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2017 Cardiovascular Research Day Abstract Book

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6<br />

Serum Amyloid A Activates the NLRP3 Inflammasome in Macrophages<br />

Preetha Shridas, PhD 1 • Maria de Beer, PhD 2 • Nancy Webb, PhD 3<br />

1Internal Medicine, University of Kentucky • 2 Physiology, University of Kentucky • 3 Molecular and<br />

Biomedical Pharmacology, University of Kentucky<br />

Faculty<br />

Objectives: Interleukin-1beta (IL-1β) has been implicated in many chronic diseases including<br />

atherosclerosis and type 2 diabetes. Production of bioactive IL-1β is controlled by the<br />

inflammasome, a multi-protein complex that regulates caspase-1 activity. Serum Amyloid A (SAA) is<br />

an acute-phase protein whose levels in circulation can increase more than 1000-fold during severe<br />

infection and tissue damage. SAA is more modestly elevated in conditions associated with chronic<br />

inflammation, including obesity. SAA has been identified as the first known physiological mediator<br />

capable of activating the NRLP3 inflammasome. The objective of this study is to investigate the<br />

mechanisms involved in SAA-mediated inflammasome activation in macrophages.<br />

Methods/Results: J774 macrophage-like cells and mouse bone-marrow derived macrophages<br />

(BMM) were stimulated with 5-25 µg/ml purified lipid-free mouse SAA, concentrations<br />

corresponding to those typically observed in obese individuals. The analyses of culture media and<br />

cell lysates demonstrated that SAA dose-dependently induced both caspase-1 activation and IL-1β<br />

secretion. The ability of SAA to induce IL-1β secretion was significantly reduced in BMM deficient in<br />

NRLP3. There was significant suppression in IL-1β secretion by SAA when J774 cells were treated<br />

with SAA in the presence of caspase-1inhibitor, Z-YVAD-FMK. A P2X7-receptor antagonist,<br />

AA38079, did not have any effect on SAA-mediated IL-1β production. Inhibition of reactive oxygen<br />

species (ROS) and cathepsin-B activation by N-acetyl-L-cysteine and CA-074, respectively, inhibited<br />

inflammasome activation by SAA. Inhibiting cellular potassium efflux by glyburide also significantly<br />

reduced SAA-mediated IL-1β secretion. Pre-incubating SAA with HDL prior to cell treatments<br />

completely inhibited its ability to trigger inflammasome activation. HDL also abrogated SAAmediated<br />

ROS generation in J774 cells.<br />

Conclusions: SAA-mediated NRLP3 inflammasome activation in macrophages is dependent on ROS<br />

generation, release of cathepsin-B, and potassium efflux, and is independent of the P2X7 receptor.<br />

Ongoing studies are investigating the cellular receptor(s) involved and the mechanism by which<br />

lipoproteins mask SAA’s effects. Blocking SAA-mediated inflammasome activation may ameliorate<br />

increased risk for type 2 diabetes and atherosclerosis in obese individuals.<br />

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