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Metabolic Signaling Drives IFN-γ
Cell Metabolism
Previews Metabolic Signaling Drives IFN-g Peter J. Siska1 and Jeffrey C. Rathmell1,* 1Department of Pathology, Microbiology and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37232-2363, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cmet.2016.10.018
IFN-g is a critical inflammatory cytokine that is closely regulated to cellular metabolic status and requires high rates of glycolysis. It has now been shown that a key mechanism to link these pathways is through maintenance of acetyl-CoA and epigenetic regulation of the IFNG locus. Activation of lymphocytes, macrophages, and dendritic cells leads to sharp increases in glucose metabolism in these cells to support their energetic and biosynthetic demands. However, these changes are much more impactful than simply supplying building blocks, as metabolites are intimately linked to fundamental signaling and differentiation processes. For example, phosphoenolpyruvate can directly regulate calcium signaling (Ho et al., 2015) and glyceraldehyde 3-phosphate and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been implicated in the regulation of interferong (IFN-g) translation (Chang et al., 2013). In cancer cells, adipocytes, and hematopoietic stem cells, it has also been shown that changes in acetyl-CoA and S-adenysyl-methionine influence gene expression through epigenetic acetylation and methylation modifications (Cluntun et al., 2015). IFN-g has emerged as particularly sensitive to cellular metabolic state, but the underlying mechanisms are only incompletely understood. Now a recent study shows that metabolic regulation of epigenetics and IFNG acetylation is a critical regulator of inflammation (Peng et al., 2016). IFN-g can be regulated on multiple levels to link cell metabolism to immune function. In addition to post-transcriptional regulation by GAPDH, the IFNG gene is under tight transcriptional control. The transcription factor T-Bet is a major driver of IFNG transcription, but IFNG-associated histones also require epigenetic modifications with histone demethylation and increased acetylation (de Arau´jo-Souza et al., 2015). Mechanisms that lead to these changes, however, are not well understood. Furthermore, it is not clear whether perturbation of metabolic pathways that provide the
substrates for epigenetic marks impact gene expression and T cell differentiation. Peng et al. (2016) add to the metabolism-epigenetic paradigm and show that lactate dehydrogenase A (LDHA) regulates glucose flux to support acetyl-CoA levels necessary for promoting acetylation of IFNG-associated histones and IFN-g expression. Starting with an effort to explore how changes in glycolysis affect immune function, Peng et al. (2016) deleted LDHA in T cells. Like Glut1-deficiency, LDHAdeficient T cells had reduced glucose consumption (Macintyre et al., 2014). Unlike T cells with impaired glucose uptake, however, LDHA-deficiency allowed glucose to flux through glycolysis and only prevented reduction of pyruvate to lactate and the coordinate oxidation of NADH to NAD+. Interestingly, T cell-specific LDHA deletion did not affect T cell activation, survival, or expression of the central CD4 T cell transcription factor T-Bet. Despite relatively normal T cells, IFN-g production was sharply reduced after LDHA deletion. Glycolytic metabolism can be linked to IFN-g expression by binding of GAPDH to the 30 UTR of IFN-g mRNA to suppress translation when glucose flux is low (Chang et al., 2013). Using a GFP reporter fused to the Ifng 30 UTR, however, Peng et al. (2016) did not find that LDHA deletion led to reduced reporter function, suggesting a potential distinct mechanism of IFN-g regulation. Indeed, T cells from mice with the Yeti allele of Ifng, in which the 30 UTR of Ifng is replaced, expressed high levels of IFN-g that were sharply reduced with LDHA deletion. The alterative 30 UTR of Ifng in the Yeti allele leads to excessive IFN-g production and an auto-inflammatory phenotype. This auto-inflammatory
