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SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation

Nature Metabolism volume 3, pages 1150–1162 (2021)Cite this article

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Abstract

Macrophages exhibit a spectrum of activation states ranging from classical to alternative activation1. Alternatively, activated macrophages are involved in diverse pathophysiological processes such as confining tissue parasites2, improving insulin sensitivity3 or promoting an immune-tolerant microenvironment that facilitates tumour growth and metastasis4. Recently, the metabolic regulation of macrophage function has come into focus as both the classical and alternative activation programmes require specific regulated metabolic reprogramming5. While most of the studies regarding immunometabolism have focussed on the catabolic pathways activated to provide energy, little is known about the anabolic pathways mediating macrophage alternative activation. In this study, we show that the anabolic transcription factor sterol regulatory element binding protein 1 (SREBP1) is activated in response to the canonical T helper 2 cell cytokine interleukin-4 to trigger the de novo lipogenesis (DNL) programme, as a necessary step for macrophage alternative activation. Mechanistically, DNL consumes NADPH, partitioning it away from cellular antioxidant defences and raising reactive oxygen species levels. Reactive oxygen species serves as a second messenger, signalling sufficient DNL, and promoting macrophage alternative activation. The pathophysiological relevance of this mechanism is validated by showing that SREBP1/DNL is essential for macrophage alternative activation in vivo in a helminth infection model.

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Fig. 1: SREBP1 activation is a feature of alternatively activated macrophages.
Fig. 2: Inhibition of SREBP1 activation impairs macrophages alternative activation and immune response to helminths.
Fig. 3: Limiting SREBP1-induced lipid synthesis reduces macrophages alternative activation without altering macrophages lipid composition.
Fig. 4: SREBP1-dependent lipid synthesis increases NADPH utilization to reduce antioxidant defences and permit macrophage alternative activation.

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Data availability

All the data presented in this manuscript are available upon reasonable request to the corresponding authors. Transfer of materials requires materials transfer agreements. The RNA-seq dataset is deposited in the Gene Expression Omnibus under accession number GSE179066. Source data are provided with this paper.

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Acknowledgements

Lyz2-Cre mice were a kind gift from S. Jackowski. We thank D. Hart, S. Grocott, C. Beresford, J. Bacon, L. McKinven, E. Rasijeff and A. Lukasik and J. Warner from the Histology core for their excellent technical assistance. This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. In particular, we thank E. Perez for advice and support in flow cytometry. We thank M. Murphy and H. Prag from the MRC Mitochondrial Biology Unit who kindly helped with the Seahorse experiments. We thank C. Frezza, L. Tronci and E. Nikitopoulou from the MRC cancer unit for their help in stable isotope tracer experimental design. We thank the IMS Genomics and Transcriptomics core for the RNA-seq analysis. This work was supported by the British Heart Foundation (RG/18/7/33636), the MRC (MC_UU_00014/2) and the FP7 MITIN (223450). K.P. was a recipient of a fellowship from the Wellcome Trust. A.N.J.M. and E.J. are supported by the Wellcome Trust (100963/Z/13/Z) and the MRC (U105178805). J.L. is a recipient fellowship of the British Heart Foundation. A.D. was a Marie-Curie Early-Stage Researcher supported by the European Union’s Horizon 2020 research and innovation programme (675585 Marie-Curie ITN ‘SymBioSys’) to J.S.-R. A.K. is supported by the Wellcome Trust (106260/Z/14/Z) and an ERC award (648889). P.F. is supported by the Science Foundation Ireland (10/IN.1/B3004). The IMS Genomics and Transcriptomics and Histology cores (B.M.-A., B.Y.H.L. and M.K.M.) are funded by the UK MRC Metabolic Disease Unit (MRC_MC_UU_12012/5) and a Wellcome Trust Strategic Award (100574/Z/12/Z). The Disease Model Core is part of the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/5) and Wellcome Trust Strategic Award (100574/Z/12/Z).

