Signaling metabolite L-2-hydroxyglutarate activates the transcription factor HIF-1α in lipopolysaccharide-activated macrophages

Abstract

Activated macrophages undergo metabolic reprogramming, which not only supports their energetic demands but also allows for the production of specific metabolites that function as signaling molecules. Several Krebs cycles, or Krebs-cycle-derived metabolites, including succinate, α-ketoglutarate, and itaconate, have recently been shown to modulate macrophage function. The accumulation of 2-hydroxyglutarate (2HG) has also been well documented in transformed cells and more recently shown to play a role in T cell and dendritic cell function. Here we have found that the abundance of both enantiomers of 2HG is increased in LPS-activated macrophages. We show that L-2HG, but not D-2HG, can promote the expression of the proinflammatory cytokine IL-1β and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated through activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) by L-2HG, a known inhibitor of the HIF prolyl hydroxylases. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also found to be decreased in LPS-stimulated macrophages and may therefore also contribute to L-2HG accumulation. Finally, overexpression of L-2HGDH in HEK293 TLR4/MD2/CD14 cells inhibited HIF-1α activation by LPS, while knockdown of L-2HGDH in macrophages boosted the induction of HIF-1α-dependent genes, as well as increasing LPS-induced HIF-1α activity. Taken together, this study therefore identifies L-2HG as a metabolite that can regulate HIF-1α in macrophages. Activated macrophages undergo metabolic reprogramming, which not only supports their energetic demands but also allows for the production of specific metabolites that function as signaling molecules. Several Krebs cycles, or Krebs-cycle-derived metabolites, including succinate, α-ketoglutarate, and itaconate, have recently been shown to modulate macrophage function. The accumulation of 2-hydroxyglutarate (2HG) has also been well documented in transformed cells and more recently shown to play a role in T cell and dendritic cell function. Here we have found that the abundance of both enantiomers of 2HG is increased in LPS-activated macrophages. We show that L-2HG, but not D-2HG, can promote the expression of the proinflammatory cytokine IL-1β and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated through activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) by L-2HG, a known inhibitor of the HIF prolyl hydroxylases. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also found to be decreased in LPS-stimulated macrophages and may therefore also contribute to L-2HG accumulation. Finally, overexpression of L-2HGDH in HEK293 TLR4/MD2/CD14 cells inhibited HIF-1α activation by LPS, while knockdown of L-2HGDH in macrophages boosted the induction of HIF-1α-dependent genes, as well as increasing LPS-induced HIF-1α activity. Taken together, this study therefore identifies L-2HG as a metabolite that can regulate HIF-1α in macrophages. Recently, much attention has been focused on how the metabolism of immune cells changes upon activation and whether these changes can be linked to specific responses. For macrophages, the remodeling of their metabolism following activation supports the adoption of different effector functions, depending on the activating stimulus. Not only does this support increased energy demands, but different activation states require distinct metabolic processes (1Kelly B. O'Neill L.A.J. Metabolic reprogramming in macrophages and dendritic cells in innate immunity.Cell Res. 2015; 25: 771-784Google Scholar). Macrophages activated with the Toll-like receptor-4 (TLR4) agonist, lipopolysaccharide (LPS) increase glycolysis to maintain production of ATP, and oxidative phosphorylation (OXPHOS) is impaired. Macrophages activated with IL-4, so-called alternatively activated macrophages, rely mainly on the Krebs cycle and OXPHOS (2Galván-Peña S. O'Neill L.A.J. Metabolic reprograming in macrophage polarization.Front. Immunol. 2014; 5: 420Google Scholar). Metabolites themselves can also act as signaling molecules, for example, roles for the Krebs cycle metabolites succinate and α-ketoglutarate (αKG) in macrophage differentiation have been described (3Ryan D.G. Murphy M.P. Frezza C. Prag H.A. Chouchani E.T. O'Neill L.A. Mills E.L. Coupling Krebs cycle metabolites to signalling in immunity and cancer.Nat. Metab. 