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PERK is a critical metabolic hub for immunosuppressive function in macrophages

Abstract

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

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Fig. 1: PERK stress signaling promotes an immunosuppressive phenotype in macrophages.
Fig. 2: Multivariate analysis of transcriptomics and metabolomics data.
Fig. 3: PERK activity is essential for metabolic reprogramming in M2 macrophages.
Fig. 4: PERK regulates intrinsic serine biosynthesis via ATF-4.
Fig. 5: Serine biosynthesis promotes an immunosuppressive phenotype in macrophages.
Fig. 6: Serine biosynthesis contributes to mitochondrial fitness independent of respiratory chain assembly.
Fig. 7: Dysregulation of SBP suppresses JMJD3-mediated histone demethylation.
Fig. 8: Therapeutic PERK inhibition suppresses tumorigenesis.

Data availability

RNA-seq and ChIP-seq results are available in the Gene Expression Omnibus database under accession nos. GSE165836 and GSE183287, respectively. Other data available on request to corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank S. Adoro and A. Huang for providing Eif2ak3fl/fl and OT-I mice, respectively. We thank A. Huang, S. Adoro and G. Dubyak for helpful discussions, and H. Fujioka from the Cleveland Center for Membrane and Structural Biology for expert technical assistance. All the individuals above are affiliated with Case Western Reserve University. L.N.R is supported by the Immunology T32 Training Program (no. AI089474). P.-C.H. is supported in part by a postdoctoral fellowship provided by Ministry of Science and Technology, Taiwan. C.-W.J.L. is funded by the National Institutes of Health’s National Cancer Institute K22 award (no. K22CA241290), and startup funds from the Department of Microbial Infection and Immunity and Pelotonia Institute of Immuno-oncology at Ohio State University. J.I. is supported by a UNIL interdisciplinary grant. P.-C.H. is funded by the European Research Council starting grant (no. 802773-MitoGuide), SNSF project grants (no. 31003A_182470), the Cancer Research Institute (CLIP investigator award and Lloyd J. Old STAR award) and University of Lausanne (UNIL) interdisciplinary grant. S.C.-C.H. is funded by the Cancer Research Institute CLIP Investigator Award, the VeloSano Pilot Award, Case Comprehensive Cancer Center American Cancer Society pilot grants (nos. IRG-91-022-19 and IRG-16-186-21), Case GI SPORE DRP grant (no. 5P50CA150964-08) and the Cleveland Digestive Research Core Center pilot grant (no. 1P30DK097948).

Author information

Authors and Affiliations

Authors

Contributions

L.N.R. and S.C-C.H. conceived the study. L.N.R., H.Z., H.-Y.C, H.G.-A., W.C. and Y.K. performed the experiments. L.N.R, H.Z., P.-S.L., J.I., C.-W.J.L., P.-C.H. and S.C.-C.H. analyzed the data. Y.W., H.G.-A, P.-C.H. and A.A.-L. performed the bioinformatics analysis. L.N.R. and S.C-C.H. wrote the manuscript.

Corresponding author

Correspondence to Stanley Ching-Cheng Huang.

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Competing interests

P.-C.H. is a member of the scientific advisory for Elixiron Immunotherapeutics. The remaining authors declare no competing interests.

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Nature Immunology thanks Navdeep Chandel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 PERK deficiency does not affect M1 activation.

a, Enrichment plot of endoplasmic reticulum (ER) stress genes in IL-4c treated mouse peritoneal macrophages compared with naïve (PBS) macrophages by GSEA analysis. b, GSEA result comparing ER stress genes between TAMs and nontumor macrophages from patients with lung carcinoma. c, Pearson correlation of TAM CD68 expression with genes encoding molecules involved in the PERK pathway in caxncer patients from the TCGA. d, Immunoblot analysis of p-PERK, PERK, and β-actin from BMDMs stimulated with IL-4, Thapsigargin, or phosphatase. Ratio of p-PERK to total PERK was determined using ImageJ. Data are representative of three independent experiments. e, Frequency of p-PERK+ in BMDMs stimulated with IL-4 or Thapsigargin (n = 3; mean ± s.e.m). Data are collected from three independent experiments. f, Percentage of CD206+CD301+ in BMDMs treated with either DMSO (vehicle), IL-4, or Thapsigargin (n = 3; mean ± s.e.m). Data are collected from three independent experiments. g, Expression of CD206 and CD301 from BMDMs treated with IL-4 (M2) in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Data are representative of three independent experiments. h, Percentage of 7AAD naïve BMDMs isolated from Eif2ak3fl/fl or Eif2ak3fl/fl x LysMCre mice (n = 6; mean ± s.e.m). Data are collected from six independent experiments. i, Western blotting analysis of PERK, iNOS, Arg1, and β-actin from PERK wild-type and deficient BMDMs treated with LPS plus IFN-γ (M1), IL-4 (M2), or Thapsigargin. Data are representative of two independent experiments. j-l, Representative histogram (left) and quantitative plot (right) of iNOS (j), TNF-α (k), or expression of CD206 and CD301 (l) in BMDMs stimulated with LPS plus IFN-γ (M1) or IL-4 (M2) (n = 3; mean ± s.e.m). Data are representative of three independent experiments. m, Immunoblot analysis of p-PERK, PERK, XBP1s, and β-actin in unstimulated (M0), LPS + IFN-γ (M1), or IL-4 (M2) stimulated BMDMs. Data are representative of three independent experiments. All data were analyzed using two-tailed unpaired Student’s t-test (e,g,j,j,k,l) or one-way ANOVA with Tukey’s multiple comparisons test (f).

