Hypoxia Increases the Potential for Neutrophil-mediated Endothelial Damage in Chronic Obstructive Pulmonary Disease.

Katharine M Lodge,Kylie B R Belchamber,Elizabeth Sapey,Victoria C Ridger,Andrew S Cowburn,Clara E Green,Wei Li,Arlette Vassallo,Alison M Condliffe,Zhen Tong,Danya Agha-Jaffar,Koralia Paschalaki,Merete Long,Bin Liu,Paul R Newby,Charlotte Summers,Robert A Stockley,Edwin R Chilvers

doi:10.1164/rccm.202006-2467oc

Abstract

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Highlights

Chronic obstructive pulmonary disease (COPD) is characterised by neutrophilic inflammation in the setting of tissue hypoxia, and by increased risk of cardiovascular disease (CVD) and pulmonary hypertension (PH)
Given the known role of the PI3K pathway in the hypoxic upregulation of degranulation from GM-CSF-primed neutrophils, and the aberrant chemotaxis of COPD patient neutrophils which could be corrected by PI3K inhibition [30], we explored whether inhibition of PI3K signalling pathways modulated the hypoxic response of Platelet activating factor (PAF)-primed neutrophils using PI3K isoform-selective small molecule inhibitors
Hypoxia increases the capacity for neutrophil supernatants to damage endothelial cells As COPD patients suffer increased cardiovascular morbidity compared to healthy controls and display a footprint of increased circulating protease activity (Fig 1), we investigated whether hypoxia increases the potential for neutrophil-mediated endothelial damage

Summary

Introduction

Chronic obstructive pulmonary disease (COPD) is characterised by neutrophilic inflammation in the setting of tissue (and often systemic) hypoxia, and by increased risk of cardiovascular disease (CVD) and pulmonary hypertension (PH). Patients with severe COPD are systemically hypoxic, significant tissue hypoxia (less than 1.3% oxygen) can occur even in mild disease, demonstrated in inflamed airways [5,6,7] and atherosclerotic vasculature [8], where neutrophils accumulate. Highly activated neutrophils can release granule contents extracellularly (degranulation), with potential for collateral tissue damage [2]. Circulating neutrophils primed for enhanced degranulation have been identified in exacerbating COPD patients [21], with potential to contribute to endothelial injury. Proteomic analysis reveals hypoxia-driven secretion of highly histotoxic proteins from healthy neutrophils, and a subset of these are further increased from COPD neutrophils. We identify increased levels of corresponding ‘hypoxic neutrophil’ histotoxic granule proteins in COPD plasma, together with endothelial injury biomarkers.

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Conclusion