Seasonal influenza virus infections may lead to debilitating disease, and account for significant fatalities annually worldwide. Most of these deaths are attributed to the complications of secondary bacterial pneumonia. Evidence is accumulating to support the notion that neutrophil extracellular traps (NETs) harbor several antibacterial proteins, and trap and kill bacteria. We have previously demonstrated the induction of NETs that contribute to lung tissue injury in severe influenza pneumonia. However, the role of these NETs in secondary bacterial pneumonia is unclear. In this study, we explored whether NETs induced during pulmonary influenza infection have functional significance against infections with Streptococcus pneumoniae and other bacterial and fungal species. Our findings revealed that NETs do not participate in killing of Streptococcus pneumoniae in vivo and in vitro. Dual viral and bacterial infection elevated the bacterial load compared to animals infected with bacteria alone. Concurrently, enhanced lung pathogenesis was observed in dual-infected mice compared to those challenged with influenza virus or bacteria alone. The intensified NETs in dual-infected mice often appeared as clusters that were frequently filled with partially degraded DNA, as evidenced by punctate histone protein staining. The severe pulmonary pathology and excessive NETs generation in dual infection correlated with exaggerated inflammation and damage to the alveolar-capillary barrier. NETs stimulation in vitro did not significantly alter the gene expression of several antimicrobial proteins, and these NETs did not exhibit any bactericidal activity. Fungicidal activity against Candida albicans was observed at similar levels both in presence or absence of NETs. These results substantiate that the NETs released by primary influenza infection do not protect against secondary bacterial infection, but may compromise lung function.

Obesity is an independent risk factor for severe outcome of influenza infection. Higher dietary fat consumption has been linked to greater morbidity and severe influenza in mouse models. However, the extent of generation of neutrophil extracellular traps (NETs or NETosis) in obese individuals during influenza pneumonia is hitherto unknown. This study investigated pulmonary NETs generation in BALB/c mice fed with high-fat diet (HFD) and low-fat diet (LFD), during the course of influenza pneumonia. Clinical disease progression, histopathology, lung reactive oxygen species, and myeloperoxidase activity were also compared. Consumption of HFD over 18 weeks led to significantly higher body weight, body mass index, and adiposity in BALB/c mice compared with LFD. Lethal challenge of mice (on HFD and LFD) with influenza A/PR/8/34 (H1N1) virus led to similar body weight loss and histopathologic severity. However, NETs were formed at relatively higher levels in mice fed with HFD, despite the absence of significant difference in disease progression between HFD- and LFD-fed mice.

Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia.

Frequent outbreaks caused by influenza viruses pose considerable public health threats worldwide. Virus-inflicted alveolar damage represents a major contributor of acute lung injury in influenza. We have previously demonstrated that hepatocyte growth factor (HGF) produced by macrophages enhances alveolar epithelial proliferation during influenza infection. Here, we investigated the therapeutic efficacy of recombinant human HGF (rhHGF) and an antiviral agent (oseltamivir) alone or in combination to treat influenza viral pneumonia in macrophage-depleted BALB/c mice. Combination therapy of infected mice significantly reduced lung pathology and mortality compared to other animal groups that received either treatment alone. Combination treatment with rhHGF induced alveolar type II (AT2) epithelial hyperplasia more prominently in the distal airways, evident by increased cells with double-positive staining for surfactant protein-C and proliferating cell nuclear antigen within the alveolar epithelial lining. Similarly, rhHGF supplementation also induced stem cell antigen-1 (SCA-1) transcriptional expression at 5 days post-infection (dpi), but mRNA levels of both SCA-1 and its receptor c-KIT were decreased by 10 dpi. Microarray and pathway analyses indicated that rhHGF administration may act by accelerating tissue repair and suppressing inflammatory processes to minimize damage by infection and to restore lung function by earlier repair. These results reveal that transient administration of rhHGF may confer synergistic effects in enhancing pulmonary repair by promoting AT2 cell proliferation. Thus, the combination of rhHGF and oseltamivir may represent a promising therapeutic option against influenza pneumonia to improve existing antiviral treatment regimens.