Our goal was to investigate the role of synergistic bacterial superinfections in exacerbation of influenza virus-associated mortality using mild, self-limiting viral and bacterial infections. Singly infected animals did not die, nor did they exhibit overt signs of disease. Both the mild virus and bacterial infections and the transient bacteremia found in about 30% of S. pneumococcus-infected mice were cleared within 7 days. In contrast, superinfected mice rapidly became sick and succumbed to infection within 2 to 4 days postsuperinfection. In all influenza virus-infected mice, type 3 pneumococcus superinfection resulted in the prolonged systemic dissemination of bacteria. The high bacterial loads observed in all major organs likely overwhelm the host immune system such that it is no longer able to control the infection, and this leads to septic shock. The in vivo studies described here using mild, self-limiting viral and bacterial infections are highly reminiscent of synergistic disease observed in the clinical setting and should provide for an informative, more clinically relevant model to evaluate the effects of antivirals, antibiotics, and vaccine approaches on controlling synergistic respiratory disease.
In order to more closely mimic clinical establishment of synergistic disease, we used a filamentous non-mouse-adapted strain of influenza virus. Original human isolates of influenza virus are highly filamentous in morphology; even the avian H5N1 strain of influenza virus exhibits filamentous morphology. In contrast, most laboratory-adapted strains, including the commonly used influenza A/PR/8/34 strain of influenza virus, produce strictly spherical virions. Previous studies examining virus-bacterium synergistic interactions in vivo have utilized mouse-adapted, spherical strains of influenza virus that are themselves highly lethal to mice (20
). Several mechanisms have been proposed that may contribute to synergistic exacerbation of respiratory disease. Early studies demonstrated that Staphylococcus aureus
adheres more efficiently to influenza virus-infected MDCK cells (11
). More recently, Streptococcus pneumoniae
has also been shown to bind more efficiently to influenza virus-infected epithelial cells in vitro (25
) and to mouse respiratory epithelium in vivo (31
). This increased binding is believed to be a result of destruction of ciliated cells and complete desquamation of the respiratory epithelium. In our studies, we also found that S. pneumoniae
colonizes the respiratory tracts of mice more efficiently in animals with an ongoing influenza virus infection. However, the observation that animals superinfected after virus clearance remained highly susceptible to synergistic fatal septicemia suggests that direct viral and bacterial interactions are not required for enhanced colonization or exacerbative disease.
A recent study has suggested that increased levels of the anti-inflammatory cytokine IL-10 may be responsible for increased severity of pneumococcal infection in mice recovering from influenza virus infection (35
). However, administration of an anti-IL-10 monoclonal antibody that was able to marginally reduce bacterial loads in superinfected animals did not protect animals and only delayed mortality (35
). In our study, IL-10 mRNA levels in the lungs and spleens of superinfected mice were upregulated by four- to fivefold in comparison to singly infected mice. However, dramatic increases in mRNA levels for a variety of proinflammatory molecules in superinfected animals, including IL-6, TNF-α, IL-1β, inducible nitric oxide synthase (not shown), and notably G-CSF, were also observed. These cytokines all result in local inflammation and tissue damage when produced in excess amounts. Thus, our results suggest that multiple inflammatory mediators are synergistically exacerbated rather than IL-10 alone, and these all contribute to septic shock and death.
Depression of the immune system has been implicated in the reduced bacterial clearance from influenza virus-predisposed mice. In vitro studies have shown that influenza virus is able to cause apoptosis in neutrophils (9
) and inhibit phagocytic and chemotaxic function of macrophages (19
). Other studies have observed a decrease in chemotactic activity of neutrophils at the time of secondary infection in an in vitro model (2
). In our synergy model, however, we did not observe overt signs of neutropenia. In contrast, superinfection resulted in a massive influx of neutrophils into the lungs. This coincided with upregulated levels of the neutrophil chemoattractants MIP-2 and KC, mouse homologues of human IL-8, in the lungs of superinfected animals. Both have been implicated to play significant roles in acute lung injury (ALI) of patients (18
), a disease characterized by accumulation of inflammatory infiltrates such as activated neutrophils in the lung tissue and BALF that leads to tissue damage and respiratory distress. Immune system deregulation predisposes patients to ALI. Our studies suggest that the massive influx of neutrophils observed in our superinfected mice is contributing to the pathogenesis of the synergistic infection. Upon activation, neutrophils produce proinflammatory cytokines, which cause fever and local inflammation as well as aid in recruitment of additional inflammatory cells, including T and B lymphocytes (14
). Activated neutrophils also produce antimicrobial agents, such as hydrogen peroxide, H2
, superoxide anion, O2−
, and nitrous oxide, NO (14
). Thus, the enhanced bacterial colonization of the lungs following influenza virus infection may lead to the sustained production of toxic compounds by neutrophils that together with the bacteria increases tissue destruction. In addition, protein levels of G-CSF, which regulates the maturation, differentiation, and proliferation of neutrophils, were highly elevated in both serum and BALF from superinfected animals.
