Abstract 4339871: Inflammasome-mediated Galectin-3 release limits its protective role in macrophage lysosomal damage response during atherosclerosis

Galectin-3 (Gal3) is a β-galactoside-binding lectin widely recognized as a secreted inflammatory biomarker in cardiovascular disease, with significantly elevated circulating levels in patients with atherosclerosis and myocardial infarction. However, the molecular mechanisms of Gal3 action and whether its intracellular role contributes to atherogenesis remain unclear. Through analysis of bulk and single-cell RNA-seq datasets, we demonstrate that Lgals3 transcripts are upregulated during plaque progression with abundance in myeloid/macrophage lineage and foamy macrophage. Furthermore, Gal3 expression strongly correlates with a network of lysosomal genes, suggesting a possible link between macrophage Gal3 and lysosomal function. Indeed, instigation of lysosome membrane damage in primary macrophages via cholesterol crystals and LLOMe triggers robust recruitment of Gal3 to lysosomes by sensing exposed carbohydrate moieties of proteins including Lamp1. This recruitment initiates a lysosomal recovery response involving lysosomal removal through autophagy/lysophagy and their replacement via TFEB-mediated lysosomal biogenesis. In Gal3-deficient macrophages, this recovery program is impaired, with diminished autophagy, impaired TFEB activation, accumulation of dysfunctional lysosomes, enhanced apoptosis and NLRP3 inflammasome/IL-1β activation. Also, the protective effects of intracellular Gal3 are recapitulated in vivo, where Gal3-null bone marrow transplanted in atherogenic LDLR-null mice yields increased lesion size as well as altered plaque composition with macrophage accumulation, apoptosis, and necrotic core formation. Moreover, previous studies have shown that serum Gal3 levels are associated with the poor outcomes in cardiovascular diseases patients. Consistent with this, we confirmed that serum Gal3 levels are significantly higher in patients with myocardial infarction compared to healthy controls. We find that the activation of inflammasome and ensuing pyroptosis leads to the release of Gal3 from macrophages which deplete intracellular Gal3 and limits its protective functions. In turn, the inhibition of inflammasome by MCC950 retains intracellular Gal3 and rescues it beneficial lysosomal roles. Together, our findings reveal a previously unrecognized protective function of Gal3 in atherosclerotic macrophages and identify the mechanism of Gal3 release that expends its traditional function as a secreted inflammatory marker.