Abstract

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.

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Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary information) and the data that support the findings of this study are available from the corresponding author upon reasonable request. Next-generation sequencing data can be accessed at Gene Expression Omnibus under accession code GSE123596.

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Acknowledgements

We thank members of the Pearce laboratories for support and discussions, and B. Sleckman and A. Morales at Cornell Weill and E. Latz and M. Lauterbach at University of Bonn for their generous advice during these studies. We also thank D. Braas and the UCLA Metabolomics Centre and the DeepSequencing facility at the Max Planck Institute of Immunobiology and Epigenetics for their technical support. This work was funded by National Institutes of Health grants AI 110481 (E.J.P.) and CA18125 (E.L.P.), a Capes Humboldt Research Fellowship (A.C.), a Sir Henry Wellcome Fellowship awarded by The Wellcome Trust (D.J.P.), and the Max Planck Society (E.J.P and E.L.P).

Author information

Affiliations

  1. Department of Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, Freiburg im Breisgau, Germany

    • Alanna M. Cameron
    • , Angela Castoldi
    • , David E. Sanin
    • , Lea J. Flachsmann
    • , Cameron S. Field
    • , Daniel. J. Puleston
    • , Ryan L. Kyle
    • , Annette E. Patterson
    • , Fabian Hässler
    • , Joerg M. Buescher
    • , Beth Kelly
    • , Erika L. Pearce
    •  & Edward J. Pearce
  2. The Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK

    • Daniel. J. Puleston
  3. Faculty of Biology, University of Freiburg, Freiburg im Breisgau, Germany

    • Edward J. Pearce

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Contributions

A.M.C., A.C., B.K., E.L.P. and E.J.P. designed experiments and provided conceptual input. A.M.C., A.C., L.J.F., C.S.F., D.J.P., R.L.K., A.E.P, F.H. and J.M.B., performed experiments and developed methodologies. A.M.C., A.C., L.J.F., D.E.S., E.L.P. and E.J.P. analyzed data. A.M.C. and E.J.P. wrote the manuscript.

Competing interests

E.J.P. and E.L.P. are founders of Rheos Medicines, and E.L.P. is a member of the Scientific Advisory Board for ImmunoMet Therapeutics.

Corresponding author

Correspondence to Edward J. Pearce.

Integrated supplementary information

  1. Supplementary Figure 1 Inflammatory macrophages are depleted of NAD+, and NAD+ salvage is required for viability.

    (a) NAD+ in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h, quantified by NAD cycling assay (n = 3 biologically independent samples, representative of three independent experiments). (b) NADH, NADP+, NADPH in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h, as quantified by LC-MS (n = 3 biologically independent samples, representative of three independent experiments). (c) Expression of NAMPT mRNA in macrophages polarized as indicated for 18h, normalized to mRNA encoding HPRT and presented relative to M0 control cells, set as 1 (n = 3 biologically independent samples, representative of three independent experiments). (d) RNA-seq analysis of expressed enzymes in the Preiss Handler Pathway or de novo NAD+ synthesis pathway in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h. Statistically significant (adjusted p value < 0.1) upregulated (> 2 fold) genes denoted by * in M(γ+LPS) polarized cells compared to M0 (n = 3 biologically independent samples). (e) NADH levels of M0, M(γ+LPS), M(LPS) and M(IL-4) cultured for 18 h in the presence or absence of 50 nM FK866, relative to M0 (n = 3 biologically independent samples, representative of two independent experiments). (f) Viability of M(γ+LPS) treated with increasing FK866 concentrations from 20 nM – 1600 nM as determined by flow cytometry, presented as percentage of vehicle treated control (n = 3 biologically independent samples, representative of three independent experiments). (g) NAD+ of bone marrow macrophages derived from C57Bl/6N mice. M0, M(γ+LPS) +/- 50 nM FK866, M(LPS) +/- 50nM FK866 and M(IL-4) cultured for 18 h, and NAD+ quantified by LC-MS (n = 3 biologically independent samples, representative of two independent experiments). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, ***p < 0.001 and ****p < 000.1.

