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A2447 - Fibroblast-Mediated Glycolytic Reprogramming of Macrophages Through IL6, Lactate and PKM2 Is a Critical Intercellular Pathway in Pulmonary Hypertension
Author Block: K. El Kasmi1, S. Riddle1, K. E. Rawlinson1, J. M. Poczobutt1, M. J. Ostaff1, H. Zhang1, A. Flockton1, M. Li1, M. G. Frid1, S. Kumar1, B. A. McKeon1, C. Mickael2, R. Kumar2, R. Moldovan3, A. D'alessandro4, B. B. Graham2, K. R. Stenmark1; 1Pediatrics and Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States, 2Pulmonary Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, United States, 3Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States, 4Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Rationale: We have recently identified metabolic reprogramming involving PKM2 as a major hallmark of activated fibroblasts (PH-Fibs) in the remodeled pulmonary artery (PA) adventitia of patients with pulmonary hypertension (PH) and calves with hypoxic PH. Further, we have previously shown that PH-Fibs polarize macrophages towards a pro-inflammatory phenotype characterized by expression of Arg1, Il4ra, Socs3, Hif1, and Vegfa. Genetic targeting of IL6 in the fibroblast significantly attenuated expression of this phenotype in macrophages. Here we tested the hypothesis that PH-Fib-mediated glycolytic reprogramming of macrophages is a critical intercellular pathway in PH.
Methods and Results: Mice with macrophage specific knockout of the IL6 receptor exhibited significant protection from hypoxia induced PH. Furthermore, expression of Arg1, Il4ra, Socs3, Hif1, and Vegfa in response to conditioned media (CM) from PH-Fibs was significantly reduced in IL6-receptor knockout bone marrow derived macrophages (BMDMs). Macrophages have recently been shown to increase Arg1 and Vegfa expression through HIF1 in response to lactate derived from glycolytic cancer cells. Here we found that metabolically reprogrammed PH-Fibs had increased lactate production and CM from PH-Fibs increased lactate transporter (MCT1/4) expression on BMDMs. In vitro, lactate synergized with IL6 in inducing Arg1 expression in BMDMs. Furthermore, pharmacological blockade of MCT1/4 in BMDMs significantly attenuated Arg1, Il4ra, Socs3, Hif1, and Vegfa expression. NMR spec, LC-MS, and FLIM techniques demonstrated that CM from PH-Fibs or IL6 induced aerobic glycolysis and increased expression of Glut1, Pkm2, Ldha/b, free NADH, and TCA cycle reprogramming with succinate accumulation in BMDMs. Moreover, the PA of calves with hypoxic PH exhibited increased GLUT1 protein and in vitro IL6, IL6/lactate, PH-Fib CM activated Glut1 expression in BMDMs. HIF1 and PKM2 are known critical regulators of glycolytic reprogramming in macrophages. Pharmacological blockade of PKM2 with 2 different strategies (TEPP, shikonin) or genetic targeting in BMDMs resulted in significantly reduced expression of Arg1, Il4ra, Socs3, Hif1, and Vegfa when exposed to CM from PH-Fibs, IL6, or IL6/lactate. Importantly, pharmacological inhibition of glycolysis (using 2DG or TEPP) or of lactate transport not only attenuated macrophage activation but increased expression of anti-inflammatory Hmox1 and Il10.
Conclusion: In PH, IL6 and lactate produced by metabolically reprogrammed adventitial fibroblasts synergize to induce a distinct pro-remodeling phenotype in macrophages. This phenotype in turn critically depends on aerobic glycolysis and TCA cycle reprograming through HIF1 and PKM2. Importantly, pharmacological targeting of lactate transport or of PKM2 has the potential to redirect macrophages towards an anti-inflammatory phenotype.