This is the first time that the role of AM during HPAIV H5N1 virus infection has been studied. This study shows that AM are more susceptible to HPAIV H5N1 infection than to seasonal H3N2 or pH1N1 virus infection but, that this infection results neither in virus production nor in an excessive immune response.
Macrophages cultured from monocytes do not respond the same way as AM to two out of three influenza viruses included in this study: there are significant differences between MM and AM in the percentages of cells infected, cytokine response and virus production after infection with HPAIV H5N1 and seasonal H3N2 virus. Therefore, MM are not suitable to study the interaction between AM and these influenza virus strains.
HPAIV H5N1 infected more AM than seasonal H3N2 or pH1N1 viruses, in both the ex vivo
lung cultures and in AM collected from BAL. This fits with the results of our virus attachment studies, in which HPAIV H5N1 attached more abundantly to AM than H3N2 virus 
and pH1N1 
. Infection of an AM likely hampers its protective function and, since approximately one AM is found in each alveolus 
, this could lead to substantial loss of protection in the alveoli.
HPAIV H5N1 infection in AM did not result in significantly higher induction of TNF-alpha mRNA compared to seasonal H3N2 or pH1N1 virus. This is in contrast with the significantly higher TNF-alpha mRNA levels in MM after HPAIV H5N1 virus infection compared to seasonal H3N2 and pH1N1 virus infection observed in our and previous studies 
. The observed difference in TNF-alpha induction can likely be explained by the activation phenotype of AM compared to that of MM. MM represent classical activated macrophages, while AM are alternative activated or deactivated macrophages. LPS is known to induce TNF-alpha production in classically activated macrophages but not in alternatively activated macrophages 
. The inhibited TNF-alpha induction of AM in this study fits with their alternatively activated phenotype. This inhibition of TNF-alpha induction in alternatively activated macrophages is thought to be regulated by the p50 subunit of NF-κB 
TNF-alpha, and the cascade it induces, is known to attract neutrophils and other leukocytes in the lung and is thought to play an important role in the pathogenesis of acute respiratory distress syndrome and multiple organ dysfunction syndrome 
. The lack of excessive TNF-alpha induction in AM after infection with the HPAIV H5N1 strain used in this study might indicate that, in contrast to previous conclusions based on studies with MM, the AM does not contribute to the excessive immune response after HPAIV H5N1 infections.
The lack of virus production in AM after infection with influenza viruses included in this study corresponds with a previous study in which no virus release in AM was detected after infection with a seasonal H1N1 or H3N2 virus 
. The lack of significant virus production indicates that AM do not release high numbers of new virus particles and thereby do not contribute to a productive infection in the human alveolus.
During influenza virus pneumonia, there is an influx of a variety of leukocytes, including monocytes 
. The monocytes that enter the alveoli during influenza virus pneumonia most likely respond differently than resident AM. We cannot exclude that these monocytes will respond in a similar way as MM, with high percentages of cells infected, a productive infection and the induction of TNF-alpha. Unfortunately, the interaction between monocytes and influenza virus has never been studied, so their role remains to be elucidated.
Interestingly, macrophages that were cultured in the presence of GM-CSF, resembled AM in shape, but differed in their response to influenza virus infection. In fact, the percentages of cells infected of GM-MM resembled that of the MM more closely. Although GM-CSF is abundantly present in the human alveolus and is thought to be responsible for the AM phenotype 
, addition of GM-CSF was not sufficient to transform the phenotype of monocytes to that of AM, with respect to their response to influenza virus infections. It is likely that other factors present in the alveolus are required for this transformation.
Overall, the pH1N1 virus used in this study did not infect high percentages of either AM or MM. Even compared to seasonal H3N2 virus, pH1N1 virus infected lower percentages of cells. Whether this is a unique feature of pH1N1 virus or a common feature of pandemic influenza viruses remains to be determined. Overall, this observation fits with the relatively mild disease caused by pH1N1 virus infection 
In conclusion, we have shown that the MM are unsuitable to study the interaction between AM and these influenza virus strains, which might be due to their difference in phenotype; alternatively activated versus classically activated. In contrast to MM, AM do not induce excessive TNF-alpha after HPAIV H5N1 infection. However, AM are more abundantly infected by HPAIV H5N1 than by seasonal H3N2 virus or pH1N1 virus. We speculate that this relatively high percentage of AM infection by HPAIV H5N1 may contribute to the unusual high pathogenicity of HPAIV H5N1 for the human lung.