Neutrophil biophysical properties and the impact of hypoxia in ANCA Associated Vasculitis
File(s)
Author(s)
Pisacano, Noelle
Type
Thesis
Abstract
The anti-neutrophil cytoplasm antibody (ANCA) associated vasculitides (AAV) are a group of systemic autoimmune diseases characterised by inflammation of small blood vessels and the presence of circulating ANCA. Neutrophils are central to disease pathogenesis; they are target of the autoantibody, ANCA, and contribute to vascular damage. Cell deformability has a marked impact on trafficking of neutrophils through microvasculature and neutrophil-endothelial interactions. Inflamed vasculature is characteristically hypoxic, a stimulus which enhances neutrophil secretion and delays apoptosis. The aim of this study was to investigate morpho-rheological phenotype of neutrophils from patients with AAV, and how this may contribute to endothelial injury in hypoxic environments.
A novel technique, Real Time-Deformability Cytometry (RT-DC), was used to identify mechanical cell signatures. Results presented identified a distinct morpho-rheological phenotype of neutrophils in patients with active AAV, compared to those in remission and healthy controls (HC), with changes in cell size, deformation, and elasticity. In keeping with this, in vitro stimulation of neutrophils with MPO- or PR3-ANCA IgG leads to decreased deformability of cells in comparison to HC-IgG stimulated cells. MPO- and PR3-ANCA promote NETosis and actin polymerisation; both mechanisms are reduced under physiological venous oxygen tension (5% O2) but are enhanced under pathological hypoxia (1% O2). MPO- or PR3-ANCA stimulation and hypoxia synergise to impact neutrophil-endothelial cell interactions, demonstrated by changes in adhesion protein secretion levels and neutrophil transendothelial migration through glomerular endothelial cells in co-culture.
In conclusion, ANCA- and hypoxia-induced morpho-rheological changes in neutrophils may influence cell trafficking through microvasculature and potential for neutrophil-mediated endothelial injury, thus contributing to the pathogenesis of AAV. It is possible distinct biophysical properties of neutrophils in patients with aAAV promote endothelial injury. Collectively these findings indicate biomechanical phenotyping can identify pro-inflammatory neutrophils with increased endothelial damage capacity in active vasculitis, with implications for diagnosis and guiding response to treatment.
A novel technique, Real Time-Deformability Cytometry (RT-DC), was used to identify mechanical cell signatures. Results presented identified a distinct morpho-rheological phenotype of neutrophils in patients with active AAV, compared to those in remission and healthy controls (HC), with changes in cell size, deformation, and elasticity. In keeping with this, in vitro stimulation of neutrophils with MPO- or PR3-ANCA IgG leads to decreased deformability of cells in comparison to HC-IgG stimulated cells. MPO- and PR3-ANCA promote NETosis and actin polymerisation; both mechanisms are reduced under physiological venous oxygen tension (5% O2) but are enhanced under pathological hypoxia (1% O2). MPO- or PR3-ANCA stimulation and hypoxia synergise to impact neutrophil-endothelial cell interactions, demonstrated by changes in adhesion protein secretion levels and neutrophil transendothelial migration through glomerular endothelial cells in co-culture.
In conclusion, ANCA- and hypoxia-induced morpho-rheological changes in neutrophils may influence cell trafficking through microvasculature and potential for neutrophil-mediated endothelial injury, thus contributing to the pathogenesis of AAV. It is possible distinct biophysical properties of neutrophils in patients with aAAV promote endothelial injury. Collectively these findings indicate biomechanical phenotyping can identify pro-inflammatory neutrophils with increased endothelial damage capacity in active vasculitis, with implications for diagnosis and guiding response to treatment.
Version
Open Access
Date Issued
2023-12-19
Date Awarded
01/06/2024
Advisor
Lodge, Katharine
Prendecki, Maria
Cowburn, Andrew
Grant Number
Edwin Chilvers-WHRL-F26143-165138
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)