Effect of succinate-induced polarization of mouse alveolar macrophages on hyperoxia epithelial-mesenchymal transition_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

FU Wei ¹ , JIANG Shang ¹ , CHEN Suheng ¹ , QU Shanshan ¹ ,  LI Yulan ²

1. The First School of Clinical Medicine, Lanzhou University, Lanzhou, Gansu 730000, P. R. China;
2. Department of Anesthesiology, The First Hospital of Lanzhou University, Lanzhou, Gansu 730000, P. R. China;

Corresponding author：

LI Yulan, Email: jasm@sina.com

Keywords：

Hyperoxia lung injury; alveolar epithelial cells; alveolar macrophages; epithelial-mesenchymal transition; succinate

DOI：

10.7507/1671-6205.202408009

Video：

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Objective To investigate the effect of succinate induced polarization of MH-S murine alveolar macrophage cells on hyperoxia-induced epithelial-mesenchymal transition (EMT) of MLE-12 mouse alveolar epithelial cells. Methods Determine the exposure time: MLE-12 cells was cultured in an incubator with 95%O₂ for different time to establish a cell model of acute hyperoxia-induced lung injury. The relative expression of EMT-related proteins (E-cadherin, N-cadherin, vimentin) was determined by Western blotting. Co-culture of MLE-12 and MH-S to explore the influence of MH-S on EMT: MLE-12 was divided into hyperoxia group for 0h, hyperoxia group for 48h and co-cultured with MH-S hyperoxia group for 48h (Co). The relative expression of EMT-related proteins was determined by Western blotting. Determination of succinate concentration and its effect on MH-S polarization and succinate receptor GPR91: MLE-12 was cultured in different concentrations of succinate medium for 24h, and the cell viability was determined by CCK-8. MH-S was divided into control group (C) and succinate group (S). Group C was cultured for 24h, and group S was added with succinate at the above concentration. The relative expression of GPR91 and polarization-related factor mRNA in MH-S was measured by RT-qPCR, and the expression of macrophage polarization-related proteins (CD11b, CD206, CD86) was measured by flow cytometry. Study on the effect of succinate on EMT by cell co-culture: MLE-12 and MH-S were co-cultured in a Transwell chamber and divided into control group (Co), succinate group (SUC) and GPR91 inhibitor group (I). Results Expression of EMT-related proteins in four groups of MLE-12 at different times: Compared with 0h, the expression of vimentin and N-cadherin in 24h and 48h increased, while the expression of E-cadherin in 48 h and 72 h decreased (P<0.05), and there was no significant difference in other groups. The follow-up experiment was conducted under hyperoxia conditions for 48h. Influence of MH-S on EMT: The expression of vimentin and N-cadherin in Co group was higher than that in 48h, and the expression of E-cadherin was lower than that in 48h (P<0.05). After 24 h of intervention with different concentrations of succinate on MLE-12, compared with the 0mmol/L, the cell viability of 2.5mmol/L, 1mmol/L and 500 μmol/L increased (P<0.05), and there was no significant difference in other groups, so the 1mmol/L succinate concentration was selected for subsequent experiment. Compared with group C, the expression of GPR91 mRNA in group S increased, and the expression of iNOS and CD86 mRNA in group S increased (P<0.05), but there was no significant difference in other groups. The analysis of flow cytometry showed that 1mmol/L succinate could increase the number and proportion of CD86⁺CD206^– alveolar macrophages. Compared with Co group, the expression of vimentin and N-cadherin in SUC group increased, while the expression of E-cadherin decreased. Compared with SUC group, the expression of vimentin and N-cadherin in group I decreased, while the expression of E-cadherin increased (P<0.05). Conclusion Succinate can induce mouse alveolar macrophages polarization to M1 through GPR91, enhance EMT of mouse alveolar epithelial cell injury model under hyperoxia, and promote the formation of pulmonary fibrosis.

