Subjects

Abstract

Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements, with mechanistic target of rapamycin (mTOR) kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here we show that an mTOR–Semaphorin 6D (Sema6D)–Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1hi macrophages and prevents development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity, and metabolism via PPARγ.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

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

References

  1. 1.

    Mosser, D. M. & Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008).

  2. 2.

    Galli, S. J., Borregaard, N. & Wynn, T. A. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils. Nat. Immunol. 12, 1035–1044 (2011).

  3. 3.

    Gordon, S. & Martinez, F. O. Alternative activation of macrophages: mechanism and functions. Immunity 32, 593–604 (2010).

  4. 4.

    Bouhlel, M. A. et al. PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metab. 6, 137–143 (2007).

  5. 5.

    Liao, X. et al. Krüppel-like factor 4 regulates macrophage polarization. J. Clin. Invest. 121, 2736–2749 (2011).

  6. 6.

    Satoh, T. et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat. Immunol. 11, 936–944 (2010).

  7. 7.

    Odegaard, J. I. et al. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature 447, 1116–1120 (2007).

  8. 8.

    Huang, S. C. et al. Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages. Nat. Immunol. 15, 846–855 (2014).

  9. 9.

    Düvel, K. et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol. Cell 39, 171–183 (2010).

  10. 10.

    Galván-Peña, S. & O’Neill, L. A. Metabolic reprograming in macrophage polarization. Front. Immunol. 5, 420 (2014).

  11. 11.

    Howell, J. J. & Manning, B. D. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends Endocrinol. Metab. 22, 94–102 (2011).

  12. 12.

    Weichhart, T., Hengstschläger, M. & Linke, M. Regulation of innate immune cell function by mTOR. Nat. Rev. Immunol. 15, 599–614 (2015).

  13. 13.

    Chen, W. et al. Macrophage-induced tumor angiogenesis is regulated by the TSC2-mTOR pathway. Cancer Res. 72, 1363–1372 (2012).

  14. 14.

    Kimura, T. et al. Polarization of M2 macrophages requires Lamtor1 that integrates cytokine and amino-acid signals. Nat. Commun. 7, 13130 (2016).

  15. 15.

    Rocher, C. & Singla, D. K. SMAD-PI3K-Akt-mTOR pathway mediates BMP-7 polarization of monocytes into M2 macrophages. PLoS One 8, e84009 (2013).

  16. 16.

    Sahin, E. et al. Macrophage PTEN regulates expression and secretion of arginase I modulating innate and adaptive immune responses. J. Immunol. 193, 1717–1727 (2014).

  17. 17.

    Kolodkin, A. L., Matthes, D. J. & Goodman, C. S. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75, 1389–1399 (1993).

  18. 18.

    Pasterkamp, R. J. & Kolodkin, A. L. Semaphorin junction: making tracks toward neural connectivity. Curr. Opin. Neurobiol. 13, 79–89 (2003).

  19. 19.

    Kang, S. & Kumanogoh, A. Semaphorins in bone development, homeostasis, and disease. Semin. Cell Dev. Biol. 24, 163–171 (2013).

  20. 20.

    Kang, S. et al. Intestinal epithelial cell-derived semaphorin 7A negatively regulates development of colitis via αvβ1 integrin. J. Immunol. 188, 1108–1116 (2012).

  21. 21.

    Kumanogoh, A. & Kikutani, H. Immunological functions of the neuropilins and plexins as receptors for semaphorins. Nat. Rev. Immunol. 13, 802–814 (2013).

  22. 22.

    Suzuki, K. et al. Semaphorin 7A initiates T-cell-mediated inflammatory responses through alpha1beta1 integrin. Nature 446, 680–684 (2007).

  23. 23.

    O'Connor, B. P. et al. Semaphorin 6D regulates the late phase of CD4+. T cell primary immune responses. Proc. Natl Acad. Sci. USA 105, 13015–13020 (2008).

  24. 24.