disease, however, was corrected through T cell LDHA deletion (Peng et al., 2016). While the immune defects of Yeti/Yeti mice clearly show a critical role for the Ifng 30 UTR, glucose metabolism holds additional sway over IFN-g that goes beyond 30 UTR-mediated regulation. In addition to post-transcriptional regulation of IFN-g expression, IFN-g is regulated on epigenetic level by histone acetylation (Denton et al., 2011). Decreased histone acetylation at the lysine 9 residue (H3K9Ac) can diminish gene expression, and this change may link glycolysis to gene expression via substrate supply. By performing chromatin immunoprecipitation sequencing, Peng et al. (2016) found that LDHA knockout (KO) T cells had a broad decrease of H3K9Ac, including acetylation of histones at Ifng loci. Histone acetylation requires acetyl coenzyme A (acetyl-CoA) (Cluntun et al., 2015), and LDHA KO T cells showed decreased levels of this metabolite, possibly due to reduced LDHA-mediated NADH oxidation to maintain glycolytic flux or a greater consumption of acetyl-CoA as an acetyl donor to oxaloacetate to maintain the tricarboxylic acid (TCA) cycle for ATP. Importantly, acetate supplementation of Ldha-deficient T cells augmented acetyl-CoA production, normalized Ifng promoter and enhancer acetylation, and increased IFN-g expression in LDHA KO T cells. These results show a new link between acetyl-coA levels and epigenetic regulation of a key immunological gene. A general question that arises from the model of gene regulation via acetylation substrate availability, however, is how genes can be selectively affected. Indeed, the effect is not global, as T-bet was not altered by LDHA-deficiency. Acetyl-CoA levels can be influenced
Cell Metabolism 24, November 8, 2016 ª 2016 Elsevier Inc. 651
Cell Metabolism
Previews
through multiple metabolic material, but also substrates IFN-ɣ Glucose Cytokine pathways, including fatty for epigenetic modifications. Glycolysis Production acid oxidation and amino Thus, metabolism is intimately acid metabolism (Etchegaray linked to gene expression. 3’UTR binding IFNG mRNA and Mostoslavsky, 2016). As Ultimately, these associations the input of each of these may influence more than GAPDH (GAPDH) Acetyl-CoA pathways and global levels IFN-g and may guide cell of acetyl-CoA increase or differentiation or ensure that Pyruvate IFNG transcription TCA decrease, local levels of specific functions can arise Histone Modulating acetyl-CoA may also vary only when cell metabolism is T-bet Acetylation DNA Flux LDHA Methylation NFAT and the KM of some histone matched to that fate. Lactate acetyl-transferases (HATs) may be sufficiently high to REFERENCES respond to these changes. Figure 1. Glycolysis Regulates Cytokine Production in Activated Selective recruitment of Brand, A., Singer, K., Koehl, G.E., T Cells through Multiple Potential Means such HATs to the Ifng and Kolitzus, M., Schoenhammer, G., GAPDH can negatively regulate IFN-g expression by binding to IFNG mRNA Thiel, A., Matos, C., Bruss, C., Kloother loci may, therefore, 30 UTR. Upregulated glycolysis disfavors the GAPDH-30 UTR binding and thus buch, S., Peter, K., et al. (2016). increases IFN-g production. Similarly, LDHA is essential to maintain high provide a dynamic means Cell Metab. Published online glycolytic flux while controlling respiration. Mitochondrial TCA is able to to link cellular metabolic staSeptember 7, 2016. release citrate that forms acetyl-CoA and allows histone acetylation at IFNG tus to transcription. In addiloci and increased IFNG transcription. Other factors that regulate IFNG tranChang, C.H., Curtis, J.D., Maggi, tion, complementary links scription, such as DNA methylation or T-bet, may also be ultimately found to L.B., Jr., Faubert, B., Villarino, A.V., be linked with cell metabolism. of glucose metabolism to O’Sullivan, D., Huang, S.C., van der Windt, G.J., Blagih, J., Qiu, J., et al. IFN-g may also exist. In (2013). Cell 153, 1239–1251. particular, the association of 0 GAPDH with the Ifng 3 UTR may suppress DNA demethylation via TET, depletion of Cluntun, A.A., Huang, H., Dai, L., Liu, X., Zhao, IFNg in glucose-deficient settings (Chang acetyl-CoA favoring deacetylation of pro- Y., and Locasale, J.W. (2015). Cancer Metab. 