Cholesterol crystals drive metabolic reprogramming and M1 macrophage polarisation in primary human macrophages

Background and aims: Metabolic reprogramming of innate immune cells is emerging as a key player in the progression of a number of chronic diseases, including atherosclerosis, where high rates of glycolysis correlate with plaque instability. This study aimed to investigate if cholesterol crystals, which are key atherosclerosisassociated DAMPs (damage/danger-associated molecular patterns), alter immune cell metabolism and whether this, in turn, impacts on macrophage phenotype and function. Methods and Results: Primary human macrophages were treated with cholesterol crystals and expression of M1 (CXCL9, CXCL10) and M2-associated (MRC1, CCL13) macrophage markers, alarmins, and inflammatory cytokines were assessed either by real-time PCR or ELISA. Cholesterol crystal-induced changes in glycolytic markers were determined using real-time PCR and western blotting, while changes in cellular respiration and mitochondrial dynamics were examined via Seahorse analysis, Fluorescence Lifetime Imaging Microscopy (FLIM) and confocal microscopy. Treatment of macrophages with cholesterol crystals upregulated mRNA levels of CXCL9 and CXCL10, while concomitantly downregulating expression of MRC1 and CCL13. Cholesterol crystal-treated macrophages also exhibited a significant shift in metabolism to favour glycolysis, accompanied by the expression of key glycolytic markers GLUT1, Hexokinase 2, HIF1 alpha, GAPDH and PFKFB3. Furthermore, we show that these effects are mediated upstream by the glycolytic enzyme, PKM2, and that direct inhibition of glycolysis or PKM2 nuclear localisation leads to a significant reduction in cholesterol crystal-induced inflammatory readouts. Conclusions: This study not only provides further insight into how atherosclerosis-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for cholesterol crystal-related inflammation.