Macrophages and neutrophils have a complicated role in protecting against high-dose lethal infection of influenza A virus. In this study, the mice were fatally infected with A/PR/8/34 (H1N1) influenza virus. We monitored the kinetics of the macrophages, neutrophils, CD4+T cell, CD8+ T cell, CD138+cell and CD38+cell in the lungs during the viral infection. We found that macrophages and neutrophils accumulated in the lungs, both in the early and late phases of the virus infection. The decreased viral load significantly correlated with the increased macrophages and neutrophils, and antibody levels both in the lung and serum. The present study suggests that macrophages and neutrophils and the antibody responses both have an important role in eliminating of influenza virus locally.
As to both induce the fatal immunopathology in the lung and ensure the enough mice to survive for further experiment, we at first established an appropriate dose of virus. We found 5 × 105 p.f.u. viruses had a decreased lethality in mice than the dose of 5 × 104 and 5 × 103 p.f.u. The reason might be that 5 × 104 p.f.u. could replicate more efficiently than 5 × 105p.f.u.viruses in the upper respiratory tract, thus came higher lethality. We understood it was not common for other influenza viruses, and did not know whether it was correlated with the characteristic of PR8 as a mouse-adapted virus strain, although there was no published article reported the similar experiments.
Observations from de Jong et al.  supported the presence of an inflammatory response role in the pathogenesis of human H5N1 disease. In addition, post-mortem studies in H5N1-infected individuals have not shown predominance of lymphocytes, but rather of macrophages, in pulmonary in-filtrates . Although CD4+T cell and CD8+T cells also have a protective effect during the pathogen infection, especially during high pathogenic avian influenza viruses infection, the protective was also seen in the absence of all T and B cells as well as in the depletion of neutrophils or NK cells . Whereas, depletion of innate lymphoid cells resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling . Furthermore, study by Tate MD  showed that neutrophil depletion early after infection with influenza virus did not alter influenza virus-derived antigen presentation or naïve CD8+T-cell expansion in the secondary lymphoid organs and of trafficking of virus-specific CD8+T cells into the infected pulmonary airways. Instead early neutrophils reduced both the overall magnitude of influenza virus-specific CD8+T cells, together with impaired cytokine production and cytotoxic effector function. In contrast to that, the depletion of macrophages lead to the death of all of the mice even those challenged with a sublethal dose of virus , therefore it was impossible to evaluate the protective effect with removal of macrophages.
The accumulation of macrophages and neutrophils in the early phase also have been observed in mice  and chickens  fatally infected with influenza viruses. In this study, we observed a significant increased frequency of macrophages in the lung at day 4 post infection. We also evaluated the kinetics of macrophages and neutrophils in the late phase infection. When the viral load in the lung significantly decreased at day 10 post infection, the macrophage and neutrophil frequencies were maintained at high levels. Previous research also found that in the late recovery phase, macrophage and neutrophil inhibition led to a marked delay in the elimination of the virus. All these observations suggest that macrophages and neutrophils may contribute to late-phase clearance of influenza viruses . Previous evidence has suggested that influenza A virus infected cells are subjected to apoptosis-dependent phagocytosis and degrade together with the invading virus within phagocytes. It has been speculated that macrophages and neutrophils accumulate in lung tissues and maximize the efficiency of the phagocytic elimination of infected cells .
IL-6 secretion significantly increased early at day 2 post-infection and quickly decreased to the level of the control, suggesting a correlation with the accumulation of macrophage. Suzuki  also reported that IL-6 mRNA was quickly induced at 24 h post-infection, but 8 h later, mRNA levels became dramatically lower. IL-6 concentrations were significantly correlated to the symptoms and signs of influenza A infection in humans [14, 15]. Elevated IL-6 values have been detected in HPAI H5N1 influenza virus infected human cells and mice –[19, 38]. As a multifunctional cytokine expressed by both lymphoid and non-lymphoid cells , IL-6 has a central role in elucidating an innate immune response and directing the transition from innate to adaptive immunity . However, IL-6 cytokine inhibition does not directly protect against death from lethal H5N1 influenza virus infection .
Our study evaluated kinetic responses and correlation of several types of immune cells, cytokines and antibodies in a mouse model, but did not reveal how they were orchestrated in the virus clearance. Further evaluation of the cooperation between macrophages, neutrophils and antibody responses in eliminating the virus with fatal infection is needed.