ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies
Lihui Wang, Zhichao Ai, Tariq Khoyratty, Kristina Zec, Hayley L. Eames, Erinke van Grinsven, Alison Hudak, Susan Morris, David Ahern, Claudia Monaco, Evgeniy B. Eruslanov, Raashid Luqmani, Irina A. Udalova
Lihui Wang, Zhichao Ai, Tariq Khoyratty, Kristina Zec, Hayley L. Eames, Erinke van Grinsven, Alison Hudak, Susan Morris, David Ahern, Claudia Monaco, Evgeniy B. Eruslanov, Raashid Luqmani, Irina A. Udalova
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Research Article Vascular biology

ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies

Abstract

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/–CD64– band neutrophils and CD66bhiCD15+CD10lo/–CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

Authors

Lihui Wang, Zhichao Ai, Tariq Khoyratty, Kristina Zec, Hayley L. Eames, Erinke van Grinsven, Alison Hudak, Susan Morris, David Ahern, Claudia Monaco, Evgeniy B. Eruslanov, Raashid Luqmani, Irina A. Udalova

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Figure 5

ER-Hoxb8–differentiated neutrophils faithfully recapitulate the phenotypes and functions of human neutrophil counterparts.

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ER-Hoxb8–differentiated neutrophils faithfully recapitulate the phenotyp...
(A) Morphology of ER-Hoxb8–differentiated neutrophils from D1 to D5 in the presence of G-CSF. Scare bars: 10 μm. One representative from 3–5 independent experiments is shown. (B) Quantification of neutrophil of different maturation stages in each ER-Hoxb8–differentiated neutrophil defined by days after G-CSF treatment. Data from at least 3 independent experiments are shown. (C) NET quantification by counting NET-forming cells stained positively with Cit-H3 and NE out of total DAPI+ cells. (D) Only mature D5 Hoxb8 neutrophils were able to perform phagocytosis. (E) Immature D3 Hoxb8 neutrophils were competent intracellular ROS producer comparable with mature D5 counterpart. (F) Immature D3 Hoxb8 neutrophils were competent to generate extracellular ROS over a period of 120 minutes. Five-minute intervals were used to plot each time point. (G) Extracellular ROS production at 120 minutes was compared across ER-Hoxb8 neutrophils of different days of maturation. (H) D3 Hoxb8 neutrophils showed higher endothelial permeability compared with neutrophils of other maturation stages in neutrophil-endothelial coculture permeability system. Data from at least 3 independent experiments are shown from C to H. (I) Detection of oxidized protein in HUVECs with Hoxb8 neutrophil coculture. Oxidized proteins potentially caused by extracellular ROS generated by D3 and D5 Hoxb8 neutrophils in the presence of 1 μM fMLP were detected via carbonylated groups in HUVEC. HUVEC without any treatment (–fMLP) and with fMLP alone (+fMLP) were used as negative controls. HUVECs treated with 10 μM H2O2 were used as the positive control. One representative from 3 independent experiments is shown. Two-way ANOVA was performed for statistical analysis in C, D, E, G, and H. Data are presented as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.