Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization

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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.

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Fig. 1: mTOR activity-dependent Sema6D expression is required for anti-inflammatory macrophage polarization.
Fig. 2: Sema6d–/– mice exhibit defective anti-inflammatory macrophage polarization and exaggerated inflammatory responses.
Fig. 3: Sema6D–PPARγ signaling is indispensable for fatty-acid uptake and metabolic reprogramming.
Fig. 4: Sema6D reverse signaling promotes anti-inflammatory macrophage polarization via the tyrosine kinase c-Abl.
Fig. 5: Sema6D signaling is critical for anti-inflammatory properties of CX3CR1hi intestinal macrophages.
Fig. 6: Sema6d–/– myeloid cells predispose mice to colitis.

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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.).

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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.

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Correspondence to Sujin Kang or Atsushi Kumanogoh.

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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).

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].

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).

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).

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].

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.

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Kang, S., Nakanishi, Y., Kioi, Y. et al. Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization. Nat Immunol 19, 561–570 (2018). https://doi.org/10.1038/s41590-018-0108-0

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