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Abstract Number: 1208

Cholesterol Loading Induces Neutrophil Extracellular Traps, and Atorvastatin Attenuates This Effect

Ming-Lin Liu1,2, Muhammad Bashir1,3, Kevin Williams4 and Victoria Werth5,6, 1Department of Dermatology, University of Pennsylvania, Philadelphia, PA, 2Department of Dermatology,, Philadelphia V.A. Hospital, Philadelphia, PA, 3Department of Dermatology, Philadelphia V.A. Hospital, Philadelphia, PA, 4Department of Medicine, Temple University School of Medicine, Philadelphia, PA, 5Dermatology, University of Pennsylvania, Philadelphia, PA, 6Dermatology, Philadelphia V.A. Hospital, Philadelphia, PA

Meeting: 2014 ACR/ARHP Annual Meeting

Keywords: cholesterol and statins, Neutrophil Extracellular Traps

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Session Information

Session Title: Innate Immunity and Rheumatic Disease: Signaling Mechanisms

Session Type: Abstract Submissions (ACR)

Background/Purpose

Neutrophils are the most common white blood cell, but their role in autoimmune and cardiovascular diseases has been underestimated. As part of host defense, neutrophils release granule proteins and chromatin DNA into the extracellular space to form neutrophil extracellular traps (NETs). Recent studies reported the presence of NETs in atherosclerotic lesions, and NETs promote thrombosis. Moreover, pharmacological inhibition of NET formation through peptidyl-arginine deiminase blockade can reduce atherosclerosis and arterial thrombosis in mice. Hypercholesterolemia is the underlying cause of atherosclerotic cardiovascular disease. Nevertheless, the effects of cholesterol loading on NET formation and the relevant cellular mechanisms have not been investigated.

Methods

Primary neutrophils were isolated from healthy donors by sequential centrifugation with Histopaque 1077 and 1119. Cultured human HL-60 cells were differentiated into neutrophil-like cells with 1.2% DMSO. Cholesterol was delivered to primary and HL-60 neutrophils as a water-soluble complex with meythl-β-cyclodextrin (MCD, Chol/MCD), which is widely used to modify the cholesterol content of cultured cells without potentially confounding effects from receptor engagement. Cells were fixed and then stained with Sytox Green, which indicates exposed nucleic acids. Formation of NETs was assessed with a fluorescence microplate reader and by fluorescence microscopy. To study the effects of atorvastatin on NET formation, selected plates were pretreated with this agent before exposure to Chol/MCD. 

Results

We found that Chol/MCD loading could induce NET formation in a time- and dose-dependent manner in primary neutrophils [4491±60 RFUs (relative fluorescent units) for control without Chol/MCD, 6553±206 RFUs with 12.5 µg/ml Chol/MCD, and 9297±223 RFUs with 25 µg/ml Chol/MCD, mean±SEM, P<0.001 by ANOVA], and in HL-60 neutrophils, detected with a fluorescence microplate reader. Both findings were confirmed by examination of the NET structure with fluorescent microscopy. Importantly, pretreatment of neutrophils with low or lower dose (10 or 25 µM), but not high dose (75 µM), atorvastatin, significantly attenuated cholesterol-induced NETosis in vitro. 

Conclusion

Our studies indicated that cholesterol loading can induce neutrophil extracellular trap formation in vitro. Low-dose atorvastatin can attenuate cholesterol-induced NETosis. Our preliminary results indicate a potential role of hypercholesterolemia in NET formation and a potential beneficial role for statins.


Disclosure:

M. L. Liu,
None;

M. Bashir,
None;

K. Williams,
None;

V. Werth,

Amgen,

9.

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