Increased levels of interferon-γ (IFN-γ) are routinely observed in the respiratory tract following influenza virus infection, yet its potential role remains unclear. We now demonstrate that influenza-induced IFN-γ restricts protective innate lymphoid cell group II (ILC2) function in the lung following challenge with the pandemic H1N1 A/CA/04/2009 (CA04) influenza virus. Specifically, IFN-γ deficiency resulted in enhanced ILC2 activity, characterized by increased production of interleukin (IL)-5 and amphiregulin, and improved tissue integrity, yet no change in ILC2 numbers, viral load or clearance. We further found that IFN-γ-deficient mice, as well as wild-type animals treated with neutralizing anti-IFN-γ antibody, exhibited decreased susceptibility to lethal infection with H1N1 CA04 influenza virus, and moreover that survival was dependent on the presence of IL-5. The beneficial effects of IFN-γ neutralization were not observed in ILC2-deficient animals. These data support the novel concept that IFN-γ can have a detrimental role in the pathogenesis of influenza through a restriction in ILC2 activity. Thus, regulation of ILC2 activity is a potential target for post-infection therapy of influenza.

Asthma is believed to be a risk factor for influenza infection, however little experimental evidence exists to directly demonstrate the impact of asthma on susceptibility to influenza infection. Using a mouse model, we now report that asthmatic mice are actually significantly more resistant to a lethal influenza virus challenge. Notably, the observed increased resistance was not attributable to enhanced viral clearance, but instead, was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm, as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, asthmatic mice had significantly increased levels of TGF-β1 and the heightened resistance of asthmatic mice was abrogated in the absence of TGF-β receptor II. We conclude that a transient increase in TGF-β expression following acute asthma can induce protection against influenza-induced immunopathology. Influenza and asthma represent the two major lung diseases in humans. While most studies have focused on exacerbation of asthma symptoms by influenza virus infection, the effects of asthma on susceptibility to influenza virus infections has been far less studied. Using a novel mouse model of asthma and influenza infection, we show that asthmatic mice are highly resistant to primary challenge with the 2009 influenza pandemic strain (CA04) compared to non-asthmatic mice. The increased resistance of asthmatic mice is not due to the enhanced T or B cell immunity but rather, to a strong anti-inflammatory TGF-beta response triggered by asthma. This study is the first to provide a mechanistic explanation for asthma-mediated protection during the 2009 influenza pandemic.

Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection.

Background. Secondary bacterial infections following influenza represent a major cause of mortality in the human population, which, in turn, has led to a call for stockpiling of bacterial vaccines for pandemic preparedness. Methods. To investigate the efficacy of bacterial vaccination for protection against secondary pneumococcal infection, mice were immunized with pneumococcal capsular polysaccharide conjugate vaccine, and then sequentially coinfected 5 weeks later with PR8 influenza virus and A66.1 Streptococcus pneumoniae. Results. In the absence of influenza virus exposure, vaccination with polysaccharide conjugate vaccine was highly effective, as indicated by 100% survival from lethal pneumococcal pneumonia and 10 000-fold greater efficiency in clearance of bacteria from the lung compared to unvaccinated mice. Enhanced clearance after vaccination was dependent upon Fc receptor (FcR) expression. However, following influenza, <40% of vaccinated mice survived bacterial coinfection and FcR-dependent clearance of antibody-opsonized bacteria reduced bacterial levels in the lungs only 5–10 fold. No differences in lung myeloid cell numbers or in FcR cell surface expression were observed following influenza. Conclusions. The results show that induction of antibacterial humoral immunity is only partially effective in protection against secondary bacterial infections that occur following influenza, and suggest that additional therapeutic strategies to overcome defective antibacterial immunity should be explored.

Methicillin-resistantStaphylococcus aureus(MRSA) is a common pathogen associated with nosocomial pneumonia and is an increasing threat for severe community-acquired pneumonia. We have now investigated the role of interleukin-12 (IL-12) in protective immunity against lung infection with MRSA. The importance of IL-12 in protection from pulmonary MRSA infection was demonstrated by the finding that IL-12p35-deficient mice had a lower survival rate, higher bacterial burdens in lung and spleen, and decreased expression of interferon gamma (IFN-γ) in the lung compared to wild-type mice. These effects were completely reversed by replacement intranasal therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of wild-type mice 24 h before pulmonary challenge with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. The IL-12-treated mice had increased numbers of lung natural killer (NK) cells and neutrophils and higher levels of IFN-γ in the lung and serum compared to untreated mice. The major source of IL-12-driven IFN-γ expression in the lung was the NK cell, and the direct target of pulmonary IFN-γ was the lung macrophage, as shown using mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice). Importantly, combination therapy with linezolid and IL-12 following intranasal MRSA infection significantly increased survival compared to that of mice receiving linezolid or IL-12 alone. These results indicate that IL-12-based immunotherapy may hold promise for treatment of MRSA pneumonia.

Francisella tularensis, the causative agent of tularemia, is most deadly in the pneumonic form; therefore, mucosal immunity is an important first line of defense against this pathogen. We have now evaluated the lethality of primaryF. tularensislive vaccine strain (LVS) pulmonary infection in mice that are defective in IgA (IgA^−/−mice), the predominant mucosal Ig isotype. The results showed that IgA^−/−mice were more susceptible than IgA^+/+mice to intranasalF. tularensisLVS infection, despite developing higher levels of LVS-specific total, IgG, and IgM antibodies in the bronchoalveolar lavage specimens following infection. In addition, the absence of IgA resulted in a significant increase in bacterial loads and reduced survival. Interestingly, IgA^−/−mice had lower pulmonary gamma interferon (IFN-γ) levels and decreased numbers of IFN-γ-secreting CD4⁺and CD8⁺T cells in the lung on day 9 postinfection compared to IgA^+/+mice. Furthermore, IgA^−/−mice displayed reduced interleukin 12 (IL-12) levels at early time points, and supplementing IgA^−/−mice with IL-12 prior to LVS challenge induced IFN-γ production by NK cells and rescued them from mortality. Thus, IgA^−/−mice are highly susceptible to primary pulmonary LVS infections not only because of IgA deficiency but also because of reduced IFN-γ responses.