Identification of a novel HIF-1α-α_Mβ₂ Integrin-NETosis axis in fibrotic interstitial lung disease

Abstract

Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) however, the underlying mechanisms remain unclear. We aimed to determine whether aberrant neutrophil activation is a feature of ILD and the relative role of hypoxia. We used lung biopsies and bronchoalveolar lavage (BAL) from ILD patients to investigate the extent of hypoxia and neutrophil activation in lungs of patients with ILD. We complemented these findings with ex vivo functional studies of neutrophils from healthy volunteers to determine the effects of hypoxia. We demonstrate for the first time HIF-1α staining in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous and phorbol 12myristate 13-acetate (PMA)-induced neutrophil extracellular trap (NET) release (NETosis), neutrophil adhesion, and trans-endothelial migration. Hypoxia also increased neutrophil expression of the α M and α X integrin subunits. Interestingly, NETosis was induced by α M β 2 integrin activation and prevented by cation chelation. Finally, NETs were demonstrated in the BAL from ILD patients, and quantification showed significantly increased cell-free DNA content and MPO-citrullinated histone H3 complexes in ILD patients compared to non-ILD controls. Our work indicates that 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-1α-Integrin-NETosis axis for future exploration in therapeutic approaches to fibrotic ILD.

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

“…We would like to acknowledge Professor Margaret Ashcroft and Dr. Luke Thomas (University of Cambridge) for advice and input on HIF, Dr. Andrew Smith and Professor Tony Segal (University College London) for their help with the neutrophil H 2 O 2 generation assay, Professor Nancy Hogg (The Francis Crick Institute) for reagents and helpful discussions, Professor Mikala Egeblad and Dr. Jean Albrengues (Cold Spring Harbor Laboratory, USA) for their help with staining lung tissue sections, and Dr. Guiseppe Ercoli (University College London) who helped with confocal imaging. This manuscript has been released as a pre-print at bioRxiv (88).…”