Innate immune response is considered as an essential process required for efficient pathogen clearance. However, deregulation of this response can lead to severe damage for the host. The regulation of host defenses to IAV infections is particularly complex and leukocyte recruitment to the inflammatory lung, which is beneficial for clearing the virus, can become deleterious to the host tissues if the reaction is excessive 
. In fact, although inflammation is essential for IAV clearance, it is becoming increasingly evident that a tight regulation of this process is necessary to avoid development of IAV-related immunopathological sequelae 
. It illustrates the ambiguity of the host response to infections. The strength of the response has to be proportionate to the pathogen attack in order to eliminate it. If the response is too weak, the pathogen will proliferate, leading to infection. If the host response is too strong, uncontrolled inflammatory reaction will lead to tissue destruction.
The present study demonstrates that PB1-F2 is endowed with pro-inflammatory activity and plays a role in the immunopathological consequences observed in severe impairment of influenza-infected lung function. By the use of reverse genetics, we obtained a mutant virus unable to express PB1-F2 and showed that this mutant was less lethal and virulent than the wt virus, consistent with a previous study 
. These differences cannot be explained by reduced replication efficiency of the ΔF2 virus since no replication variation was observed between the two viruses. Besides, the use of mutants deleted for N40 or PB1-F2 exclude a potential role of N40 in this process.
Our previous study identified IFN-β gene as a PB1-F2-mediated exacerbated gene during IAV infection of a human respiratory epithelial cell line 
. Based on these previous results, we hypothesized that PB1-F2 expression may stimulate in vivo
the host immune response, and as a consequence, be a key element that could explain the higher virulence of a virus expressing PB1-F2. IFN-β measurement within infected-mice lungs confirmed our preliminary in vitro
observation. To further study whether PB1-F2 could cause immunopathology, we compared the global gene expression patterns of wt- and ΔF2-infected mouse lungs. Our data demonstrate that expression of PB1-F2 during IAV infection enhances the host defense responses by strongly increasing the transcriptional profile of genes involved in inflammation, granulocyte migration and apoptotic pathways. No apoptotic pathways were identified when these two viruses were used to infect human pulmonary epithelial cells in vitro 
. This may reflect the cell-specific behavior of PB1-F2 that only induces apoptotic process within immune cells and not in epithelial cells 
. Thus, the use of the wt/ΔF2 virus couple showed the ability of PB1-F2 to modulate the host response, contributing to virulence in vivo
. This is supported by a recent work by McAuley et al.
characterizing the immunomodulatory properties of several variants of the PB1-F2 protein 
Using IPA software, we were able to analyze the functional relationships between the genes differentially regulated when PB1-F2 was expressed or not during infection. The analysis revealed the central role of IFN-γ, which could act as a relay between PB1-F2 and the genes implicated in respiratory disease, inflammatory response, immune cell trafficking, tissue remodeling and apoptosis induction. The PB1-F2-specific increase of IFN-γ expression was confirmed at the protein level in BAL fluids of infected mice. IFN-γ is a member of the type II interferon family and is a potent activator of macrophages and neutrophils. It is worth noting that in the IAV-infected pulmonary environment, IFN-γ represses the innate immunity developed by alveolar macrophages to enhance their adaptive antiviral response through increased MHC gene expression 
. Consequently, the scavenger receptor MARCO is down-regulated, and innate defense against secondary bacterial infections is suppressed. The link that we identified between PB1-F2 and IFN-γ over-expression appears of first importance since PB1-F2 has been previously described as facilitating the development of opportunistic pneumococcal and staphylococcal infections 
. Collectively, our data provide a rationale to understand how PB1-F2 exacerbate secondary bacterial infections and post-influenza pneumonia: PB1-F2 induces an apoptosis increase of recruited leucocytes together with a switch of their activation state from innate immunity to adaptive antiviral response through an exacerbation of the IFN-γ expression in the lung.
The mechanism by which PB1-F2 mediates inflammatory increase by itself is still unknown. However, NF-κB pathway exacerbation is involved in this process as shown by the dramatic decrease of NF-κB activity in lungs of mice infected by the ΔF2 virus when compared to the wt infection (). The targeting of PB1-F2 to mitochondria could explain the NF-κB pathway exacerbation through a membrane destabilization of this organelle and activation of the RIG-I/MAVS signaling pathway. However PB1-F2 mitochondrial localization is a matter of debate since several studies described differential localization depending on the strain of the virus and the sequence polymorphism of the protein 
. The capability of PB1-F2 to perforate or target other cellular membranes could also explain its inflammatory and apoptotic activities 
. Considering the membrane affinity of PB1-F2, it could form protein pores in endoplasmic reticulum and subsequently induce the release of Ca2+
within the cytosol. Such increase in concentration of Ca2+
in the cytosol is described to activate the NF-κB pathway and to play a pivotal role in inducing proinflammatory gene transcription in airway epithelial cells 
. Another important characteristic of PB1-F2 is its propensity to form or promote the formation of amyloïdic fibers in infected monocytes 
. These fibrillar aggregates are believed to associate to the membrane, disrupt its integrity and lead to perturbation of the cellular compartmentalization. This suggests that PB1-F2 could contribute to the pulmonary dysfunctions observed throughout IAV infections by several pathways.
A striking feature of the inflammation exacerbated by PB1-F2 expression is the enhanced neutrophil recruitment within infected airways. There is evidence, based on their function, that neutrophils play an important role in mediating acute injury characteristic of highly pathogenic IAV infections. Circulating neutrophils migrate to the site of infection and participate in the destruction of pathogens. However this process has to be tightly regulated since the beneficial effect for eliminating microbes can become deleterious to host tissues through the development of lesions. Indeed neutrophils have the potential to damage airspaces by releasing serine proteases and by generating reactive oxygen species 
. Neutrophil recruitment within lung tissue also increases protein permeability across the endothelial and epithelial barriers of the lung. This leads to the flooding of alveoli by plasma liquid and proteins and is characteristic of early lung injury 
. However, depletion of neutrophils prior to influenza infection increased viral load and mortality compared to non-treated mice 
, confirming the importance of this type of leucocyte during IAV infection, and underlying the complexity of the host-IAV interactions. A fine balance between inflammation and immunity is necessary to eliminate IAV. Our study shows that PB1-F2 is implicated in the dysfunction of this balance and that it induces a massive recruitment of neutrophils within airspaces through deregulation of the innate host defense.
In summary, the present study demonstrated that PB1-F2 expression significantly increases the expression of genes associated with inflammation in the airways of IAV-infected mice. We identified IFN-γ as a pivotal host component implicated in this process, orchestrating immune cell apoptosis induction, granulocyte recruitment and tissue remodeling observed in infected lungs. The PB1-F2 specific NF-κB pathway exacerbation that we revealed is probably involved in this IFN-γ expression increase. Further studies are required to correlate PB1-F2 sequence variability and IAV virulence to help in the prediction of the pathogenicity of emerging virus strains in the human population.