Cy Cheung

Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?

BACKGROUND In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role. METHODS We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor alpha (TNF alpha) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-alpha response. FINDINGS The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta. The concentration of TNF-alpha protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF alpha induced by the virus. INTERPRETATION The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF alpha. This characteristic may contribute to the unusual severity of human H5N1 disease.

Re-emergence of fatal human influenza A subtype H5N1 disease

Summary Human disease associated with influenza A subtype H5N1 reemerged in January, 2003, for the first time since an outbreak in Hong Kong in 1997. Patients with H5N1 disease had unusually high serum concentrations of chemokines (eg, interferon induced protein-10 [IP-10] and monokine induced by interferon γ [MIG]). Taken together with a previous report that H5N1 influenza viruses induce large amounts of proinflam-matory cytokines from macrophage cultures in vitro, our findings suggest that cytokine dysfunction contributes to the pathogenesis of H5N1 disease. Development of vaccines against influenza A (H5N1) virus should be made a priority.

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.

BackgroundFatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.MethodsWe used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.ResultsWe demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.ConclusionThe H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid α2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

p38 mitogen-activated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus H5N1

ABSTRACT Avian influenza A virus subtype H5N1 can infect humans to cause a severe viral pneumonia with mortality rates of more than 30%. The biological basis for this unusual disease severity is not fully understood. We previously demonstrated that in contrast to human influenza A virus subtypes including H1N1 or H3N2, the H5N1 virus associated with the “bird flu” outbreak in Hong Kong in 1997 (H5N1/97) hyperinduces proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), in primary human macrophages in vitro. To delineate the molecular mechanisms involved, we analyzed the role of transcription factor NF-κB and cellular kinases in TNF-α dysregulation. H5N1 and H1N1 viruses did not differ in the activation of NF-κB or degradation of IκB-α in human macrophages. However, we demonstrated that unlike H1N1 virus, H5N1/97 strongly activates mitogen-activated protein kinase (MAPK), including p38 MAPK and extracellular signal-regulated kinases 1 and 2. Specific inhibitors of p38 MAPK significantly reduced the H5N1/97-induced TNF-α expression in macrophages. Taken together, our findings suggest that H5N1/97-mediated hyperinduction of cytokines involves the p38 MAPK signaling pathway. These results may provide insights into the pathogenesis of H5N1 disease and rationales for the development of novel therapeutic strategies.

Induction of Proinflammatory Cytokines in Primary Human Macrophages by Influenza A Virus (H5N1) Is Selectively Regulated by IFN Regulatory Factor 3 and p38 MAPK

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-α, IFN-β, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease. Here we show that TNF-α, IFN-β, and IFN-λ1 are the key mediators directly induced by the H5N1 virus in primary human macrophages. In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3). IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-β, IFN-λ1, and MCP-1 but only to a partial reduction of TNF-α. IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus. We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-β, IFN-λ1, and MCP-1 but only partly control TNF-α induction. A more precise identification of the differences in the regulation of TNF-α and IFN-β could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.

Human herpesvirus 6 and human herpesvirus 7 infections in bone marrow transplant recipients

Human cytomegalovirus (HCMV), human herpesvirus‐6 (HHV‐6), and human herpesvirus‐7 (HHV‐7) DNA in peripheral blood leukocytes (PBL) of 61 bone marrow transplant recipients was monitored weekly during the first 12 weeks post‐transplantation by a nested polymerase chain reaction (PCR). Thirty‐seven (61%), 17 (28%), and 32 (53%) of patients had one or more PBL specimens positive for HCMV, HHV‐6 or HHV‐7 DNA, respectively. HHV‐7 DNA in PBL during the early post‐transplant period was associated with a longer time to neutrophil engraftment (mean 28.8 days vs 19.8 days; P = 0.01). In two patients who failed to engraft, HHV‐6 DNA and HHV‐7 DNA was detected in plasma and PBL, respectively, early in their post‐transplant period. Patients with HCMV disease were more likely to have concurrent HHV‐7 DNA in PBL prior to onset of disease than were patients with asymptomatic HCMV infection, suggesting that HHV‐7 may be a cofactor in the progression from HCMV infection to HCMV disease. In the 17 patients (179 specimens) in whom viral DNA in plasma was studied (in addition to PBL), a positive result was found only in 3. In each, viral DNA in plasma appeared to correlate with clinically significant disease. HHV‐7 DNA in plasma was associated with encephalitis in an allograft recipient. J. Med. Virol. 53:295–305, 1997.

Systems-Level Comparison of Host-Responses Elicited by Avian H5N1 and Seasonal H1N1 Influenza Viruses in Primary Human Macrophages

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-α genes. A network-based analysis suggests that the synergy between IFN-β and TNF-α results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.

Hyperinduction of Cyclooxygenase-2-Mediated Proinflammatory Cascade: A Mechanism for the Pathogenesis of Avian Influenza H5N1 Infection

The mechanism for the pathogenesis of H5N1 infection in humans remains unclear. This study reveals that cyclooxygenase-2 (COX-2) was strongly induced in H5N1-infected macrophages in vitro and in epithelial cells of lung tissue samples obtained during autopsy of patients who died of H5N1 disease. Novel findings demonstrated that COX-2, along with tumor necrosis factor alpha and other proinflammatory cytokines were hyperinduced in epithelial cells by secretory factors from H5N1-infected macrophages in vitro. This amplification of the proinflammatory response is rapid, and the effects elicited by the H5N1-triggered proinflammatory cascade are broader than those arising from direct viral infection. Furthermore, selective COX-2 inhibitors suppress the hyperinduction of cytokines in the proinflammatory cascade, indicating a regulatory role for COX-2 in the H5N1-hyperinduced host proinflammatory cascade. These data provide a basis for the possible development of novel therapeutic interventions for the treatment of H5N1 disease, as adjuncts to antiviral drugs.

Early Diagnosis of Primary Human Herpesvirus 6 Infection in Childhood: Serology, Polymerase Chain Reaction, and Virus Load

Qualitative and quantitative polymerase chain reaction (PCR) for human herpesvirus 6 (HHV-6) DNA in whole blood and plasma was correlated with serology and clinical assessment in 143 children hospitalized for undifferentiated febrile illness to evaluate options for diagnosis of primary HHV-6 infection on the acute blood specimen. PCR and serology for HHV-7 were done in parallel to define serologic cross-reactions. Using HHV-6 seroconversion as the reference standard, detection of HHV-6 DNA in whole blood in the absence of antibody in the plasma was the most reliable evidence of primary HHV-6 infection. Detection of HHV-6 DNA in plasma and a high virus load in whole blood (>3.3 log10 copies/5 microL) had a sensitivity of 90% and 100%, respectively, in diagnosing primary HHV-6 infection. However, both were occasionally found in patients with other infections, possibly associated with HHV-6 reactivation. Maternal antibody may confound interpretation of serology in patients under 3 months of age.