research, Madison, WI, USA

    Steven V. John, Gretchen L. Seim, James A. Votava, Uzziah S. Urquiza, Nicholas L. Arp, John Steill, Jack Freeman, Isaiah Roberts, Xin Qing, Ron Stewart & Jing Fan

  2. Cellular and Molecular Biology Graduate Program, University of Wisconsin–Madison, Madison, WI, USA

    Steven V. John, Nicholas L. Arp & Jing Fan

  3. Nutrition and Metabolism Graduate Program, University of Wisconsin–Madison, Madison, WI, USA

    Gretchen L. Seim, Xin Qing & Jing Fan

  4. Cellular and Molecular Pathology Graduate Program, University of Wisconsin–Madison, Madison, WI, USA

    Billy J. Erazo-Flores & Laura J. Knoll

  5. Department of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, WI, USA

    Billy J. Erazo-Flores, Laura J. Knoll & Jing Fan

  6. Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA

    Uzziah S. Urquiza & Jing Fan

  7. Neurodegeneration New Medicines Center and Department of Molecular & Cellular Biology, The Scripps Research Institute, La Jolla, CA, USA

    Lauren N. Carnevale & Stuart A. Lipton

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Contributions

S.V.J., G.L.S. and J.Fan designed most of the experiments, other than specifically noted below. S.V.J., G.L.S., J.A.V., N.L.A. and U.S.U. conducted most of the experiments other than those specifically noted below. S.V.J. and G.L.S. conducted the experiments for the RNA-seq. S.V.J., G.L.S., J.S., J.Freeman, R.S. and J.Fan. analysed the RNA-seq data. S.V.J., B.J.E.-F., L.J.K. and J.Fan conceptualized the T. gondii experiments and analysed the data. S.V.J., N.L.A., U.S.U., L.N.C., S.A.L. and J.Fan contributed to the study of ATIC inhibition. I.R. contributed to immunoblotting and X.Q. contributed to the forodesine treatment experiment. S.V.J., G.L.S., J.A.V., N.L.A., U.S.U., I.R. and J.Fan analysed the data for the other experiments. S.V.J. and J.Fan wrote and edited the manuscript with input from all authors. J.Fan supervised the entire project.

Corresponding author

Correspondence to Jing Fan.

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Nature Metabolism thanks Justin Perry, Dylan Ryan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Reprogramming of nucleotide metabolism is independent of changes in cell proliferation or viability.

a) Relative cell number of BMDMs over a timecourse with or without continual stimulation. Mean ± SD (n = 4 independent samples). b) Cell viability in a population of BMDMs that are unstimulated and cultured for 72 h (n = 4 independent samples), continually stimulated for 72 h (n = 4), or acutely stimulated for 96 h (n = 3). Mean ± SD. c) Metabolomic changes in pentose phosphates, nucleotides, nucleosides, and nitrogenous bases in RAW 264.7 cells over a timecourse of continual stimulation. d) Relative cell number of RAW 246.7 cells over a timecourse with or without continual stimulation. Mean ± SD (n = 3 independent samples). e) The top 10 genes enriched along principal component 1 of the transcriptomic changes in BMDMs over a timecourse of acute stimulation. c, e) Relative (c) metabolite abundances or (e) gene expression are compared to unstimulated cells (0 h) and displayed on a heatmap, with saturating color representing 10-fold change (or 3.32 on log2 scale). Each box represents the mean of n = 3 independent samples. a, d) Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated to stimulated conditions at 72 h. b) Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett’s test comparing all groups to unstimulated cells. ns indicates not significant (p > 0.01).

Source data

Extended Data Fig. 2 Kinetic glutamine tracing in nucleotide de novo synthesis pathway.

a) Labeling kinetics of UDP and UTP in unstimulated or stimulated BMDMs. b) Relative intracellular abundance of UMP in unstimulated or stimulated BMDMs. Mean ± SD (n = 3 independent samples). c) Labeling kinetics of dCTP in unstimulated or stimulated BMDMs. d–e) Labeling kinetics of (d) glutamine, carbamoyl aspartate, orotate, UMP and (e) dTMP, CTP in unstimulated or stimulated RAW 264.7 macrophages. f) Labeling kinetics of ADP and ATP in unstimulated or stimulated BMDMs. g) Labeling kinetics of AICAR, IMP, AMP, and XMP in unstimulated or stimulated RAW 264.7 macrophages. a, c–g) Cells were labeled with γ-15N-glutamine for various time points as indicated on x-axis. Mean ± SD (n = 3 independent samples). a–c, f) Stimulated BMDM cells are continually stimulated for 48 h. d–e, g) Stimulated RAW 264.7 cells are continually stimulated for 24 h. a–g) Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated to stimulated cells at the final time point. ns indicates not significant (p > 0.01).

