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α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming

Abstract

Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming.

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Figure 1: Glutamine metabolism modulates macrophage activation via αKG production.
Figure 2: αKG promotes metabolic changes induced by M2 activation.
Figure 3: αKG promotes IL-4-induced epigenetic changes in a Jmjd3-dependent manner.
Figure 4: Integration of αKG/succinate ratio in macrophage determines macrophage immune responses.
Figure 5: αKG suppresses M1 activation through a PHD-dependent post-translational regulation of IKKβ.
Figure 6: Glutamine metabolism supports induction of endotoxin tolerance in an αKG-dependent manner.
Figure 7: αKG supports endotoxin tolerance in macrophages by modulating NF-κB signal and Jmjd3-dependent regulation.

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Acknowledgements

We thank F. Cottard and C.-P. Lin for technical help and P. Romero and C. Hess for discussion. Supported by Swiss National Science Foundation project grant (31003A_163204), the Swiss Institute for Experimental Cancer Research (26075483), the Harry J. Lloyd Charitable Foundation, the Swiss Cancer Foundation (KFS-3949-08-2016) and a Melanoma Research Alliance Young Investigator Award to P.-C.H. S.-M.F. is supported by a Flanders Research Foundation (FWO) research grant and by Eugène Yourassowsky Schenking. J.I. is supported by the University of Lausanne. H.-D.H. is supported by the Ministry of Science and Technology of Taiwan (MOST105-2627-M-009-007 and MOST103-2628-B-009-001-MY3). T.C. is supported by a University of Lausanne FBM PhD fellowship. M.V. is supported by the National scholarship program of the Slovak Republic.

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P.-S.L., H.W., X.L., T.C., T.T., S.C., G.D.C.,W.-C.C., M.V., C.M., K.D. and J.I. performed experiments. P.-S.L., H.W., S.C., C.-H.C., M.M., H.-D.H., S.-M.F., J.I. and P.-C.H. analyzed results. P.-S.L., H.W. and P.-C.H. designed the studies. P.-S.L. and P.-C.H. wrote the manuscript.

Corresponding author

Correspondence to Ping-Chih Ho.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 αKG produced from glutaminolysis supports M2 phenotype but inhibits M1 activation.

(a) An illustration of α-ketoglutarate metabolic pathway controlled by genes listed in (Fig. 1d). (b, c) Quantitative PCR (qPCR) analysis of relative mRNA expression of M2 marker genes in BMDMs stimulated with IL-4 (b) and M1 marker genes in BMDMs stimulated with LPS (c) in glutamine-replete media supplemented with indicated treatments for 6h. Compound 968 (10μM); Dimethyl-α-ketoglutarate (DM-αKG; 1mM). (d, e) qPCR analysis of the relative mRNA expression of M2 markers genes (d) and arginase activity (e) in BMDMs stimulated with or without IL-4 in glutamine-depleted media plus escalating doses of DM-αKG for 6h. (f, g) qPCR analysis of the relative mRNA expression of M1 markers genes (f) and ELISA of IL-1β and TNFα cytokine production (g) in BMDMs stimulated with or without LPS in glutamine-depleted media plus escalating doses of DM-αKG for 6h. (h) The effects of glutaminase 1 inhibitor (BPTES; 10mM) and supplementation of DM-aKG (1mM) on IL-1β secretion in LPS stimulated macrophages in the presence of Nigericin (left panel) or MSU (monosodium urate crystal) (right panel) were analyzed by ELISA. *P < 0.05 is determine by unpaired, two-tailed Student's t-test. Data shown are representative from three independent experiments (b, c, d, e, f; mean ± s.d.), from two independent experiments (g; mean ± s.d.) and cumulative results of two independent experiments (h; mean ± s.d.).

Supplementary Figure 2 αKG does not modulate IL-4-induced STAT6 activation.

(a) Immunoblot analysis of phosphor-STAT6 and STAT6 in BMDMs stimulated with IL-4 in glutamine-replete (+Gln.) or glutamine-depleted (-Gln) media for 0-2h. β-actin served as loading control. (b) Immunoblot analysis of phosphor-STAT6 and STAT6 in BMDMs stimulated with IL-4 in the presence of absence of BPTES in glutamine-replete media for 0-2h. (c) Immunoblot analysis of phosphor-STAT6 and STAT6 in BMDMs activated with IL-4 in in glutamine-depleted media supplemented with control vehicle (Ctrl) or DM-αKG (1mM). (d, e) qPCR analysis of relative mRNA expression of M2 marker genes (d) and arginase activity (e) in BMDMs stimulated with IL-4 in the indicated cultures for 6h. (f) Immunoblot analysis of Jmjd3 and β-actin 6 in BMDMs derived from Cas9-LysM-Cre mice transduced with lentivirus harboring control sgRNAs or Jmjd3-targeting sgRNAs. Data shown are representative from three independent experiments (a, b, c, d, e) and from two independent experiments (f).

Supplementary Figure 3 αKG/succinate ratio generated by macrophage activation regulates macrophage immune responses.