2019; 1: 16-33Google Scholar). LPS promotes the oxidation of succinate by succinate dehydrogenase (SDH), which can drive the production of mitochondrial reactive oxygen species (mtROS), or following its mitochondrial export, stabilize HIF-1α (4Mills E.L. Kelly B. Logan A. Costa A.S.H. Varma M. Bryant C.E. Tourlomousis P. Dabritz J.H.M. Gottlieb E. Latorre I. Corr S.C. McManus G. Ryan D. Jacobs H.T. Szibor M. et al.Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages.Cell. 2016; 167: 457-470.e13Google Scholar, 5Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. Zheng L. Gardet A. Tong Z. Jany S.S. Corr S.C. et al.Succinate is an inflammatory signal that induces IL-1β through HIF-1α.Nature. 2013; 496: 238-242Google Scholar, 6Ryan D.G. O'Neill L.A.J. Krebs cycle reborn in macrophage immunometabolism.Annu. Rev. Immunol. 2020; 38: 289-313Google Scholar, 7Liu P.S. Wang H. Li X. Chao T. Teav T. Christen S. Di Conza G. Cheng W.C. Chou C.H. Vavakova M. Muret C. Debackere K. Mazzone M. Huang H.D. Fendt S.M. et al.α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming.Nat. Immunol. 2017; 18: 985-994Google Scholar). However, the balance between succinate and αKG must be sufficiently skewed in favor of αKG in order for alternative macrophage activation to occur, as αKG is required for the Jmjd3-dependent epigenetic regulation of specific genes (7Liu P.S. Wang H. Li X. Chao T. Teav T. Christen S. Di Conza G. Cheng W.C. Chou C.H. Vavakova M. Muret C. Debackere K. Mazzone M. Huang H.D. Fendt S.M. et al.α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming.Nat. Immunol. 2017; 18: 985-994Google Scholar). 2HG is a metabolite derived from αKG, which has yet to be characterized in macrophages. Since 2HG is a chiral metabolite, two distinct isoforms of 2HG exist, L-2HG and D-2HG. Their accumulation has been linked to the metabolic disorders termed 2-hydroxyglutaric acidurias (2HGA) and certain cancers (8Ye D. Guan K.-L. Xiong Y. Metabolism, activity, and targeting of D- and L-2-hydroxyglutarates.Trends Cancer. 2018; 4: 151-165Google Scholar, 9Struys E.A. 2-Hydroxyglutarate is not a metabolite; d-2-hydroxyglutarate and l-2-hydroxyglutarate are!.Proc. Natl. Acad. Sci. U. S. A. 2013; 110E4939Google Scholar). Increased D-2HG is associated with tumors possessing mutant isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2), which are frequently found in low-grade glioma and acute myeloid leukaemia (10Dang L. Su S.M. Isocitrate dehydrogenase mutation and (R)-2-hydroxyglutarate: From basic discovery to therapeutics development.Annu. Rev. Biochem. 2017; 86: 305-331Google Scholar), while an increase in L-2HG has been seen in renal cell carcinoma (11Shim E.H. Livi C.B. Rakheja D. Tan J. Benson D. Parekh V. Kho E.Y. Ghosh A.P. Kirkman R. Velu S. Dutta S. Chenna B. Rea S.L. Mishur R.J. Li Q. et al.L-2-Hydroxyglutarate: An epigenetic modifier and putative oncometabolite in renal cancer.Cancer Discov. 2014; 4: 1290-1298Google Scholar). In healthy tissues, accumulation is controlled by enantiomer-specific dehydrogenases (D-2HGDH and L-2HGDH), which oxidize 2HG back in to αKG. Both D-2HG and L-2HG have been shown to act as competitive inhibitors of multiple αKG-dependent dioxygenases, including the ten-eleven translocation (TET) family of DNA hydroxylases, the Jmjj-domain containing histone lysine demethylases (KDMs), and the HIF prolyl hydroxylases (PHDs), where L-2HG has been shown to be a much more effective inhibitor than D-2HG (12Koivunen P. Lee S. Duncan C.G. Lopez G. Lu G. Ramkissoon S. Losman J.A. Joensuu P. Bergmann U. Gross S. Travins J. Weiss S. Looper R. Ligon K.L. Verhaak R.G. et al.Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation.Nature. 2012; 483: 484-488Google Scholar). Here, we have examined a role for 2HG in macrophages. Our data suggests that the accumulation of L-2HG driven by LPS stimulation increases HIF-1α stability and therefore activity. The subsequent increase in HIF-target gene expression contributes to a proinflammatory activation state, including the adoption of a highly glycolytic metabolism and expression of the HIF-1α -dependent gene, notably that encoding interleukin 1β (IL-1β). LC-MS was used to analyze changes in metabolite abundance in control and LPS-stimulated bone-marrow-derived macrophages (BMDMs) (Fig. 1, A and B). Consistent with previously published reports, aspartate was decreased in LPS-stimulated cells, while abundance of fumarate, itaconate, and succinate was increased (Fig. 1, A and B). 