UvA-DARE ( Digital Academic Repository ) Bench-to-bedside review : Bacterial pneumonia with influenza-pathogenesis and clinical implications

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post-infl uenza pneumonia, but Streptococcus pneumoniae is the most prominent pathogen involved [15]. A recent report on the new H1N1 infl uenza strain indicates that 29% of fatal H1N1 cases between May 2009 and August 2009 in the United States were associated with a secondary bacterial infection [16], which is markedly less than for previous infl uenza pandemics [17,18]. In addition to S. aureus and S. pneumoniae, Streptococcus pyogenes was also frequently isolated [16,18]. Primary infections with these pathogens are usually less severe than secondary infections. Th e incidence of invasive pneu mococcal disease closely correlates with the infl uenza season [19], and pneumococcal vaccination not only results in an overall reduced number of pneumonia cases, it also leads to markedly reduced cases of virusassociated pneumonia [20]. Although secondary bacterial pneumonia has been described for other respiratory viruses as well, the morbidity and mortality is much lower than observed for infl uenza [21,22].

Pathogenesis of bacterial pneumonia with infl uenza
Bacterial respiratory infection during infl uenza virus infection can be divided into combined viral/bacterial pneumonia or secondary bacterial infection following infl uenza. Clinical symptoms do not distinguish between bacterial and viral pneumonia early in the course of disease, rendering early clinical distinction a challenge. Critically ill patients with viral pneumonia present with bilateral interstitial infi ltrates on the chest radiograph indistinguishable from bacterial pneumonia [23]. Other markers of infl ammation are also not specifi c. Distinction between viral and bacterial pneumonia by microbiological and/or molecular techniques, however, is highly relevant in terms of initiating antimicrobial therapy, as 32% of patients with viral pneumonia develop a concomitant bacterial pneumonia [23]. Secondary bacterial infections following infl uenza are more easily recognized clinically compared to combined viral/bacterial pneu monia, since these bacterial infections tend to occur during the recovery phase from infl uenza [24]. Epidemio logical studies indicate that individuals infected with infl uenza virus are most susceptible to secondary bacterial pneumonia between 4 and 14 days after the onset of infl uenza symptoms [25].
Although the incidence of a secondary bacterial infection does not show a clear distinction between combined viral/bacterial pneumonia and secondary bacterial infection following infl uenza, the processes leading to severe bacterial pneumonia in conjunction with infl uenza virus infections are multifactorial and diff er between early and late bacterial infection. During combined viral/ bacterial infection, the virus not only interacts with the host response, it also interacts with bacterial-induced infl ammation, increasing bacterial colonization and outgrowth as well as viral replication ( Figure 1). Conversely, the host response to both patho gens will aff ect viral replication and bacterial growth [26,27]. From a mechanistic point of view, post-infl uenza pneumonia is less complicated than combined viral/bacterial pneumonia, since the virus has been cleared ( Figure 1). Th e patho genesis of post-infl uenza pneumonia involves virus-induced changes to the host [28,29]. Th ese diff erences are important to take into consideration when studying the mechanisms of secondary bacterial complica tions and may also have an impact on thera peutic strategies to be followed when patients are hospitalized for infl uenza complicated by pneumonia.
Th e severity of combined viral/bacterial infection or post-infl uenza pneumococcal pneumonia is classically attributed to infl uenza-induced damage to the airway epithelium, which leads to increased colonization of bacteria at the basal membrane [30]. Infl uenza virus preferentially infects and replicates in airway epithelial cells, leading to the induction of an antiviral process in order to eradicate the virus. Besides limiting viral replication by means of transcriptional and translational Combined viral/bacterial pneumonia Post-influenza pneumonia inhibi tion, epithelial cells are instructed to undergo apoptosis [31]. Th e apoptotic bodies containing the virus are subsequently removed by (alveolar) macrophages [32]. Major drawbacks of this antiviral mechanism include not only the increased risk of bacterial colonization, but also enhanced invasion by bacteria. In addition to epithelial injury, mucociliary clearance has recently been shown to be impaired during infl uenza virus infection, leading to an enhanced burden of S. pneumoniae already at 2 hours after bacterial challenge [33].