Source data

Extended Data Fig. 2 ISR in macrophage activation and metabolism.

a,b, Expression of CD206, CD301 (a), PD-L2 and Relmα (b) in macrophages stimulated with IL-4 in the presence or absence of ISRIB (n = 3; mean ± s.e.m). Data is representative of three independent experiments. c, Representative histogram (left) and quantitative analysis (right) of iNOS expression in macrophages stimulated with LPS + IFN-γ in the presence or absence of ISRIB (n = 3; mean ± s.e.m). Data is representative of three independent experiments. d, Expression of genes associated with ISR, assessed by RNA-Seq analysis. e,f, Expression of CD206, CD301 (e), PD-L2 and Relmα (f) in Gcn2+/+ and Gcn2-/- macrophages in the presence or absence of IL-4 (n = 3; mean ± s.e.m). Data is representative of two independent experiments. g,h, Representative histograms (left) and quantitative analysis (right) of iNOS (g) or TNF-α (h) in Gcn2+/+ and Gcn2-/- macrophages in the presence or absence of LPS + IFN-γ (n = 3; mean ± s.e.m). Data is representative of two independent experiments. i,j, Basal OCR (i) and ECAR (j) of Gcn2+/+ and Gcn2-/- M0, M1, and M2 macrophages (n = 3; mean ± s.e.m). Data is representative of two independent experiments. k, Basal OCR of M0 and M2 BMDMs in the presence or absence of ISRIB (n = 3; mean ± s.e.m). Data is representative of two independent experiments. l, Intracellular serine content from M0, M1 and M2 macrophages in the presence of absence of ISRIB (n = 3; mean ± s.e.m). Data is representative of two independent experiments. All data were analyzed using two-tailed unpaired Student’s t-test (a,b,c,e,f,g,h,I,j,k,l).

Source data

Extended Data Fig. 3 Pharmacological inhibition of PERK deviates cellular metabolism in M2 macrophages.

a, Basal OCR of naïve (M0) and M1 (LPS + IFN-γ) BMDMs from PERK sufficient or deficient mice (n = 3; mean ± s.e.m). Data collected from three independent experiments. b, Basal ECAR of naïve (M0), M1 (LPS + IFN-γ), and M2 (IL-4) BMDMs from PERK wild-type or null animals (n = 3; mean ± s.e.m). Data are collected from three independent experiments. c,d,e Basal OCR (c), ECAR (d), or ATP production (e) from wild-type BMDMs treated with IL-4 in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Dashed line indicates wild-type M0. Data are collected from three independent experiments. f, Representative histogram (left) and quantitative analysis (right) of BODIPY FL C16 staining in BMDMs treated with IL-4 in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Data representative of three independent experiments. g, Representative histogram (left) and the frequency (right) of BODIPY (493/503) staining in BMDMs stimulated with IL-4 in the presence or absence of GSK2656157 (n = 3; mean ± s.e.m). Data representative of three independent experiments. h, Representative TEM images of PERK wild-type and PERKcKO M2 (IL-4) macrophages. Red arrows indicate mitochondria. Data representative of three biological replicates. i, Representative histogram (left) and the frequency (right) of MitoTracker Green+ staining in BMDMs treated IL-4 (n = 3; mean ± s.e.m). Data representative of three independent experiments. j, Representative histogram (left) and the frequency (right) of MitoTracker Orange+ staining in BMDMs treated with IL-4 (n = 3; mean ± s.e.m). Data representative of three independent experiments. k, RNA-seq analysis of genes associated with mitochondrial calcium transport. l, Mitochondrial calcium flux (Rhod-2) from BMDMs treated with IL-4 in the presence or absence of GSK2656157 was determined and normalized by wild-type naïve M0 macrophages. Arrow, stimulation using 10 μM ionomycin (n = 6 for wild-type M0, M2; n = 4 for GSK-treated cells; mean ± s.e.m). Data are representative of three independent experiments. All data were analyzed using two-tailed unpaired Student’s t-test (a,b,c,d,e,f,g,i,j,l) or two-tailed paired Student’s t-test (l).

