Glutamine mitigates murine burn sepsis by supporting macrophage M2 polarization through repressing the SIRT5-mediated desuccinylation of pyruvate dehydrogenase

Abstract

Background: Alternative (M2)-activated macrophages drive the anti-inflammatory response against sepsis, a leading cause of death in patients suffering from burn injury. Macrophage M2 polarization is intrinsically linked with dominant oxidative phosphorylation (OXPHOS). Glutamine serves as a major anaplerotic source to fuel OXPHOS, but it remains unknown whether glutamine can modulate metabolic checkpoints in OXPHOS that favour M2 polarization. The study aims to explore whether glutamine essentially supports M2 polarization in IL-4-stimulated murine macrophages by sustaining the activity of PDH and whether glutamine augments macrophage M2 polarization and thus alleviates inflammation and organ injury in a murine burn sepsis model. Methods: To understand how glutamine promotes M2 activation in interleukin (IL-4)-treated murine macrophages, we detected glutamine-dependent M2 polarization and its relationship with the pyruvate dehydrogenase (PDH) complex by RT-PCR, flow cytometry and western blot. To explore how glutamine modulates PDH activity and thus supports M2 polarization, we compared the expression, phosphorylation and succinylation status of PDHA1 and then examined sirtuin SIRT5-dependent desuccinylation of PDHA1 and the effects of SIRT5 overexpression on M2 polarization by RT-PCR, flow cytometry and western blot. To determine whether glutamine or its metabolites affect M2 polarization, macrophages were cocultured with metabolic inhibitors, and then SIRT5 expression and M2 phenotype markers were examined by RT-PCR, flow cytometry and western blot. Finally, to confirm the in vivo effect of glutamine, we established a burn sepsis model by injecting Pseudomonas aeruginosainto burn wounds and observing whether glutamine alleviated proinflammatory injuries by RT-PCR, flow cytometry, western blot, immunofluorescent staining, hematoxylin-eosin staining and enzyme-linked immuno sorbent assay. Results: We showed that consumption of glutamine supported M2 activation in IL-4-treated murine macrophages by upregulating the activity of PDH. Mechanistically, glutamine did not affect the expression or alter the phosphorylation status of PDHA1 but instead downregulated the expression of SIRT5 and repressed SIRT5-dependent desuccinylation on PDHA1, which in turn recovered PDH activity and supported M2 polarization. This effect was implemented by its secondary metabolite α-ketoglutarate (αKG) rather than glutamine itself. Finally, we demonstrated that glutamine promoted macrophage M2 polarization in a murine burn sepsis model, thereby repressing excessive inflammation and alleviating organ injury in model mice. Conclusions: Glutamine mitigates murine burn sepsis by essentially supporting macrophage M2 polarization, with a mechanism involving the repression of the SIRT5-mediated desuccinylation of pyruvate dehydrogenase that replenishes OXPHOS and sustains M2 macrophages.

Keywords

Glutamine / SIRT5 / Desuccinylation / PDH / Macrophages polarization / Burn / Sepsis

References

[1] Zhang P, Zou B, Liou YC, Huang C. The pathogenesis and diagnosis of sepsis post burn injury. Burns Trauma. 2021; 9: tkaa047. https://doi.org/10.1093/burnst/tkaa047.

[2] Kamolz LP.Burns: learning from the past in order to be fit for the future. Crit Care. 2010; 14: 106. https://doi.org/10.1186/cc8192.

[3] Stanojcic M, Vinaik R, Abdullahi A, Chen P, Jeschke MG. NLRP3 knockout enhances immune infiltration and inflammatory responses and improves survival in a burn sepsis model. Immunology. 2022; 165: 195-205.

[4] Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol. 2020; 15: 123-47.

[5] Luck ME, Herrnreiter CJ, Choudhry MA. Gut microbial changes and their contribution to post-burn pathology. Shock. 2021; 56: 329-44.

[6] Sierawska O, Małkowska P, Taskin C, Hrynkiewicz R, Mertowska P, Grywalska E, et al. Innate immune system response to burn damage-focus on cytokine alteration. Int J Mol Sci. 2022; 23: 716. https://doi.org/10.3390/ijms23020716.

[7] Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki J, Sajti E, et al. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017; 356: eaal3222. https://doi.org/10.1126/science.aal3222.

[8] Murray PJ. Macrophage polarization. Annu Rev Physiol. 2017; 79: 541-66.

[9] Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014; 41: 14-20.

[10] Liu PS, Wang H, Li X, Chao T, Teav T, Christen S, et al. α-Ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol. 2017; 18: 985-94.

