1Universitätsklinikum Würzburg Deutsches Zentrum für Herzinsuffizienz Würzburg, Deutschland; 2Institute of Systems Immunology University of Würzburg Würzburg, Deutschland
Background: The physiological immune response to myocardial infarction (MI) relies on cells of the innate and adaptive immune system, which play key roles during cardiac injury, healing, and tissue remodeling. Activation and differentiation of immune cells into pro- and anti-inflammatory subsets are associated with substantial changes in cellular and mitochondrial metabolism. Mitochondria are central metabolic organelles of eukaryotic cells interconnecting various metabolic conduits as well as serving as platforms for diverse signaling pathways during immune responses. Cardiolipin (CL) plays a crucial role in mitochondrial metabolism and signaling, but a specific role for this phospholipid in macrophage polarization has not been explored. The acyl-transferase TAFFAZIN catalyses the last maturation step in the biogenesis of CL. To investigate the role of mature CL in macrophage polarization we generated a mouse model with a macrophage specific knockout of TAFAZZIN (Taz-mKO). Bone marrow-derived macrophages (BMDM) from this mouse model and from a mouse with a systemic small hairpin (sh) RNA-mediated knockdown (Taz-KD) were analyzed.
Methods: Bone marrow-derived macrophages (BMDM) were isolated from the Taz-mKO and Taz-KD mice. These cells were stimulated with lipopolysaccharide (LPS) to induce a pro-inflammatory phenotype. Nigericin was used to induce the activation of the NLRP3-inflammasome. Subsequently, inflammatory markers were analyzed via quantitative PCR (qPCR), while ETC and NLRP3-inflammasome components, mitochondrial function, and IL-1b production were investigated via Blue Native PAGE, Western blot, Seahorse extracellular flux analysis, and ELISA.
Results: We show that immune cell activation with pro-inflammatory stimuli induces a decrease in the oxygen consumption rate (OCR) and an increase in the extracellular acidification rate (ECAR) in BMDM, indicating a shift from oxidative phosphorylation towards glycolysis. LPS-stimulated TAFAZZIN deficient macrophages show decreased maximal respiratory capacity. We characterize gene expression profiles of pro- and anti-inflammatory cytokines after stimulation with LPS and IL-4 in Taz-knockout and -knockdown BMDM. We determine IL-1b cytokine release after NLRP3-inflammasome activation in BMDM with deficiency or reduction in TAFAZZIN. Our data indicate a novel role for mature CL during a pro-inflammatory immune response of macrophages.
Conclusion: This study reveals the role of CL remodeling in innate immune responses. Conclusions of this study will help to develop novel therapeutic strategies to modulate inflammatory processes in ischemic diseases such as MI.