Influenza A virus (IAV) is of major concern worldwide, causing 3-5 million cases of severe disease and up to 600,000 deaths annually. Inflammation and cell death pathways play an important role in the induction of immune responses and viral clearance, however excess inflammation and epithelium damage are also associated with severe IAV infections. Ninjurin-1 (NINJ1) is a transmembrane protein that was initially identified in screen for spinal cord injury effectors and has since been implicated in several diseases such as atherosclerosis, sepsis, and various cancers. Most recently, NINJ1 has been identified as an executioner of cell lysis during pyroptosis and secondary necrosis in macrophages, through oligomerisation and the formation of large rings in in the cell membrane. As these cell death pathways are also active in IAV infection, we were interested in determining if NINJ1 contributed to IAV-induced cell lysis in epithelial cells, which are the main sites of viral replication.
Human normal bronchial epithelial cells (HBEC3-KT) infected with different strains of IAV (seasonal H1N1 and H3N2) all showed productive viral replication and virion release, as well as plasma membrane rupture (PMR) as demonstrated by LDH release. Inhibition of NINJ1 oligomerisation and PMR via glycine treatment reduced both virion and LDH release, although effects were strain dependent. We then generated NINJ1 KO cells using CRISPR/Cas9 and observed a partial reduction in LDH release after infection with IAV, which also correlated with decreased virus release. Work is ongoing to determine how and when NINJ1 is activated during IAV infection of epithelial cells and to further investigate the differences in PMR between the IAV strains in our HBEC3-KT model. Overall, we suggest that NINJ1 activation and induction of PMR is important during IAV infection both in driving inflammation and cell death but also potentially allowing dissemination throughout the lung epithelium.