Influenza is an infectious disease whose impact and consequences are often underestimated¹ given that it annually causes approximately 500,000 deaths worldwide,² a number that can further increase during influenza pandemics. Bacterial coinfections are frequent occurrences in patients hospitalized for influenza.³The most frequent bacterial agent involved is Streptococcus pneumoniae,^3-5 but the bacterial spectrum may be quite diverse. Nasopharyngeal colonization with S. pneumoniae has been reported in about 5-20% of adult patients and 40-60% of children,^6,7 with percentages as high as 85% in children with ages below 4 years.⁸ Experimental studies performed in murine models of pneumococcal colonization show that infection with influenza A virus can associate a significant increase in bacterial load throughout the respiratory tract, from the nasopharyngeal to the pulmonary level.⁹

In the 1918 influenza pandemic, mortality was largely attributed to bacterial super-infection,¹⁰ and epidemiological data subsequently recorded have confirmed that influenza virus and S. pneumoniae display an important synergy. Therefore, it has become of utmost importance to exhaustively understand the mechanisms involved in influenza-pneumococcus co-pathogeny. Although numerous studies have already been performed, and several types of mechanisms have been identified and may potentially explain the increased susceptibility to pneumococcal infections secondary to influenza, a complete understanding of the interactions between the immune system, the virus and the bacterium still eludes us.

Influenza infection alters the pulmonary physiology and the immune responses, rendering the host more vulnerable to pneumococcal invasion. Decreased mucociliary clearance,¹¹ immune cells dysfunction and dysregulated cytokine milieu¹² are some well-known mechanisms that diminish the host’s defense against S. pneumoniae. This brief review summarizes recent discoveries that increase our understanding of the mechanisms of synergy between influenza virus and S. pneumoniae.

By using the search terms “Streptococcus pneumoniae” or “S. pneumoniae” or “pneumococcal” and “influenza” on PubMed and narrowing the search down to the publication timespan January 2016 - January 2017, we have identified 8 fundamental studies that describe the pathogenic mechanisms between these two microbial agents.

