Differential pulmonary transcriptomic profiles in murine lungs infected with low and highly virulent influenza H3N2 viruses reveal dysregulation of TREM1 signaling, cytokines, and chemokines.

Investigating the relationships between critical influenza viral mutations contributing to increased virulence and host expression factors will shed light on the process of severe pathogenesis from the systems biology perspective. We previously generated a mouse-adapted, highly virulent influenza (HVI) virus through serial lung-to-lung passaging of a human influenza H3N2 virus strain that causes low virulent influenza (LVI) in murine lungs. This HVI virus is characterized by enhanced replication kinetics, severe lung injury, and systemic spread to major organs. Our gene microarray investigations compared the host transcriptomic responses of murine lungs to LVI virus and its HVI descendant at 12, 48, and 96 h following infection. More intense expression of genes associated with cytokine activity, type 1 interferon response, and apoptosis was evident in HVI at all time-points. We highlighted dysregulation of the TREM1 signaling pathway (an amplifier of cytokine production) that is likely to be upregulated in infiltrating neutrophils in HVI-infected lungs. The cytokine gene expression changes were corroborated by elevated levels of multiple cytokine and chemokine proteins in the bronchoalveolar lavage fluid of infected mice, especially at 12 h post-infection. Concomitantly, the downregulation of genes that mediate proliferative, developmental, and metabolic processes likely contributed to the lethality of HVI as well as lack of lung repair. Overall, our comparative transcriptomic study provided insights into key host factors that influence the dynamics, pathogenesis, and outcome of severe influenza.

Doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza H3N2 virus: involvement of matrix metalloproteinases.

Acute respiratory distress syndrome, a severe form of acute lung injury (ALI), is a major cause of death during influenza pneumonia. We have provided evidence for the involvement of recruited neutrophils, their toxic enzymes such as myeloperoxidase and matrix metalloproteinases (MMPs), and neutrophil extracellular traps in aggravating alveolar-capillary damage. In this study, we investigated the effects of doxycycline (DOX), an inhibitor of MMPs, on influenza-induced ALI. BALB/c mice were infected with a sublethal dose of mouse-adapted virulent influenza A/Aichi/2/68 (H3N2) virus, and administered daily with 20mg/kg or 60 mg/kg DOX orally. The effects of DOX on ALI were determined by measuring inflammation, capillary leakage, and MMP activities. Furthermore, levels of T1-α (a membrane protein of alveolar type I epithelium) and thrombomodulin (an endothelial protein) in the bronchoalveolar lavage fluid were evaluated by Western blot analysis. Our results demonstrate significantly decreased inflammation and protein leakage in the lungs after DOX treatment. Levels of MMP-2 and MMP-9 activity, T1-α and thrombomodulin were also diminished in the DOX-treated group. These findings were corroborated by histopathologic analyses, which demonstrated significant reduction in lung damage. Although DOX treatment reduced ALI, there were no effects on virus titers and body weights. Taken together, these results demonstrate that DOX may be useful in ameliorating ALI during influenza pneumonia. Further studies are warranted to determine whether DOX can be used in combination with anti-viral agents to alleviate severe influenza pneumonia.

MCP-1 antibody treatment enhances damage and impedes repair of the alveolar epithelium in influenza pneumonitis.

Recent studies have demonstrated an essential role of alveolar macrophages during influenza virus infection. Enhanced mortalities were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis resulted in enhanced alveolar epithelial damage and apoptosis, as evaluated by histopathology, immunohistochemistry, Western blot, RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment was achieved by treatment with monoclonal antibody against monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus. Interestingly, elevated levels of hepatocyte growth factor (HGF), a mitogen for alveolar epithelium, were detected in bronchoalveolar lavage samples and in lung homogenates of control untreated and nonimmune immunoglobulin (Ig)G-treated mice after infection compared with anti-MCP-1-treated infected mice. The lungs of control animals also displayed strongly positive HGF staining in alveolar macrophages as well as alveolar epithelial cell hyperplasia. Co-culture of influenza virus-infected alveolar epithelial cells with freshly isolated alveolar macrophages induced HGF production and phagocytic activity of macrophages. Recombinant HGF added to mouse lung explants after influenza virus infection resulted in enhanced BrdU labeling of alveolar type II epithelial cells, indicating their proliferation, in contrast with anti-HGF treatment showing significantly reduced epithelial regeneration. Our data indicate that inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that HGF produced by macrophages in response to influenza participates in the resolution of alveolar epithelium.

Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis.

Most pandemic influenza virus strains undergo adaptation or reassortment before they acquire the ability to cause fatal infections in a new host species. The pathologic changes and tissue tropism during virus adaptation are not fully understood. Here we investigated pathologic changes and tissue tropism by serial lung-to-lung passaging of human influenza virus strain A/Aichi/2/68 (H3N2) in a BALB/c mouse model. Enhanced pulmonary lesions and systemic virus infection were observed during adaptation. Late passage 10 (P10) virus caused extra-pulmonary spread with necrotic and inflammatory lesions in the brain, heart, spleen and intestine of infected animals, in contrast to infection with earlier passage viruses which were restricted to lungs. Non-conservative mutations in the hemagglutinin (Gly218Glu) and non-structural 1 (Asp125Gly) proteins were identified in P10 virus which exhibited high virulence. Virus growth kinetics showed enhanced replication ability of P10 virus in different cell lines. P10 virus also exhibited the ability to bind to erythrocytes of different host species. These results demonstrate extra-pulmonary spread of influenza virus during adaptation with enhanced replication ability in a new host. This mouse adaptation model may provide a basis for understanding cross-species adaptability corresponding to increased virulence of the influenza A virus, a phenomenon of relevance to the emergence of future highly pathogenic strains.

Characterization of cytotoxic T-lymphocyte epitopes and immune responses to SARS coronavirus spike DNA vaccine expressing the RGD-integrin-binding motif.

Integrins are critical for initiating T-cell activation events. The integrin-binding motif Arg-Gly-Asp (RGD) was incorporated into the pcDNA 3.1 mammalian expression vector expressing the codon-optimized extracellular domain of SARS coronavirus (SARS-CoV) spike protein, and tested by immunizing C57BL/6 mice. Significant cell-mediated immune responses were characterized by cytotoxic T-lymphocyte (51)Cr release assay and interferon-gamma secretion ELISPOT assay against RMA-S target cells presenting predicted MHC class I H2-Kb epitopes, including those spanning residues 884-891 and 1116-1123 within the S2 subunit of SARS-CoV spike protein. DNA vaccines incorporating the Spike-RGD/His motif or the Spike-His construct generated robust cell-mediated immune responses. Moreover, the Spike-His DNA vaccine construct generated a significant antibody response. Immunization with these DNA vaccine constructs elicited significant cellular and humoral immune responses. Additional T-cell epitopes within the SARS-CoV spike protein that may contribute to cell-mediated immunity in vivo were also identified.

Combination therapy with hepatocyte growth factor and oseltamivir confers enhanced protection against influenza viral 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.

In vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection.

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.