Over the past few years it has become increasingly clear that epithelial injury is not the only factor that contributes to the severe outcome resulting from bacterial complications during infl uenza infection [27-29, 33, 34]. Mouse studies have revealed additional mechanisms that play a critical role in either combined viral/bacterial infection or post-infl uenza pneumococcal pneumonia (sum marized in Table 1). Most mouse models that are currently used focus on combined viral/bacterial pneumonia (bacterial challenges up to 7 days after infl uenza) [25,[33][34][35], while other models are used to investigate post-infl uenza pneumonia [28,29] (bacterial challenges ranging from 14 days up to 35 days after infl uenza infection).

Viral factors contributing to secondary bacterial complications
Several viral factors have been identifi ed as critical for the development of secondary bacterial pneumonia. Viral neuraminidase has been shown to enhance bacterial growth as well as bacterial dissemination in a mouse model for secondary pneumococcal pneumonia. Studies with recombinant infl uenza strains containing diff erent neuraminidase genes indicate that neuraminidase activity correlates with increased adhesion of pneumococci to airway epithelial cells, which could be reversed by adding neuraminidase inhibitors [36]. Infl uenza strains with relatively high neuraminidase activity, such as the 1957 pandemic infl uenza strain, were associated with an increased incidence of pneumococcal pneumonia and higher mortality rates in mice after bacterial challenge [37]. In addition, mice treated with neuraminidase inhibitors for up to 5 days after viral exposure showed markedly increased survival rates. Nevertheless, neuraminidase inhibitors were only partially protective in this model for bacterial complications following infl uenza virus infection [38].
In addition to neuraminidase, PB1-F2, a pro-apoptotic protein expressed by most infl uenza A strains, has been implicated in the pathogenesis of secondary bacterial pneumonia as well. Mice infected with viral strains lacking PB1-F2 were largely protected against secondary bacterial complications. In line with this, mice infected with a viral strain that expresses the PB1-F2 protein from the 1918 pandemic infl uenza strain appeared to be highly susceptible to pneumococcal pneumonia [39]. Since PB1-F2 did not have an impact on bacterial loads and since it has been implicated in the pathogenesis of primary infection with infl uenza virus, it may be concluded that PB1-F2 induces lung pathology during viral infection, which may enhance the infl ammatory response to a secondary challenge. Th e underlying mechanism of PB1-F2-induced lung pathology is largely unknown.

Bacterial factors contributing to secondary bacterial pneumonia
Bacterial components that contribute to secondary bacterial pneumonia have been poorly investigated. In contrast to viral neuraminidase, bacterial neuraminidase has not been implicated in combined viral/bacterial pneumonia or post-infl uenza pneumonia [34,37,40]. Th e fact that bacterial neuraminidase does not contribute to enhanced replication of infl uenza is most likely due to poor enzymatic activity compared to viral neuraminidase and the strict sialic acid substrate requirements of bacterial neuraminidase. In contrast, pneumococcal surface protein A (PspA) has been shown to increase bacterial colonization in mice infected with infl uenza virus [40]. PspA is known to interfere with complement-mediated phagocytosis and lactoferrin-mediated killing. However, it is also identifi ed as a virulence factor for primary pneumococcal pneumonia [41]. As such, PspA seems to have a limited contribution to the severe outcome of bacterial pneumonia with infl uenza. Similarly, pneumococcal hyaluro nidase has been identifi ed as a virulence factor for primary pneumococcal pneumonia, but did not have an impact on pneumococcal pneumonia following infl uenza [40].
