Abstract
Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) particularly in the most severe form, idiopathic pulmonary fibrosis (IPF), although the underlying mechanisms remain unclear. Neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. Hypoxia has an important role in inflammation and may also contribute to pulmonary disease. We aimed to determine how neutrophil activation occurs in ILD and the relative importance of hypoxia. Using lung biopsies and bronchoalveolar lavage (BAL) fluid from ILD patients we investigated the extent of hypoxia and neutrophil activation in ILD lungs. Then we used ex vivo neutrophils isolated from healthy volunteers and BAL from patients with ILD and non-ILD controls to further investigate aberrant neutrophil activation in hypoxia and ILD. We demonstrate for the first time using intracellular staining, HIF-1α stabilization in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (PMA)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (NET) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). Hypoxia also increased neutrophil expression of the αM (+3.1-fold, p < 0.001) and αX (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by αMβ2 integrin activation and prevented by cation chelation. Finally, we observed NET-like structures in IPF lung sections and in the BAL from ILD patients, and quantification showed increased cell-free DNA content (+5.5-fold, p < 0.01) and MPO-citrullinated histone H3 complexes (+21.9-fold, p < 0.01) in BAL from ILD patients compared to non-ILD controls. In conclusion, HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β2 integrins. Our results identify a novel HIF-1α- αMβ2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD.
Highlights
The interstitial lung diseases (ILD) are a group of diffuse parenchymal lung disorders that can result in pulmonary fibrosis (PF) [1]
This staining pattern suggests that tissue-specific hypoxia and neutrophil recruitment may be a feature of the ILD lung
More recently the release of neutrophil extracellular trap (NET), essential for robust immune defense against pathogens, has been linked to increased immunopathology in patients with COVID-19, a disease characterized by neutrophilic inflammation and endothelial activation [53, 54]
Summary
The interstitial lung diseases (ILD) are a group of diffuse parenchymal lung disorders that can result in pulmonary fibrosis (PF) [1]. Despite recent advances in diagnostics and therapeutics, ILD is still associated with substantial morbidity and mortality [2]. The pathogenesis of IPF is unknown but is thought to involve a “frustrated repair” response to repetitive epithelial injury, with associations to genes and proteins linked to epithelial function, integrity and repair. Progressive epithelial damage, and abnormal wound repair leads to extensive scar formation and correlates, clinically, with worsening hypoxia. Further evidence from animal models suggests that hypoxia may contribute to a vicious cycle of disease progression [5]. This evidence has led to the view that hypoxia itself may contribute to worsening of PF but the mechanistic pathway is unknown
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