[11] Nelson VL, Nguyen HCB, Garcìa-Cañaveras JC, Briggs ER, Ho WY, DiSpirito JR, et al. PPA γis a nexus controlling alternative activation of macrophages via glutamine metabolism. Genes Dev. 2018; 32: 1035-44.

[12] Kieler M, Hofmann M, Schabbauer G. More than just protein building blocks: how amino acids and related metabolic pathways fuel macrophage polarization. FEBS J. 2021; 288: 3694-714.

[13] Viola A, Munari F, Sanchez-Rodriguez R, Scolaro T, Castegna A. The metabolic signature of macrophage responses. Front Immunol. 2019; 10: 1462. https://doi.org/10.3389/fimmu.2019.01462.

[14] Ren W, Xia Y, Chen S, Wu G, Bazer FW, Zhou B, et al. Glutamine metabolism in macrophages: a novel target for obesity/type 2 diabetes. Adv Nutr. 2019; 10: 321-30.

[15] Tavakoli S, Downs K, Short JD, Nguyen HN, Lai Y, Jerabek PA, et al. Characterization of macrophage polarization states using combined measurement of 2-Deoxyglucose and glutamine accumulation: implications for imaging of atherosclerosis. Arterioscler Thromb Vasc Biol. 2017; 37: 1840-8.

[16] Palmieri EM, Menga A, Martin-Perez R, Quinto A, Riera-Domingo C, De Tullio G, et al. Pharmacologic or genetic targeting of glutamine Synthetase skews macrophages toward an M1-like phenotype and inhibits tumor metastasis. Cell Rep. 2017; 20: 1654-66.

[17] Wang F, Zhang S, Vuckovic I, Jeon R, Lerman A, Folmes CD, et al. Glycolytic stimulation is not a requirement for M2 macrophage differentiation. Cell Metab. 2018; 28: 463-475.e4.

[18] Jha AK, Huang SC, Sergushichev A, Lampropoulou V, Ivanova Y, Loginicheva E, et al. Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization. Immunity. 2015; 42: 419-30.

[19] Van den Bossche J, O’Neill LA, Menon D. Macrophage Immunometabolism: where are we (going)? Trends Immunol. 2017; 38: 395-406.

[20] Zeng Z, Huang Q, Mao L, Wu J, An S, Chen Z, et al. The pyruvate dehydrogenase complex in sepsis: metabolic regulation and targeted therapy. Front Nutr. 2021; 8: 783164. https://doi.org/10.3389/fnut.2021.783164.

[21] Tan Z, Xie N, Cui H, Moellering DR, Abraham E, Thannickal VJ, et al. Pyruvate dehydrogenase kinase 1 participates in macrophage polarization via regulating glucose metabolism. J Immunol. 2015; 194: 6082-9.

[22] Park J, Chen Y, Tishkoff DX, Peng C, Tan M, Dai L, et al. SIRT5-mediated lysine Desuccinylation impacts diverse metabolic pathways. Mol Cell. 2013; 50: 919-30.

[23] Min BK, Park S, Kang HJ, Kim DW, Ham HJ, Ha CM, et al. Pyruvate dehydrogenase kinase is a metabolic checkpoint for polarization of macrophages to the M1 phenotype. Front Immunol. 2019; 10: 944. https://doi.org/10.3389/fimmu.2019.00944.

[24] Li N, Hu X, Liu Y, Wang Y, Wang Y, Liu J, et al. Systemic inflammatory responses and multiple organ dysfunction syndrome following skin burn wound and Pseudomonas aeruginosa infection in mice. Shock. 2013; 40: 152-9.

[25] Shah AM, Wang Z, Ma J. Glutamine metabolism and its role in immunity, a comprehensive review. Animals. 2020; 10: 326. https://doi.org/10.3390/ani10020326.

[26] Zhu Y, Fan S, Lu Y, Wei Y, Tang J, Yang Y, et al. Quercetin confers protection of murine sepsis by inducing macrophage M2 polarization via the TRPM2 dependent calcium influx and AMPK/ATF3 activation. J Funct Foods. 2019; 56: 1-13.

[27] Pyonteck SM, Akkari L, Schuhmacher AJ, Bowman RL, Sevenich L, Quail DF, et al. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat Med. 2013; 19: 1264-72.

[28] Chen X, Liu Y, Gao Y, Shou S, Chai Y. The roles of macrophage polarization in the host immune response to sepsis. Int Immunopharmacol. 2021; 96: 107791. https://doi.org/10.1016/j.intimp.2021.107791.

[29] Cruzat V, Macedo RM, Noel KK, Curi R, Newsholme P. Glutamine: metabolism and immune function, supplementation and clinical translation. Nutrients. 2018; 10: 1564. https://doi.org/10.3390/nu10111564.