S. pneumoniae has been shown to bind to the plateletactivating factor receptor (PAFR) through phosphatidylcholine in the bacterial cell wall [42], which has been suggested to increase colonization of bacteria and/or to mediate transition from the lung to the blood [43]. Th e impact of this interaction was further investigated using PAFR knockout mice [44,45] and pharmacological inhibitors of PAFR [35]. Although infl uenza virus has been shown to upregulate the expression of PAFR [43], no studies have identifi ed a more pronounced role for it in secondary pneumococcal pneumonia compared to primary pneumococcal infection [35,44,45]. PAFR appears to mediate invasive pneumococcal disease during primary and secondary pneumococcal pneumonia, while colonization within the lung seems to be dependent on the bacterial strain [43][44][45].
In conclusion, there is little evidence that bacterial virulence plays an important role in the pathogenesis of secondary pneumococcal pneumonia after infl uenza. Protease activity by S. aureus has been shown to increase the virulence of infl uenza A virus in mice by cleaving virus hemagglutinin. However, protease inhibitors have not been further investigated in models of secondary bacterial pneumonia [46].

Host factors contributing to secondary bacterial pneumonia
Most studies on the mechanism underlying bacterial pneumonia following infl uenza have focused on impaired host defense against secondary infection with an unrelated pathogen. Infl uenza virus infection has been shown to impair neutrophil function at multiple levels [28,34,[47][48][49][50][51][52][53][54]. Initial studies indicated that infl uenza virus reduces chemotaxis and chemokinesis of neutro phils in vitro and in vivo [55], which appeared to be strain-dependent in subsequent studies with patients infected with infl uenza virus [52]. In addition to this direct inhibitory mechanism, a recent study identifi ed type I interferon (IFN), an antiviral cytokine, as an impor tant factor in the downregulation of relevant chemokines, such as keratinocyte-derived chemokine and macrophage infl ammatory protein 2, thereby inhibit ing the migration of neutrophils [53]. However, several studies reported increased, rather than reduced, numbers of neutrophils after secondary bacterial challenge in mice infected with infl uenza virus [28,34,56]. Th e increased number of neutrophils may correlate with higher bacterial loads in these models of secondary bacterial pneumonia. Th e higher bacterial loads might be explained by a reduced phagocytic capacity of neutrophils [28,34,45,57,58]. In vitro studies with ultraviolet irradiated and heat killed infl uenza virus indicated that the reduction in phagocytic capacity is mediated, at least in part, by viral neuraminidase activity [58]. Nevertheless, the impaired eff ector function is still present after the virus has been cleared [28], indicating that host factors contribute to impaired bacterial killing. IL-10 production is synergistically enhanced in mice infected with S. pneumoniae during viral infection [38,56] as well as after clearance [28] of infl uenza virus. Inhibition of IL-10 markedly improved survival in a mouse-model for post-infl uenza pneumococcal pneumonia, which was associated with reduced bacterial loads. Th e role of IL-10 in combined viral/ bacterial pneumonia seems to be limited, since IL-10 knockout mice did not show an improved response to secondary bacterial infection [59]. It should be noted, however, that IL-10 knockout mice respond diff erently to primary viral infection as well, leading to a more pronounced proinfl ammatory state [60]. Together, these fi ndings not only illustrate the complexity of secondary bacterial pneumonia, they also stress that combined viral/bacterial infection is intrinsically diff erent from post-infl uenza pneumonia.
Th e tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been shown to enhance IL-10 levels in a mouse model for post-infl uenza pneumococcal pneumonia [61]. Inhibition of IDO, which is expressed during the recovery phase of infl uenza infection, reduced bacterial loads during secondary, but not primary, pneumococcal infection. Despite a clear reduction in bacterial loads as well as markedly reduced levels of IL-10 and TNF-α, it did not have an impact on survival. It is unlikely, therefore, that IDO predisposes for bacterial pneumonia by means of enhancing IL-10 production.