3, 10. et al., 2013). While Peng et al. (2016) did teins involved in cellular metabolism, such not directly identify this 30 UTR-mediated as AMPK, or NAD activation of Sirtuins de Arau´jo-Souza, P.S., Hanschke, S.C., and Viola, J.P. (2015). J. Immunol. Res. 2015, 849573. mechanism, their data do not formally (Kinnaird et al., 2016). The roles of these exclude this model (Figure 1). In partic- metabolic links to immune regulation Denton, A.E., Russ, B.E., Doherty, P.C., Rao, S., ular, the Yeti mice show a clear role for have not been well explored but may and Turner, S.J. (2011). Proc. Natl. Acad. Sci. USA 108, 15306–15311. the 30 UTR to regulate Ifng levels and be exploited in treatment of autoimmune 0 auto-inflammation. Whether the 3 UTR and inflammatory diseases. Conversely, Etchegaray, J.P., and Mostoslavsky, R. (2016). or epigenetic modifications primarily accumulation of lactate in the tumor Mol. Cell 62, 695–711. impact cytokine expression remains an microenvironment and subsequent inhi- Ho, P.C., Bihuniak, J.D., Macintyre, A.N., Staron, open question, but these models are not bition T cell IFN-g production may sup- M., Liu, X., Amezquita, R., Tsui, Y.C., Cui, G., Micevic, G., Perales, J.C., et al. (2015). Cell 162, mutually exclusive. press anti-tumor immunity (Brand et al., 1217–1228. Epigenetic regulations by availability 2016). These metabolic links to gene of metabolic intermediates have been expression and epigenetic regulation Kinnaird, A., Zhao, S., Wellen, K.E., and Michelakis, E.D. (2016). Nat. Rev. Cancer 16, 694–707. extensively studied in cancer models. likely represent fundamental mechanisms Many chromatin-modifying enzymes can of immune regulation and a new class of Macintyre, A.N., Gerriets, V.A., Nichols, A.G., R.D., Rudolph, M.C., Deoliveira, D., Anuse metabolic intermediates as both co- targets to modify the immune response. Michalek, derson, S.M., Abel, E.D., Chen, B.J., Hale, L.P., factors and substrates and are regulated The findings of Peng et al. (2016) parallel and Rathmell, J.C. (2014). Cell Metab. 20, 61–72. by their availability (Kinnaird et al., 2016). findings in cancer biology that metaboPeng, M., Yin, N., Chhangawala, S., Xu, K., Leslie, Examples include a-ketoglutarate and lites and flux through specific pathways C.S., and Li, M.O. (2016). Science. Published succinate as substrates and products in provide not only energy or biosynthetic online September 29, 2016.
652 Cell Metabolism 24, November 8, 2016
Previews Metabolic Signaling Drives IFN-g Peter J. Siska1 and Jeffrey C. Rathmell1,* 1Department of Pathology, Microbiology and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37232-2363, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cmet.2016.10.018
IFN-g is a critical inflammatory cytokine that is closely regulated to cellular metabolic status and requires high rates of glycolysis. It has now been shown that a key mechanism to link these pathways is through maintenance of acetyl-CoA and epigenetic regulation of the IFNG locus. Activation of lymphocytes, macrophages, and dendritic cells leads to sharp increases in glucose metabolism in these cells to support their energetic and biosynthetic demands. However, these changes are much more impactful than simply supplying building blocks, as metabolites are intimately linked to fundamental signaling and differentiation processes. For example, phosphoenolpyruvate can directly regulate calcium signaling (Ho et al., 2015) and glyceraldehyde 3-phosphate and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been implicated in the regulation of interferong (IFN-g) translation (Chang et al., 2013). In cancer cells, adipocytes, and hematopoietic stem cells, it has also been shown that changes in acetyl-CoA and S-adenysyl-methionine influence gene expression through epigenetic acetylation and methylation modifications (Cluntun et al., 2015). IFN-g has emerged as particularly sensitive to cellular metabolic state, but the underlying mechanisms are only incompletely understood. Now a recent study shows that metabolic regulation of epigenetics and IFNG acetylation is a critical regulator of inflammation (Peng et al., 2016). IFN-g can be regulated on multiple levels to link cell metabolism to immune function. In addition to post-transcriptional regulation by GAPDH, the IFNG gene is under tight transcriptional control. The transcription factor T-Bet is a major driver of IFNG transcription, but IFNG-associated histones also require epigenetic modifications with histone demethylation and increased acetylation (de Arau´jo-Souza et al., 2015). Mechanisms that lead to these changes, however, are not well understood. Furthermore, it is not clear whether perturbation of metabolic pathways that provide the