Citation： FU Wei, JIANG Shang, CHEN Suheng, QU Shanshan, LI Yulan. Effect of succinate-induced polarization of mouse alveolar macrophages on hyperoxia epithelial-mesenchymal transition. Chinese Journal of Respiratory and Critical Care Medicine, 2025, 24(1): 33-41. doi: 10.7507/1671-6205.202408009 Copy

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19.	Connors J, Dawe N, Van Limbergen J. The role of succinate in the regulation of intestinal inflammation. Nutrients, 2018, 11(1): 25.
20.	Yue L, Lu X, Dennery PA, Yao H. Metabolic dysregulation in bronchopulmonary dysplasia: Implications for identification of biomarkers and therapeutic approaches. Redox Biol, 2021, 48: 102104.
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25.	Kannan S, Pang H, Foster DC, et al. Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity. Cell Death Differ, 2006, 13(2): 311-323.
26.	Sugahara K, Tokumine J, Teruya K, et al. Alveolar epithelial cells: Differentiation and lung injury. Respirology, 2006, 11 Suppl: S28-S31.
27.	Cabrera-Benítez NE, Parotto M, Post M, et al. Mechanical stress induces lung fibrosis by epithelial-mesenchymal transition. Crit Care Med, 2012, 40(2): 510-517.
28.	Gorowiec MR, Borthwick LA, Parker SM, et al. Free radical generation induces epithelial-to-mesenchymal transition in lung epithelium via a TGF-β1-dependent mechanism. Free Radic Biol Med, 2012, 52(6): 1024-1032.
29.	Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest, 2009, 119(6): 1420-1428.
30.	Phan SH. Genesis of the myofibroblast in lung injury and fibrosis. Proc Am Thorac Soc, 2012, 9(3): 148-152.
31.	Tanjore H, Blackwell TS, Lawson WE. Emerging evidence for endoplasmic reticulum stress in the pathogenesis of idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol, 2012, 302(8): L721-L729.
32.	Vyas-Read S, Wang W, Kato S, et al. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition. Am J Physiol Lung Cell Mol Physiol, 2014, 306(4): L326-L340.
33.	Willis BC, duBois RM, Borok Z. Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc, 2006, 3(4): 377-382.
34.	Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest, 2003, 112(12): 1776-1784.
35.	Wynn TA. Integrating mechanisms of pulmonary fibrosis. J Exp Med, 2011, 208(7): 1339-1350.
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37.	Jiang D, Liang J, Fan J, et al. Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med, 2005, 11(11): 1173-1179.
38.	Cassel SL, Eisenbarth SC, Iyer SS, et al. The NALP3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci USA, 2008, 105(26): 9035-9040.
39.	Ingram JL, Rice AB, Geisenhoffer K, et al. IL-13 and IL-1β promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGF-Rα. FASEB J, 2004, 18(10): 1132-1134.
40.	Wilson MS, Madala SK, Ramalingam TR, et al. Bleomycin and IL-1β-mediated pulmonary fibrosis is IL-17A dependent. J Exp Med, 2010, 207(3): 535-552.
41.	Hurskainen M, Mižíková I, Cook DP, et al. Single-cell transcriptomic analysis of murine lung development on hyperoxia-induced damage. Nat Commun, 2021, 12(1): 1565.
42.	Mills E, O'Neill LA. Succinate: A metabolic signal in inflammation. Trends Cell Biol, 2014, 24(5): 313-320.
43.	Rubic T, Lametschwandtner G, Jost S, et al. Triggering the succinate receptor GPR91 on dendritic cells enhances immunity. Nat Immunol, 2008, 9(11): 1261-1269.
44.	Saraiva AL, Veras FP, Peres RS, et al. Succinate receptor deficiency attenuates arthritis by reducing dendritic cell traffic and expansion of T(h)17 cells in the lymph nodes. FASEB J, 2018: fj201800285.
45.	Tannahill GM, Curtis AM, Adamik J, et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature, 2013, 496(7444): 238-242.
46.	Tretter L, Patocs A, Chinopoulos C. Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis. Biochim Biophys Acta, 2016, 1857(8): 1086-1101.
47.	He W, Miao FJ, Lin DC, et al. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature, 2004, 429(6988): 188-193.
48.	Ariza AC, Deen PM, Robben JH. The succinate receptor as a novel therapeutic target for oxidative and metabolic stress-related conditions. Front Endocrinol, 2012, 3: 22.
49.	Diehl J, Gries B, Pfeil U, et al. Expression and localization of GPR91 and GPR99 in murine organs. Cell Tissue Res, 2016, 364(2): 245-262.
50.	Gilissen J, Jouret F, Pirotte B, Hanson J. Insight into SUCNR1 (GPR91) structure and function. Pharmacol Ther, 2016, 159: 56-65.
51.	de Castro Fonseca M, Aguiar CJ, da Rocha Franco JA, Gingold RN, Leite MF. GPR91: Expanding the frontiers of Krebs cycle intermediates. Cell Commun Signal, 2016, 14: 3.
52.	Li Y, Arita Y, Koo HC, Davis JM, Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003, 29(6): 779-783.

