Background/Purpose: Neutrophil extracellular traps (NETs) have recently been implicated in vascular damage and atherothrombosis. Extruded DNA fibers containing multiple proinflammatory and thrombotic molecules induce the activation and recruitment of monocytes and dendritic cells to the vessel wall. Enhanced NETosis occurs in rheumatoid arthritis (RA), which has been shown related to the pathogenesis of this disorder. However, the relevance of this abnormality in the development of atherosclerosis, and the effects of new therapeutic approaches has not been elucidated yet. The aims of this study were: 1) To evaluate the association of netosis-derived products with the development of atherothrombosis in RA. 2) To study the role of biologic therapies in inhibiting NETosis.

Methods: One hundred RA patients and 30 healthy donors were included. Carotid intima media thickness (CIMT) was used as atherosclerosis marker. Inflammatory and prothrombotic molecules and oxidative stress markers were analyzed in plasma. Neutrophils were isolated and spontaneous and induced NETs formation was assessed through fluorescence microscopy. Oxidative stress status (JC1 and DCFH), myeloperoxidase (MPO), and neutrophil elastase (NE) protein expression were measured in neutrophils by flow cytometry. Cell-free DNA plasma levels were analyzed using specific ELISA-base kits. mRNA expression of peptidyl arginine deiminase 4 (PAD4) and various proatherothrombotic molecules were analyzed by RT-PCR.

Results: NETosis was found increased in RA patients, alongside MPO and NE protein expression in neutrophils. Cell free DNA plasma levels were further elevated, and strongly correlated with clinical parameters such as DAS28, CRP and ESR, and autoimmunity state (RF and anti-CCPs positivity). In addition, high levels of cell-free DNA were associated with elevated levels of IL-6, MCP-1, spSelectin and tPA in plasma, as well as with increased NTyr and decreased TAC and NO. At cellular level, cell free DNA plasma levels correlated with increased oxidative status in RA neutrophils (JC1 and DCFH) and mRNA expression of PADI4. Those patients having pathologic CIMT showed increased cell-free DNA plasma levels. Two new cohorts of 60 RA patients, treated either with Infliximab (n=40) or Tocilizumab (n=20) for six months were further evaluated. Both drugs promoted decreased generation of NETs, along with reduction of MPO, NE, PAD4, and the percentage of low density granulocytes, as well as of the proatherothrombotic profile of RA patients.

Conclusion: 1) Elements associated with the extrusion of NETs are significantly enhanced in RA patients. 2) NETosis-derived products, such as cell-free DNA, strongly correlated with clinical parameters, inflammatory and oxidative markers. Thus, NETosis-derived products demonstrated diagnostic potential for disease activity and atherosclerosis, as well as for analysis of therapeutic effectiveness in RA patients. Funded by CTS7940, PI15/01333, PI2013-0191, CP15/00158.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/netosis-derived-products-might-have-diagnostic-potential-for-disease-activity-atherosclerosis-and-analysis-of-therapeutic-effectiveness-in-rheumatoid-arthritis-patients/