Recent observations in our laboratory indicate that local IDO activity induces apoptosis of neutrophils during bacterial infection of the airways (submitted for publication). IDO-mediated apoptosis, which has been extensively studied for T lymphocytes, is particularly mediated by metabolites such as kynurenine and 3-hydroxy anthranilic acid, rather than depletion of tryptophan. Tryptophan metabolites have been implicated in monocyte and macrophage apoptosis as well [62,63]. Together, these data indicate that IDO functions as a natural mechanism to remove infl ammatory cells. Th is mechanism to resolve infl ammation prevents excessive damage to the airways after viral infection, but increases the susceptibility to secondary bacterial pneumonia.
In addition to neutrophils, macrophages and monocytes [58,64] have also been shown to have a reduced phagocytic capacity during infl uenza infection. IFN-γ has been shown to play a critical role in macrophage dysfunction through downregulation of 'macrophage receptor with collagenous structure' (MARCO) expression on alveolar macrophages [65]. MARCO can be classifi ed as a scavenger receptor involved in the innate recognition and subsequent killing of bacteria. MARCO knockout mice have been shown to be more susceptible to pneumococcal pneumonia, which was associated with higher bacterial loads, enhanced lung pathology and increased mortality rates [63]. Although other factors that mediate opsonization or phagocytosis of bacteria have been extensively studied for primary bacterial pneumonia [66][67][68], their roles in either combined viral/ bacterial pneumonia or post-infl uenza pneumonia are largely unknown.
Knowledge about the role of other pattern recognition receptors, such as Toll-like receptors (TLRs), is limited. A recent study indicated that infl uenza virus infection resulted in sustained desensitization of TLRs for up to 6 weeks after infl uenza virus infection [29]. Mice exposed to infl uenza virus exert a poor response to lipopolysaccharide, lipoteichoic acid and fl agellin, ligands for TLR4, TLR2 and TLR5, respectively, as refl ected by reduced neutrophil numbers in bronchoalveolar lavage fl uid. Th ese data are supported by the fact that TLR2 knockout mice were equally susceptible to secondary bacterial pneumonia following infl uenza virus infection compared to wild-type mice [69]. It is worth noting that TLR4 can compensate for a defect in TLR2 during primary pneumococcal pneumonia [70]. In addition to TLR desensitization, CD200R expression has been proposed to impair the host response towards bacteria during infl uenza virus infection [71]. Although CD200-CD200R interactions have been shown to negatively regulate infl ammation through induction of IDO [72], its role in secondary bacterial pneumonia has not been investigated yet.
Taken together, these host factors contributing to severe post-infl uenza pneumonia all relate to altered innate immune mechanisms that are supposed to resolve or dampen virus-induced infl ammation and related tissue damage. It should be noted that most studies have been performed using mouse models for combined viral/ bacterial pneumonia or post-infl uenza bacterial pneumonia and require confi rmation in humans.

Current treatment options
Vaccination against infl uenza has been shown to reduce mortality rates during infl uenza epidemics [73]. Seasonal infl uenza epidemics are primarily caused by antigenic drift (that is, single-point mutations that are caused by the high mutation rate of infl uenza virus strains). Although single-point mutations occur at random, genetic changes can be predicted in advance [74]. Th ese predictions provide the opportunity to develop vaccines to prevent seasonal infl uenza and therefore also the risk of secondary bacterial infections. Vaccination of elderly patients has been shown to reduce hospitalizations by 52%. In contrast to seasonal infl uenza, pandemic infl uenza, such as caused by the recently emerged H1N1 strain [3,75], results from antigenic shift. It is hard to predict when these changes occur and which strains are involved. It is virtually impossible, therefore, to develop vaccines directed against pandemic infl uenza strains in advance. Vaccines against new infl uenza strains only become available when the vaccine has been validated extensively.
