Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5 days later re-stimulated with Pam3CSK for 24 h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5 days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1β, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.

Graphical Abstract

Ischemia provokes only part of the immune training repertoire in cardiac fibroblasts. Trained immunity in myeloid and non-myeloid cells is triggered by certain infectious and sterile triggers like β-glucan or oxidized LDL, respectively. Key characteristics of immune training are as follows: stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, transcriptional induction of lactate dehydrogenase (LDH), phosphoglycerate kinase (PGK)1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), increased glycolysis and lactate production, and enhanced cytokine response to a secondary stimulus such as the toll-like receptor agonist Pam3CSK4. Simulated ischemia/reperfusion (SI/R) reproduces some but not all of these features in human cardiac fibroblasts (CF) as indicated with asterisk (*).