2HG also accumulated in LPS-stimulated cells (Fig. 1C), and the intracellular concentration reached 0.4 μM (Fig. 1D). Enantiomer-specific analysis combining chiral derivatization with LC-MS (13Cheng Q.Y. Xiong J. Huang W. Ma Q. Ci W. Feng Y.Q. Yuan B.F. Sensitive determination of onco-metabolites of D- and L-2-hydroxyglutarate enantiomers by chiral derivatization combined with liquid chromatography/mass spectrometry analysis.Sci. Rep. 2015; 5: 15217Google Scholar) revealed that abundance of both L-2HG and D-2HG was increased by LPS (Fig. 1E), with D-2HG making a greater contribution to the total pool of 2HG than L-2HG. Expression of the enzymes responsible for degradation of both D-2HG and L-2HG, D-2HGDH and L-2HGDH, respectively, both decreased following LPS stimulation, though the kinetics of this differed. Downregulation of D2hgdh was seen early after LPS stimulation, and by 24 h, poststimulation expression had returned to basal levels (Fig. 1F). Downregulation of L2hgdh was a later event, with expression reduced by half at 24 h (Fig. 1G). An unbiased quantitative proteomic screen confirmed decreased L-2HGDH in LPS-stimulated BMDMs (Fig. 1H), though D-2HGDH was not detected in this screen. The increase in D-2HG and L-2HG may therefore in part be due to a decrease in their respective dehydrogenases. A schematic of 2-HG synthesis and degradation is shown in Figure 1I. Figure S1 illustrates the strategy for derivatizing the 2-HG enantiomers and the chromatograph of labeled D-2HG and L-2HG. As it has been reported that L-2HG can inhibit the PHDs, we next used a cell-permeable analogue of each enantiomer to examine the effects on HIF-1α and several gene (12Koivunen P. Lee S. Duncan C.G. Lopez G. Lu G. Ramkissoon S. Losman J.A. Joensuu P. Bergmann U. Gross S. Travins J. Weiss S. Looper R. Ligon K.L. Verhaak R.G. et al.Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation.Nature. 2012; 483: 484-488Google Scholar, R. L. E.A. Li M. R.J. et oncometabolite 2-hydroxyglutarate histone lysine Rep. Scholar). increased both HIF-1α and in LPS-stimulated BMDMs (Fig. with in the of LPS, HIF-1α be detected in BMDMs with In in cells with in HIF-1α or was seen (Fig. These that the two enantiomers of 2HG play distinct roles in macrophage function. The increase in HIF-1α in cells with was confirmed to be (Fig. and at h after (Fig. (Fig. and (Fig. in LPS-stimulated BMDMs confirmed that increased both LPS-induced HIF-1α and while had on The expression of several HIF-target genes was In cells with of and and in the of and this with L-2HG or in with LPS (Fig. had on its or in with LPS (Fig. was by with or (Fig. in the proinflammatory driven by with increased both glycolysis and glycolytic in BMDMs (Fig. and while with had (Fig. and These data that L-2HG, D-2HG, can modulate HIF-1α in macrophages. immune cells, as both and T cells, dendritic cells and cell have been reported to 2HG from their though whether macrophages are of this is M. K. R. K. S. K. A. M. D. with HIF-1α stability metabolism oxidative phosphorylation and 2018; Scholar, L. S. T. K. M. D. E. K. M. C. S. T. A. et of T cell immunity by the oncometabolite 2018; Scholar, I. K. M. C. K. K. E. J. P. K. K. M. and L-2-hydroxyglutarate inhibit by dendritic J. Sci. 2019; Scholar). We therefore next 2HG increased in macrophages with 2HG (Fig. and with enantiomer-specific a for D-2HG L-2HG was (Fig. In with the increase in HIF-1α seen in cells with L-2HG not only increased HIF-1α in LPS-stimulated macrophages (Fig. and with but also for the of HIF-1α in cells (Fig. 1 and of the HIF-target genes, and also increased in LPS-stimulated macrophages with L-2HG but not D-2HG (Fig. was also boosted in LPS-stimulated macrophages with L-2HG (Fig. and these show that L-2HG, but not D-2HG, can increase HIF-1α in macrophages. We next examined HIF-1α BMDMs with the in order to degradation of HIF-1α and and to a in the of (Fig. and with The known and inhibited HIF-1α and with not an increase in total HIF-1α in cells where its degradation was that the accumulation of HIF-1α in the of L-2HG is not as a of increased synthesis (Fig. with with this cells with in the of increase in HIF-1α was seen (Fig. with The of an in cells with both and L-2HG that L-2HG can increase HIF-1α by its degradation to as L-2HG