1. Angus DC. Oxygen therapy for the critically ill. N Engl J Med, 2020, 382(11): 1054-1056.
2. Girardis M, Busani S, Damiani E, et al. Effect of conservative vs conventional oxygen therapy on mortality among patients in an intensive care unit: The Oxygen-ICU randomized clinical trial. JAMA, 2016, 316(15): 1583-1589.
3. Kallet RH, Matthay MA. Hyperoxic acute lung injury. Respir Care, 2013, 58(1): 123-141.
4. Chu DK, Kim LH, Young PJ, et al. Mortality and morbidity in acutely ill adults treated with liberal versus conservative oxygen therapy (IOTA): a systematic review and meta-analysis. Lancet, 2018, 391(10131): 1693-1705.
5. Capellier G, Panwar R. Is it time for permissive hypoxaemia in the intensive care unit?. Crit Care Resusc, 2011, 13(3): 139-141.
6. Simoes DC, Psarra AM, Mauad T, et al. Glucocorticoid and estrogen receptors are reduced in mitochondria of lung epithelial cells in asthma. PLoS One, 2012, 7(6): e39183.
7. Adamson IY, Young L, Bowden DH. Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am J Pathol, 1988, 130(2): 377-383.
8. Bishop AE. Pulmonary epithelial stem cells. Cell Prolif, 2004, 37(1): 89-96.
9. Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radic Biol Med, 2009, 47(4): 333-343.
10. Carnesecchi S, Pache JC, Barazzone-Argiroffo C. NOX enzymes: potential target for the treatment of acute lung injury. Cell Mol Life Sci, 2012, 69(14): 2373-2385.
11. Porzionato A, Sfriso MM, Mazzatenta A, et al. Effects of hyperoxic exposure on signal transduction pathways in the lung. Respir Physiol Neurobiol, 2015, 209: 106-114.
12. Hafner C, Wu J, Tiboldi A, et al. Hyperoxia induces inflammation and cytotoxicity in human adult cardiac myocytes. Shock, 2017, 47(4): 436-444.
13. Huang X, Xiu H, Zhang S, Zhang G. The role of macrophages in the pathogenesis of ALI/ARDS. Mediators Inflamm, 2018, 2018: 1264913.
14. Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity, 2014, 41(1): 14-20.
15. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity, 2016, 44(3): 450-462.
16. Laskin DL, Sunil VR, Gardner CR, Laskin JD. Macrophages and tissue injury: Agents of defense or destruction? Annu Rev Pharmacol Toxicol, 2011, 51: 267-288.
17. Suresh MV, Aktay S, Yalamanchili G, et al. Role of succinate in airway epithelial cell regulation following traumatic lung injury. JCI Insight, 2023, 8(18).
18. Akram M. Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys, 2014, 68(3): 475-478.
19. Connors J, Dawe N, Van Limbergen J. The role of succinate in the regulation of intestinal inflammation. Nutrients, 2018, 11(1): 25.
20. Yue L, Lu X, Dennery PA, Yao H. Metabolic dysregulation in bronchopulmonary dysplasia: Implications for identification of biomarkers and therapeutic approaches. Redox Biol, 2021, 48: 102104.
21. Gong J, Feng Z, Peterson AL, et al. The pentose phosphate pathway mediates hyperoxia-induced lung vascular dysgenesis and alveolar simplification in neonates. JCI Insight, 2021, 6(5): e137594.
22. Antonioli L, Blandizzi C, Pacher P, Haskó G. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer, 2013, 13(12): 842-857.
23. Serhan CN, Chiang N, Dalli J, Levy BD. Lipid mediators in the resolution of inflammation. Cold Spring Harb Perspect Biol, 2014, 7(2): a016311.
24. Wang YH, Yan ZZ, Luo SD, et al. Gut microbiota-derived succinate aggravates acute lung injury after intestinal ischemia/reperfusion in mice. Eur Respir J, 2023, 61(2): 2200840.
25. Kannan S, Pang H, Foster DC, et al. Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity. Cell Death Differ, 2006, 13(2): 311-323.
26. Sugahara K, Tokumine J, Teruya K, et al. Alveolar epithelial cells: Differentiation and lung injury. Respirology, 2006, 11 Suppl: S28-S31.
27. Cabrera-Benítez NE, Parotto M, Post M, et al. Mechanical stress induces lung fibrosis by epithelial-mesenchymal transition. Crit Care Med, 2012, 40(2): 510-517.