    Toyofuku, T. et al. Dual roles of Sema6D in cardiac morphogenesis through region-specific association of its receptor, Plexin-A1, with off-track and vascular endothelial growth factor receptor type 2. Genes Dev. 18, 435–447 (2004).

  25. 25.

    Watarai, H. et al. PDC-TREM, a plasmacytoid dendritic cell-specific receptor, is responsible for augmented production of type I interferon. Proc. Natl Acad. Sci,. USA 105, 2993–2998 (2008).

  26. 26.

    Toyofuku, T. et al. Guidance of myocardial patterning in cardiac development by Sema6D reverse signalling. Nat. Cell Biol. 6, 1204–1211 (2004).

  27. 27.

    Byles, V. et al. The TSC-mTOR pathway regulates macrophage polarization. Nat. Commun. 4, 2834 (2013).

  28. 28.

    Zhu, L. et al. TSC1 controls macrophage polarization to prevent inflammatory disease. Nat. Commun. 5, 4696 (2014).

  29. 29.

    Martinez, F. O., Sica, A., Mantovani, A. & Locati, M. Macrophage activation and polarization. Front. Biosci. 13, 453–461 (2008).

  30. 30.

    Parsa, R. et al. Adoptive transfer of immunomodulatory M2 macrophages prevents type 1 diabetes in NOD mice. Diabetes 61, 2881–2892 (2012).

  31. 31.

    Kreider, T., Anthony, R. M., Urban, J. F. Jr. & Gause, W. C. Alternatively activated macrophages in helminth infections. Curr. Opin. Immunol. 19, 448–453 (2007).

  32. 32.

    Reese, T. A. et al. Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 447, 92–96 (2007).

  33. 33.

    Lawrence, T. & Natoli, G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat. Rev. Immunol. 11, 750–761 (2011).

  34. 34.

    Angela, M. et al. Fatty acid metabolic reprogramming via mTOR-mediated inductions of PPARγ directs early activation of T cells. Nat. Commun. 7, 13683 (2016).

  35. 35.

    Laplante, M. & Sabatini, D. M. An emerging role of mTOR in lipid biosynthesis. Curr. Biol. 19, R1046–R1052 (2009).

  36. 36.

    Takegahara, N. et al. Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis. Nat. Cell Biol. 8, 615–622 (2006).

  37. 37.

    Wen, H., Lei, Y., Eun, S. Y. & Ting, J. P. Plexin-A4-semaphorin 3A signaling is required for Toll-like receptor- and sepsis-induced cytokine storm. J. Exp. Med. 207, 2943–2957 (2010).

  38. 38.

    Zigmond, E. et al. Ly6C hi monocytes in the inflamed colon give rise to proinflammatory effector cells and migratory antigen-presenting cells. Immunity 37, 1076–1090 (2012).

  39. 39.

    Hu, X. et al. Integrin CD11b attenuates colitis by strengthening Src-Akt pathway to polarize anti-inflammatory IL-10 expression. Sci. Rep. 6, 26252 (2016).

  40. 40.

    Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T. & Mulligan, R. C. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153–1163 (1991).

  41. 41.

    Liberatore, R. A., Goff, S. P. & Nunes, I. NF-kappaB activity is constitutively elevated in c-Abl null fibroblasts. Proc. Natl Acad. Sci. USA 106, 17823–17828 (2009).

  42. 42.

    Keshet, R. et al. c-Abl tyrosine kinase promotes adipocyte differentiation by targeting PPAR-gamma 2. Proc. Natl Acad. Sci. USA 111, 16365–16370 (2014).

  43. 43.

    Jenkins, S. J. et al. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 332, 1284–1288 (2011).

Download references

Acknowledgements

We thank H. Inoue for technical support. This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to S. Kang); grants from AMED-CREST (JP17gm0410015) and AMED (to A.K.); COI stream and SRIP grants from MEXT (to A.K.); and grants from the Ministry of Health, Labour and Welfare of Japan (to A.K.).