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Authors and Affiliations

  1. University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, MDU MRC, Addenbrooke’s Hospital, Cambridge, UK

    Guillaume Bidault, Samuel Virtue, Kasparas Petkevicius, Anne-Claire Guénantin, Jennifer Leggat, Betania Mahler-Araujo, Brian Y. H. Lam, Marcella K. Ma, Martin Dale, Stefania Carobbio & Antonio Vidal-Puig

  2. Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK

    Helen E. Jolin & Andrew N. J. McKenzie

  3. Institute for Computational Biomedicine, Heidelberg University, Faculty of Medicine, and Heidelberg University Hospital, BioQuant, Heidelberg, Germany

    Aurélien Dugourd & Julio Saez-Rodriguez

  4. Faculty of Medicine, Institute of Experimental Medicine and Systems Biology, RWTH Aachen University, Aachen, Germany

    Aurélien Dugourd

  5. Wellcome Trust Sanger Institute, Cambridge, UK

    Anne-Claire Guénantin, Stefania Carobbio & Antonio Vidal-Puig

  6. Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), and Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge, UK

    Arthur Kaser

  7. School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

    Padraic G. Fallon

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  1. Guillaume Bidault
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Contributions

G.B. conceived the study, designed, performed and analysed the results of experiments; S.V. provided advice on design and discussion and analysed the lipidomic experiments; K.P. significantly helped with experiments; A.D. performed the bioinformatics pathway analysis; H.E.J. performed the helminths infection and collected the tissues; A.-C.G. helped with the bioinformatics analysis; B.M.-A. scored the lungs histology; B.L. analysed the RNA-seq; M.K.M. performed the library preparation; J.L. and M.D. provided technical assistance; S.C. provide expertise in the lipid synthesis assay; A.K. provided expertise on the manuscript; P.G.F. and A.N.J.M. provided expertise and designed the helminth infection study; A.V.-P. supervised the entire study; and G.B., S.V. and A.V.-P. prepared the manuscript. All authors contributed to and approved the final manuscript.

Corresponding authors

Correspondence to Guillaume Bidault or Antonio Vidal-Puig.

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Competing interests

K.P. is currently employed by AstraZeneca. J.S.-R. has received funding from GSK and Sanofi and consultant fees from Travere Therapeutics. The other authors declare no competing interests.

Additional information

Peer review information Primary handling editors: George Caputa & Elena Bellafante. Nature Metabolism thanks Yu Li, Di Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 IL-4 induced SREBP1 activation is downstream of AKT and STAT6 signalling.

a, Protein expression of phosphorylated (Ser473) and total Akt in Vehicle (DMSO) or MK-2206-treated BMM in response to IL-4. Representative picture of n = 4 biological replicates. b, mRNA expression over BK of the SREBP1 target genes Fasn and Scd2 AKT in Vehicle (DMSO) or MK-2206-treated BMMФ in response to IL-4. Data are presented as the mean ± SEM of n = 8 biological replicates from 2 independent experiments. c, Gene expression analysis of the SREBP target genes in WT and Stat6-/- BMMФ. Data analysed from the publicly available dataset (GSE106706)8 with FDR<0.05 and Fc>2. Data has been analysed using a 2-way ANOVA followed by Sidak post-hoc test.

Source data

Extended Data Fig. 2 SIRT1 and AMPK are not responsible for SREBP1 activation in response to IL-4.

a-d, mRNA expression of the SREBP1 target genes Fasn and Scd2 and of the macrophages alternative activation markers Retnla and Mgl2 in BMMФ treated with SIRT1 activator II (a) or SIRT1 inhibitor (EX-527, b) or the AMPK inhibitor Compound C (c) or the AMPK activator AICAR (d) in response to IL-4. Data are presented as the mean ± SEM of n = 4 biological replicates per group. e and f, mRNA expression over BK of the SREBP1 target genes in 25-hydroxycholesterol-treated (25HC, n = 8 biological replicates from 2 independent experiments) (e) or SREBP-1c KO (n = 3 biological replicates) (f) BMMФ in response to IL-4. Data has been analysed using a 2-way ANOVA followed by a Dunnett (a-d) or Sidak (e and f) post-hoc test.