  2. Supplementary Figure 2 Inhibition of NAMPT affects glycolytic metabolism of inflammatory macrophages due to diminished limiting of GAPDH activity by NAD+.

    (a-f) M(γ+LPS) or M(LPS) polarized for 18 h with or without FK866 and subsequently analyzed. (a,b) ECAR after glucose injection (a: n = 5 technical replicates, b: n = 4 technical replicates, both representative of over ten independent experiments). (c,d) Lactate production (n = 3 biologically independent samples, representative of two independent experiments). (e,f) basal OCR (e: n = 4 technical replicates, f: n = 4 technical replicates, both are representative of over ten independent experiments. (g-j) M(γ+LPS) or M(LPS) polarized for 18 h with vehicle control, FK866, GPP78 or STF118804 and subsequently analyzed. Real time changes in the ECAR (g) and ECAR after glucose injection (h) (n = 5 technical replicates, representative of three independent experiments). NAD+ as quantified by LC-MS (i) and ATP levels (j) (n = 4 biologically independent samples, representative of two independent experiments). (k, l) Volcano plots showing differentially expressed genes in M(γ+LPS) (k) or M(LPS) (l) untreated cells compared to FK866 treated, highlighting key genes in the glycolytic pathway as obtained from the Kyoto Encyclopedia of genes and genomes (pathway identifier: 00010) (n = 3 biologically independent samples). (m) LC-MS quantitation of glycolytic metabolites in control M(LPS) or M(LPS) + FK866 (n = 4 biologically independent samples, representative of three independent experiments). (n-q) Macrophages were polarized for 18h with or without 1 μM heptelidic acid (HA). (n) Basal ECAR, and basal OCR of M(γ+LPS) (n = 4 technical replicates, representative of two independent experiments). (o) ATP quantification of M(γ+LPS) (n = 3 biologically independent samples, representative of two independent experiments). (p) Basal ECAR, and basal OCR of M(LPS) (n = 4 technical replicates, representative of two independent experiments). (q) ATP quantification of M(LPS) (n = 3 biologically independent samples, representative of two independent experiments). (r) Western Blot showing NAMPT and β-actin expression in macrophages transduced with empty vector (EV) shRNA or Nampt shRNA. The blot was cropped to show relevant bands, and is representative of four independent experiments with similar results. (s) Viability of macrophages transduced with EV shRNA or Nampt shRNA, polarized for 18 h with γ+LPS and assessed by flow cytometry (n = 3 biologically independent samples, representative of three independent experiments). Error bars are mean ± SEM. Data were analyzed in c,d,m,o,q,s by unpaired, two-sided Student’s t-test and in h-j by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, ***p < 0.001 and ****p < 000.1

  3. Supplementary Figure 3 Modulation of inflammatory macrophage phenotype by an NAMPT inhibitor is linked to changes in glycolytic metabolism.

    (a) Example flow cytometric gating strategy used to identify live, CD11b+, F4/80+ macrophages. (b) Intracellular staining for TNF production in M0, M(γ+LPS) and M(γ+LPS) + FK866 as assessed by flow cytometry after 8 h of polarization, representative flow cytomtery plots with indicated percentage of TNF+ cells and SEM (n = 3 biologically independent samples, representative of two independent experiments). (c) Heatmap of antigen presentation and pro-inflammatory gene expression in macrophages polarized for 18 h as indicated. Statistically significant (adjusted p value < 0.1) up- or down-regulated (>2 fold) genes denoted by * in M(γ+LPS) compared to M(γ+LPS) + FK (n = 3 biologically independent samples). (d,e) M(γ+LPS) polarized with vehicle control or 50 nM FK866, GPP78 or STF118804 for 18h and assessed by flow cytometry. (d) Viability relative to control treated M(γ+LPS) (n = 4 biologically independent samples, representative of three independent experiments). (e) CD80, NOS2, and MHC class II expression (n = 3 biologically independent samples, representative of three independent experiments). (f) Expression of NOS2 and production of IL-6 and TNFα of M(γ+LPS) polarized for 18h with or without 1 μM HA and measured by flow cytometry and ELISA, respectively (n = 3 biologically independent samples, representative of two independent experiments). (g) Expression of markers RELMα, CD206 and CD301 in M(IL-4) polarized for 18h with or without FK866 treatment, analyzed by flow cytometry (n = 3 biologically independent samples, representative of three independent experiments). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.