Author information

Author notes

  1. These authors contributed equally: Sujin Kang, Yoshimitsu Nakanishi.

Affiliations

  1. Department of Immunopathology, Immunology Frontier Research Center, Osaka University, Suita City, Osaka, Japan

    • Sujin Kang
    • , Yoshimitsu Nakanishi
    • , Yoshiyuki Kioi
    • , Tetsuya Kimura
    • , Hyota Takamatsu
    • , Shohei Koyama
    • , Satoshi Nojima
    • , Masayuki Nishide
    • , Yoshitomo Hayama
    • , Yuhei Kinehara
    • , Yasuhiro Kato
    • , Takeshi Nakatani
    • , Toshihiko Toyofuku
    •  & Atsushi Kumanogoh
  2. Department of Respiratory Medicine and Clinical Immunology, Graduate School of medicine, Osaka University, Suita City, Osaka, Japan

    • Sujin Kang
    • , Yoshimitsu Nakanishi
    • , Yoshiyuki Kioi
    • , Hyota Takamatsu
    • , Shohei Koyama
    • , Masayuki Nishide
    • , Yoshitomo Hayama
    • , Yuhei Kinehara
    • , Yasuhiro Kato
    • , Takeshi Nakatani
    •  & Atsushi Kumanogoh
  3. Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka, Japan

    • Daisuke Okuzaki
  4. Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita City, Osaka, Japan

    • Junichi Takagi
  5. Department of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita City, Osaka, Japan

    • Sujin Kang
  6. Department of Immunology and Regenerative Medicine, Graduate School of Medicine, Osaka University, Suita City, Osaka, Japan

    • Toshihiko Toyofuku
  7. Department of Pathology, Graduate School of Medicine, Osaka University, Suita City, Osaka, Japan

    • Satoshi Nojima
  8. RIKEN Brain Science Institute, Laboratory for Molecular Mechanisms of Thalamus Development, Saitama, Japan

    • Tomomi Shimogori

Authors

  1. Search for Sujin Kang in:

  2. Search for Yoshimitsu Nakanishi in:

  3. Search for Yoshiyuki Kioi in:

  4. Search for Daisuke Okuzaki in:

  5. Search for Tetsuya Kimura in:

  6. Search for Hyota Takamatsu in:

  7. Search for Shohei Koyama in:

  8. Search for Satoshi Nojima in:

  9. Search for Masayuki Nishide in:

  10. Search for Yoshitomo Hayama in:

  11. Search for Yuhei Kinehara in:

  12. Search for Yasuhiro Kato in:

  13. Search for Takeshi Nakatani in:

  14. Search for Tomomi Shimogori in:

  15. Search for Junichi Takagi in:

  16. Search for Toshihiko Toyofuku in:

  17. Search for Atsushi Kumanogoh in:

Contributions

S. Kang designed and performed most of the experimental work. S. Kang and A.K. wrote the manuscript. A.K. provided important suggestions. Y.N and Y. Kioi performed the experiments and analyzed data. T.K., H.T., S. Koyama, S.N., M.N., Y.H., Y. Kato, Y. Kinehara, T.S., and T.N. performed some experiments. J.T. designed Sema6D mutant constructs. T.T the full-length of Sema6D expression vector and assisted with Seahorse analysis. D.O. performed gene-expression microarrays and deposited the data. All authors participated in discussion of the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Sujin Kang or Atsushi Kumanogoh.

