Michelle D. Tate

Neutrophils Ameliorate Lung Injury and the Development of Severe Disease during Influenza Infection

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.

Critical Role of Airway Macrophages in Modulating Disease Severity during Influenza Virus Infection of Mice

ABSTRACT 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.

Antiviral Activity of the Long Chain Pentraxin PTX3 against Influenza Viruses

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.

The Role of Neutrophils during Mild and Severe Influenza Virus Infections of Mice

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.

The role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice

BackgroundNeutrophils have been shown to play a role in host defence against highly virulent and mouse-adapted strains of influenza virus, however it is not clear if an effective neutrophil response is an important factor moderating disease severity during infection with other virus strains. In this study, we have examined the role of neutrophils during infection of mice with influenza virus strain HKx31, a virus strain of the H3N2 subtype and of moderate virulence for mice, to determine the role of neutrophils in the early phase of infection and in clearance of influenza virus from the respiratory tract during the later phase of infection.MethodsThe anti-Gr-1 monoclonal antibody (mAb) RB6-8C5 was used to (i) identify neutrophils in the upper (nasal tissues) and lower (lung) respiratory tract of uninfected and influenza virus-infected mice, and (ii) deplete neutrophils prior to and during influenza virus infection of mice.ResultsNeutrophils were rapidly recruited to the upper and lower airways following influenza virus infection. We demonstrated that use of mAb RB6-8C5 to deplete C57BL/6 (B6) mice of neutrophils is complicated by the ability of this mAb to bind directly to virus-specific CD8+ T cells. Thus, we investigated the role of neutrophils in both the early and later phases of infection using CD8+ T cell-deficient B6.TAP-/- mice. Infection of B6.TAP-/- mice with a low dose of influenza virus did not induce clinical disease in control animals, however RB6-8C5 treatment led to profound weight loss, severe clinical disease and enhanced virus replication throughout the respiratory tract.ConclusionNeutrophils play a critical role in limiting influenza virus replication during the early and later phases of infection. Furthermore, a virus strain of moderate virulence can induce severe clinical disease in the absence of an effective neutrophil response.

Playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to 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.

Activation of the NLRP3 Inflammasome by IAV Virulence Protein PB1-F2 Contributes to Severe Pathophysiology and Disease

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.

Pandemic H1N1 influenza A viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies.

Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily.

Specific Sites of N-Linked Glycosylation on the Hemagglutinin of H1N1 Subtype Influenza A Virus Determine Sensitivity to Inhibitors of the Innate Immune System and Virulence in Mice

Oligosaccharides on the hemagglutinin (HA) and neuraminidase of influenza A virus (IAV) are a target for recognition by lectins of the innate immune system, including soluble surfactant protein-D and the macrophage mannose receptor on airway macrophages. Glycans attached to the head of H1 subtype of IAV differ markedly in number and location. A reverse genetic approach was used to define the importance of particular N-glycosylation sites on H1 in determining sensitivity to innate immune defenses and virulence in mice. The HA of A/PR/8/34 (PR8, H1N1) and A/Brazil/11/78 (Brazil, H1N1) express zero and four glycosylation sites on the head of HA, respectively. Site-directed mutagenesis was used to add (PR8) or delete (Brazil) glycosylation sites, and IAV expressing wild-type or mutant HA were generated on a PR8 backbone. Addition or removal of particular glycans modulated sensitivity to mouse lung fluids but was not a major factor determining susceptibility of airway macrophages to infection. PR8 is a mouse-adapted virus, and mutations in multiple IAV genes have been shown to contribute to virulence, yet addition of glycosylation to PR8 HA was sufficient to attenuate disease. In contrast, removal of glycans from Brazil HA resulted in severe disease and death. These studies provide insight regarding the mechanisms by which IAV can induce disease in mice. Moreover, reduced glycosylation of HA is likely to be an important factor associated with adaptation of human IAV to growth in mouse lung.

N-Linked Glycosylation Facilitates Sialic Acid-Independent Attachment and Entry of Influenza A Viruses into Cells Expressing DC-SIGN or L-SIGN

ABSTRACT It is widely recognized that sialic acid (SA) can mediate attachment of influenza virus to the cell surface, and yet the specific receptors that mediate virus entry are not known. For many viruses, a definitive demonstration of receptor function has been achieved when nonpermissive cells are rendered susceptible to infection following transfection of the gene encoding a putative receptor. For influenza virus, such approaches have been confounded by the abundance of SA on mammalian cells so that it has been difficult to identify cell lines that are not susceptible to infection. We examined influenza virus infection of Lec2 Chinese hamster ovary (CHO) cells, a mutant cell line deficient in SA. Lec2 CHO cells were resistant to influenza virus infection, and stable cell lines expressing either DC-SIGN or L-SIGN were generated to assess the potential of each molecule to function as SA-independent receptors for influenza A viruses. Virus strain BJx109 (H3N2) bound to Lec2 CHO cells expressing DC-SIGN or L-SIGN in a Ca2+-dependent manner, and transfected cells were susceptible to virus infection. Treatment of Lec2-DC-SIGN and Lec2-L-SIGN cells with mannan, but not bacterial neuraminidase, blocked infection, a finding consistent with SA-independent virus attachment and entry. Moreover, virus strain PR8 (H1N1) bears low levels of mannose-rich glycans and was inefficient at infecting Lec2 CHO cells expressing either DC-SIGN or L-SIGN, whereas other glycosylated H1N1 subtype viruses could infect cells efficiently. Together, these data indicate that human C-type lectins (DC-SIGN and L-SIGN) can mediate attachment and entry of influenza viruses independently of cell surface SA.