Secondary bacterial infection often occurs after pulmonary virus infection and is a common cause of severe disease in humans, yet the mechanisms responsible for this viral-bacterial synergy in the lung are only poorly understood. We now report that pulmonary interferon-gamma (IFN-gamma) produced during T cell responses to influenza infection in mice inhibits initial bacterial clearance from the lung by alveolar macrophages. This suppression of phagocytosis correlates with lung IFN-gamma abundance, but not viral burden, and leads to enhanced susceptibility to secondary pneumococcal infection, which can be prevented by IFN-gamma neutralization after influenza infection. Direct inoculation of IFN-gamma can mimic influenza infection and downregulate the expression of the class A scavenger receptor MARCO on alveolar macrophages. Thus, IFN-gamma, although probably facilitating induction of specific anti-influenza adaptive immunity, suppresses innate protection against extracellular bacterial pathogens in the lung.

Secondary pulmonary infections by encapsulated bacteria including Streptococcus pneumoniae and Staphylococcus aureus following influenza represent a common and challenging clinical problem. The reasons for this polymicrobial synergy are still not completely understood, hampering development of effective prophylactic and therapeutic interventions. While it has been commonly thought that viral-induced epithelial cell damage allows bacterial invasiveness, recent studies by several groups have now implicated dysfunctional innate immune defenses following influenza as the primary culprit for enhanced susceptibility to secondary bacterial infections. Understanding the immunological imbalances that are responsible for virus-bacteria synergy will ultimately allow the design of effective, broad-spectrum therapeutic approaches for prevention of enhanced susceptibility to these pathogens.

Methicillin-resistant S. aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. Here we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, despite the fact that MRSA super-infection had no significant effect on viral burdens. Compared to mice infected with bacteria alone, post-influenza MRSA infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species (ROS) levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for ROS production during phagocytic bacterial killing, a process also known as oxidative burst. We found that gp91phox-containing NADPH oxidase activity in macrophages and neutrophils was essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to WT animals, gp91phox−/− mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91phox+/− mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection.

The ability of exogenous interleukin-12 (IL-12) to elicit protective innate immune responses against the extracellular pathogen Streptococcus pneumoniae was tested by infecting BALB/c mice intranasally (i.n.) with S. pneumoniae after i.n. administration of IL-12. It was found that administration of IL-12 resulted in lower bacterial burdens in the infected mice and significantly improved survival rates. All IL-12-treated mice contained higher levels of pulmonary gamma interferon (IFN-␥) after infection and significantly more neutrophils than infected mice not treated with IL-12. IFN-␥ was found to be essential for IL-12-induced resistance and for neutrophil influx into the lungs, and the observed changes correlated with increased levels of the IL-8 homologue keratinocyte-derived chemokine (KC). In addition, in vitro tumor necrosis factor alpha (TNF-␣) production by alveolar macrophages stimulated with heat-killed pneumococci was enhanced by IFN-␥, and TNF-␣ in turn could enhance production of KC by lung cells. Finally, IL-12-induced protection was dependent upon the presence of neutrophils and the KC receptor CXCR2. Taken together, the results indicate that exogenous IL-12 can improve innate defense in the lung against S. pneumoniae by inducing IFN-␥ production, which in turn enhances chemokine expression, and promotes pulmonary neutrophil recruitment into the infected lung. The findings show that IL-12 and IFN-␥ can mediate a protective effect against respiratory infection caused by extracellular bacterial pathogens.Streptococcus pneumoniae is a human pathogen that infects the host mainly through the respiratory tract. Once it crosses natural barriers, it can cause life-threatening diseases, such as pneumonia, bacteremia, and meningitis (10, 11). This grampositive bacterial pathogen remains a leading cause of serious illness among infants, immunocompromised patients, and individuals over 60 years of age (11). The large number of pneumococcal serogroups limits the utility of vaccines, suggesting that alternative approaches that target innate host defense mechanisms may be necessary for optimal protection against this pathogen (10). Elimination of extracellular bacteria from the lung of a naive host is typically thought to be mediated primarily through phagocytosis and intracellular killing by alveolar macrophages and neutrophils recruited to the site of infection (2).The importance of gamma interferon (IFN-␥) and the IFN-␥-inducing cytokine interleukin-12 (IL-12) in phagocyte activation and protection against intracellular pathogens is well accepted, but little is understood about the significance of these cytokines in protection against extracellular microbes (5). Indeed, it has been reported that IL-12 p40 Ϫ/Ϫ BALB/c mice (mice genetically deficient in IL-12 p40 production) and IFN-␥ Ϫ/Ϫ BALB/c mice (mice genetically deficient in IFN-␥ production) are more resistant to pneumococcal infection than wild-type animals are (12, 16). We have now addressed the roles of IL-12 and IFN-␥ in local protection against pulmo...

Interleukin-10 (IL-10) is an important anti-inflammatory molecule that can cause immunosuppression and long-term pathogen persistence during chronic infection of mice with viruses such as lymphocytic choriomeningitis virus. However, its specific role in immunity to acute viral infections is not fully understood. We found that IL-10 plays a detrimental role in host responses to acute influenza A virus since IL-10 ؊/؊ mice had improved viral clearance and survival after infection compared to wild-type mice. Enhanced viral clearance in IL-10 ؊/؊ mice was not correlated with increased CD4 ؉ or CD8 ؉ T-cell recruitment into the lung but was correlated with increased pulmonary anti-influenza virus antibody titers, and this was dependent upon the presence of T cells, primarily CD4 ؉ T cells. In addition, virus-specific antibody produced during the early stages of infection in the respiratory tract of IL-10 ؊/؊ but not wild-type mice was sufficient to mediate passive protection against viral challenge of naïve mice. Complement was necessary for this antibody-mediated passive protection, but Fc␥R or neutrophil deficiency did not significantly influence viral clearance. Our results show that an absence of IL-10 at the time of primary infection leads to enhanced local virus-specific antibody production and, thus, increased protection against influenza A virus infection.