Integrated supplementary information

  1. Supplementary Figure 1 Sema6D deficiency does not affect macrophage differentiation or immune cell development.

    (a) Representative flow cytometry data for Sema6D expression in IL-4 stimulated WT BMDMs after Torin1 treatment. (b) Expression of Sema6d mRNA in IL-4–stimulated WT BMDMs after rapamycin treatment, evaluated by quantitative real-time PCR. Expression levels of target genes were normalized to the corresponding level of Gapdh mRNA. (c) Expression of Sema6d mRNA in various immune subpopulations, including bone marrow dendritic cells (BMDCs), peritoneal macrophages (PEC M), GM-CSF–derived macrophages (GM-CSF M), M-CSF–derived macrophages (M-CSF M), activated T cells stimulated with anti-CD3 and anti-CD28 (mature T), and B220 + cells. Expression levels of target genes were normalized to the corresponding level of Gapdh mRNA. (d, e) Representative flow cytometry data for immune populations in spleen (d) and bone marrow (e). Eight-week-old mice were used. (f) Representative flow cytometry data for CD11b, F4/80, and TLR4 expression in M-CSF–derived BMDMs at steady state. *P < 0.05, **P < 0.01, ***P < 0.005. P-values were determined using one-way ANOVA (b). Data are representative of independent experimental replicates, and are presented as means ± SD (n = 3 (b) samples per group).

  2. Supplementary Figure 2 Sema6d/− BMDMs are refractory to alternative activated macrophage polarization.

    (a) Pparg, Retnla, Arg1, and Chi3l1 mRNA expression in WT and Sema6d/ BMDMs after stimulation with 100 ng/ml IL-4 for the indicated times, as determined by quantitative real-time PCR. (b) Mrc1, Retnla, and Bcl3 mRNA expression in WT and Sema6d/ BMDMs stimulated with 10 ng/ml IL-10 for 48 hrs. (c) Arg1, Retnla, and Chi3l1 mRNA expression in WT and Sema6d/ BMDMs stimulated with 100 ng/ml IL-13 for 48 hrs. (d) Arg1, Relm, and Il10 mRNA expression in freshly isolated PECs from WT and Sema6d/ mice after stimulation with IL-4 for 48 hrs, as determined by quantitative real-time PCR. (e) IL-6, IL-10, and IL-12p40 production in freshly isolated PECs from WT and Sema6d/ mice after stimulation with LPS + IFN-γ for 4 hrs, as detected by ELISA. Expression of IL-4 receptor (Cd124) (f) and Jak1 and Jak3 (g) was detected by quantitative real-time PCR in IL-4–induced WT and Sema6d/ BMDMs. (h) Immunoblot analysis of the levels of phospho-STAT6 in WT and Sema6d−/− BMDMs after IL-4 treatment. *P < 0.05, **P < 0.01, ***P < 0.005. P-values were determined using unpaired, two-tailed Student's t-tests (b–e). Data are representative of three independent experimental replicates, and are presented as means ± SD [n = 1 (a), n = 3 (b–g) samples per group].

  3. Supplementary Figure 3 PPARγ signaling is a downstream pathway of Sema6D reverse signaling in macrophages.

    (a) Canonical pathway analysis using differentially expressed genes (DEGs) based on RNA-seq data from WT and Sema6d/ BMDMs under LPS + IFN-γ stimulation. Dot size relates to the number of differentially expressed genes. (b) Upstream regulator analysis of genes downregulated in Sema6d/ BMDMs under IL-4 stimulation. All data sets were examined by Ingenuity Pathway Analysis (Qiagen Bioinformatics).

  4. Supplementary Figure 4 STAT1 and mTOR activities are normal, but PPARγ expression is impaired, in inflammatory Sema6d−/− macrophages.

    (a, b) Activity of phospho-STAT1 in WT and Sema6d/ BMDMs after LPS (a) or IFN-γ (b) stimulation was detected by western blotting. (c) Phospho-AKT and phopho-S6K in WT and Sema6d/ BMDMs after LPS stimulation for the indicated times were detected by western blotting. (d) Pparg mRNA expression in WT and Sema6d−/− BMDMs was measured by quantitative real-time PCR after stimulation with LPS, IFN-γ, or LPS + IFN-γ for 4 hrs. (e) Quantitative real-time PCR analyses were performed to measure Nos2 and Il6 mRNA expression in Sema6d−/− BMDMs expressing empty vector, Sema6D (full length), or PPARγ (full length) after LPS + IFN-γ stimulation for 6 hrs. *P < 0.05, **P < 0.01, ***P < 0.005. P-values were determined using unpaired, two-tailed Student's t-test (d) and one-way ANOVA (e). Data are representative of two (e) or three (a–d) independent experimental replicates, and are presented as means ± SD (n = 3 (d, e) samples per group).

