ADIPONECTIN LIMITS MONOCYTIC MICROPARTICLE-INDUCED INFLAMMASOME ACTIVATION AND ENDOTHELIAL DYSFUNCTION

Abstract

Monocytic microparticles (mono-MPs) are small vesicles (100-1000 nm) that are shed from monocytes following cellular activation or apoptosis. Levels of mono-MPs are heightened in the inflammatory state and mono-MPs have been reported to play a fundamental role at all stages of the atherothrombotic process. Alterations in monocyte-endothelial cell (EC) interaction mediate immune activation and propagate inflammation within the developing atheroma. Adiponectin is an anti-inflammatory adipokine that primes monocyte differentiation into anti-inflammatory M2 macrophages vs. pro-inflammatory M1 macrophages and inhibits EC activation thereby retarding plaque development. The goal of this work was to uncover the mechanistic root underlying the pathophysiological and clinical benefits of adiponectin. Specifically, we hypothesized that mono-MPs trigger NLRP3 inflammasome-dependent EC activation and adiponectin inhibits this phenomenon, in part, through limiting mono-MP production and activation. Mono-MPs were characterized using electron microscopy, flow cytometry and NanoSight microscopy. Exosomes were excluded based on size and absence of the MP-specific marker phosphatidylyserine. Studies were conducted with THP-1 monocytes and peritoneal monocytes isolated from adiponectin knockout (Adipoq-/-) mice. We evaluated the effects of lipopolysaccharide (LPS; 5 μg/ml, 24 h) and recombinant adiponectin (10 μg/ml) on mono-MP production, expression of the NLRP3 inflammasome and its components, and key downstream inflammatory factors. We also studied how MPs alter the expression of critical adhesion molecules and inflammatory transcription factors in human umbilical vein endothelial cell (HUVEC) cultures. Inflammasome activation was examined in mono-MPs derived from THP-1 cells. Imaging analysis confirmed that the mono-MPs isolated were sized 100-1000 nm and expressed phosphatidylyserine. Absence of exosomes in the final mono-MP samples was further confirmed by a lack of CD63 expression. LPS concentration-dependently increased MP generation by THP-1 monocytes. Adiponectin suppressed MP release in untreated and LPS-stimulated monocytes. LPS-treated mono-MPs exhibited a marked upregulation in the NLRP3 inflammasome and its components - ASC, caspase-1, pro-IL1β and IL1β. Adiponectin attenuated these changes. Mono-MP-HUVEC co-cultures revealed significant increases in markers of endothelial activation (ICAM-1, VCAM-1) and inflammatory transcription factors (p-IkB, p-NFkB and p-ERK-1). Adiponectin inhibited these outcomes. Compared to monocytes from Adipoq+/+ mice, those from Adipoq-/- littermates showed a ∼6-fold greater production of MPs (Cd11b+/Ly6Chi/AnnexinV+) following LPS stimulation. We report a novel mechanism through which mono-MPs sustain NLRP3 inflammasome activation and endothelial dysfunction. These data reveal a previously unknown atheroprotective mechanism for adiponectin that may account for the inverse association observed between circulating adiponectin levels and the risk of diabetes and obesity.