Background/Purpose: Neutrophils contribute to the innate immune response of killing pathogens through the formation of neutrophil extracellular traps (NETs), which is also called NETosis. NETosis is aberrantly increased in many autoimmune diseases such as lupus and rheumatoid arthritis (RA) and may drive or worsen disease. NETosis is induced by cytokines and chemokines leading to formation of reactive oxygen species (ROS) and isolevuglandins (IsoLGs). IsoLGs accumulate and disrupt chromatin structure leading to the citrullination of histones and release of myeloperoxidase (MPO) and neutrophil elastase. 2-HOBA is a scavenger of reactive dicarbonyls such as IsoLGs. In this study, we tested the hypothesis that scavenging IsoLGs with 2-HOBA reduces in vitro NET formation in neutrophils from patients with RA.

Methods: Blood samples were collected from 12 patients with RA. Isolated neutrophils were untreated or treated with phorbol 12-myristate 13-acetate (PMA) or 2-HOBA plus PMA. The neutrophils were plated and stained for MPO using an Alexa Fluor555 tagged antibody, and DNA using Hoechst 33342 and imaged by confocal microscopy. Imaris Microscope Image Analysis Software was used for analysis. NETs were quantified as the colocalization of extracellular DNA and MPO and normalized to neutrophil area and separately to neutrophil count. Neutrophils were determined based on shape and size to exclude extracellular DNA in neutrophil count and neutrophil area. The average of 3-4 images were used per patient for each condition. Geometric mean of NET area for each condition was compared by Mann-Whitney U test.

Results: Patients had moderate disease activity based on DAS28 score (median = 4.15, Table). As expected, PMA treatment significantly increased NETosis 2.8 to 5.9-fold compared to the untreated control (Figures 1 and 2). Treatment with 2-HOBA plus PMA reduced NETosis 15.7 to 27.9-fold compared to PMA alone (p< 0.0001), and reduced NETosis 4.7 to 5.5-fold compared to the untreated control (p< 0.001) (Figures 1 and 2).

Conclusion: 2-HOBA, a scavenger of reactive dicarbonyls like IsoLGs, prevented PMA-induced NETosis in neutrophils from patients with RA, and even reduced NETosis compared to baseline levels. Future studies will be dedicated to examining the in vivo effect of 2-HOBA on NETosis and clinical effect on disease in patients with RA and other autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/scavenging-isolevuglandins-with-2-hoba-decreases-in-vitro-neutrophil-extracellular-traps-in-cells-from-patients-with-rheumatoid-arthritis/