  5. Supplementary Figure 5 Sema6D and PPARγ act sequentially, but not in parallel, to regulate anti-inflammatory macrophage polarization.

    (a) Immunoblot analysis of PPARγ in Sema6d−/− BMDMs expressing empty vector or Sema6D (full length) after IL-4 stimulation for 48 hrs. (b) Immunoblot analysis of PPARγ expression in WT and Sema6d−/− BMDMs expressing empty vector or PPARγ (full length) after IL-4 stimulation for 24 hrs. (c) Chi3l1 and Arg1 mRNA expression in WT and Sema6d/ BMDMs expressing empty vector (−) or PPARγ-full length (+) after stimulation with IL-4 for 24 hrs, as determined by quantitative real-time PCR. (d) Experimental protocol for Supplementary Figure 5e. (e) Chi3l1 and Pparg mRNA expression in WT and Sema6d/ BMDMs expressing empty (−) or Sema6D-full length (+), pre-treated with GW9662 for 3 days following stimulation with IL-4 for 24 hrs, as determined by quantitative real-time PCR. (f) Quantitative real-time PCR analysis of relative expression of Acsl3 and Fads2 in WT and Sema6d−/− IL-4 stimulated BMDMs. (g) Extracellular acidification rate (ECAR) of indicated cells at baseline and after glucose, oligomycin, and 2-deoxyglucose treatment. *P < 0.05, **P < 0.01, ***P < 0.005. P-values were determined using one-way ANOVA (c, e). Data are representative of two (a–e, g) or three (f) independent experimental replicates, and are presented as means ± SD [n = 3 (c, e, f) samples per group].

  6. Supplementary Figure 6 Sema6D reverse signaling controls macrophage polarization.

    (a) Retnla, Chi3l1, and Pparg mRNA expression in WT, Sema6d/, and Plxna1/ BMDMs after IL-4 stimulation for 48 hrs, as determined by quantitative real-time PCR. (b) IL-6, IL-10, and IL-12p40 production in WT, Sema6d/, and Plxna1/ BMDMs after stimulation with LPS plus IFN-γ for 4 hrs, as determined by ELISA. (c) Immunoblot analysis of PPARγ in Sema6d/ BMDMs expressing empty vector, ∆IC, or Sema6D after IL-4 stimulation. (d) Representative flow cytometry data of Sema6D expression in wild-type- BMDM, LPS stimulated BMDM, and IL-4 stimulated BMDMs. (e) Flow cytometry analysis of neuropilin-1 (NP-1) expression in BMDM and LPS BMDMs. (f) Immunoblot analysis of phosphorylation of c-Abl in WT and Sema6d/ BMDMs after IL-4 stimulation, followed by starvation. Lysates of IL-4 stimulated WT or Sema6d/ BMDMs were immunoprecipitated with anti-c-Abl antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies. (g) Schematic model of the mechanism of Sema6D reverse signaling leading to lipid metabolism during macrophage polarization. During IL-4–induced anti-inflammatory activation, mTOR is activated and upregulates Sema6D, which in turn activates c-Abl kinase to promote PPARγ expression. Activated PPARγ mediates fatty acid uptake and metabolic reprogramming, which are essential events for full anti-inflammatory activation. This Sema6D–PPARγ axis signaling confers anti-inflammatory properties on CX3CR1hi intestinal macrophage and prevents development of colitis.

  7. Supplementary Figure 7

    Uncropped blots.

Supplementary information

  1. Supplementary Figures and Tables

    Supplementary Figures 1–7 and Supplementary Tables 1 and 2

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41590-018-0108-0

Further reading