[30] Zhang J, Pavlova NN, Thompson CB. Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine. EMBO J. 2017; 36: 1302-15.

[31] Puschel F, Favaro F, Redondo-Pedraza J, Lucendo E, Iurlaro R, Marchetti S, et al. Starvation and antimetabolic therapy promote cytokine release and recruitment of immune cells. Proc Natl Acad Sci U S A. 2020; 117: 9932-41.

[32] Park S, Jeon JH, Min BK, Ha CM, Thoudam T, Park BY, et al. Role of the pyruvate dehydrogenase complex in metabolic Remodeling: differential pyruvate dehydrogenase complex functions in metabolism. Diabetes Metab J. 2018; 42: 270-81.

[33] Kumar S, Lombard DB. Functions of the sirtuin deacylase SIRT5 in normal physiology and pathobiology. Crit Rev Biochem Mol Biol. 2018; 53: 311-34.

[34] Sun R, Zhang Z, Bao R, Guo X, Gu Y, Yang W, et al. Loss of SIRT5 promotes bile acid-induced immunosuppressive microenvironment and hepatocarcinogenesis. J Hepatol. 2022; 77: 453-66.

[35] Du J, Zhou Y, Su X, Yu JJ, Khan S, Jiang H, et al. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science. 2011; 334: 806-9.

[36] Hirschey MD, Zhao Y. Metabolic regulation by lysine Malonylation, Succinylation, and Glutarylation. Mol Cell Proteomics. 2015; 14: 2308-15.

[37] Bharathi SS, Zhang Y, Gong Z, Muzumdar R, Goetzman ES. Role of mitochondrial acyl-CoA dehydrogenases in the metabolism of dicarboxylic fatty acids. Biochem Biophys Res Commun. 2020; 527: 162-6.

[38] Westin MA, Hunt MC, Alexson SE. The identification of a succinyl-CoA thioesterase suggests a novel pathway for succinate production in peroxisomes. J Biol Chem. 2005; 280: 38125-32.

[39] Karunanidhi A, Land CV, Rajasundaram D, Grings M, Vockley J, Mohsen AW. Medium branched chain fatty acids improve the profile of tricarboxylic acid cycle intermediates in mitochondrial fatty acid beta-oxidation deficient cells: a comparative study. J Inherit Metab Dis. 2022; 45: 541-56.

[40] Gibson GE, Xu H, Chen HL, Chen W, Denton TT, Zhang S. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines. J Neurochem. 2015; 134: 86-96.

[41] Wang ZE, Zheng JJ, Bin FJ, Wu D, Su S, Yang YJ, et al. Glutamine relieves the hypermetabolic response and reduces organ damage in severe burn patients: a multicenter, randomized controlled clinical trial. Burns. 2021 S0305-4179(21)00360-0. https://doi.org/10.1016/j.burns.2021.12.005.

[42] Wang ZE, Wu D, Zheng LW, Shi Y, Wang C, Chen ZH, et al. Effects of glutamine on intestinal mucus barrier after burn injury. Am J Transl Res. 2018; 10: 3833-46.

[43] Shi Y, Wang Z, Wu W, Wu D, Wang C, Peng X. Glutamine protects intestinal mucosa and promotes its transport after burn injury in rats. Int J Clin Exp Pathol. 2018; 11: 1825-35.

[44] Yang YJ, Liu MM, Zhang Y, Wang ZE, Dan-Wu, Fan SJ, et al. Effectiveness and mechanism study of glutamine on alleviating hypermetabolism in burned rats. Nutrition. 2020; 79-80: 110934. https://doi.org/10.1016/j.nut.2020.110934.

[45] Wischmeyer PE.Glutamine in burn injury. Nutr Clin Pract. 2019; 34: 681-7.

[46] Moriguti EKU, Rosique MJ, Tirapelli LF, Tirapelli DPC, Jordão AA, Evora PRB, et al. Oral glutamine dipeptide or oral glutamine free amino acid reduces burned injury progression in rats. Braz J Biol. 2024; 84: e250936. https://doi.org/10.1590/1519-6984.250936.

[47] Chen J, Zhang S, Wu J, Wu S, Xu G, Wei D. Essential role of nonessential amino acid glutamine in atherosclerotic cardiovascular disease. DNA Cell Biol. 2020; 39: 8-15.

[48] Zhu Y, Zhang S, Sun J, Wang T, Liu Q, Wu G, et al. Cigarette smoke promotes oral leukoplakia via regulating glutamine metabolism and M2 polarization of macrophage. Int J Oral Sci. 2021; 13: 25.