Source data

Extended Data Fig. 3 LPS + IFNγ stimulation-induced changes in nucleotide degradation and salvage.

a–b) Labeling kinetics of (a) IMP and (b) inosine in unstimulated or 48 h continually stimulated BMDMs after labeling with U-13C-glucose for 0–4 hours. c) Extracellular metabolites released into the media by BMDMs over a timecourse of continual stimulation. Cells were incubated in the media for 22 h before analysis. Data are presented relative to the level in spent media incubated with 24 h stimulated BMDMs on a log scale. N.d. indicates not detected. Mean + SD (n = 3 independent samples). d) Relative intracellular abundance of inosine in BMDMs over a timecourse of continual stimulation, with or without treatment of 10 µM forodesine (FD) throughout the timecourse. e) Fraction of M + 1 labeled GMP from 24 h γ-15N-glutamine labeling in BMDMs over a timecourse of continual stimulation, with or without treatment of 10 µM forodesine throughout the timecourse. f) Relative intracellular PRPP abundance in BMDMs over a timecourse of continual stimulation, with or without treatment of 10 µM forodesine throughout the timecourse. a–b, d–f) Mean ± SD (n = 3 independent samples). a–b) Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated to stimulated cells at each time point. d–f) Statistical analysis was performed with unpaired t-test comparing untreated to FD treated cells at each time point. ns indicates not significant (p > 0.01).

Source data

Extended Data Fig. 4 One-carbon metabolism supplements do not rescue the stimulation-induced inhibition of nucleotide de novo synthesis.

a) Transcriptional changes of detected genes in one carbon metabolism and serine de novo synthesis in BMDMs over a timecourse of acute stimulation. Relative expression of each gene is presented as relative to unstimulated cells (0 h) on a log2 scale as a heatmap, with saturating color representing 10-fold change (or 3.32 on log2 scale). Each box represents the mean of n = 3 independent samples. b–e) Labeled fraction of (b) M + 1 labeled AICAR, (c) M + 2 labeled IMP, (d) M + 2 labeled ATP, and (e) M + 2 labeled GTP in unstimulated or stimulated BMDMs supplemented with 1 mM 13C-formate for 24 h. f) Relative total intracellular dTTP abundance in unstimulated or stimulated BMDMs supplemented with 1 mM 13C-formate for 24 h. Data are presented relative to abundance in unstimulated cells. g–i) Labeled fraction of (g) IMP (M + 2) and (h) dTTP (M + 1), and (i) relative intracellular dTTP abundance in RAW 264.7 cells that are unstimulated, stimulated, or unstimulated and treated with 200 nM methotrexate (MTX), labeled with γ-15N-glutamine for 24 h. Cells were additionally supplemented with or without 1 µM folinate throughout the timecourse. b–i) Stimulated cells are continually stimulated for 48 h. Mean ± SD (n = 3 independent samples). b–f) Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated to stimulated cells. g–i) Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test. Bars with different lower-case letters (a, b, c, or d) indicate a statistically significant difference with p < 0.05.

Source data

Extended Data Fig. 5 Rescuing altered redox ratio and aspartate level is not sufficient to rescue the stimulation-induced inhibition of nucleotide de novo synthesis.

a) Basal oxygen consumption rate of unstimulated or stimulated BMDMs. Mean ± SD (n = 10 independent samples). b) Intracellular NAD to NADH ratio in unstimulated or stimulated BMDMs with or without supplementation of 2.5 mM pyruvate. c) Relative intracellular abundance of aspartate in BMDMs over a timecourse of continual stimulation. d) Relative intracellular abundance of aspartate in unstimulated or stimulated BMDMs, with or without supplementation of 2.5 mM pyruvate or 5 mM aspartate throughout the timecourse. e–i) Labeled fraction of (e) M + 2 labeled IMP, (f) M + 2 labeled AMP, (g) M + 1 and M + 3 labeled GMP, (h) M + 1 and M + 2 labeled CDP, and (i) M + 1 labeled UMP after 24 h γ-15N-glutamine labeling in unstimulated or stimulated BMDMs, with or without supplementation of 2.5 mM pyruvate or 5 mM aspartate throughout the timecourse. N.d. indicates not detected. a–b, d–i) Stimulated cells are continually stimulated for 48 h. b–i) Mean ± SD (n = 3 independent samples). a) Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated to stimulated cells. c) Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett’s test comparing all groups to unstimulated cells (0 h). b, d–i) Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test. Bars with different lower-case letters (a, b, c, d, or e) indicate a statistically significant difference with p < 0.05.

