Identification of a Novel HIF-1α-αMβ2 Integrin-NET Axis in Fibrotic Interstitial Lung Disease

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 α M (+3.1-fold, p < 0.001) and α X (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by α M β 2 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 β 2 integrins. Our results identify a novel HIF-1αα M β 2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD.

Pathobiology of Airway Remodeling in Asthma: The Emerging Role of Integrins

“… 155 , 156 Furthermore, neutrophils and their products have been implicated in lung fibrogenesis in other chronic lung diseases such as interstitial lung disease (ILD). For example αMβ2 integrins can regulate neutrophil extracellular trap (NET) formation in ILD, 157 and secretory leukocyte protease inhibitor (SLPI), which inhibits neutrophil elastase, has differential effects on collagen expression in mouse lung tissue. 158 Previous work has shown that integrin expression by sputum neutrophils in asthmatic patients is aberrant compared with healthy controls, 159 however, whether such changes in integrin expression affect the overall activity of neutrophils in asthma and the impact this has on airway remodeling is yet to be elucidated.…”

A persistent neutrophil-associated immune signature characterises post-COVID19 pulmonary sequelae

“…to have functional roles in driving bleomycin-induced brosis in mice [45][46][47][48] Evidence of increased neutrophil activation and NET formation in lung tissue and BAL uid from brotic ILD patients has also been shown, possibly driven by hypoxia and upregulation of HIF-1α 49 . Furthermore, upregulation of genes related to pulmonary brosis, as well as those for neutrophil activation and NETosis have been demonstrated in acute fatal COVID-19 50 , further emphasising the central role of these processes in acute severe disease.…”

Pulmonary fibrosis from molecular mechanisms to therapeutic interventions: lessons from post-COVID-19 patients

“…Recently, Khawaja et al demonstrated that the hypoxic condition and HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium. Of note, in both PF and COVID-19, a progressive loss of lung functionality is evidenced that can lead to hypoxia phenomena sustaining the vicious loop of neutrophil activation [48] .…”