IsoLGs (Isolevuglandins) Drive Neutrophil Migration in Hypertension and Are Essential for the Formation of Neutrophil Extracellular Traps

Abstract

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.

Keywords: Angiotensin II; extracellular traps; hypertension; isolevuglandins; neutrophil.

Figure 1:. Ang II-induced neutrophil expansion is inhibited by scavenging isoLGs.

Spleens from C57Bl/6 mice treated with sham, Ang II, or Ang II+2HOBA were harvested, T-cells were depleted and remaining cells were submitted for single cell sequencing. (A) Volcano plot identifying differentially expressed genes from total myeloid cells between Ang II+2HOBA and Ang II treated mice. (B) Genes downregulated by 2HOBA are enriched in granulocyte cells. (C) Expression of neutrophil specific genes Ly6G, Ngp, Lrg1, Camp, S100a8, and S100a9 are enriched in myeloid cells in Ang II treated mice and are reduced with 2HOBA co-treatment. (D) The percent of neutrophils is increased by Ang II and reduced by 2HOBA. (E) Dot plots of neutrophil clusters.

Figure 2:. 2HOBA increases bone marrow and splenic neutrophils and reduces circulating neutrophils.

C57Bl/6 mice were treated with sham, Ang II, or Ang II + 2HOBA. Tissue was harvested following perfusion of tissues. (A) Representative flow cytometry plots of neutrophils from peripheral blood. Quantitation of neutrophils in (B) peripheral blood, (C) bone marrow, and (D) spleen. Data is represented as averages ± SEM. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc test (n = 7-9, *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001). (E) Human neutrophil migration in response to CXCL8 was determined in the presence or absence of Et2HOBA by transwell assay. Quantitation of migrated neutrophils is represented. (n = 3-5, *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001)

Figure 3:. Ang II-induced peripheral tissue NETosis is reduced with 2HOBA in mice.

C57Bl/6 mice were treated with sham, Ang II, or Ang II + 2HOBA. Plasma and kidney were harvested. Plasma was analyzed for the presence of (A) H3-cit and (B) dsDNA. (C) Representative flow cytometry plots of NETs in aorta. (D) Quantitation of NETs/Aorta. (E) Representative image of renal tissue from an Angiotensin II treated mouse stained for DNA, myeloperoxidase (MPO), and H3-Cit. White bar = 80.2 μm (F) Quantitation of NET area per 10x field per animal. Data is represented as averages ± SEM. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc test (n = 4-8, *P < 0.05, **P < 0.01, **** P < 0.0001).

Figure 4:. IsoLGs are increased in ionomycin treated neutrophils and this is prevented with isoLG scavenging.

Neutrophils from healthy humans subjects were isolated and treated with control, ionomycin, or ionomycin and Et2HOBA were examined for isoLG adducts by flow cytometry using the D11 ScFv single chain antibody. The isoLG adduct fluorescence minus one (FMO) control is also represented. (A) Representative histograms of isoLG adduct accumulation. (B) Representative flow cytometry plots of isoLG-adduct accumulation. (C) Quantitation of isoLG-adduct⁺ neutrophils. (D) Mean fluorescence intensity of IsoLG adducts. Data is represented as averages ± SEM. Data were analyzed by paired 1-way ANOVA with Tukey’s post-hoc test (n = 5, *P < 0.05, **P < 0.01).

Figure 5:. Scavenging isoLGs prevents NETosis in isolated human neutrophils.

Neutrophils from healthy humans subjects were isolated and treated with control, Et2HOBA, 3 μM ionomycin, or 3 μM ionomycin+Et2HOBA. Cells were fixed and stained for H3-Cit, MPO, NE, and IsoLG to determine NETosis. (A) Representative images of colocalized H3-Cit, MPO, NE, and DNA representing NETosis in cultured human neutrophils. White bar = 100 μm. Quantitation of (B) total NET area /field and (C) MPO/cell count (D) NE/cell count (E) H3-Cit/cell count and (F) IsoLG/cell count. Data is represented as averages ± SEM. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc test (n = 5, **** P < 0.0001).

Figure 6:. IsoLGs induce neutrophil nuclear expansion and nucleosome disruption.

Isolated human neutrophils were imaged live every 300 seconds for 5400 seconds following treatment with ionomycin or ionomycin + Et2HOBA. (A) Confocal images of the individual neutrophils over time. White bar = 10 μm (B) Quantitation of nuclear area over time. Nuclear area of individual cells are represented as connected lines. (C) Quantitation of nuclear area over time represented as averages ± SEM. Data were analyzed by 2-way ANOVA. (n = 15-20, **** P time x nuclear area < 0.0001). (D) Nucleosome assembly assay with isoLG adducted components (E) Disruption of pre-formed nucleosomes with addition of increasing concentration of isoLG.