Extended Data Fig. 3 SREBP1 but not SREBP2 activation is required for macrophage alternative activation.

a and b, mRNA expression of Retnla and Mgl1 (a) and Tnf and Il1b (b) in WT and SCAP-KO BMMФ, 4h and 24h post IL-4 stimulation. mRNA expression over BK as mean ± SEM of n = 8 biological replicates from 2 independent experiments. c, Alternative activation of Vehicle (EtOH) or 25-hydroxycholesterol (25HC)-treated BMMФ in response to IL-4. Alternative activation was assessed by the expression of RELMα and CD206 by flow cytometry. The quantification of the number of alternatively activated macrophages is presented as mean ± SEM in d. Data of n = 4 biological replicates per group. e, Expression of the macrophage alternative activation markers Mrc1, Mgl1 Retlna and Arg1 in Vehicle (EtOH) or 25-hydroxycholesterol (25HC)-treated BMMФ in response to IL-4. mRNA expression over BK as mean ± SEM of n = 4 biological replicates per group. f, Expression of the macrophage alternative activation markers of Mrc1, Arg1 and Mgl1 in WT and SREBP1c-KO BMMФ in response to IL-4. mRNA expression over BK of n = 3 biological replicates. g, mRNA expression over BK of RAW264.7 macrophages transfected with siRNA against SREBP1, SREBP2 or both in response to IL-4. Data is expressed as mean ± SEM of n = 6 different experiments. Data has been analysed using a 2-way ANOVA followed by Sidak (a, b, d-f) or Tukey (g) post-hoc test.

Extended Data Fig. 4 Macrophage SREBP1 activation is required for immune response to helminth infection.

a, Neutrophil number presented as mean ± SEM in the lungs of naïve or 5- and 7-days post N. brasiliensis inoculation in WT and SCAP-KO mice. b, Eosinophil number presented as mean ± SEM in the lungs of naïve or 5- and 7-days post N. brasiliensis inoculation in WT and SCAP-KO mice. c, Percentage of alternatively activated alveolar macrophages in the lungs of naïve 5- and 7-days post N. brasiliensis inoculation in WT and SCAP-KO mice within the alveolar macrophage population. Alternative polarization was quantified by the expression of RELMα and CD206 by flow cytometry. d-f, Interstitial macrophages number (d), percentage of alternative activation (e) and number of alternatively activated interstitial macrophages (f) presented as mean ± SEM in the lungs of naïve or 5- and 7-days post N. brasiliensis inoculation in WT and SCAP-KO mice. g and h, Alveolar (g) and interstitial (h) neutrophil histological score in the lungs of naïve or 5- and 7-days post N. brasiliensis inoculation in WT and SCAP-KO mice. i, Correlation between the alveolar proteinaceous debris score and the number of alveolar macrophages 5 days post N. brasiliensis inoculation in WT and SCAP-KO mice. Pooled data as mean ± SEM n = 6-12 mice per group from 2 independent experiments. Data was analyzed using a two-way ANOVA followed by Sidak post-hoc test for comparison between genotypes at different days of post inoculation (a-h) or linear regression modelling (i).

Extended Data Fig. 5 Fatty acid synthesis in response to IL-4 requires SREBP1 activation.

a and b, Gene enrichment analysis of the pathways associated with de novo lipogenesis and SREBP activation from RNA sequencing comparing CTR and IL-4-treated BMMФ or of the interaction effect of IL-4 in WT and SCAP-KO BMMФ. Data from n = 6 biological replicates per group. c, Lipid synthesis rate in Lipopolysaccharide (LPS) or IL-4-treated BMMФ. The data represents the incorporation of radiolabelled 14C-acetate in the lipid fraction as mean ± SEM of n = 4 biological replicates per group. d, Proportion of newly synthesized palmitate per hour in control or IL-4 stimulated BMMФ. The data are presented as mean ± SEM of n = 4 biological replicates. e, Exogenous palmitate uptake rate of control or IL-4 stimulated BMMФ. The data are presented as mean ± SEM of n = 4 biological replicates. f, FAME composition in order of increasing chain length and desaturation of WT and SCAP-KO BMMФ in response to IL-4. Data is presented as mean ± SEM of n = 4 biological replicates. g, Total, essential and non-essential fatty acid content of WT and SCAP-KO BMMФ in response to IL-4. Data is presented as mean ± SEM of n = 4 biological replicates. h, Exogenous palmitate uptake rate of WT and SCAP-KO BMMФ in response to IL-4. The data are presented as mean ± SEM of n = 4 biological replicates. Statistical analysis of the RNAseq data is detailed in the methods section. Data has been analysed using a 2-way ANOVA followed by a Dunnett (c) or Sidak post-hoc test (f-h) or a two-tailed Student’s t-test (d and e).