Influenza virus mainly targets the respiratory tract-based cells and especially the ciliate epithelial cells.13 As with any type of infection, the innate immune mechanisms are typically activated first, and, if elimination of the viral pathogen is unsuccessful or incomplete, the adaptive immune mechanisms shortly ensue.14 This immune conflict during infection with influenza virus leads to changes in the physical and immunologic defense in the entire  respiratory tract, which consequently predisposes the host to bacterial superinfections. McCullers et al. have shown that lethality is high in simultaneous coinfection with S. pneumoniae and influenza virus, and maximum when secondary pneumococcal infection occurs at about seven days following the primary infection with influenza virus.15 Among the best studied mechanisms of influenza and pneumococcal co-pathogenicity is the deterioration and limited restoration of the respiratory tract ciliate epithelial cells, processes which in turn associate a decrease in mucociliary clearance,11,16 and consequently lead to a heightened risk of bacterial pneumonia. Influenza virus compromises the innate immune system and additionally involves the dysfunction of the immune cells, i.e., neutrophils and/or macrophages, which would normally be involved in bacterial clearance through phagocytosis.12 Moreover, the inflammatory response may be further exacerbated by the influenza virus through release of mediators such as cytokines and chemokines thus determining tissue lesions, increasing susceptibility to secondary bacterial infections, and stimulating bacterial adhesion to epithelial respiratory cells.12,17 These three mechanisms (deficit of mucociliary clearance, dysfunction of immune cells and abnormal secretion of inflammatory mediators) appear to be the main drivers of the co-pathogenicity between influenza virus and S. pneumoniae, as field literature published during the last year clearly shows. The inhibition of mucociliary clearance (measured as decreased tracheal mucociliary velocity) may become apparent two hours after influenza virus infection, and may allow the accumulation of S. pneumoniae in the trachea, as a first essential step in the pathogenesis of pneumococcal superinfection.11 Macrophages play an essential role in host defense, being responsible for phagocytosis and clearance of pathogens and of inflammatory  debris, respiratory tract included.18 In a study on human monocyte-derived macrophages challenged with S. pneumoniae, Cooper et al.19 showed a significant increase in interferon-β (IFN-β) expression in macrophages primed with influenza virus, and an association with a decreased bacterial phagocytosis. The authors also identified a significant decrease in CD36 expression, a non-opsonic receptor generally involved in phagocytosis,20 but in Cooper’s in vitro study this decreased expression did not directly impact phagocytosis, and the authors concluded that the increased expression of IFNβ remained the main driver of impaired pneumococcal phagocytosis in influenza A virusinfected macrophages.19 Another category of receptors contributing to the innate immune response are Toll-like receptors (TLRs), and recognition of influenza virus is performed by members of at least three distinctive pattern recognition receptor (PRRs) classes, among which TLRs play a leading role.21 Endosomal TLRs detect viral genetic material, while TLRs located on the surface of antigenpresenting cells or epithelial cells are responsible for sensing bacterial components.22  In a study conducted on human monocytederived dendritic cells, Spelmink et al.23 showed that influenza virus increases the expression of TLR3, which recognize pneumococcal genetic material (RNA) and consequently increase the secretion of type I interferons (IFN-α, IFN-β).23 These changes have been, in turn, demonstrated to be linked to an increased lethality if pneumococcal superinfection is associated.24 The role of interferon in the pathogenesis of respiratory viral infections is also correlated with its effect on inducing the activation of the JAKSTAT signaling pathway. Hoffmann et al.25 have shown that coinfection with influenza A virus and S. pneumoniae significantly impacts the JAKSTAT pathway, and that coinfected human monocyte-derived macrophages exhibit an increased expression of interferon-γ-inducible protein 10 (IP-10).25 These results have also been replicated in the clinic, where a 3.6-fold higher level of IP-10 was found in the serum of children with mixed viral and bacterial pneumonia, and a 4.2-fold higher level in those with severe pneumonia, suggesting that IP-10 can be further explored for its potential role as maker for mixed infection or severe disease.25 Influenza-driven cellular immunity is also regulated by Th1 lymphocytes and their production of interferon-γ.26 Duvigneau et al.27 have studied the contribution of several cytokines to the pathogenesis of pneumococcal infection following influenza on murine models, showing that IFN-γ, interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were significantly increased. The authors showed that IFN-γ affects bacterial clearance, facilitating bacterial growth and systemic dissemination following acute viral infection.27 While a similar role in impairing bacterial clearance has been attributed to IL-6, its impact is not as  important as that of IFN-γ, but may be positively associated with an increase in the duration of bacterial colonization in the lungs.27 Duvigneau et al. have also shown that bacterial clearance following influenza is not influenced by TNF-α levels.27 This study is in accordance with previous research, which revealed that IFN-γ suppresses the phagocytosis of alveolar macrophages and encourages bacterial multiplication.28 The exact role of IL-6 and TNF-α has, however, been subject to discussion in previous studies. IL-6 has been acknowledged as a severity marker in pneumococcal infection, due to its strong proinflammatory functions.29 In addition, other studies have suggested a protective role of TNFα during pneumococcal infection, 30 despite its
