Introduction and Objectives Idiopathic pulmonary fibrosis is a chronic progressive lung disease of unknown cause. Its pathogenesis is poorly understood but activation of latent TGFß on lung epithelium is an important factor. TGFß must be activated, as it is secreted in a latent complex with its propeptide, the latency associated peptide, and the avß6 integrin is a key activator in the lung. The Influenza A virus is a single-stranded segmented RNA virus that infects epithelial cells leading to cell death and injury. Toll-like receptors (TLRs) detect pathogens, such as influenza. TLR3 activation has been found to increase RhoA activity. We previously showed that RhoA is a key intermediary in avß6 integrin-mediated TGFß activation. The aim of this study is to investigate whether influenza can activate TGFß and stimulate TLR3 leading to activation of TGFß through the avß6 integrin in epithelial cells.
Materials and Methods Immortalised human bronchial epithelial cells (iHBECs) were infected with influenza A (H2N3) virus at a multiplicity of infection 1 with, or without, the avß6 blocking antibody 6.3G9. iHBECs were also stimulated with the synthetic TLR3 ligand poly(I:C). TGFß activity was determined by: (1) immunoblotting for phosphorylated (phospho-) Smad2, and (2) Transformed mink lung cells (TMLC)-iHBEC cocultures. Infection efficiency was measured by Interferon ß mRNA levels by real-time qPCR.
Results Infection with H2N3 and stimulation with poly(I:C) led to increase in phospho-smad2 and luciferase activity in coculture indicating increase in TGFß activation levels in a dose- and time-dependent manner. In both cases this was blocked with the addition of 6.3G9. qPCR data following infection showed increased IFNß1 and PAI-1, indicating the ability of the virus to infect the cells and activate TGFß.
Conclusions Influenza infection and poly(I:C) activates TGFß in iHBECs in an avß6 integrin dependent manner. The data suggests a novel mechanism by which influenza infection of epithelial cells may promote lung fibrosis.
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