Source data

Extended Data Fig. 4 Inhibition of PHGDH or PSAT1 impairs M2 macrophage function.

a, Expression of genes encoding the serine one-carbon metabolic pathway in naïve (M0), M1 (LPS + IFN-γ), or M2 (IL-4) macrophages, assessed by RNA-Seq analysis. b,c, Western blotting analysis of PHGDH, PSAT1 and β-actin in BMDMs transduced with either retrovirus expressing Luc (b,c), PHGDH (b) or PSAT1 (c) shRNA and stimulated with IL-4. Data are representative of three independent experiments. d,e, Expression of CD206, CD301, PD-L2 and Relmα by IL-4 stimulated BMDMs treated with CBR-5884 (d) or transduced with PHGDH shRNA (e; middle), or transduced with PSAT1 shRNA (e; bottom) (n = 3; mean ± s.e.m). Data are representative of three independent experiments. f, Expression of CD206 and CD301 in M0 or LPS + IFN-γ (M1) stimulated BMDMs from Psat1fl/fl or Psat1fl/fl x LysMCre mice (n = 3; mean ± s.e.m). Data are representative of three independent experiments. g, Relative histogram (left) and gMFI (right) of either naïve (M0) or M1 macrophages from PSAT1 wild-type and knockout mice (n = 3; mean ± s.e.m). Data are representative of three independent experiments. h,i, Basal OCR and ECAR of wild-type BMDMs treated with IL-4 in the presence or absence of CBR-5884 (h) or transduced with shRNA targeting Phgdh or Psat1 (n = 3; mean ± s.e.m) (i). Data are collected from three independent experiments. j, ATP production from BMDMs stimulated with IL-4 in the presence or absence of CBR-5884 (n = 3; mean ± s.e.m). All data were analyzed using two-tailed unpaired Student’s t-test (d,f,g,h,j), or ordinary one-way ANOVA with Dunnett’s multiple comparisons test (e,i).

Source data

Extended Data Fig. 5 Loss of PERK does not result in H3K27m3 histone hypermethylation in pro-inflammatory M1 genes.

a,b, RT-qPCR analysis of Jmjd3 expression from PERK wild-type/knockout (a), or PSAT1 wild-type/knockout (b) BMDMs stimulated with IL-4 (n = 2; mean ± s.e.m). Data are collected from two independent experiments. c,d, Western blotting analysis of PERK, Jmjd3 and β-actin in M0, M1, and M2 BMDMs from PERK wild-type and knockout mice (c) or from PSAT1 wild-type and knockout mice (d). Data are representative two independent samples. e, Changes in gene expression of targets associated with H3K27me3 from PERK wild-type or knockout BMDMs stimulated with IL-4 (M2; left) or LPS + IFN-γ (M1; right). f, H3K27me3 histone modifications of Irf4, Pparg, Phgdh and Mgl2 from PERK wild-type and knockout BMDMs in the presence or absence of LPS + IFN-γ. g, H3K27me3 histone modifications of selected genes associated with pro-inflammation from naïve (M0), LPS + IFN-γ (M1) or IL-4 (M2) stimulated PERK wild-type or knockout macrophages. All data were analyzed using two-tailed unpaired Student’s t-test (a,b).

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Extended Data Fig. 6 Therapeutic PERK inhibition in mouse melanoma model.

a, Schematic of the experimental design for small molecule inhibitor treatments. b, Body weight of tumor-bearing mice given the indicated treatments (n = 9 mice per group; mean ± s.e.m). Data are collected from two independent experiments. c, Frequency of tumor-infiltrating immune cells (TAMs, TILs, DCs, MDSCs, and NK cells) from B16-F10 tumor-bearing mice with treatment of either DMSO (Vehicle), GSK2656157, or NCT-503. Tumors were harvested on day 16. (n = 10 mice per group; mean ± s.e.m). Data are collected from two independent experiments. d, Schematic of the experimental design for the treatment of αPD-1, GSK2656157, and αPD-1 + GSK2656157. Data were analyzed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test (b,c).

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Raines, L.N., Zhao, H., Wang, Y. et al. PERK is a critical metabolic hub for immunosuppressive function in macrophages. Nat Immunol 23, 431–445 (2022). https://doi.org/10.1038/s41590-022-01145-x

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