Therapeutic in vitro and in vivo approach of influenza virus infection by simultaneous reduction of virus titre and cytokine expression through inhibition of virus-induced NF-kappaB and Raf-MEK-ERK activation

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PintoRuth-2008-12-09.pdf (5.81 MB)

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2008

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DOI:
http://dx.doi.org/10.22029/jlupub-13381

Abstract

Influenza A virus (IAV) infection can cause severe pneumonia and lead to acute respiratory distress syndrome and ultimately death. Despite damage by viral replication, imbalanced production of anti-viral cytokines ( cytokine storm ) resulting in inflammation can also lead to severe lung destruction.Therapeutics as vaccines and anti-viral drugs target components of the virus itself resulting in resistant variants. Therefore new therapeutic measures are urgently needed. IAV has been shown to activate the NF-kappaB and MAPK (Raf/EK/ERK) signalling pathway. These pathways seem to have both pro- and anti-viral effects, by promoting nuclear export of the viral genome and by inducing expression of anti-viral pro-inflammatory factors. Therefore it was postulated that the inhibition of these signalling pathways will simultaneously reduce virus replication as well as modulate cytokine production without affecting host defence. Using specific IKK- (Bay-11-7082) and MEK- (U0126) inhibitors at non-toxic concentrations, I analysed the effect of NF-kappaB and MAPK pathway inhibition on propagation of a highly pathogenic avian influenza virus (strain A/FPV/Bratislava/79, H7N7) and a human influenza virus (strain A/PR/8/34, H1N1) and the virus-induced cytokine induction in infected human lung epithelia cells (A549) and mice primary alveolar epithelial cells (AECs), in vitro. Experiments were also performed in an in vivo mouse model for proof of principle applying the above inhibitors and the A/PR/8/34 virus for infection. Results show, (1) by western blot and transcription factor assay that both pathways (NF-kappaB and MAPK) are activated upon IAV infection in A549 cells; (2) that both inhibitors (IKK-, MEK-inhibitor), used at non-toxic concentrations, lead to decrease in signalling, virus titres (FFU assay) and (3) reduced cytokine expression (multiplex cytokine assay and ELISA), in vitro (A549 and AECs) as well as in vivo (mice). I also observed differences in the virus-induced (FPV and PR8) cytokine release when comparing different cell types (A549, AECs) as well as in the mouse model. The results demonstrate that inhibition of NF-kappaB and Raf/MEK/ERK pathway can be used to simultaneously reduce virus titres and modulate pro-inflammatory cytokine expression in vitro as well as in vivo. This could be of importance for future therapeutic strategies to treat influenza pneumonia and virus induced Cytokine Storm .

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