(a) The expression of intracellular succinate (Suc.) and α-ketoglutarate (αKG) in BMDMs with IL-4 or LPS for 18h, followed by metabolite extraction mentioned in methods and then measured with mass spectrometry. (b) Arginase activity in BMDMs stimulated with IL-4 in glutamine-depleted media supplemented with diethyl-succinate (DE-Suc.; 5mM) plus indicated concentration of DM-αKG for 6h. (c) ELISA of IL-1β in BMDMs stimulated with LPS in glutamine-depleted media supplemented with diethyl-succinate (DE-Suc.; 5mM) plus indicated concentration of DM-αKG for 6h. (d) Arginase activity in BMDMs stimulated with IL-4 in glutamine-depleted media supplemented with DM-αKG plus indicated concentration of DE-Suc. for 6h. (e) ELISA of IL-1β in BMDMs stimulated with LPS in glutamine-depleted media supplemented with DM-αKG plus indicated concentration of DE-Suc. for 6h. *P < 0.05 is determine by unpaired, two-tailed Student's t-test. Data shown are representative from three independent experiments (a, b, d; mean ± s.d.) and from two independent experiments (c, e; mean ± s.d.).

Supplementary Figure 4 Glutaminolysis suppresses NF-κB nuclear translocation upon LPS treatment.

(a) Immunoblot analysis of nuclear NF-κB p65 in BMDMs stimulated with LPS plus control vehicle (Ctrl) or BPTES in glutamine-replete media for 0-60 mins, followed by nuclear fraction isolation. Lamin A/C served as loading control for nuclear fraction lysate and β-actin served as loading control for whole cell lysate (WCL). (b) Immunoblot analysis of IKKβ in BMDMs left untransduced (Ctrl) or transduced with lentivirus harboring green fluorescence protein (GFP)-fused wild type (Wt) or P191A mutant form (P191A) of IKKβ. β-actin served as loading control. Data shown are representative from three independent experiments (a) and from two independent experiments (b).

Supplementary Figure 5 Glutaminolysis during LPS priming is essential for induction of endotoxin tolerance via impairment of the NF-κB pathway.

(a) A schematic diagram of in vitro endotoxin tolerance experimental design. (b) qPCR analysis of relative mRNA expression in BMDMs stimulated with or without LPS (10ng/ml) in glutamine-replete media supplemented with control vehicle (Ctrl) or BPTES for 18h and then re-challenged with LPS (10ng/ml) for another 6h. (c) A schematic diagram of in vitro endotoxin tolerance experimental design. BMDMs were stimulated with or without LPS (10ng/ml) in glutamine-depleted (Gln.-depleted) media supplemented with or without DM-αKG for 18h and then re-challenged with LPS (10ng/ml). (d, e) IL-1β (d) and TNFα (e) production in BMDMs stimulated with LPS as described in (c), followed by ELISA analysis. (f) A heat map showing expression of genes encoding tissue repairing and antimicrobial molecules in BMDMs stimulated with LPS for 18h in glutamine-replete media (ET w/ Gln.), glutamine-depleted media (ET w/o Gln.) or glutamine-depleted media supplemented with 1mM DM-αKG (ET w/o Gln.+ DM-αKG), assessed by RNA-sequencing. (g) A heat map showing metabolome changes associated with induction of endotoxin tolerance, based on metabolite expression between non-tolerant macrophage (LPS w/ Gln.; LPS stimulation for 6h in glutamine-replete media) and tolerant macrophages (ET w/ Gln.; endotoxin tolerant macrophages stimulated with LPS for 6h in glutamine-replete media). The metabolites of this list in BMDMs stimulated with LPS in glutamine-depleted media (ET w/o Gln.) or glutamine-depleted media supplemented with DM-αKG (ET w/o Gln.+ DM-αKG) for 18h and then re-activated with LPS for another 6h were also included in this heat map. (h) The expression of lactate and pyruvate in BMDMs of indicated groups were determined by mass spectrometry. *P < 0.05 is determine by unpaired, two-tailed Student's t-test. Data shown are representative from three independent experiments (b; mean ± s.d.), cumulative results from two independent experiments (d, e, f; mean ± s.d.) and combined from three independent experiments and normalize to LPS w/ Gln. group (g, h; mean ± s.d.).

Supplementary Figure 6 Glutamine metabolism during LPS priming prevents reactivation of proinflammatory genes in a Jmjd3-independent manner.

(a, b, c) Immunoblots of proximal signaling pathways of toll-like receptor 4 in BMDMs treated as described in figure 6a (a) or treated in glutamine-replete media supplemented with control vehicle (Ctrl) or BPTES (b), or treated in glutamine-depleted media supplemented with or without DM-αKG (c), for 18h and then re-stimulated with second exposure of LPS for 0-60 mins. β-actin served as loading control. (d) Immunoblots of NF-κB p65 in BMDMs treated with LPS in glutamine-replete media supplemented with or without BPTES for 18h, followed by re-stimulated with second exposure of LPS for 0-60 mins. (e) Immunoblots of NF-κB p65 in BMDMs treated with first exposure of LPS in glutamine-replete (+Gln.) or glutamine-depleted (-Gln.) media plus control vehicle or DM-αKG for 18h. After washing, BMDMs were then re-stimulated with second exposure of LPS for indicated period. Those BMDMs did not receive first LPS treatment represent control groups for intact responsiveness to LPS-induced nuclear translocation of NF-κB. (f) qPCR analysis of relative mRNA expression of M1 marker genes in Cas9-expressing BMDMs transduced with lentivirus harboring control sgRNAs or Jmjd3-targeting sgRNAs. These BMDMs were treated with or without LPS in the indicated conditions for 18h and then re-challenged with LPS re-exposure in glutamine-replete media for another 6h. (g) A schematic diagram of in vivo endotoxin tolerance assay. *P < 0.05 is determine by unpaired, two-tailed Student's t-test. Data shown are representative from three independent experiments (a, b, c, d, e, f; mean ± s.d.).

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Liu, PS., Wang, H., Li, X. et al. α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol 18, 985–994 (2017). https://doi.org/10.1038/ni.3796

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