Alveolar macrophages produce a robust innate immune response to influenza. This includes a significant induction of cytokines and chemokines, pathogen recognition, and apoptotic responses, which are similar to the responses of human monocyte derived macrophages 
. Consistent with other studies of avian or human influenza infections in humans and animals 
, PR/8 stimulated an early and prominent IFN response in human AM despite of the failure to release infectious viral particles. Human AM produce both type I and type III interferons ( and ). In contrast, alveolar epithelial cells do not produce any type I interferon IFN-α in response to influenza 
. These results indicate a cell-specific pattern in producing IFN in response to viral infection. It is well known that RIG-I like RNA helicases (RLHs) and TLRs are the two main PRRs responsible for IFN production against RNA viruses including influenza. RLHs (RIG-I and MDA-5) recognize cytoplasmic viral double-stranded RNA, whereas TLRs (TLR3 and TLR7) sense viral nucleic acid in the endosomal compartment 
. In the current study, PR/8 infection up-regulated mRNA levels of RIG-I and MDA-5 mainly at 4 hpi, but the mRNAs of TLR3 and 7 mainly at 24 hpi (), which suggests that RLHs might be the early sensors and TLRs might be the late sensors for PR/8 in human AM. These results correlate well with those reported by Takeuchi and Thompson that RLHs were responsible for local production of IFNs, whereas TLRs were mainly involved in the late stages of systemic infection 
. At early times PR/8 triggered mainly pro-inflammatory responses, whereas at later times PR/8 also activated pathways involved in the maintenance of homeostasis such as the activation of IL-10 and IL-6, as well as up-regulation of SOCS genes (Data S1
). Therefore, therapeutic regulation of the inflammatory response in acute lung injury should consider both strategies to inhibit secreted cytokines but also strategies to dampen the innate immune response by stimulating IL-10 and SOCS genes. We were able to confirm the results found with PR/8 in contemporary influenza virus NY/238-infected human AM with the exception of an increase in TLR7 mRNA. This might be due to a lower MOI of virus used in the experiments because of the limitation of the viral titer, but it could also be due to differences in the natures of these two viruses or the difference in methods for propagating these two viruses.
CXCL9–11 were the most highly induced chemokines by influenza viruses as verified at both mRNA and protein levels ( and ). These three chemokines bind to a common receptor CXCR3, and the importance of CXCR3 signaling has been shown in the pathogenesis of several viruses including influenza 
. CXCL10 is highly induced in avian flu (H5N1)-infected ferrets, non-human primates, and human cells including alveolar epithelial cells and monocyte-derived macrophages 
, and has been viewed as a prognostic marker for several viral infections 
. In mice, the peak level of CXCL11 mRNA coincides with the peak of the viremia 
, and the CXCL11 protein has been reported to have anti-viral activity 
. In addition, all three CXCR3 ligands can induce epithelial cell chemotaxis and proliferation and perhaps accelerate epithelial wound repair during the resolution of viral infections 
. The robust induction of CXCL9, 10, and 11 in both AM ( and ) and human alveolar type II cells 
as well as the distinct CXCL10 response induced by both live and UV-inactivated influenza virus PR/8 and contemporary virus NY/238 ( and ) suggest that this family of proteins likely plays an important role in the human lung alveolar defense against influenza infection, which will require further study.
