The therapeutic role of tyrosine kinases in highly pathogenic influenza A/H5N1 virus infection

Abstract

Highly pathogenic avian influenza (HPAI) viruses such as the influenza A/H5N1 strain are able to cause severe illness upon infection, and it is believed that the “cytokine storm” induced during HPAI infection contributes, at least partially, to viral pathogenicity. During virus infection, intricate cell signalling pathways are stimulated which involve the activation of host receptor tyrosine kinases (RTKs), as well as non-receptor tyrosine kinases (nRTKs). Also, due to an increased emergence of antiviral drug resistant influenza viruses, commonly administered antivirals such as oseltamivir are ineffective against these resistant strains.

The development of new drugs for influenza treatment is time-consuming and cost intensive, hence the repurposing of readily available drug compounds especially those that have reached the phase of clinical trials with known pharmacokinetics and pharmacodynamics is considered an attractive alternative as medication for influenza illness. Here, we demonstrate the use of a drug compound that targets the kinase activity of nRTK focal adhesion kinase (FAK), and is able to exert an immunomodulatory effect upon the reduction of its kinase activity during influenza A virus (IAV) infection of the human monocyte-derived human macrophages and human primary alveolar epithelial cells (hAECs). Also, through the use of the PathScan® Intracellular Signalling Array, signalling molecules STAT1, AKT1S1, BAD, HSPB1, and RPS6, were found to be activated upon H5N1 infection in human macrophages but only STAT1 was observed to be activated upon infection in hAECs. In addition, their activation could be significantly reduced upon FAKi treatment, except for HSPB1. As well, siRNA treatment of a select number of previously mentioned targets in H5N1-infected human macrophages led to the reduction of pro-inflammatory cytokine and chemokine mRNA levels, and viral M gene levels. Additionally, the therapeutic effects of FAKi treatment during HPAI H5N1 virus infection was evaluated using in vivo model. Out of all treatments, mice administered combination treatment (antiviral and FAK inhibitor) had the highest endpoint body weight, reduced levels of pro-inflammatory cytokines and chemokines, and reduced immune cell populations in the bronchoalveolar lavage fluid. Lastly, the roles of RTKs during IAV were investigated, in particular the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases and platelet-derived growth factor receptor (PDGFR). Out of all members of the TAM family, only Axl expression was significantly upregulated upon H5N1 virus infection in both types of primary cells. Also, there was selective upregulation of PDGFR expression and its ligands during IAV infection in human macrophages. PDGFRB expression was differentially upregulated compared to its counterpart, PDGFRA, whereas PDGFA and PDGFD mRNA expression were upregulated compared to mock infection. As well, PDGFR inhibitors tyrphostin A9 and imatinib, which was originally developed for the treatment of cancers, were both found to exhibit immunomodulatory activity upon early treatment of macrophages infected with H5N1 virus.

In conclusion, nRTKs and its counterpart RTKs play important roles during influenza infection through the dysregulation of host cell signalling pathways. The reduction of tyrosine kinase activity through treatment of repurposed drug compounds provides therapeutic benefits, leading to the identification of possible novel treatments for HPAI virus infection.