Acetylcholine Inhibits Monomeric C-Reactive Protein Induced Inflammation, Endothelial Cell Adhesion, and Platelet Aggregation; A Potential Therapeutic?

Mark Slevin,Vittorio Caprio,Yasmin Zeinolabediny,Baoqiang Guo,Mario Di Napoli,Wen-Hui Fang,Xianwei Zeng,Donghui Liu,Rocco S Iemma,Nicola Phillips,Garry Mcdowell,Raid Albaradie,Naif K Binsaleh,Glenn R Ferris

doi:10.3389/fimmu.2018.02124

Abstract

Objectives: In this study, we examined the possibility of using targeted antibodies and the potential of small molecular therapeutics (acetylcholine, nicotine and tacrine) to block the pro-inflammatory and adhesion-related properties of monomeric C-reactive protein (mCRP).Methods: We used three established models (platelet aggregation assay, endothelial leucocyte binding assay and monocyte inflammation via ELISA and Western blotting) to assess the potential of these therapeutics.Results: The results of this study showed that monocyte induced inflammation (raised tumor necrosis factor-alpha-TNF-α) induced by mCRP was significantly blocked in the presence of acetylcholine and nicotine, whilst tacrine and targeted antibodies (clones 8C10 and 3H12) had less of or no significant effects. Western blotting confirmed the ability of acetylcholine to inhibit mCRP-induced cell signaling phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), p38 and nuclear factor-kappa B (NF-κB). There was no evidence of direct binding between small molecules and mCRP. mCRP also induced endothelial cell-monocyte adhesion in a dose dependent fashion, however, both acetylcholine and nicotine as well as targeting antibodies notably inhibited adhesion. Finally, we investigated their effects on mCRP-induced platelet aggregation. All three small molecules significantly attenuated platelet aggregation as did the antibody 8C10, although 3H12 had a weaker effect.Discussion: Acetylcholine and to a lesser extent nicotine show potential for therapeutic inhibition of mCRP-induced inflammation and cell and platelet adhesion. These results highlight the potential of targeted antibodies and small molecule therapeutics to inhibit the binding of mCRP by prevention of membrane interaction and subsequent activation of cellular cascade systems, which produce the pro-inflammatory effects associated with mCRP.

Highlights

Activated platelets and endothelial leucocyte interactions represent an important link between pro-thrombotic and pro-inflammatory association of monomeric C-reactive protein
MCRP significantly decreased the levels of anti-inflammatory cytokine IL-10 by 25%, opposite to LPS which augmented the production of IL-10 by 2.2-fold (P < 0.001, Figure 1C)
We investigated whether the small molecules and anti-monomeric C-reactive protein (mCRP) antibodies (3H12 and 8C10) would affect macrophage cytokine profiles induced by mCRP

Summary

Introduction

Activated platelets and endothelial leucocyte interactions represent an important link between pro-thrombotic and pro-inflammatory association of monomeric C-reactive protein (mCRP). These interactions may play a significant role in atherogenesis of cardiovascular diseases leading to acute ischemic stroke and a more chronic role in the development of brain lesions in vascular dementia and associated diseases [1]. The main proposed mechanism of mCRP-associated interaction with cellular membranes and receptors is via its cholesterol binding domain (cystathionine-βsynthase; CBS; a.a 35–47). In this mechanism, the hydrophobic region of the mCRP inserts into lipid rafts of the plasma membrane binding to cholesterol molecules [2]. Deposition of mCRP on to endothelial cell (EC)-circulating micro particles seems to be associated with chronic inflammation and linked to macrophage activation and T cell polarization [3]

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