Pro-inflammatory cytokines/ chemokines production after infection of A549 Human epithelial cell line with different strains of human influenza viruses

BACKGROUND. Influenza virus infection causes early release of cytokines and chemokines that are important for controlling the spread of the virus. However, an excessive release of mediators may lead to tissue damage. Generally, disease severity correlates strongly with cytokine levels and markedly elevated levels of cytokines and chemokines have been observed following infection with highly pathogenic avian H5N1 virus and with pandemic H1N1 virus of 1918 Spanish flu. Respiratory epithelial cells, the primary targets for influenza virus infection, have been shown to up-regulate pro-inflammatory mediators following in vitro infection with influenza viruses. Because of the importance of knowing factors contributing to the pathogenesis of influenza viruses, we investigated the abilities of different A and B human influenza viruses isolated during the past years in Italy to induce pro-inflammatory cytokine and chemokine expression in the human epithelial cell line A549. METHODS. MDCK cells were used for propagating viruses and virus titrations. For the measurement of pro-inflammatory cytokine/chemokines levels A549 cells were infected at an m.o.i. of 1. After 1 hour of incubation, the inoculum was removed, fresh culture medium with 2% fetal bovine serum was added, and cells were incubated at 37°C in 5% CO2. Cell-culture supernatants were harvested at 4, 12, 24 and 48 hours post-infection and analyzed for the presence of IL-6, IL-8, TNF-alpha, RANTES and MCP-1 using specific immunoenzymatic assays. Mock infected cells served as control. The results reported are those obtained from three different experiments and the values are expressed as percent increase in cytokine production respect to mock-infected cells. RESULTS AND CONCLUSIONS. All influenza viruses tested were capable of inducing increases in the production of IL-6, IL-8, TNF-alpha, RANTES and MCP-1 after infection of A549 cells. Moreover higher levels of some of these factors were produced upon viral infection with the pandemic influenza virus (H1N1/2009) as compared with an antigenically related epidemic descendent virus or with an influenza B virus isolated from a patient with severe influenza as compared with B virus responsible of mild illness. Thus, our in vitro studies show an influenza virus regulated expression of pro-inflammatory cytokines/chemokines and suggest the contribution of these mediators to the entity of clinical manifestation.