  4. Supplementary Figure 4 Inhibition of NAMPT affects macrophage metabolism but does not modulate the frequency of immune cells in peritoneal lavage fluid or adipose tissue.

    Frequency of eosinophils (a), neutrophils (b), and dendritic cells (c) in peritoneal lavage 12 hours after i.p. administration of PBS, FK866, LPS or LPS and FK866 (n = 5 mice, representative of two independent experiments). (d) Basal ECAR and basal OCR of peritoneal macrophages isolated from mice given i.p. PBS, FK866, LPS or LPS and FK866 (n = 5 technical replicates derived from 5 pooled biological samples, representative of three independent experiments). (e) Basal ECAR and basal OCR of macrophages isolated from epididymal adipose tissue 12 hours after i.p. administration of PBS, FK866, LPS or LPS and FK866 (n = 5 technical replicates derived from 5 pooled biological samples, representative of two independent experiments). Frequency of macrophages (f) eosinophils (g), neutrophils (h), and dendritic cells (o) in epididymal adipose tissue and basal ECAR and basal OCR of macrophages isolated from epididymal adipose tissue 12 hours after i.p. administration of PBS, FK866, LPS or LPS and FK866 (n = 5 mice, representative of two independent experiments). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.

  5. Supplementary Figure 5 The addition of exogenous pyruvate does not ‘rescue’ the metabolic or phenotypic effects of NAMPT inhibition in inflammatory macrophages.

    Real-time changes in the ECAR (a) and ECAR after glucose injection (b) measured using Seahorse, of M(γ+LPS) untreated or treated with 50 nM FK866, 5 mM pyruvate or both FK and pryuvate for 18 h (n = 6 technical replicates, data are representative of two independent experiments). NAD+ (c), and F1,6BP and 2PG/3PG (d) levels measured by LC-MS in macrophages polarized to M(γ+LPS) untreated or treated with 50 nM FK866, 5 mM pryuvate or both FK and pyruvate for 18 h (n = 4 biologically independent samples, representative of one experiment). Viability (e) and expression of markers CD80, NOS2 and MHC class II (f) M(γ+LPS) with or without FK866, pyruvate or both FK and pyruvate for 18 h analyzed by flow cytometry (n = four biologically independent samples, representative of two independent experiments). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05 and ****p < 000.1.

  6. Supplementary Figure 6 Depletion of NAD+ is associated with PARP expression and DNA damage.

    (a) NADH, NADP+, NADPH in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h, as quantified by LC-MS (n = 3 biologically independent samples, representative of three concordant experiments). (b) Expression of NAMPT mRNA in macrophages polarized as indicated for 1 h, normalized to mRNA encoding HPRT and presented relative to M0, set as 1 (n = 3 biologically independent samples, representative of two concordant experiments). (c) NAMPT and β-actin (loading control) expression as shown by immunoblot in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h. The blot was cropped to show relevant bands, and is representative of three independent experiments with similar results. (d) CD38 expression of M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h or 18 h, analysed by flow cytometry (n = 3 biologically independent samples, representative of three concordant experiments). Heatmaps of PARP expression at 1 h (e) and 18 h (f), and (g) genes annotated with the Gene Ontology term ‘cellular response to DNA damage stimulus’, at 1 h, in M0, M(γ+LPS), M(LPS) and M(IL-4). Statistically significant (adjusted p value < 0.1) up or down-regulated (> 2 fold) genes denoted by * in M(γ+LPS) polarized cells compared to M0 (n = 3 biologically independent samples). (h,i) C57Bl/6N M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h. (h) NAD+ measured by LCMS (n = 3 biologically independent samples, representative of three concordant experiments). (i) γ-H2AX by flow cytometry (n = 3 biologically independent samples, representative of two concordant experiments). (j) Viability of M(γ+LPS) polarized with increasing concentrations of Rucaparib, assessed by flow cytometry at 18 h (n = 3 biologically independent samples, representative of five concordant experiments). (k,l) M0 and M(γ+LPS) treated with 10 μM Ruc or vehicle control for 18 h and subsequently analyzed. (k) Expression of CD80, NOS2 and MHC class II measured by flow cytometry (n = 3 biologically independent samples, representative of five concordant experiments). (l) IL-6 and TNF production by ELISA (n = 4 biologically independent samples, representative of three concordant experiments). (m-o) M0, M(LPS) or M(IL-4) were treated with or without 10 μM Ruc for 4 h, and γ-H2AX expression was assessed by flow cytometry. (n = 3 biologically independent samples, representative of two independent experiments). (p) ECAR after glucose injection of M(γ+LPS) polarized with or without 50 nM FK866, 10 μM Ruc or both inhibitors in combination (n = 6 technical replicates, representative of five concordant experiments). Error bars are mean ± SEM. Data were analyzed in a,b,d,h-k by one-way ANOVA with Tukey’s multiple comparison test and in l-o by unpaired, two-tailed students t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.