Extended Data Fig. 6 Fatty acid but not cholesterol synthesis is required for macrophage alternative activation.

a, Lipid synthesis in BMMФ pre-treated with increasing doses of the FASN inhibitors C75 and cerulenin (Cer) 30 minutes prior 24h IL-4 stimulation. The data represents the incorporation of radiolabelled 14C-acetate in the lipid fraction as mean ± SEM of n = 7 biological replicates from 2 independent experiments. b, Expression of the macrophage alternative activation markers Mrc1, Mgl2, Arg1, Retlna and Il4i1 in Vehicle (DMSO) or C75 (10µM)-treated BMMФ in response to IL-4. mRNA expression over BK as mean ± SEM of n = 4 biological replicates per group. c, Alternative activation of BMMФ in response to IL-4 and pre-treated with increasing doses of Cerulenin (Cer). Alternative activation was assessed by flow cytometry using the co-expression of RELMα and CD301. The quantification of the number of M(IL-4) macrophages as mean ± SEM of n = 4 biological replicates is presented in d. e, Expression of the macrophage alternative activation markers Retlna and Mgl1 in Vehicle (DMSO) or cerulenin (2.5µg/mL)-treated BMMФ in response to IL-4. mRNA expression over BK as mean ± SEM of n = 4 biological replicates per group. f, Expression of the inflammatory cytokine Tnf and Il1b in Vehicle (DMSO) or C75 (10µM)-treated BMMФ in response to IL-4. mRNA expression over BK as mean ± SEM of n = 4 biological replicates per group. g and h, Expression of the SREBP2-target genes (g) and macrophage activation markers Tnf, Retlna and Mgl2 (h) in Vehicle (DMSO) or Simvastatin (10µM)-treated BMMФ in response to IL-4. mRNA expression over BK as mean ± SEM of n = 4 biological replicates per group. i, mRNA expression of the macrophage activation markers Retlna and Mgl2 in Vehicle (DMSO) or C75 (10µM)-treated BMMФ in response to IL-4 supplemented or not with HMG-CoA (1mM). mRNA expression over BK as mean ± SEM of n = 4 biological replicates per group. Data has been analysed using a 2-way ANOVA followed by a Dunnett (a and d) or Sidak post-hoc test (e-h) or one-way ANOVA followed by Tukey post-hoc test (i).

Extended Data Fig. 7 Fatty acid or cholesterol supplementation does not rescue the alternative activation of SCAP-KO or C75-treated macrophages.

mRNA expression of the SREBP1 target genes Fasn and Scd2 in C75-treated (a) or SCAP-KO (c) macrophages and of the macrophages alternative activation markers in C75-treated (b) or SCAP-KO (d) macrophages in response to IL-4 and/or palmitic acid (PA, 10 or 50 µM), oleic acid (OA, 10 or 50 µM) or water-soluble cholesterol (50 µM). mRNA expression of BK of n = 4 (C75) or n = 3 (SCAP-KO) biological replicates. Data has been analysed using a 2-way ANOVA followed by a Tukey post-hoc test.

Extended Data Fig. 8 Sources of the ROS in M(IL-4) cells.

