ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies
Lihui Wang, … , Raashid Luqmani, Irina A. Udalova
Lihui Wang, … , Raashid Luqmani, Irina A. Udalova
Published September 22, 2020
Citation Information: JCI Insight. 2020;5(20):e139163. https://doi.org/10.1172/jci.insight.139163.
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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 4

CD10lo LDNs are potent ROS producers but deficient in some innate immune functions.

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CD10lo LDNs are potent ROS producers but deficient in some innate immune...
(A) NET quantification by counting NET-forming cells stained positively with Cit-H3 and NE out of total DAPI+ cells. Three independent experiments were performed on FACS purified neutrophil populations from 3 GCA patients. (B) Phagocytosis was FACS quantified by the intake of pHrodo red E. coli bioparticles across the neutrophil populations. Mature but not immature neutrophils were capable of efficient phagocytosis. Three independent experiments were performed on FACS purified neutrophil populations from 3 GCA patients. (C) Intracellular ROS production was FACS quantified by green fluorescence converted from dihydrorhodamine 123 (DHR123) in the presence of ROS. Both mature and immature LDNs could generate ROS intracellularly, comparable with mature NDNs under PMA stimulation. Four independent experiments were performed on FACS purified neutrophil populations from 4 GCA and 4 HC donors. (D) Extracellular ROS was measured using OxyBURST Green H2HFF BSA. In the presence of 1 μM fMLP, immature CD10loCD64–CD16hi LDNs showed an increased and sustained release of ROS in comparison with CD10hi LDNs and CD10hi NDNs up to 120 minutes. (E) Extracellular ROS production at 120 minute with or without fMLP treatment. (F) Vascular damage was quantified by a permeability assay of endothelial barrier in a neutrophil-endothelial coculture system. Both CD10loCD64–CD16hi and CD10loCD64+CD16lo immature LDNs showed higher permeability compared with CD10hi NDNs, indicating their potential roles associated with vascular damage in vasculitis. Three independent experiments were performed on FACS purified neutrophil populations from 3 GCA patients in D, E, and F. Two-way ANOVA was used for statistical analysis in A, B, C, E, and F. Data are presented as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.