potentially pro-apoptotic actions in the lung.31 The role of another cytokine, IL-35, was highlighted by Chen et al.32 in a study on murine models of post-influenza pneumococcal pneumonia. They showed that coinfected mice presented a significant increase in IL-35 levels, but that this induction was considerably lower for interferon α/β receptor (IFANR)-deficient mice, indicating that type I IFN signaling is involved in the expression of IL-35.32 IL-10 has also been shown to be involved in the pathogenesis of pneumococcal infection following influenza.33 Barthelemy et al. showed that influenza-associated increased IL-10 levels  coupled with decreased IL-12 levels suppress the activation of invariant natural killer T cells, thereby rendering the host more susceptible to secondary pneumococcal infection.33 These cells’ protective role in viral and bacterial infection has also been previously studied, as they stimulate the production of IL-22 (which is involved in damage control and restoration of pulmonary tissue following influenza  infection)34,35 and IL-33 (which activates group 2 innate lymphoid cells, with beneficial role on tissue repair during influenza infection).36 Apart from the priming effect of influenza virus, S. pneumoniae per se may directly contribute to the induction of inflammation. Among the most studied pneumococcal enzymes, the neuraminidase is widely known for its involvement in virulence, and three different enzyme types have been described, coded by NanA, NanB and NanC.37,38 The most active and highly expressed is NanA, a gene detected in virtually all strains of S. pneumoniae. 38 An important function of the neuraminidase is to cleave sialic acid from cellular receptors;37,38 by doing so, it facilitates bacterial access to the host cell. In addition, NanA catalyzes the cleavage of sialic acid residues from host proteins such as lactoferrin and immunoglobulin A2, which are essential to bacterial clearance.38 Wren et al.39 have highlighted the significance of NanA in pneumococcal colonization and in the pathogenesis of pneumococcus-influenza coinfection. By performing a murine model study, they concluded that pneumococcal NanA promotes pathological mechanisms by both enzymatic (sialidase activity) and non-enzymatic processes (such as biofilm formation), its expression allowing the full cooperation of the two microorganisms. Furthermore, Wren et al. have shown that neuraminidase inhibitors can decrease pneumococcal adhesion to host epithelial cells without impacting biofilm formation,39 while a previous study in mice concluded that viral neuraminidase inhibitors confer protection against pneumococcal pneumonia due to their inhibition of enzymatic activity.40 The interaction between influenza and pneumococcal neuraminidases has also been explored by Whalter et al.,41 who showed that the interaction between the two neuraminidase types is fundamental for disease pathogenesis, and that it can be disrupted by administering viral neuraminidase inhibitors.41 Timing of infection The order of the bacterial and viral challenge also appears to significantly impact copathogenicity. McCullers et al. have shown that in murine models baseline nasopharyngeal colonization with S. pneumoniae may essentially protect the host from developing influenza and from lethal outcomes of infection, whereas lethality is quantifiably higher when simultaneous coinfection occurs, and maximum when secondary pneumococcal infection ensues at roughly seven days following primary infection with influenza virus. 15 In another murine study, McCullers et al. have shown that colonization with S. pneumoniae and subsequent infection with influenza virus leaves the host more prone (63%) to developing otitis media, due to virallyinduced Eustachian tube dysfunction, or disruption of mucosal surfaces.42 When influenza virus precedes pneumococcal infection, Cooper et al. have shown that macrophages lose their ability to successfully clear S. pneumoniae through phagocytosis, by an IFN-β-related mechanism19 and Duvigneau et al. have shown that IFN-γ affects bacterial clearance, facilitating bacterial growth and systemic dissemination.27 Barthelemy et al. showed that increased IL-10 and decreased IL-12 levels suppress the activation of the otherwise protective invariant  natural killer T cells, and leave the host more susceptible to secondary pneumococcal infection following influenza.33 And finally, Siegel et al showed that influenza infection followed by pneumococcal colonization seven days later stimulates the multiplication of S. pneumoniae and facilitates its access to the inferior respiratory tract by increasing the availability of sialic acid in the nasopharynx and by stimulating the secretion of Muc5ac, a eavily sialylated mucin from the respiratory tract. 

In conclusion, influenza virus and S. pneumoniae have developed multiple collaborative mechanisms, and the strongest synergy is apparent when influenza virus precedes S. pneumoniae infection by approximately one week. This sequence ensures a decrease in pulmonary defenses, including bacterial clearance and phagocytosis, and primes the respiratory tract for a new infection with potentially severe consequences.

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