Impact of Type I and III Interferons on Respiratory Superinfections Due to Multidrug-Resistant Pathogens

Abstract

The increased morbidity and mortality associated with bacterial pneumonias that are acquired following influenza infection are well appreciated by clinicians. One of the major components of the immune response to influenza is the induction of the types I and III interferon cascades, which encompasses the activation of over 300 genes. The immunological consequences of IFN activation, while important for viral clearance, modify the host proinflammatory responses through effects on the inflammasome, Th17 signaling and recruitment of phagocytic cells. IFN signaling affects both susceptibility to subsequent Streptococcus pneumoniae and Staphylococcus aureus infection as well as the intensity of the immune responses associated with pulmonary damage. Appreciation for the effects of IFN activation on anti-bacterial pulmonary defense mechanisms should help to inform therapeutic strategies in an ICU setting.

It has been shown throughout history that bacterial infection contributes significantly to overall mortality associated with major influenza epidemics. Analysis of specimens from the 1918 pandemic identified Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae in individuals who died from influenza [1], and, likewise, morbidity and mortality in the era of modern intensive care has been correlated with coinfections in major influenza outbreaks of 1957, 1968, and, more recently, with H1N1 in 2009–2010 [2–9]. A 2016 retrospective study of S. aureus/influenza virus coinfection documented mortality rates 4 times greater than those due to S. aureus alone [10].

Influenza increases the susceptibility to subsequent bacterial infection by many mechanisms. These mechanisms can be both physical and immunological. Physical mechanisms can relate to the overall health of the host, such as increased body temperature [11]. Influenza can facilitate bacterial adherence to the airway epithelium due to cellular damage [12–14] or via the upregulation of receptors for adherence, such as ICAM-1 and CEACAM1 [12]. In the case of S. pneumoniae, influenza virus infection can also act as a signal to disperse cells from biofilms [15]. Bacterial infection is also aided by decreases in airway function due to factors such as mucus, disruption of surfactant, decreased ciliary function, and pulmonary edema [16–19].

Immunological mechanisms of superinfection can be related to dysregulated cytokine production, adherent leukocyte chemotaxis, and desensitization of innate immune receptors [20]. Influenza virus infection in mice has been shown to influence the function of neutrophils and alveolar macrophages—2 important immune cells of the airway. Influenza increases their frequency of apoptosis [21–24], which can lead to the release of intracellular contents and to further inflammation [21]. Phagocytic function and chemotaxis of alveolar macrophages and neutrophils is decreased by influenza virus infection [25–27]. Influenza can also influence the function of cells, such as T cells. T cells have been shown to produce more interferon γ (IFN-γ) during influenza virus infection, which suppresses macrophage phagocytosis [27]. Suppression of the T-helper type 17 (Th17) response has also been documented [28, 29]. The ability of the innate immune system to response to bacterial productions has also been shown to be reduced by influenza virus infection. After influenza virus infection Toll-like receptors (TLRs) were shown to be desensitized to their ligands, leading to reduced chemokine production and nuclear factor κB dependent gene expression in alveolar macrophages [26]. A major component of effective viral clearance, especially in response to influenza, is the participation of type I and III IFNs, which are increasingly appreciated for their diverse roles in host defenses against bacterial as well as viral infections [30–32].

TYPE I AND III IFN SIGNALING AND THEIR ROLES IN INFECTION

The complexity and redundancies of the IFN family suggest their importance in host defense. As they are predominantly activated through receptors that are intracellular, the IFN cascades are readily activated in immune cells that phagocytose and recognize bacterial pathogen-associated molecular patterns (PAMPs), as well as by stromal cells that take up bacterial components [33]. The type I IFN family consists of 13 IFN-α subtypes, IFN-β, IFN-ε, and IFN-ω, while type III IFN is composed of IFN-λ1 (also known as interleukin 29 [IL-29]), IFN-λ2 (also known as interleukin 28A [IL-28A]), IFN-λ3 (also known as IL-28B), and IFNL4 (IL-29 is a pseudogene in mice) [34, 35]. Interaction of a type I IFN with its heterodimeric receptor (IFN α/β receptor [IFNAR]) results in dimerization and phosphorylation of STAT1/2 via Jak1 and Tyk2, leading to the downstream transcription of hundreds of genes [36]. Many bacterial pathogens, both intracellular and extracellular, are able to induce a type I IFN response via recognition of PAMPs and signaling messengers such as DNA, RNA, peptidoglycan, lipopolysaccharide, and cyclic diadenosine monophosphate [31, 37, 38]. TLR2, TLR3, TLR4, TLR7, TLR8, TLR9, NOD2, RNA polymerase III, and stimulator of IFN genes (STING) are among the many sensors involved in activating the type I IFN response [31, 39, 40]. Type III IFN (IFN-λ) signals through the IL-28 receptor (IL-28R; also known as IFNLR), also a heterodimer, consisting of IL-28R and interleukin 10 receptor β, that is primarily located on epithelial cells and neutrophils, in contrast to IFNAR, which is ubiquitous [41, 42]. Type III IFN signaling uses many of the same receptors as type I IFN signaling, and both pathways converge to phosphorylate STAT1/2 and thus activate similar transcripts [43, 44]. What differs in the transcriptome of type I and III IFN is the intensity and duration of the response. Type I is activated early and strongly, and type III is induced over time at lower levels [45, 46].