The response of alveolar macrophages was different in a several ways from that reported for human monocyte derived macrophages. The major difference is that alveolar macrophages infected with human influenza viruses do not release much infectious virus, whereas human monocyte-derived macrophages do (
and ). The mechanism for the non-productive infection was not investigated in this study and is likely complicated. One of the possible mechanisms might be related to the lack of gene expression of transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT) by human AM (microarray data not shown). Both TMPRSS2 and HAT are type II transmembrane serine proteases 
possessing trypsin-like activity and are known to be important for cleaving influenza HA required for productive infection 
. In recent studies Bottcher et al suggest that TMPRSS2 is mainly responsible for cleavage of newly synthesized HA, whereas HAT cleaves both endocytosed and newly synthesized HA 
. Therefore, lack of these two gene products in human AM may partially explain the lack of released infectious virus by these cells. In addition, both PR/8 and NY/238 viruses induced an early activation of type I IFN, especially IFN-α ( and , , and ). The strong anti-viral property of type I IFN 
may also contribute to the non-productive infection in these cells. Further studies will be required to understand the mechanism for the failure of release of infectious viral particles by human AM. In addition, inactivation of influenza by UV did not abolish the influenza viruses-stimulated CXCL10 secretion by AM ( and ), which is different from studies with human monocyte-derived macrophages 
and with human alveolar type II epithelial cells isolated from the same donors (
and data not shown). In those studies, release of CXCL10 is totally dependent on viral replication. The mechanism for the distinct CXCL10 response in human AM will require additional and carefully designed studies. The differences between human AM and monocyte-derived macrophages indicate the importance of investigating the response of AM to influenza infection during the initial phases of infection in the lung because AM are main targets for both human and avian influenza viruses 
Chemokine and cytokine responses are required for protection of the host against viral infection. However, an exuberant response contributes to the influenza-induced morbidity and mortality, especially in severe pandemic and avian influenza infections 
. In the current study, PR/8 infection induced an increase in TNF-α and IL-1β, well-known paracrine proinflammatory factors. Therefore, we hypothesized that inhibiting these factors might reduce the influenza-induced-inflammatory response. Since the contemporary virus NY/238 induced a similar cytokine and chemokine response as PR/8, it would be reasonable to expect that the regulation of chemokine and cytokine in contemporary influenza infection might also be similar to PR/8 infection. As shown in , inhibiting TNF and/or IL-1 decreased more than 50% of the PR/8-induced secretion of inflammatory chemokines CXCL8 and CCL5 but did not truly affect type I interferon or CXCL10 response, although we observed a decrease of CXCL10 in the presence of both inhibitors (). TNF and IL-1 signaling are known to be regulated by NF-κB and there are several NF-κB binding sites in the promoter of CXCL10 
, despite of the fact that CXCL10 is an IFN-induced protein 
. This may explain why inhibiting both pathways slightly decreased the amount of CXCL10 from infected AM. Our results suggest that short term targeting the critical paracrine factors might be beneficial for controlling the excessive infiltration of inflammatory cells and acute lung injury during pandemic or avian flu infection in vivo
. Of course, this would require careful consideration of time and dose so as not to increase secondary bacterial infections.
Influenza infection significantly decreased mRNA level of macrophage receptors CLEC7A, MSR1, CD36, and MRC1 ( and ). CLEC7A belongs to the C-type lectin family and functions as a PRR that recognizes a variety of beta-1, 3-linked and beta-1, 6-linked glucans from fungi. A decrease of CLEC7A in infected AM suggests that these cells might not efficiently recognize and engulf fungi after influenza infection. As shown in , the uptake of zymosan, a yeast cell wall component containing beta-1-3-glycosolic linkeages, was decreased in a dose-dependent manner in PR/8-infected human AM. This effect was not associated with cell loss or cytopathic effect because we did not observe a significant cytopathic effect () even at a MOI of 1 (data not shown). However, the explanation of the decreased uptake might be more complicated than simply the loss of this receptor. In addition, other macrophage receptors MSR1, MARCO, CD36, as well as mannose receptor MRC1 are important for bacterial and particle uptake 
. Mice with deletions of MSR1 or CD36 have increased susceptibility to pneumococcal or staphylococcal pneumonia 
. Although impairment of macrophage phagocytosis of bacteria after influenza in mice is well recognized 
and secondary bacterial infection after influenza is a common clinical problem, we were not able to detect a significant decrease in uptake of heat-inactivated S. aureus
in human AM until 72 hpi, at which time the cytopathic effect was significant. We did not observe a consistent decrease of MSR1 protein by flow cytometry in PR/8-infected AM, which might explain why the infection did not impair the bacterial uptake (data not shown). We were also not able to verify the decrease of mRNA level of MARCO, another important macrophage scavenger receptor for influenza infections in mice and human cells 
. Nine of 11 donors showed a decrease in mRNA levels of MARCO after infection with PR/8 (). Two other donors had an increase in levels of MARCO mRNA. Therefore, changes of bacteria-related receptors in human AM after influenza require additional studies, and there may be variations in response among individuals.
In summary, we performed a global profiling of innate immune response and regulation with a focus on chemokine and cytokine response in influenza-infected human AM. Human AM are apparently different from human monocyte derived macrophages in their ability to release infectious virus and the CXCL10 response to UV inactivated virus. Future studies should compare these responses in peripheral and alveolar macrophages from the same donors. In addition, during acute lung injury, short term targeting of paracrine inflammatory factors such as TNF and IL-1 as well as targeting IL-10 and SOCS genes might decrease the acute injury and allow for better gas exchange.