Background The role of anti-dsDNA in the pathogenesis of the systemic lupus erythematosus (SLE) has been clearly established. However, the influence of these autoantibodies in the atherothrombotic status of SLE patients has not yet been evaluated
Objectives 1. To analyse in vivo the involvement of anti-dsDNA antibodies in the development of CVD in SLE patients. 2. To evaluate in vitro the mechanisms underlying the effects of anti-dsDNA antibodies in these processes
Methods The study was conducted in 50 SLE patients and 38 healthy donors. Endothelial function was assessed by measuring the post-occlusive hyperaemia using Laser-Doppler. Various markers of oxidative stress, inflammatory cytokines, prothrombotic mediators and NETosis, were quantified in purified leukocytes and plasma from SLE patients and controls. Activation of intracellular pathways was analyzed in monocytes using pathscan intracellular signaling array. In vitro, purified neutrophils, monocytes and lymphocytes from healthy donors and endothelial cells (ECs) were treated separately and in a trans-well co-culture system with anti-dsDNA antibodies isolated from the serum of SLE patients. Then, markers of inflammation, thrombosis, oxidative stress and NETosis were evaluated by flow cytometry (protein), RT-PCR (mRNA) and electron microscopy
Results SLE patients showed impaired micro-vascular endothelial function (reduction of hyperaemia post occlusion area) and altered expression levels of pro-inflammatory proteins (IL6, IL8, MCP-1 and PCR), prothrombotic molecules (TF), oxidative stress markers (peroxides and mitochondrial membrane potential) and netosis-related molecules (NE, MPO and cell free-DNA). Monocytes from anti-dsDNA-positive SLE patients showed activation of various pathways related to inflammation, thrombosis and apoptosis (ErK, STAT-3, p38, JNK, GSK, Bad and Caspase-3). Association studies demonstrated that molecules related to inflammation and thrombosis, endothelial dysfunction, oxidative status and netosis were linked to the occurrence of thrombotic events, as well as to the presence of anti-dsDNA antibodies. In vitro treatment of purified leukocytes with anti-dsDNA antibodies promoted an increase in the production of NETosis, levels of peroxides and percentage of cells with altered mitochondrial membrane potential, as well as enlarged expression of a number of proinflammatory and prothrombotic molecules. In vitro treatment of HUVEC with anti-dsDNA antibodies promoted an increase in endothelial activation molecules (ICAM-1, VCAM-1 and E-selectin).
Conclusions 1. Positivity for anti-dsDNA antibodies is linked to an increased pro-atherothrombotic status in SLE patients. 2. Anti-dsDNA antibodies, in vitro, promote NETosis on neutrophils, apoptosis on monocytes, modulate the expression of molecules related to inflammation and thrombosis, and induce endothelial activation. Together, that data suggest the involvement of such autoantibodies on atherothrombosis development in SLE
Acknowledgements CTS-794 and ISCIII (FIS15/1333;RIER RD16/0012/0015)
Disclosure of Interest None declared