Abstract 130: Isolevuglandins Drive Neutrophil Migration In Hypertension And Are Essential For The Formation Of Neutrophil Extracellular Traps

Abstract

Introduction: Neutrophil extracellular traps (NETs) consist of decondensed chromatin and proteins including citrullinated histone 3, myeloperoxidase, and neutrophil elastase . Previous studies describe a role of neutrophils and NETs in hypertension and associated end-organ damage. Isolevuglandins (IsoLGs) are products of lipid peroxidation that play a role in immune activation in hypertension. We hypothesized that isoLGs are necessary for the process of NETosis and neutrophil migration in hypertension. Methods: C57BL/6 mice were treated with 490 ng/kg/min angiotensin (Ang) II and vehicle or the isoLG scavenger, 2-hydroxybenzylamine (2HOBA). Single cell sequencing was performed on spleens. NETs were quantified by flow cytometry and immunofluorescence. Human neutrophils were isolated and treated with ionomycin and vehicle or ethyl-2HOBA (Et2HOBA). Neutrophil chromatin expansion was determined by live cell confocal microscopy. Results: Single cell sequencing revealed increased splenic neutrophil accumulation following Ang II treatment. This was attenuated with 2HOBA co-treatment (Ang II 7.78%, Ang II + 2HOBA 2.46%, n=3). Flow cytometry revealed a marked increase in aortic NET accumulation that was reduced with 2HOBA (Sham 304 ± 110.6, Ang II 3,522 ± 928.6, Ang II + 2HOBA 1,007 ± 340.9 NETs, n=6-7). Immunofluorescence staining of kidneys revealed an increase in NET area in Ang II treated mice and a reduction with 2HOBA co-treatment (Sham 75.64 ± 27.2, Ang II 636.9 ± 105.0, Ang II + 2HOBA 14.98 ± 7.5 μm 2 , n=4-6). Ionomycin induced NETosis of isolated human neutrophils was associated with isoLG adduct accumulation that was attenuated with Et2HOBA co-treatment (54.24 ± 11.77% vs 1.78 ± 0.41%, n=5). Live cell confocal microscopy revealed NET area was increased 9.2-fold with ionomycin treatment compared to control. Pretreatment with Et2HOBA attenuated this response. Finally, isoLGs directly disrupt nucleosome assembly in vitro . Conclusion: These findings demonstrate an essential role of isoLGs in NETosis and neutrophil migration in hypertension. Moreover, they suggest a role of isoLG scavengers in the treatment of hypertension and additional NET-associated diseases including atherosclerosis and lupus.