boosted synthesis of with both and a in HIF-1α with with (Fig. L-2HG (Fig. increased In macrophages, the two that control HIF-1α of oxygen are and signaling (1Kelly B. O'Neill L.A.J. Metabolic reprogramming in macrophages and dendritic cells in innate immunity.Cell Res. 2015; 25: 771-784Google Scholar, P. S. of hypoxia-inducible by J. Scholar). In BMDMs with or was on or signaling (Fig. Both and D-2HG also had on transcription this to that increased HIF-1α driven by L-2HG was likely to be due to the of the enzymes to the subsequent of examine a role for 2-HG in HIF-1α we HEK293 TLR4/MD2/CD14 cells in which we L-2HGDH and HEK293 cells can both L-2HG and D-2HG C.B. Y. J. increases in L-2-hydroxyglutarate the metabolic to Metab. 2015; Scholar), and overexpression of the enzymes for each enantiomer be to abundance of 2HG in an enantiomer-specific LPS in increased to LPS-activated BMDMs (Fig. with L-2HGDH was the LPS-induced increase in HIF-1α was while in cells where D-2HGDH was in HIF-1α was seen from the LPS-stimulated control (Fig. and with whether the accumulation of L-2HG was to modulate knockdown of L-2HGDH in BMDMs was also In BMDMs with targeting an increase in LPS-induced HIF-1α was seen (Fig. An increase in expression of and was also seen in LPS-stimulated BMDMs (Fig. These by L-2HGDH expression and therefore L-2HG HIF-1α and HIF-1α -dependent genes also be by L-2HG. that changes in levels of L-2HG are to HIF-1α activity, L-2HG in HIF-1α activation in macrophages with Metabolic reprogramming in macrophages is well to be for an immune D.G. O'Neill L.A.J. Krebs cycle reborn in macrophage immunometabolism.Annu. Rev. Immunol. 2020; 38: 289-313Google Scholar). the production of metabolites with signaling as the Krebs cycle metabolites succinate, and αKG. Our identifies a role for L-2HG in HIF-1α activation in LPS-activated macrophages. HIF-1α and the increase in glycolytic has been shown to be for inflammatory macrophage activation T. Y. I. R. R. Corr M. V. is for myeloid Scholar, T. H. G. Wang X. C. glycolysis metabolism is to the activation of inflammatory 2017; Scholar). Several have shown L-2HG to be a more inhibitor of certain αKG-dependent enzymes than D-2HG R. L. E.A. Li M. R.J. et oncometabolite 2-hydroxyglutarate histone lysine Rep. Scholar, W. H. Y. Y. Wang P. S. C. Wang P. J. Wang B. et 2-hydroxyglutarate is a competitive inhibitor of Scholar). of these reported that the for was μM in the of L-2HG, but for D-2HG R. L. E.A. Li M. R.J. et oncometabolite 2-hydroxyglutarate histone lysine Rep. Scholar). A was seen with factor HIF L-2HG had a much than D-2HG. However, for two histone demethylases between the two supports that D-2HG does not have an on metabolites have been reported to increase HIF-1α activity, the in the of macrophage function succinate (4Mills E.L. Kelly B. Logan A. Costa A.S.H. Varma M. Bryant C.E. Tourlomousis P. Dabritz J.H.M. Gottlieb E. Latorre I. Corr S.C. McManus G. Ryan D. Jacobs H.T. Szibor M. et al.Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages.Cell. 2016; 167: 457-470.e13Google Scholar, 5Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. Zheng L. Gardet A. Tong Z. Jany S.S. Corr S.C. et al.Succinate is an inflammatory signal that induces IL-1β through HIF-1α.Nature. 2013; 496: 238-242Google Scholar). accumulation and subsequent from the mitochondria can to while by at may also contribute to HIF-1α The glycolytic metabolism in macrophages therefore allows the mitochondria to be from and Krebs cycle to Our study that L-2HG is HIF-1α in macrophages. Our data also the of macrophages to both D-2HG and L-2HG, as immune cells have been shown to L. S. T. K. M. D. E. K. M. C. S. T. A. et of T cell immunity by the oncometabolite 2018; Scholar). A in T cells for D-2HG has been but this does not to be on BMDMs L. S. T. K. M. D. E. K. M. C. S. T. A. et of T cell immunity by the oncometabolite 2018; Scholar). is also a of the of L-2HG. As L-2HGDH and D-2HGDH are both mitochondrial this suggests that it is this that enantiomer Both D-2HG and L-2HG are from αKG by enzyme activity, an enzyme is of a in to its in enzyme 2016; 4: Scholar). For L-2HG the enzymes responsible for this are both dehydrogenases and and dehydrogenase A C.B. Y. J. increases in L-2-hydroxyglutarate the metabolic to Metab. 2015; Scholar, A.M. R.G. S. Lu C. C.B. induces production of Metab. 