Final Report Summary - FLU-MACROPHAGE (Alveolar macrophage involvement in the pathogenesis of influenza A virus)
Summary report
% L FP7-PEOPLE-IEF-2008
Alveolar macrophage(AM) involvement in the pathogenesis of influenza A virus
The project as initially designed aimed to determine the role of macrophages in the early response and the pathology of influenza.
Aim one: To determine the role of AM as a host for influenza during the early steps of lung infection.
In the early spring of 2009 influenza virus H1N1 was detected in the human population in North America. Within a few months, this novel virus had spread to both humans and pigs on all continents, attaining pandemic status in June 2009. This pandemic underscored the importance of the pig population both as a reservoir and a mixing vessel for influenza strains, and provided added impetus for comparing the biology of influenza in human and pig populations. In view of determining the relevance of the pig model of influenza infection to human biology, we undertook in vitro studies of macrophage infection. We found that pig alveolar macrophages were resistant to replication of a human H3N2 viral strain (A/Udorn/72), mimicking the selective resistance of human lung macrophages compared to blood cells. Although a complete replicative cycle was not supported by pig AM, H3N2 did replicate within the cells as shown by viral nucleoprotein (NP) staining, suggesting that H3N2 could still hijack and alter the cell biology even though AM do not release functional viral particles.
The availability of recently isolated human pandemic H1N1 virus (A/England/195/2009) allowed us to compare susceptibility of pig AM to different viral strains. Like human, pig AM were resistant to pandemic H1N1 infection, as has been shown in human AM.
Aim two: To determine the response of AM to influenza infection and their role in the evolution of disease.
We participated in an important in vivo pig study of pandemic H1N1 infection led by the veterinary laboratories agency. Correlates of infection, including clinical signs, virology, molecular biology, pathology and host responses supported the idea that the pig has a dual role as a valuable model for studying human disease and as a primary reservoir host. These studies essentially confirmed the low virulence of the pandemic virus. In parallel, through collaboration with the RIKEN omics science centre in Yokohama, the gene expression responses of human macrophages to flu infection was compared by cap-analysis of gene expression (CAGE). In this study a large number of profoundly susceptible flu cases (previously-well young people who required intensive care) were recruited. The objective was to determine differences in the regulation of expression of innate immune genes in these individuals that would underlie their enhanced susceptibility to severe disease. Human monocyte-derived macrophages cells from these donors were treated with two strains of virus, H3N2 (Udorn) and pandemic H1N1, and gene expression was determined over 48 hours.
The massive amount of data retrieved from the CAGE project is currently being analysed using cutting-edge bioinformatics tools. Key insights are already beginning to be apparent. One is the inter-individual variety in human response to infection, which can range from varying levels of activation of specific genes to different time courses of activation. We will be interested in correlating the host response to the actual medical history of the donors and determine whether gene expression signatures in macrophages can be predictive of the course of the pathology in the organism as a whole. Parallel studies in the pig are directed towards understanding the similarities and differences that allow the pig to act as a reservoir
Aim three: To evaluate the impact of alveolar influenza infection on the susceptibility to bacterial co-infection and to investigate the underlying mechanisms.
The CAGE and microarray studies are also allowing us to compare detailed bacterial lipopolysaccharides (LPS) and influenza-induced time courses in multiple individuals. The initial data indicates that although influenza infection and LPS induce many genes in common early after infection/stimulation, this initial response is curtailed dramatically during flu infection while continuing unabated in the LPS time course. This important difference suggests that genes required in the host response to bacterial infection are specifically suppressed during influenza infection. We have also noted distinct promoter preferences between influenza and LPS stimulation. Further analysis is underway to elucidate the interaction between influenza and bacterial infections, as well as to determine the inter-individual variability of host response.
Aim four: To define ways in which alveolar macrophages can be protected from influenza infection and limit systemic damage caused by the massive cytokine response.
Because of the shift in the model and ambition of the project, this objective has not yet been explored. The understanding of the role of macrophages gained from the studies described in Aims one-three should reveal therapeutic avenues to test in the context of both human and pig flu infection.
Conclusions
The comparative analysis of human and pig response to influenza and bacterial infection is key to understanding how host response is specifically tailored to diverse types of infection, as well as defining influenza-specific targets for therapeutic intervention. We anticipate major publications describing the outcomes of this project within the next 12 months.
As a personal outcome, following the completion of this Fellowship I was recruited to a position expanding on previous work on candidate susceptibility loci significantly associated with severe influenza in humans. Working on both the pig and human influenza models, we aim to identify and understand the functional role of variants associated with inadequate host response to flu infection, and define options for intervention.
Socio-economic impact
Translating genomic information into therapeutic avenues of intervention was a major aspect of the studies undertaken. While we have not yet reached the understanding required to explore therapeutic intervention, it is expected that the follow-up studies to this Fellowship will allow us to identify specific targets predictive of severe outcome of influenza infection.
A better understanding of host susceptibility determinants is paramount to the control of influenza mortality in extremely susceptible hosts, be they human or animal. Considering the important economic impact of both endemic and pandemic influenza, development of susceptibility assays that will help focus the preventative efforts during flu season or in the event of another global pandemic are essential for a more efficient use of available human and material resources.
