Abstract 14023: PRG4 in Calcified Atherosclerotic Plaque Induces Immune Tolerance in Macrophages, and is Implicated in Inflammation-Driven Vascular Calcification

Abstract

Atherosclerotic calcification is an independent predictor for a cardiovascular event, and its development is driven by macrophage-led inflammation. PRG4 was previously shown to be the most upregulated gene in microarray data of carotid endarterectomy-derived, high vs low-calcified atherosclerotic plaques. It was shown to be a pro-calcifying factor in the disease, modulating the pro-calcifying switch of vascular smooth muscle cells (VSMC). PRG4’s role in calcified plaque is largely unknown, however, we are able to show a profound effect on macrophage immune response. Transcriptomic data of a highly calcified plaque cohort (N=18 patients) shows PRG4 expression is correlated with inflammatory gene pathways, including toll-like receptor signalling; and immunohistochemistry of this cohort displays co-localization with CD68 + cells. Despite this, PRG4 is not expressed in macrophages in plaque single-cell datasets or transcriptomics datasets, and plaque origin is likely fibroblast and VSMC. Human peripheral blood mononuclear cell (PBMC)-derived macrophage incubation with 100μg/ml recombinant human (rh)PRG4 shows strong NF-κB activation, and secretion of TNF, IL-6, IL-12p70 and IL-10. rhPRG4 stimulation also significantly enhances inflammasome activation, increasing ASC activation (p<0.003) and IL-1β secretion (p<0.001). Moreover, it suppressed macrophage phagocytosis of both zymosan particles (p>0.001), and hydroxyapatite particles (p=0.004). Subsequently, cells stimulated with rhPRG4 develop immune tolerance, a suppressed inflammatory response, to secondary stimulation with either rhPRG4 or lipopolysaccharide - with almost complete ablation of TNF secretion (p<0.001). This phenomenon is accompanied by a 5-fold A20 upregulation (p<0.001), a well-established key regulator of endotoxin tolerance. This immunosuppressive effect was coupled with increased resistance to mitochondrial stress (p<0.001). In conclusion, we see a PRG4-specific phenotype in macrophages; suppressed phagocytosis capability, and acute pro-inflammatory activation of NF-κB and inflammasome pathways, that over longer exposure develops into immunosuppression. This may have profound implications for the overall disease mechanism.