a, Gene enrichment analysis of the pathways associated with redox homeostasis and response to oxidative stress in IL-4 treated BMMФ. Data from n = 6 biological replicates per group. Data of n = 6 biological replicates from 2 independent experiments per group. b, ROS accumulation in Vehicle (EtOH) or 25-hydroxycholesterol (25HC)-treated BMMФ in response to IL-4. ROS were quantified by the fluorescence ratio of CM-H2DCFDA over DNA (Hoechst). Data as mean ± SEM of n = 7 biological replicates from 2 independent experiments. c, ROS accumulation in Vehicle (DMSO) or cerulenin (1µg/mL)-treated BMMФ in response to IL-4. ROS were quantified by the median fluorescence intesnity of CM-H2DCFDA by flow cytometry. Data as mean ± SEM of n = 4 biological replicates. d, Reactive oxygen species (ROS) levels in BMMФ pre-treated with DPI (NADPH oxidase inhibitor), Allopurinol (xanthine oxidase inhibitor) or L-NAME (nitric oxide synthase inhibitor) prior 24h stimulation with IL-4 or LPS. ROS were quantified by the fluorescence ratio of CM-H2DCFDA over DNA (Hoechst). Data are presented as mean ± SEM of n = 4 biological replicates. e, Mitochondrial ROS production in response to IL-4. Mitochondrial ROS levels were determined by the fluorescence ratio of MitoSox over DNA (Hoechst). Data are presented as mean ± SEM of n = 8 biological replicates from 2 independent experiments. f, Time-course of fatty acid oxidation in response to IL-4 (10ng/mL). Data of n = 4 biological replicates. g and h, Oxygen consumption rate (OCR) in WT and SCAP-KO BMMФ (g) or in Vehicle (DMSO) or C75 (10 μM)-treated BMMФ (h) in control or IL-4 stimulated macrophages. OCR was monitored using an XF-96 Extracellular Flux Analyzer following the sequential treatments with oligomycin (oligo), FCCP and rotenone/antimycin (R/A). Data are presented as mean ± SEM of n = 4 (WT vs KO) and n = 8 from 2 independent experiments (DMSO vs C75) biological replicates per group. i, Fatty acid oxidation in IL-4 treated BMMФ in response to the AMPK activator AICAR (100 or 500 µM). Data of n = 4 biological replicates. j, Lipid synthesis in BMMФ treated with increasing doses of the AMPK activator AICAR in response to IL-4 (10ng/mL, 24h). Data of n = 4 biological replicates. k, Fatty acid oxidation assay of SCAP-KO macrophages in response to IL-4 (10ng/mL, 24h). Etomoxir (40 µM) was used as a negative control for FAO. Data of n = 4 biological replicates. l, Fatty acid oxidation assay in control or IL-4 stimulated macrophages treated or not with C75 (10 μM) or Cerulenin (1µg/mL) for 24h. Data of n = 4 biological replicates. m, ROS levels in C75 (10 μM) and/or Etomoxir (ETO, 40 μM)-treated BMMФ in response to IL-4. ROS were quantified by the fluorescence ratio of CM-H2DCFDA over DNA (Hoechst). Data of n = 4 biological replicates. n and o, Mitochondrial ROS production in WT and SCAP-KO BMMФ (n = 12 biological replicates from 3 independent experiments) (n) or in Vehicle (DMSO) or C75 (10 μM)-treated (n = 12 biological replicates from 3 independent experiments) BMMФ (o) in M(IL-4) macrophages. Mitochondrial ROS levels were determined by the fluorescence ratio of MitoSox over DNA (Hoechst). p, Reduced glutathione (GSH) levels in C75 (10 μM)-treated BMMФ in response to IL-4. Data presented as mean ± SEM of n = 4 biological replicates. Data has been analysed using a 2-way ANOVA followed by a Sidak (b, c, k, l and n-p) or Dunnett (d and j) or Tukey (g and h) post-hoc test or a two-tailed Student’s t-test (e) or a one-way ANOVA followed by Dunnett post-hoc test (f and i).

Extended Data Fig. 9 ROS scavenging impairs macrophage alternative activation.

a, ROS levels in N-acetyl cysteine (NAC, 10 mM)-treated BMMФ in response to IL-4. ROS were quantified by the fluorescence ratio of CM-H2DCFDA over DNA (Hoechst). Data as mean ± SEM of n = 8 biological replicates from 2 independent experiments. b and c, Alternative activation of NAC-treated BMMФ in response to IL-4. Alternative activation was assessed by the expression of RELMα and CD206 by flow cytometry. The quantification of the number of M(IL-4) macrophages is presented as mean ± SEM in c. Data of n = 8 biological replicates from 2 independent experiments. d, mRNA expression over BK of Arg1, Mgl1 and Retnla in NAC-treated BMMФ in response to IL-4. Data as mean ± SEM of n = 4 biological replicates. Data was analysed using a two-way ANOVA followed by Sidak post-hoc test.

Extended Data Fig. 10

Schematic representation of the mechanism by which DNL is activated and sensed in alternatively activated macrophages.

Supplementary information

Supplementary Information

Gating strategy.

Reporting Summary

Supplementary Table 1

Pathway analysis of WT BMDMs in response to IL-4 and pathway analysis of the interaction effect of IL-4 in WT and SCAP-KO BMDMs.

Supplementary Tables 2–4

Mouse models details and antibody and primers lists.

Source data

Source Data Fig. 1

Uncropped western blots Fig. 1c.

Source Data Fig. 3

Uncropped western blots Fig. 3.

Source Data Extended Data Fig. 1

Uncropped western blots Extended Data Fig. 1a.

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Bidault, G., Virtue, S., Petkevicius, K. et al. SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation. Nat Metab 3, 1150–1162 (2021). https://doi.org/10.1038/s42255-021-00440-5

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