28. Gorowiec MR, Borthwick LA, Parker SM, et al. Free radical generation induces epithelial-to-mesenchymal transition in lung epithelium via a TGF-β1-dependent mechanism. Free Radic Biol Med, 2012, 52(6): 1024-1032.
29. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest, 2009, 119(6): 1420-1428.
30. Phan SH. Genesis of the myofibroblast in lung injury and fibrosis. Proc Am Thorac Soc, 2012, 9(3): 148-152.
31. Tanjore H, Blackwell TS, Lawson WE. Emerging evidence for endoplasmic reticulum stress in the pathogenesis of idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol, 2012, 302(8): L721-L729.
32. Vyas-Read S, Wang W, Kato S, et al. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition. Am J Physiol Lung Cell Mol Physiol, 2014, 306(4): L326-L340.
33. Willis BC, duBois RM, Borok Z. Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc, 2006, 3(4): 377-382.
34. Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest, 2003, 112(12): 1776-1784.
35. Wynn TA. Integrating mechanisms of pulmonary fibrosis. J Exp Med, 2011, 208(7): 1339-1350.
36. Wynn TA, Barron L. Macrophages: Master regulators of inflammation and fibrosis. Semin Liver Dis, 2010, 30(3): 245-257.
37. Jiang D, Liang J, Fan J, et al. Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med, 2005, 11(11): 1173-1179.
38. Cassel SL, Eisenbarth SC, Iyer SS, et al. The NALP3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci USA, 2008, 105(26): 9035-9040.
39. Ingram JL, Rice AB, Geisenhoffer K, et al. IL-13 and IL-1β promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGF-Rα. FASEB J, 2004, 18(10): 1132-1134.
40. Wilson MS, Madala SK, Ramalingam TR, et al. Bleomycin and IL-1β-mediated pulmonary fibrosis is IL-17A dependent. J Exp Med, 2010, 207(3): 535-552.
41. Hurskainen M, Mižíková I, Cook DP, et al. Single-cell transcriptomic analysis of murine lung development on hyperoxia-induced damage. Nat Commun, 2021, 12(1): 1565.
42. Mills E, O'Neill LA. Succinate: A metabolic signal in inflammation. Trends Cell Biol, 2014, 24(5): 313-320.
43. Rubic T, Lametschwandtner G, Jost S, et al. Triggering the succinate receptor GPR91 on dendritic cells enhances immunity. Nat Immunol, 2008, 9(11): 1261-1269.
44. Saraiva AL, Veras FP, Peres RS, et al. Succinate receptor deficiency attenuates arthritis by reducing dendritic cell traffic and expansion of T(h)17 cells in the lymph nodes. FASEB J, 2018: fj201800285.
45. Tannahill GM, Curtis AM, Adamik J, et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature, 2013, 496(7444): 238-242.
46. Tretter L, Patocs A, Chinopoulos C. Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis. Biochim Biophys Acta, 2016, 1857(8): 1086-1101.
47. He W, Miao FJ, Lin DC, et al. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature, 2004, 429(6988): 188-193.
48. Ariza AC, Deen PM, Robben JH. The succinate receptor as a novel therapeutic target for oxidative and metabolic stress-related conditions. Front Endocrinol, 2012, 3: 22.
49. Diehl J, Gries B, Pfeil U, et al. Expression and localization of GPR91 and GPR99 in murine organs. Cell Tissue Res, 2016, 364(2): 245-262.
50. Gilissen J, Jouret F, Pirotte B, Hanson J. Insight into SUCNR1 (GPR91) structure and function. Pharmacol Ther, 2016, 159: 56-65.
51. de Castro Fonseca M, Aguiar CJ, da Rocha Franco JA, Gingold RN, Leite MF. GPR91: Expanding the frontiers of Krebs cycle intermediates. Cell Commun Signal, 2016, 14: 3.
52. Li Y, Arita Y, Koo HC, Davis JM, Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003, 29(6): 779-783.

Chinese Journal of Respiratory and Critical Care Medicine

Effect of succinate-induced polarization of mouse alveolar macrophages on hyperoxia epithelial-mesenchymal transition

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