substrates for epigenetic marks impact gene expression and T cell differentiation. Peng et al. (2016) add to the metabolism-epigenetic paradigm and show that lactate dehydrogenase A (LDHA) regulates glucose flux to support acetyl-CoA levels necessary for promoting acetylation of IFNG-associated histones and IFN-g expression. Starting with an effort to explore how changes in glycolysis affect immune function, Peng et al. (2016) deleted LDHA in T cells. Like Glut1-deficiency, LDHAdeficient T cells had reduced glucose consumption (Macintyre et al., 2014). Unlike T cells with impaired glucose uptake, however, LDHA-deficiency allowed glucose to flux through glycolysis and only prevented reduction of pyruvate to lactate and the coordinate oxidation of NADH to NAD+. Interestingly, T cell-specific LDHA deletion did not affect T cell activation, survival, or expression of the central CD4 T cell transcription factor T-Bet. Despite relatively normal T cells, IFN-g production was sharply reduced after LDHA deletion. Glycolytic metabolism can be linked to IFN-g expression by binding of GAPDH to the 30 UTR of IFN-g mRNA to suppress translation when glucose flux is low (Chang et al., 2013). Using a GFP reporter fused to the Ifng 30 UTR, however, Peng et al. (2016) did not find that LDHA deletion led to reduced reporter function, suggesting a potential distinct mechanism of IFN-g regulation. Indeed, T cells from mice with the Yeti allele of Ifng, in which the 30 UTR of Ifng is replaced, expressed high levels of IFN-g that were sharply reduced with LDHA deletion. The alterative 30 UTR of Ifng in the Yeti allele leads to excessive IFN-g production and an auto-inflammatory phenotype. This auto-inflammatory
disease, however, was corrected through T cell LDHA deletion (Peng et al., 2016). While the immune defects of Yeti/Yeti mice clearly show a critical role for the Ifng 30 UTR, glucose metabolism holds additional sway over IFN-g that goes beyond 30 UTR-mediated regulation. In addition to post-transcriptional regulation of IFN-g expression, IFN-g is regulated on epigenetic level by histone acetylation (Denton et al., 2011). Decreased histone acetylation at the lysine 9 residue (H3K9Ac) can diminish gene expression, and this change may link glycolysis to gene expression via substrate supply. By performing chromatin immunoprecipitation sequencing, Peng et al. (2016) found that LDHA knockout (KO) T cells had a broad decrease of H3K9Ac, including acetylation of histones at Ifng loci. Histone acetylation requires acetyl coenzyme A (acetyl-CoA) (Cluntun et al., 2015), and LDHA KO T cells showed decreased levels of this metabolite, possibly due to reduced LDHA-mediated NADH oxidation to maintain glycolytic flux or a greater consumption of acetyl-CoA as an acetyl donor to oxaloacetate to maintain the tricarboxylic acid (TCA) cycle for ATP. Importantly, acetate supplementation of Ldha-deficient T cells augmented acetyl-CoA production, normalized Ifng promoter and enhancer acetylation, and increased IFN-g expression in LDHA KO T cells. These results show a new link between acetyl-coA levels and epigenetic regulation of a key immunological gene. A general question that arises from the model of gene regulation via acetylation substrate availability, however, is how genes can be selectively affected. Indeed, the effect is not global, as T-bet was not altered by LDHA-deficiency. Acetyl-CoA levels can be influenced
Cell Metabolism 24, November 8, 2016 ª 2016 Elsevier Inc. 651
Cell Metabolism
Previews