% L FP7-PEOPLE-IEF-2008
Alveolar macrophage(AM) involvement in the pathogenesis of influenza A virus
The project as initially designed aimed to determine the role of macrophages in the early response and the pathology of influenza.
Aim one: To determine the role of AM as a host for influenza during the early steps of lung infection.
In the early spring of 2009 influenza virus H1N1 was detected in the human population in North America. Within a few months, this novel virus had spread to both humans and pigs on all continents, attaining pandemic status in June 2009. This pandemic underscored the importance of the pig population both as a reservoir and a mixing vessel for influenza strains, and provided added impetus for comparing the biology of influenza in human and pig populations. In view of determining the relevance of the pig model of influenza infection to human biology, we undertook in vitro studies of macrophage infection. We found that pig alveolar macrophages were resistant to replication of a human H3N2 viral strain (A/Udorn/72), mimicking the selective resistance of human lung macrophages compared to blood cells. Although a complete replicative cycle was not supported by pig AM, H3N2 did replicate within the cells as shown by viral nucleoprotein (NP) staining, suggesting that H3N2 could still hijack and alter the cell biology even though AM do not release functional viral particles.
The availability of recently isolated human pandemic H1N1 virus (A/England/195/2009) allowed us to compare susceptibility of pig AM to different viral strains. Like human, pig AM were resistant to pandemic H1N1 infection, as has been shown in human AM.
Aim two: To determine the response of AM to influenza infection and their role in the evolution of disease.
We participated in an important in vivo pig study of pandemic H1N1 infection led by the veterinary laboratories agency. Correlates of infection, including clinical signs, virology, molecular biology, pathology and host responses supported the idea that the pig has a dual role as a valuable model for studying human disease and as a primary reservoir host. These studies essentially confirmed the low virulence of the pandemic virus. In parallel, through collaboration with the RIKEN omics science centre in Yokohama, the gene expression responses of human macrophages to flu infection was compared by cap-analysis of gene expression (CAGE). In this study a large number of profoundly susceptible flu cases (previously-well young people who required intensive care) were recruited. The objective was to determine differences in the regulation of expression of innate immune genes in these individuals that would underlie their enhanced susceptibility to severe disease. Human monocyte-derived macrophages cells from these donors were treated with two strains of virus, H3N2 (Udorn) and pandemic H1N1, and gene expression was determined over 48 hours.
The massive amount of data retrieved from the CAGE project is currently being analysed using cutting-edge bioinformatics tools. Key insights are already beginning to be apparent. One is the inter-individual variety in human response to infection, which can range from varying levels of activation of specific genes to different time courses of activation. We will be interested in correlating the host response to the actual medical history of the donors and determine whether gene expression signatures in macrophages can be predictive of the course of the pathology in the organism as a whole. Parallel studies in the pig are directed towards understanding the similarities and differences that allow the pig to act as a reservoir
Aim three: To evaluate the impact of alveolar influenza infection on the susceptibility to bacterial co-infection and to investigate the underlying mechanisms.
The CAGE and microarray studies are also allowing us to compare detailed bacterial lipopolysaccharides (LPS) and influenza-induced time courses in multiple individuals. The initial data indicates that although influenza infection and LPS induce many genes in common early after infection/stimulation, this initial response is curtailed dramatically during flu infection while continuing unabated in the LPS time course. This important difference suggests that genes required in the host response to bacterial infection are specifically suppressed during influenza infection. We have also noted distinct promoter preferences between influenza and LPS stimulation. Further analysis is underway to elucidate the interaction between influenza and bacterial infections, as well as to determine the inter-individual variability of host response.
Aim four: To define ways in which alveolar macrophages can be protected from influenza infection and limit systemic damage caused by the massive cytokine response.
Because of the shift in the model and ambition of the project, this objective has not yet been explored. The understanding of the role of macrophages gained from the studies described in Aims one-three should reveal therapeutic avenues to test in the context of both human and pig flu infection.
Conclusions
The comparative analysis of human and pig response to influenza and bacterial infection is key to understanding how host response is specifically tailored to diverse types of infection, as well as defining influenza-specific targets for therapeutic intervention. We anticipate major publications describing the outcomes of this project within the next 12 months.
As a personal outcome, following the completion of this Fellowship I was recruited to a position expanding on previous work on candidate susceptibility loci significantly associated with severe influenza in humans. Working on both the pig and human influenza models, we aim to identify and understand the functional role of variants associated with inadequate host response to flu infection, and define options for intervention.
Socio-economic impact
Translating genomic information into therapeutic avenues of intervention was a major aspect of the studies undertaken. While we have not yet reached the understanding required to explore therapeutic intervention, it is expected that the follow-up studies to this Fellowship will allow us to identify specific targets predictive of severe outcome of influenza infection.
A better understanding of host susceptibility determinants is paramount to the control of influenza mortality in extremely susceptible hosts, be they human or animal. Considering the important economic impact of both endemic and pandemic influenza, development of susceptibility assays that will help focus the preventative efforts during flu season or in the event of another global pandemic are essential for a more efficient use of available human and material resources.