Besides vaccination, treatment options to prevent a complicated course of infl uenza is to inhibit viral replication with antiviral agents, such as amantadine (Symmetrel®), or neuraminidase inhibitors, such as oseltamivir (Tamifl u®) and zanamivir (Relenza®). Th ese agents have been shown to reduce infl uenza-related symptoms [76][77][78], but their effi cacy against bacterial complications remains to be determined [79]. Viral neuraminidase has been shown to be involved in the enhanced response to bacteria in a mouse model for post-infl uenza pneumococcal pneumonia [37]. Moreover, mice treated with neuraminidase inhibitors were less susceptible to secondary bacterial infections. However, neuraminidase inhibitors did not completely prevent mortality in mice with infl uenza complicated by bacterial pneumonia, which may relate to the relatively small timewindow in which neuraminidase inhibitors can reduce viral replication [80]. In addition, the effi cacy of neuraminidase inhibitors in established viral/bacterial pneumonia was not tested. Rimantadine, an amantadine analogue, did not improve mortality in mice with postinfl uenza pneumococcal pneumonia [33]. Th e effi cacy of these inhibitors in the treatment of bacterial compli cations in humans has not been established yet. Th ese approaches mainly focus on the prevention of secondary bacterial pneumonia.
Patients with community-acquired pneumonia who demonstrate or have demonstrated signs and symptoms of illness compatible with infl uenza in the days or weeks before should be empirically treated with antibiotics targeting S. pneumoniae and S. aureus in order to cover the most common pathogens causing the most severe secondary infections, and coverage of H. infl uenzae is also recommended [81]. Appropriate antimicrobial agents therefore include cefotaxime, ceftriaxone and respira tory fl uoroquinolones. As mentioned above, combined infection needs to be confi rmed by microbiological and molecular techniques. When samples from respiratory tract are proven culture negative, antibiotics can be stopped. Treatment targeted at methicillinresistant S. aureus (by vancomycin or linezolid) should be limited to patients with confi rmed infection or a compatible clinical presentation (shock and necrotizing pneumonia) [80]. Of note, mouse studies indicate that ampicillin treatment is insuffi cient to prevent mortality in a model for secondary bacterial pneumonia, while the bacteriostatic protein synthesis inhibitors clindamycin or azithromycin improve the outcome after streptococcal pneumonia in infl uenza-infected mice [82]. Th is protective eff ect is likely mediated by inhibition of toxin release [82], but it may be associated with the antiinfl ammatory properties of these latter antimicrobial agents as well [83,84]. Although ampicillin alone did not have an impact on survival in infl uenza-infected mice with secondary pneumococcal pneumonia, it did improve mortality rates in mice previously treated with oseltamivir compared to mice treated with oseltamivir alone [37].

Future perspectives
Secondary bacterial complications are the result of an altered host response due to infl uenza virus infection. Most factors that have been identifi ed to play a critical role in post-infl uenza pneumococcal pneumonia are in fact mechanisms to prevent excessive infl ammation and/ or to promote resolution of infl ammation, which are initiated to restore tissue homeostasis after clearance of the primary infection. At the same time, these mechanisms greatly impair the host response towards secondary unrelated pathogens. Cytokines and chemokines appear to play a critical role in dampening virus-induced immunopathology. IFN-γ and IL-10 have been shown to alter macrophage and neutrophil function, respectively, while type I IFN seems to impair neutrophil recruitment after secondary bacterial infection. In addition, IDO expression is induced by proinfl ammatory cytokines such as TNF-α, IFN-γ, IL-12 and IL-18, leading to apoptosis of infl ammatory cells. Although the contribution of these mediators needs to be confi rmed in humans, targeting cytokines may be an alternative approach to trigger an eff ective host response to bacteria. Although it is practically not feasible to neutralize these infl ammatory mediators as prophylactic treatment to prevent secondary bacterial pneumonia in all infl uenza-infected subjects, it may be a useful approach in hospitalized subjects, especially those that are admitted to the intensive care unit.

Conclusion
Infl uenza may be complicated by bacterial pneumonia. It is important to consider the time interval between viral and bacterial infection. At present, antibiotic treatment appears to be the only therapeutic option for postinfl uenza pneumonia. Further insight into the underlying mechanisms in combined viral/bacterial infection and post-infl uenza pneumonia may provide new targets for the treatment of these complicated infections.