through multiple metabolic material, but also substrates IFN-ɣ Glucose Cytokine pathways, including fatty for epigenetic modifications. Glycolysis Production acid oxidation and amino Thus, metabolism is intimately acid metabolism (Etchegaray linked to gene expression. 3’UTR binding IFNG mRNA and Mostoslavsky, 2016). As Ultimately, these associations the input of each of these may influence more than GAPDH (GAPDH) Acetyl-CoA pathways and global levels IFN-g and may guide cell of acetyl-CoA increase or differentiation or ensure that Pyruvate IFNG transcription TCA decrease, local levels of specific functions can arise Histone Modulating acetyl-CoA may also vary only when cell metabolism is T-bet Acetylation DNA Flux LDHA Methylation NFAT and the KM of some histone matched to that fate. Lactate acetyl-transferases (HATs) may be sufficiently high to REFERENCES respond to these changes. Figure 1. Glycolysis Regulates Cytokine Production in Activated Selective recruitment of Brand, A., Singer, K., Koehl, G.E., T Cells through Multiple Potential Means such HATs to the Ifng and Kolitzus, M., Schoenhammer, G., GAPDH can negatively regulate IFN-g expression by binding to IFNG mRNA Thiel, A., Matos, C., Bruss, C., Kloother loci may, therefore, 30 UTR. Upregulated glycolysis disfavors the GAPDH-30 UTR binding and thus buch, S., Peter, K., et al. (2016). increases IFN-g production. Similarly, LDHA is essential to maintain high provide a dynamic means Cell Metab. Published online glycolytic flux while controlling respiration. Mitochondrial TCA is able to to link cellular metabolic staSeptember 7, 2016. release citrate that forms acetyl-CoA and allows histone acetylation at IFNG tus to transcription. In addiloci and increased IFNG transcription. Other factors that regulate IFNG tranChang, C.H., Curtis, J.D., Maggi, tion, complementary links scription, such as DNA methylation or T-bet, may also be ultimately found to L.B., Jr., Faubert, B., Villarino, A.V., be linked with cell metabolism. of glucose metabolism to O’Sullivan, D., Huang, S.C., van der Windt, G.J., Blagih, J., Qiu, J., et al. IFN-g may also exist. In (2013). Cell 153, 1239–1251. particular, the association of 0 GAPDH with the Ifng 3 UTR may suppress DNA demethylation via TET, depletion of Cluntun, A.A., Huang, H., Dai, L., Liu, X., Zhao, IFNg in glucose-deficient settings (Chang acetyl-CoA favoring deacetylation of pro- Y., and Locasale, J.W. (2015). Cancer Metab. 3, 10. et al., 2013). While Peng et al. (2016) did teins involved in cellular metabolism, such not directly identify this 30 UTR-mediated as AMPK, or NAD activation of Sirtuins de Arau´jo-Souza, P.S., Hanschke, S.C., and Viola, J.P. (2015). J. Immunol. Res. 2015, 849573. mechanism, their data do not formally (Kinnaird et al., 2016). The roles of these exclude this model (Figure 1). In partic- metabolic links to immune regulation Denton, A.E., Russ, B.E., Doherty, P.C., Rao, S., ular, the Yeti mice show a clear role for have not been well explored but may and Turner, S.J. (2011). Proc. Natl. Acad. Sci. USA 108, 15306–15311. the 30 UTR to regulate Ifng levels and be exploited in treatment of autoimmune 0 auto-inflammation. Whether the 3 UTR and inflammatory diseases. Conversely, Etchegaray, J.P., and Mostoslavsky, R. (2016). or epigenetic modifications primarily accumulation of lactate in the tumor Mol. Cell 62, 695–711. impact cytokine expression remains an microenvironment and subsequent inhi- Ho, P.C., Bihuniak, J.D., Macintyre, A.N., Staron, open question, but these models are not bition T cell IFN-g production may sup- M., Liu, X., Amezquita, R., Tsui, Y.C., Cui, G., Micevic, G., Perales, J.C., et al. (2015). Cell 162, mutually exclusive. press anti-tumor immunity (Brand et al., 1217–1228. Epigenetic regulations by availability 2016). These metabolic links to gene of metabolic intermediates have been expression and epigenetic regulation Kinnaird, A., Zhao, S., Wellen, K.E., and Michelakis, E.D. (2016). Nat. Rev. Cancer 16, 694–707. extensively studied in cancer models. likely represent fundamental mechanisms Many chromatin-modifying enzymes can of immune regulation and a new class of Macintyre, A.N., Gerriets, V.A., Nichols, A.G., R.D., Rudolph, M.C., Deoliveira, D., Anuse metabolic intermediates as both co- targets to modify the immune response. Michalek, derson, S.M., Abel, E.D., Chen, B.J., Hale, L.P., factors and substrates and are regulated The findings of Peng et al. (2016) parallel and Rathmell, J.C. (2014). Cell Metab. 20, 61–72. by their availability (Kinnaird et al., 2016). findings in cancer biology that metaboPeng, M., Yin, N., Chhangawala, S., Xu, K., Leslie, Examples include a-ketoglutarate and lites and flux through specific pathways C.S., and Li, M.O. (2016). Science. Published succinate as substrates and products in provide not only energy or biosynthetic online September 29, 2016.
652 Cell Metabolism 24, November 8, 2016