  7. Supplementary Figure 7 LPS-induced mitochondrial ROS drives oxidative DNA damage.

    (a) Nitrite production by M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h or 18 h. Samples for which readings were non-detectable indicated as n.d. (n = 3 biologically independent experiments, representative of two independent experiments).(b) NRF2 and β-actin (loading control) expression in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h or 18 h as indicated. The blot was cropped to show relevant bands, and is representative of two independent experiments with similar results. Real-time changes in OCR (c), basal OCR (d) and SRC (e) of macrophages unstimulated, or stimulated with LPS for 0.5-4 h as indicated (n = 5 technical replicates, representative of three independent experiments). Macrophages were treated with 10 mM NAC (f, g) or MitoTempol (MT; h) for 1 h, then polarized with LPS. Subsequently MitoSox staining (f) and γ-H2AX expression (g,h) were assessed by flow cytometry (n = 3 biologically independent samples, representative of three (g,h) or four (f) independent experiments). (i) 8OHG immunoflouresence of cells treated with etoposide (positive control), or stained with an IgG control or secondary antibody only, and analysed by confocal microscopy. Scale bar: 5 μm, white arrows indicate nuclear co-localization of 8OHG staining, insert shows an enlargement of nuclei with 8OHG staining (data are representative of three independent experiments. Each experiment was performed with 3 biologically independent samples with 10 images collected per sample, per condition). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.

  8. Supplementary Figure 8 Inhibition of complex III and ROS scavenging affect inflammatory macrophage function.

    (a) Viability of M(γ+LPS) polarized in the presence of CI inhibitor (Rotenone), CIII Qi inhibitor (Antimycin A) or a Complex III Qo inhibitor (Myx) for 1 h then harvested and assessed by flow cytometry. Viability presented as percentage control M(γ+LPS) (n = 3 biologically independent samples, representative of five independent experiments). (b) NAD+ and NADH quantification by LCMS in M(γ+LPS) polarized for 1 h with or without Myx (n = 3 biologically independent samples, representative of three independent experiments). (c) Viability of M(γ+LPS) at 18 h, after treatment with Myx or vehicle control for the first hour of polarization. Viability shown relative to vehicle control M(γ+LPS) (n = 3 biologically independent samples, representative of three independent experiments). (d,e) M(γ+LPS) were polarized with or without 10 mM NAC for 18 h then analyzed. (d) CD80, NOS2, MHC class II expression (n = 4 biologically independent samples, representative of three independent experiments). (e) IL-6 and TNF production (n = 3 biologically independent samples, representative of three independent experiments). (f,g) Macrophages were pre-treated for 2 h with NAC then stimulated with γ+LPS, 1 h later cells were washed and fresh γ+LPS added. 18 h later cells were analysed. (f) CD80, NOS2, MHC class II expression (n = 4 biologically independent samples, representative of two independent experiments). (g) IL-6 and TNF production (n = 4 biologically independent samples, representative of two independent experiments). Error bars are mean ± SEM. Data were analyzed by unpaired, two-tailed students t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.

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https://doi.org/10.1038/s41590-019-0336-y