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Extended Data Fig. 6 Dynamic changes in NO production in relation to dynamic changes in nucleotide metabolism upon LPS + IFNγ stimulation.

a) Relative intracellular abundance of citrulline in BMDMs over a timecourse of continual LPS + IFNγ stimulation. Data are presented as log2 fold change compared to abundance in unstimulated cells (0 h). b) Transcriptional changes of Nos2 and Tyms in BMDMs over a timecourse of acute LPS + IFNγ stimulation. Relative expression is displayed as log2 fold change compared to unstimulated cells (0 h). c) Relative intracellular abundance of citrulline in wildtype or Nos2−/− BMDMs over a timecourse of continual stimulation. N.d. indicates not detected. d) Fraction of M + 1 labeled glutamine from 24 h γ-15N-glutamine labeling in unstimulated or 48 h continually stimulated wildtype or Nos2−/− BMDMs, with or without treatment of 200 µM NO donor DETA-NONOate throughout the timecourse. e) Relative intracellular AICAR abundance in BMDMs over a timecourse of continual stimulation. a–e) Mean ± SD (n = 3 independent samples). e) Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett’s test comparing all groups to unstimulated cells (0 h). ns indicates not significant (p > 0.01).

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Extended Data Fig. 7 NO is a key regulator mediating the rewiring in nucleotide metabolism that is specific to classical activation.

a) Arginine uptake, as measured by the changes in arginine level in spent media compared to fresh media after a 22 h incubation period, by BMDMs over a timecourse of continual and acute stimulation. Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett’s test comparing all groups to unstimulated cells (0 h). ns indicates not significant (p > 0.05). b) p-mTOR, mTOR, p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1 protein levels in unstimulated Nos2−/− BMDMs over a timecourse of 200 µM DETA-NONOate treatment. c) p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1 protein levels in wildtype or Nos2−/− BMDMs that are unstimulated or continually stimulated with LPS + IFNγ, with or without treatment of 200 µM DETA-NONOate for 48 h. d) Schematic of differential arginine metabolism by classically activated and alternatively activated macrophages. e) Citrulline and ornithine abundance in RAW 264.7 cells over a timecourse of continual LPS + IFNγ (red) or IL-4 + IL-13 (blue) treatment. Data are presented relative to abundance in unstimulated cells (0 h). Statistical analysis was performed with unpaired two-tailed student’s t-test comparing stimulated groups at each time point. ns indicates not significant (p > 0.01). a, e) Mean ± SD (n = 3 independent samples). f) Schematic of the in vivo stimulation experiment. g–h) The fold changes of (g) dTTP and (h) IMP in CD11b+F4/80+ cells harvested from wildtype or Nos2−/− mice treated with LPS + IFNγ. Data are presented relative to abundance in CD11b+F4/80+ cells harvested from PBS injection controls from each trial. Mean ± SEM (n = 9 independent mice for PBS injection controls. n = 5 for LPS + IFNγ samples, with each replicate consisting of cells pooled together from two stimulated mice). g–h) Statistical analysis was performed with paired two-tailed student’s t-test comparing stimulated cells. i–j) The fold changes of (i) dTTP and (j) IMP in wildtype or Nos2−/− peritoneal macrophages after 48 h continual stimulation with LPS + IFNγ after isolation (ex vivo), compared to unstimulated control. Mean ± SD (n = 3 independent mice). Statistical analysis was performed with unpaired two-tailed student’s t-test comparing stimulated cells.

