Mechanisms of pathogenic avian influenza-induced immune responses in human cells

by Mok, Ka-pun

(Uncorrected OCR) Abstract of thesis entitled ?echanisms of pathogenic avian influenza-induced immune responses in human cells?submitted by Mok Ka Pun Chris for the Degree of Master of Philosophy at The University of Hong Kong in December 2004 Since the first avian influenza virus transmissions to humans were documented in 1997, there have been repetitive resurgences of the infection in Hong Kong and South East Asia. However, the pathogenesis of this highly pathogenic virus, which causes high morbidity and mortality in the infected patients, has not been fully investigated. It is well known that apoptosis and cytokine induction are the two major cellular events in influenza virus-infected monocytes/macrophages. Investigation on the mechanisms of these two processes in avian influenza virus-infected macrophages may provide new insights into the immunological responses and pathogenesis of the pathogenic avian influenza viruses in humans. Previous reports showed that human strains of influenza virus could induce apoptosis in primary human monocytes. Here, we investigated whether there is a differential onset of apoptosis in H5N1/97-infected human cells in comparison with H1N1 infections. By using Western analysis and electron microscopy, we demonstrated - 1 - that the H5N1/97-infected macrophages had a slower onset of apoptosis when compared to the cells infected by H1N1 viruses. Concomitantly, there was a delay in the activation of the caspase cascades, initiators of apoptosis, in H5N1 infection versus that of H1N1. Similarly, we have observed deferred onset of apoptosis in cells infected by other avian influenza virus subtypes, including those that are known precursors of H5N1/97. H5N1/97 causes hyper-induction of cytokine expression including interferons (IFNs), chemokines and TNF-a in primary human macrophages, as compared to the human influenza H1N1 virus. However, the mechanisms on the induction of these cytokines in human macrophages by avian influenza viruses are still not known. I hypothesize that the induction of IFN-?by avian influenza viruses, including H5N1/97 and its precursor H9N2 (G1/97), involves the activation of NF-?B and IRF-7. By using PCR, electrophoretic mobility shift assay (EMSA) and Western analysis, the roles of NF-?B and IRF-7 were shown to be involved in the induction of IFN-? The results showed that the activation level of NF-?B is similar in cells infected with avian or human strains of influenza viruses. Moreover, the transcription of IFN-?mRNA in avian influenza virus-infected macrophages was significantly abrogated by a NF-?B inhibitor, caffeic acid phenethyl ester (CAPE), suggesting that the induction is through NF-?B. In contrast, significant transcriptional activation of IRF-7 was observed in - 2 - avian influenza virus-infected cells, as compared to the human influenza virus-infected ones. Taken together, our results suggest that the levels of cellular activation, including cytokine induction and apoptosis, in avian influenza virus-infected human macrophages are different to that of the human influenza virus-infected cells. Interestingly, on the one hand, cytokine expression in avian-influenza virus-infected human macrophages is super-induced. On the other hand, the apoptosis including the caspase family triggered by the avian virus is delayed. The differential onset of apoptosis in H5N1-infected cells, as compared to H1N1, may account for longer cell survival resulting in super-induction of cytokine synthesis. - 3 -