The clinical response to influenza infection ranges from mild disease to severe pneumonia and it remains unclear whether the inflammatory response to infection is protective or pathogenic. We have defined a novel role for neutrophils in ameliorating lung injury during influenza infection, thereby limiting development of severe disease. Infection of neutrophil-depleted mice with influenza virus HKx31 (H3N2) led to rapid weight loss, pneumonia, and death. Neutropenia was associated with enhanced virus replication in the respiratory tract; however, viral titers were declining at the time of death, leading us to investigate other factors contributing to mortality. In addition to thymic atrophy, lymphopenia, and viremic spread, depletion of neutrophils led to exacerbated pulmonary inflammation, edema, and respiratory dysfunction. Thus, while it is well established that neutrophils contribute to lung injury in a range of pathological conditions, reduced numbers or impaired neutrophil function can facilitate progression of mild influenza to severe clinical disease.

Airway macrophages provide a first line of host defense against a range of airborne pathogens, including influenza virus. In this study, we show that influenza viruses differ markedly in their abilities to infect murine macrophages in vitro and that infection of macrophages is nonproductive and no infectious virus is released. Virus strain BJx109 (H3N2) infected macrophages with high efficiency and was associated with mild disease following intranasal infection of mice. In contrast, virus strain PR8 (H1N1) was poor in its ability to infect macrophages and highly virulent for mice. Depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in BJx109-infected mice but did not modulate disease severity in PR8-infected mice. The severe disease observed in macrophage-depleted mice infected with BJx109 was associated with exacerbated virus replication in the airways, leading to severe airway inflammation, pulmonary edema, and vascular leakage, indicative of lung injury. Thymic atrophy, lymphopenia, and dysregulated cytokine and chemokine production were additional systemic manifestations associated with severe disease. Thus, airway macrophages play a critical role in limiting lung injury and associated disease caused by BJx109. Furthermore, the inability of PR8 to infect airway macrophages may be a critical factor contributing to its virulence for mice.

The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen ‘danger’ signals. Elucidating immunostimulatory PB1-F2 mediation of NLRP3-inflammasome activation is a major step forward in our understanding of the aetiology of disease attributable to exuberant inflammatory responses to IAV infection.

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3−/− mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

The inflammasome NLRP3 is activated by pathogen associated molecular patterns (PAMPs) during infection, including RNA and proteins from influenza A virus (IAV). However, chronic activation by danger associated molecular patterns (DAMPs) can be deleterious to the host. We show that blocking NLRP3 activation can be either protective or detrimental at different stages of lethal influenza A virus (IAV). Administration of the specific NLRP3 inhibitor MCC950 to mice from one day following IAV challenge resulted in hypersusceptibility to lethality. In contrast, delaying treatment with MCC950 until the height of disease (a more likely clinical scenario) significantly protected mice from severe and highly virulent IAV-induced disease. These findings identify for the first time that NLRP3 plays a detrimental role later in infection, contributing to IAV pathogenesis through increased cytokine production and lung cellular infiltrates. These studies also provide the first evidence identifying NLRP3 inhibition as a novel therapeutic target to reduce IAV disease severity.

Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6high neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1+ cells, not neutrophils, limit disease severity during mild influenza infections.