Recombinant human G-CSF (rhG-CSF) has been utilized in conjunction with antibiotics to prevent neutropenia-associated infections in cancer patients receiving chemotherapy. However, recent clinical studies have examined a possible correlation between the administration of G-CSF and acute lung injury and acute respiratory distress syndrome (37
). Filgrastim (Roche), a commonly used rhG-CSF cancer therapeutic, when administered subcutaneously leads to serum levels of 48 to 56 ng/ml (10
). Due to the short half-life of rhG-CSF, patients receive daily administration of filgrastim until neutropenia subsides (10
). In neutropenic rats, lipopolysaccharide administered following treatment with recombinant G-CSF resulted in exacerbated lung injury compared to control rats that received no G-CSF therapy (5
). This suggests that high G-CSF levels in the serum may be sufficient to predispose animals and/or patients to heightened bacterial infections. Interestingly, in our study G-CSF levels in excess of 4,600 pg/ml in the serum and over 2,400 pg/ml in the BALF following bacterial superinfection were determined, levels within the range at which rats and humans are predisposed to exacerbated lung disease by rG-CSF treatment. Together, these studies and the data presented here suggest that the release of G-CSF by the inflamed endothelium and alveolar monocytes and macrophages likely contributes significantly to the synergistic pathogenesis leading towards fatal septicemia.
The lung is not the only organ subject to neutrophil-induced tissue damage. Dysfunction of the blood-brain barrier in traumatic brain injury patients is associated with an increase of IL-8 production by brain microvascular endothelial cells and astrocytes (28
). The production of IL-8 in the brain is induced by the synergistic activity of TNF-α and IL-1β (28
). IL-1β was consistently upregulated at the mRNA level in the brains of superinfected mice. In a rat model of bacterial meningitis, administration of neutralizing antibodies targeting MIP-2 significantly decreased the influx of neutrophils into the meninges, further illustrating the contributing role of neutrophils in disease progression (13
). KC, MIP-2, and G-CSF mRNA levels were all upregulated in the brains of the superinfected mice. Both KC and G-CSF mRNA expression were upregulated in the spleens from superinfected animals, suggesting that neutrophils may also be infiltrating the spleens and causing damage as well. KC and G-CSF were also elevated in the serum, which further implies the presence of circulating activated neutrophils.
Clearly evident from our study and the reports by others is that influenza virus infection establishes an environment in the respiratory tract, “the predisposed state,” that allows for an exacerbative response to subsequent bacterial infections. Singly infected animals did not display any adverse upregulation of proinflammatory cytokines that could be directly attributed to the synergistic effect. However, virus infection did result in an influx of predominantly lymphocytes and monocytes into the bronchoalveolar spaces, which were maintained well after the virus had been cleared. This suggests that these inflamed, or activated, lymphocytes/monocytes are predisposed to secrete massive levels of chemokines and proinflammatory cytokines once a superinfection results. Future studies will focus on identifying and characterizing these cells and how they respond to different stimuli.
In summary, we have established a murine model of fatal septicemia that closely mimics clinical exacerbation of influenza virus-associated respiratory disease by superinfection with serotype 3 pneumococci. The use of mild, nonlethal doses of each organism is sufficient to establish a synergistic and highly virulent relationship between the two pathogens. Further, our results suggest that bacteremia and the accompanying systemic tissue damage is of paramount importance in virus-bacterium synergistic pathogenesis. Whereas the neutrophil is likely to be the major host cellular infiltrate responsible for the exacerbation of disease in predisposed animals, it is the virus-induced lymphocytes infiltrating the lungs that are likely predisposed for massive upregulation of neutrophil chemoattractants and activators, such as MIP-2, KC, and G-CSF. G-CSF may be acting to prolong neutrophil survival, allowing for sustained secretion of tissue-damaging molecules in the lungs, spleens, and brains of superinfected animals. The present study further illustrates the complexity of factors that synergistically contribute to exacerbative influenza virus-associated illness. As we learn more about the nature of the influenza virus-induced predisposed state, we can then begin to design new therapeutic treatment strategies to reduce this synergistic exacerbation of disease. Future studies will focus on evaluating whether other bacterial pathogens frequently associated with influenza virus infections, i.e., Staphylococcus aureus and Haemophilus influenzae, also exacerbate disease in a similar fashion.