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Extended Data Fig. 8 HGPRT knockout effectively modulates nucleotide metabolism and significantly impacts macrophage gene expression.

a) Cell viability in wildtype and Hprt1 KO RAW 264.7 cells, either unstimulated or continually stimulated for 48 h. b) M + 1 labeled fraction of GTP in wildtype or Hprt1 KO (2 clones) RAW 264.7 cells, either unstimulated or continually stimulated for 48 h. Cells were labeled with γ-15N-glutamine for 24 h before analysis. c) Expression levels of Hprt1 and Aprt (encoding adenine phosphoribosyltransferase) in unstimulated wildtype or Hprt1 KO RAW 264.7 cells. Data are presented as fold change relative to wildtype unstimulated. d) Metabolomic changes in pentose phosphates, nucleotides, nucleosides, and nitrogenous bases in 48 h stimulated wildtype or Hprt1 KO (2 clones) RAW 264.7 cells. Metabolite abundances are relative to wildtype cells and displayed on a log2 scale as a heatmap. Each box represents the mean of n = 3 independent samples. e) Labeling pattern of M + 1 GTP in BMDMs over a timecourse of continual stimulation treated with or without 6-mercaptopurine. Cells were labeled with γ-15N-glutamine for 24 h before analysis. a–c, e) Mean ± SD (n = 3 independent samples). a, c, e) Statistical analysis was performed using unpaired two-tailed student’s t-test comparing WT to Hprt1 KO or untreated to 6-MP treated cells at each time point. ns indicates not significant (p > 0.01). b) Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test. Bars with different lower-case letters (a, b, c, or d) indicate a statistically significant difference with p < 0.05. f–g) Volcano plot showing differentially expressed genes (up-regulated in red and down-regulated in green) in Hprt1 KO RAW 264.7 cells compared to wildtype cells in f) unstimulated state, and g) 48 h stimulated state.

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Extended Data Fig. 9 HGPRT knockout suppresses macrophage migration.

a) Gene ontology enrichment of the pathways that are most downregulated in Hprt1 KO macrophages compared to wildtype. b) Schematic of a macrophage migration assay. c) Migration of wildtype and Hprt1 KO (2 clones) RAW 264.7 cells across a Matrigel-coated transwell towards C5a. Mean ± SD (n = 5 independent samples). d) Labeled fraction of left: 3PG (M + 3) and right: serine (M + 3) in wildtype or Hprt1 KO (2 clones) RAW 264.7 cells over a timecourse of continual LPS + IFNγ stimulation. Cells were labeled with U-13C-glucose for 24 h before analysis. e) Relative intracellular abundance of 3-phosphoserine in wildtype or Hprt1 KO (2 clones) RAW 264.7 cells over a timecourse of continual stimulation. f–g) Transcript levels of (f) Phgdh, Psat1, Psph, and (g) Shmt1, and Shmt2 in unstimulated or 48 h continually stimulated wildtype or Hprt1 KO RAW 264.7 cells. d–g) Mean ± SD (n = 3 independent samples). c, f–g) Statistical analysis was performed using unpaired two-tailed student’s t-test comparing wildtype to Hprt1 KO. ns indicates not significant (p > 0.05). d–e) Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test. Bars with different lower-case letters (a, b, or c) indicate a statistically significant difference with p < 0.01.

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Extended Data Fig. 10 Various fates of nucleotides in unstimulated or LPS + IFNγ stimulated macrophages, and in macrophages infected with T. gondii.

a) Schematic showing T. gondii replication in a macrophage and its dependence on salvaging host purine. Specifically identified is the parasitophorous vacuole that separates host cell cytosol from T. gondii. b–c) Labeled fractions of (b) guanosine and adenosine units from digested RNA, compared to (c) free GTP and ATP, after 24 h U-13C-glucose labeling in unstimulated or 24 h continually stimulated wildtype BMDMs. d–e) Labeling kinetics of intracellular (d) NAD+ (M + 4) and (e) NADP+ (M + 4) compared to their precursor ATP (M + 4) in unstimulated or 48 h stimulated BMDMs supplemented with 50 µM U-15N-inosine for 0–4 hours as indicated on x-axis. Mean ± SD (n = 3 independent samples). Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated and stimulated cells at the final timepoint.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–4.

Reporting Summary

Supplementary Data 1

Table 1: Source Data for Supplementary Fig. 1; Table 2: Uncropped immunoblot for Supplementary Fig. 4

Supplementary Tables 1–3

Supplementary Table 1: Cell culture compounds; Supplementary Table 2: qPCR primers; Supplementary Table 3: Primary antibodies

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John, S.V., Seim, G.L., Erazo-Flores, B.J. et al. Classically activated macrophages undergo functionally significant nucleotide metabolism remodelling driven by nitric oxide. Nat Metab 7, 1681–1702 (2025). https://doi.org/10.1038/s42255-025-01337-3

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