Background: IsoLGs (isolevuglandins) are electrophilic products of lipid peroxidation formed in the presence of reactive oxygen species. IsoLGs contribute to hypertension by an unknown mechanism. Studies have shown that reactive oxygen species production drives the formation of neutrophil extracellular traps (NETs) and that NETs accumulate within the aorta and kidneys of patients with hypertension. The purpose of this study was to determine the role of isoLGs in neutrophil migration and NET formation (NETosis) in hypertension. Methods: Mice were treated with Ang II (angiotensin II) and the specific isoLG scavenger 2-hydroxybenzylamine and examined for tissue neutrophil and NET accumulation by single-cell sequencing and flow cytometry. Isolated human neutrophils were studied to determine the role of isoLGs in NETosis and neutrophil chromatin expansion by immunofluorescence and live cell confocal microscopy. Results: Single-cell sequencing performed on sham, Ang II, and Ang II+2-hydroxybenzylamine treated mice revealed neutrophils as a primary target of 2-hydroxybenzylamine. Peripheral neutrophil migration, aortic NET accumulation, and renal NET accumulation is blocked with 2-hydroxybenzylamine treatment. In isolated human neutrophils, isoLGs accumulate during NETosis and scavenging of isoLGs prevents NETosis. IsoLGs drive neutrophil chromatin expansion during NETosis and disrupt nucleosome structure. Conclusions: These observations identified a critical role of isoLGs in neutrophil migration and NETosis in hypertension and provide a potential therapy for NET-associated diseases including hypertension and associated end organ damage.

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.

Isolevuglandins (isoLGs) are lipid aldehydes that form in the presence of reactive oxygen species (ROS) and drive immune activation. We found that isoLG-adducts are presented within the context of major histocompatibility complexes (MHC-I) by an immunoproteasome dependent mechanism. Pharmacologic inhibition of LMP7, the chymotrypsin subunit of the immunoproteasome, attenuates hypertension and tissue inflammation in the angiotensin II (Ang II) model of hypertension. Genetic loss of function of all immunoproteasome subunits or conditional deletion of LMP7 in dendritic cell (DCs) or endothelial cells (ECs) attenuated hypertension, reduced aortic T cell infiltration, and reduced isoLG-adduct MHC-I interaction. Furthermore, isoLG adducts structurally resemble double-stranded DNA and contribute to the activation of STING in ECs. These studies define a critical role of the immunoproteasome in the processing and presentation of isoLG-adducts. Moreover they define a role of LMP7 as a regulator of T cell activation and tissue infiltration in hypertension.

Autoimmune and inflammatory diseases such as obesity and cancer are highly linked to altered bone growth and osteoporosis. Hypertension (HTN) is an inflammatory condition associated with reduced bone mineral density and increased risk for fragility fractures in adults. As cases of pediatric HTN rise in the United States, HTN may increase bone loss in children and future risk for osteoporosis. We hypothesized that HTN-induced osteoporosis in young mice reduces bone quality like that of aging. To assess this, 3.5- or 16-month-old male C57BL/6J mice received Angiotensin (Ang) II (490 ng/kg/day) or vehicle infusion by subcutaneous osmotic mini-pumps. After 6-weeks of infusion, the lumbar vertebral bodies (VB) were harvested, and analyzed by high-resolution micro-computed tomography and finite element analysis. In young mice, Ang-II induced HTN caused reductions in bone volume fraction (33.4 ± 0.8 vs 25.4 ± 1.1 %, p<0.0001) and trabecular thickness (66.4 ± 1.1 vs 54.3 ± 1.5 μm, p<0.0001). HTN reduced the estimated failure force (24.3 ± 0.7 vs 16.1 ± 1.1 N, p<0.0001) in young mice. In contrast to young mice, in aged mice Ang-II infusion did not induce bone loss; however, at baseline, the old mice exhibited reduced bone quality like that of hypertensive young mice: bone volume fraction (22.5 ± 0.7 vs 25.4 ± 1.1 %, p=0.2928), trabecular thickness (58.0 ± 1.5 vs 54.3 ± 1.1 μm, p=0.3866), and estimated failure force (17.6 ± 0.8 vs 16.1 ± 1.1 N, p=0.6517). To ascertain the impact of inflammation in the bone marrow microenvironment, the bone marrow was analyzed by flow cytometry. The number of CD4 + T cells producing IL-17A was increased in hypertensive vs normotensive young mice (230.0 ± 8.9 vs 167.3 ± 16.6 cells per 1 million events, p=0.0035), as were dendritic cells (408.3 ± 29.1 vs. 229.1 ± 20.5 cells per 1 million events, p<0.0001). CD4 + T cells producing IFN-γ also rose (2845 ± 129 vs 2437 ± 174 cells per 1 million events, p=0.07) in young hypertensive mice. Both normotensive and hypertensive old mice experienced overactive inflammatory responses compared to the young group. Thus, HTN mimics aspects of aging on bone health, causing inflammation in the bone marrow and osteoporosis. Efforts to detect and treat HTN in children may deter untoward events related to bone loss in adulthood.