Elsevier

Atherosclerosis

Volume 251, August 2016, Pages 197-205
Atherosclerosis

Cholesterol crystals activate Syk and PI3 kinase in human macrophages and dendritic cells

https://doi.org/10.1016/j.atherosclerosis.2016.06.035Get rights and content

Highlights

  • Cholesterol crystals activate Syk and PI3 kinase in human innate immune cells.

  • Cholesterol crystals drive IL-1 production in a Syk- and PI3K-dependent manner.

  • Cholesterol crystals activate MAP kinases downstream of Syk and PI3 kinase.

  • Cholesterol crystals induce S100 and MMP1 expression via tyrosine kinases.

Abstract

Background and aims

Cholesterol crystals are a key component of atherosclerotic lesions where they promote pro-inflammatory cytokine production and plaque destabilization. Antagonists of inflammatory mediators and agents that dissolve or prevent the formation of cholesterol crystals are being explored as potential therapeutics for atherothrombosis. We sought to identify signalling molecules activated following exposure of immune cells to cholesterol crystals with the view to identifying novel therapeutic targets.

Methods

Human macrophages and dendritic cells (DC) were exposed to cholesterol crystals and activation of signalling molecules was assessed by immunoblotting. The role of Syk and PI3K in crystal-induced interleukin (IL)-1 production was determined by ELISA using specific kinase inhibitors. Real-time PCR was employed to examine the role of Syk/PI3K in cholesterol crystal-induced expression of S100 proteins and MMPs.

Results

Exposure of human macrophages and DC to cholesterol crystals induced robust activation of Syk and PI3K within 2–5 min. Pharmacological inhibition of Syk/PI3K reduced crystal-induced IL-1α/β production by approximately 80%. Activation of the downstream MAP kinases, MEK and ERK, was suppressed following inhibition of Syk and PI3K. Finally, inhibition of both Syk and PI3K significantly reduced cholesterol crystal-induced S100A8 and MMP1 gene expression by >70% while inhibition of PI3K also reduced S100A12 expression.

Conclusion

Cholesterol crystals activate specific cell signalling pathways which drive the production of inflammatory cytokines and degradative enzymes known to contribute to disease initiation and progression. These molecular events are dependent on activation of Syk and PI3K, hence, they represent potential therapeutic targets for the treatment of cholesterol crystal-related pathologies.

Introduction

Cholesterol crystals found in atherosclerotic plaques arise as a result of excessive cholesterol intake and hypercholesterolemia. While originally considered to be markers of advanced atherosclerosis, recent studies have demonstrated that cholesterol crystals form early on in diet-induced atherogenesis and their appearance coincides with that of immune cells in the subendothelial space where they can contribute to arterial inflammation and plaque destabilization [1], [2]. The presence of macrophages and dendritic cells (DC) in atheromatous plaques has led to atherosclerosis being classified as a chronic inflammatory disease as innate immune cells together with the cytokines they produce can influence plaque stability which eventually leads to myocardial infarction [3]. Cytokines of the IL-1 family as well as TNF-α and IL-6 are all highly expressed in advanced plaques [4]. IL-1 signalling has long been associated with the pathogenesis of atherosclerosis as the appearance and activation of IL-1β, in particular, correlates with disease severity [1], [5]. IL-1β is detected in early human atherosclerotic plaques, as well as murine models of atherosclerosis, suggesting that it plays a role in the initiation of the disease [6].

Cholesterol crystals have been shown to directly drive the secretion of IL-1β via activation of Nod-like receptor related protein 3 (NLRP3), which forms a large multi-protein complex, called the inflammasome. The precise mechanism of inflammasome activation has yet to be elucidated; however, it is believed to involve lysosomal rupture following crystal uptake, release of lysosomal proteases and potassium efflux [1], [5]. Hence, inflammasome components are now considered potential targets for the treatment of cholesterol crystal-induced inflammation. In addition to IL-1β production, inflammasome activation also leads to the secretion of IL-1α via pyroptotic cell death [7] and, while much emphasis has been placed on the former, it has been demonstrated that deletion of IL-1α confers greater protection from disease in Apolipoprotein-E-deficient mice than IL-1β deletion [8].

It has recently been demonstrated that cholesterol crystals bind to the C-type lectin receptor (CLR), Mincle, on the surface of human DC and trigger innate immune responses [9]. Mincle activates immune cells by coupling with the FcRγ chain, an immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein [10]. Given that ITAM-mediated signals can activate NLRP3 in a Syk-dependent manner [11], we sought to determine whether cholesterol crystals themselves activate Syk and its downstream signalling partners in primary human macrophages and DC. We demonstrate that Syk and PI3K are activated by cholesterol crystals in both cell types in a receptor independent manner and that crystal-induced IL-1 production is suppressed following treatment with Syk and PI3K inhibitors. We also demonstrate that cholesterol crystals activate MAP kinases downstream of Syk and PI3K. Finally, we show that the treatment of macrophages with cholesterol crystals leads to the production of the damage-associated molecules, S100A8 and S100A12, and the up-regulation of the matrix metalloprotease (MMP)1 in a Syk- and PI3K-dependent manner.

Section snippets

Reagents

Ultrapure lipopolysaccharide (LPS) and the PI3K inhibitor, LY294002, were from Invivogen (Toulouse, France). The Syk inhibitor, R788, was from AdooQ BioScience (Irvine, CA). Recombinant human M-CSF was from PeproTech (Rocky Hill, NJ). Recombinant human IL-4 and GM-CSF were from Immunotools (Friesoythe, Germany). Lymphoprep was from Stemcell Technologies (Grenoble, France). Primary antibodies were obtained from Cell Signalling Technology (Beverly, MA). Cholesterol, the Syk inhibitor,

Cholesterol crystals activate Syk and PI3K in primary macrophages and DC

We have previously demonstrated that the membrane-associated tyrosine kinase, Syk, is activated by osteoarthritis-associated basic calcium phosphate (BCP) crystals [12]. Similar findings have also been reported for MSU crystals and alum particles [11], [13]. In order to determine if cholesterol crystals can induce the activation of Syk, primary macrophages and DC were stimulated with cholesterol crystals, at doses reported in previous published studies, over the course of 30 min. Activation of

Discussion

It is now well established that cholesterol crystals contribute to atherogenesis through the induction of inflammatory cytokines [1], [5], however, little is known about cholesterol crystal-induced cell signalling. In this study, we provide further insight into the mechanism by which cholesterol crystals activate intracellular signalling pathways and induce pro-inflammatory cytokine production. We show that cholesterol crystals activate the membrane-proximal kinases, Syk and PI3K, in primary

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

This work is supported by the Health Research Board, Ireland (Grant no: HRA-POR 2012/20).

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