Apoptosis, cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Abstract

Background: Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods: Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results: The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions: In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

Figure 1

Cytokines/chemokines induced by transfection of NS1 mRNA of different influenza subtypes. NCI-H292 cells were transfected with NS1 mRNA derived from seven subtypes of influenza A including seasonal H1N1 (H1), H2N2, H3N2, H5N1, H7N3, H9N2 and 2009 pandemic H1N1 (pdmH1). Cytokine/chemokine levels are expressed as fold-changes compared to the non-transfected cell controls ± SD (p* < 0.05) measured at the same time points.

Figure 2

Proportion of overall apoptotic cells induced by transfection of NS1 mRNA of different influenza subtypes. NCI-H292 cells were transfected with NS1 mRNA derived from seven subtypes of influenza A including seasonal H1N1 (H1), H2N2, H3N2, H5N1, H7N3, H9N2 and 2009 pandemic H1N1 (pdmH1). CC represents non-transfected controls. The overall proportion of death cells was measured by propidium iodide staining using flow cytometry with the sub-G1 cell proportion counted ± SD (p* < 0.05).

Figure 3

Proportion of early apoptotic cells induced by transfection of NS1 mRNA of different influenza subtypes. NCI-H292 cells were transfected with NS1 mRNA derived from seven subtypes of influenza A including seasonal H1N1 (H1), H2N2, H3N2, H5N1, H7N3, H9N2 and 2009 pandemic H1N1 (pdmH1). CC represents non-transfected controls. Cells at early apoptotic phase as stained positive for annexin V but not propidium iodide were identified by flow cytometry ± SD (p* < 0.05).