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. Metabolic changes are key drivers of immune cell differentiation. Mitochondria are central metabolic organelles of eukaryotic cells interconnecting various metabolic pathways including the electron transport chain (ETC), the Krebs cycle, fatty acid oxidation (FAO), and redox regulation. Here, we investigate the role of a structural remodeling of the mitochondrial respiratory chain in macrophage polarization.

Methods: The macrophage cell line J774A.2 as well as bone marrow-derived macrophages (BMDM) were stimulated with lipopolysaccharide (LPS) to induce a pro-inflammatory phenotype. Subsequently, inflammatory markers were analyzed via quantitative PCR (qPCR), while ETC components and mitochondrial function were investigated via Blue Native PAGE, Western Blot, and Seahorse Analysis. The integrated stress response inhibitor (ISRIB) was used to modulate ATF4 signaling and 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid (CDDO) was used to inhibit LONP1 activity.

Results: We show in J774A.2 cells and in BMDM that immune cell activation with pro-inflammatory stimuli induces changes in the subunit composition of the respiratory chain complex I. These structural alterations result in a decrease in the oxygen consumption rate (OCR) and an increase in the extracellular acidification rate (ECAR), indicating a shift from oxidative phosphorylation towards glycolysis. Instrumental for these changes is the upregulation of the specific mitochondrial matrix Lon protease (LONP1) upon LPS stimulation. We observe that LONP1 upregulation is dependent on the integrated stress response (ISR) and driven by the transcription factor ATF4. ISR silencing by ISRIB and inhibiting LONP1 by the specific inhibitor CDDO prevented macrophage polarization to a pro-inflammatory phenotype. Moreover, we found that LONP1 inhibition prevented the structural alteration of the respiratory chain. Our data indicate a novel role for structural alterations of the mitochondrial respiratory chain during the differentiation of macrophages towards a pro-inflammatory phenotype.

Conclusion: This study reveals novel molecular mechanisms, by which mitochondria control innate immunity. Insights from this study will help to identify novel therapeutic strategies to modulate inflammatory processes in ischemic diseases such as MI.