2015; Scholar). Both and are while is in the on the enzyme responsible for L-2HG it may be for L-2HG to be of the mitochondria to on the be required in order to the enzyme responsible for L-2HG synthesis in macrophages and whether mitochondrial Our study that for 2-HG accumulation in macrophages is of L-2HGDH and show that L-2HG is a HIF-1α However, as the concentration of L-2HG seen in LPS-stimulated macrophages is the as to whether this increase in L-2HG is to modulate HIF-1α activity. The data that this is in the as L-2HGDH expression was to HIF-1α expression and expression of Metabolic reprogramming in macrophages several that in accumulation of metabolites that may is therefore likely that L-2HG accumulation in with succinate, and fumarate, to this both through of the and also by αKG levels G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. Zheng L. Gardet A. Tong Z. Jany S.S. Corr S.C. et al.Succinate is an inflammatory signal that induces IL-1β through HIF-1α.Nature. 2013; 496: 238-242Google Scholar, Lee S. C. M. J. B. J. L. HIF overexpression with of in renal role of in regulation of HIF Scholar). this study has focused on the role of L-2HG in HIF-1α it is likely that the increase in both L-2HG and D-2HG seen in LPS-activated macrophages to inhibit αKG-dependent of function of the in cells has been shown to L-2HG accumulation with increases in DNA and histone which their to E. S. X. Y. Z. M. J. The mitochondrial is for cell 2017; Scholar). in cells, of inhibit their and is associated with increases in 2HG and succinate and a E.M. I. P. H. L.A. L.A. is for function of T 2019; Scholar). D-2HG has been shown to promote cell differentiation from cells an epigenetic in the and of the transcription factor known to drive differentiation T. Wang X. K. M. Li K. Ma T. Wang H. L. S. D. Y. K. et control of and cell balance by an epigenetic 2017; Scholar). whether enantiomer of 2HG has an on macrophage function epigenetic In for the regulation of HIF-1α by L-2HG. These data that L-2HG through the PHDs, increase the stability of The increased abundance of L-2HG in LPS-stimulated macrophages may be for the of HIF-1α and the induction of proinflammatory of HIF-1α the adoption of a glycolytic and the expression of D-2HG abundance is also increased in LPS-stimulated macrophages, it not HIF-1α L-2HG may therefore be as an that has a signaling role in inflammatory macrophage used for the of BMDMs from and specific in with and HEK293 cells from in a and was confirmed by was from the and a and in cells and for and from L-2HG D-2HG and from LPS from was from and used at was from was from with with the after the was 1 and for on The was to for at in a at for 1 h, at for at The was a and at for at For metabolite of each was to and at to analysis by The of the from each was used for chiral in metabolite and a and at was in and was In was to each and for at with a and in the as chiral LC-MS analysis was a to a The liquid was with a and both from and at The was of with and The was at with the described by et G.M. Zheng L. N.J. Gottlieb E. of cell metabolism LC-MS and 2015; Scholar). The was in and to due to and The and LC-MS analysis of was as described by Cheng et (13Cheng Q.Y. Xiong J. Huang W. Ma Q. Ci W. Feng Y.Q. Yuan B.F. Sensitive determination of onco-metabolites of D- and L-2-hydroxyglutarate enantiomers by chiral derivatization combined with liquid chromatography/mass spectrometry analysis.Sci. Rep. 2015; 5: 15217Google Scholar). of metabolites was by of the from of with the of concentration of metabolites was cell as previously described E.L. Ryan D.G. Prag H.A. D. D. Z. M.P. Costa A.S.H. M. E. J. R. et is an metabolite that of 2018; Scholar). analysis of metabolite abundance was with cells in and In 2 and in a containing and for at in the to and In was to stabilize and at and for at at The was with and in 2 containing by to to and with in a and at a for h at to a and at and at a and at to a and was to a for h at with of to a and to In was to each for at The was of to an an with a on a with of a h of in with a of analysis used an as described previously M.P. M. E.L. R. W. and of expression cell 2014; 86: Scholar). 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W. and D. G. R. A. S. H. C. and M. P. J. S. M. C. and L. A. C. D. G. A. S. H. E. L. and M. P. J. C. W. C. L. A. C. W. L. A. and was by from the and the by L. A. as well as from by S. M. C.

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