The importance of IFN signaling in host responses to infection is also evident in the multiple bacterial components and pathways that activate the type I and III IFN pathway. Conserved bacterial PAMPs such as DNA stimulate IFN signaling, but individual pathogens elicit responses through discrete signaling pathways. DNA is capable of activating type I IFN for both S. aureus and S. pneumoniae, TLR9 mediates IFN responses associated with S. aureus, and STING mediates IFN responses to S. pneumoniae [36, 47, 48]. S. aureus activates TLR9 through the normal endocytosis and digestion of bacteria leading to intracellular release of DNA and is activated regardless of the bacterial viability. S. pneumoniae requires live organisms, as it creates a pore through the major virulence factor pneumolysin, which facilitates DNA being introduced into the cell [36, 48, 49]. Different strains of S. aureus use discrete mechanisms of IFN signaling. Strain 502A, used in colonization interference during the S. aureus nursery outbreaks in the 1960s [50–53], potently stimulates type I IFNs through NOD2 and bacterial autolysis [54]. Mice lacking IFNAR have improved survival and bacterial clearance [47, 54, 55], while strains that induce greater amounts of IFN-β have increased virulence in models of acute pneumonia [54]. Studies have shown that influenza increases the susceptibility to S. aureus or S. pneumoniae infection and that type I and III IFNs play a major role in this observation [56–60].

TYPE I AND III IFN SIGNALING INFLUENCE THE NASAL MICROBIOME AND COLONIZATION DENSITY

Upper airway tract colonization with gram-positive organisms is a major risk factor for subsequent aspiration and pneumonia. A significant proportion (20%–50%) of healthy individuals, both adults and infants, are colonized at any one time with S. aureus [61, 62], while the prevalence of colonization with S. pneumoniae is higher among children as compared to adults [63]. There is significant evidence that carriage increases the risk of infection [64]. Individuals who are chronic carriers typically have higher rates of disease [65–67], and the strains isolated from infection sites are usually the carriage isolates [61, 67, 68]. When the colonization prevalence is decreased, a concomitant decrease in the incidence of disease is also observed [69, 70].

The commensal flora plays a significant role on local immunity, and influenza alters the nasal microbiome. When this balance is upset, the propensity for colonization with specific pathogens is increased, and the incidence of pneumonia is likewise increased. Antecedent influenza virus infection has been shown to increase colonization and aspiration of S. aureus and S. pneumoniae into the airway from the nasopharynx [49, 60, 71, 72] and has been linked to multiple consequences of the IFN cascade. Treatment of mice with attenuated influenza vaccine, while not influencing invasive disease, increased the colonization density of S. aureus and S. pneumoniae, as well as their duration of carriage [71]. A separate study also observed increases in staphylococci and alterations in the nasal microbiome after administration of live attenuated influenza vaccine [72]. This alteration in microbiome correlated with increased type I IFN levels that were produced in response to the vaccine [72]. Both type I and III IFN signaling were similarly found to inhibit clearance of S. aureus from the nasopharynx in the context of superinfection [60].

EFFECTS OF TYPE III IFN ON THE NASAL MICROBIOME

Influenza is able to alter the mouse nasal microbiome via a type III IFN-STAT1–dependent mechanism. Influenza led to an expansion of the nasal microbiome, as well as a restructuring of the abundance of specific genera, such as Klebsiella and Aerococcus [60]. These changes were observed to be type III IFN dependent. In the absence of IL-28R, no changes in the flora were evident, while purified IFN-λ alone was able to alter the nasal microbiome. The alterations in the flora led to increased colonization with S. aureus. Colonization studies with S. aureus in influenza virus–infected mice showed that influenza increases the ability of S. aureus to persist in the nasopharynx, and this was diminished in the absence of type III IFN signaling (Figure 1). These studies suggested that IFN-dependent expression of antimicrobial peptides in the nasopharyx was associated with the selective changes in the nasopharyngeal flora and correlated with increases in pathogens, such as S. aureus, that naturally infect influenza virus–infected patients but not with increases in other ubiquitous pathogens, such as Pseudomonas aeruginosa [60].

Figure 1.

Influence of type I and III interferon (IFN) signaling on the pathogenesis of Staphylococcus aureus and Streptococcus pneumoniae infection after influenza. Abbreviations: IFNAR, interferon α/β receptor; IL-17, interleukin 17; IL-23, interleukin 23; IL-27, interleukin 27; IL-28R, interleukin 28 receptor; MIP-2, macrophage inflammatory protein 2; Th17, T-helper type 17; TLR, Toll-like receptor.

RNA and proteomic analysis of the upper respiratory tract identified increases in the antimicrobial proteins RegIIIγ and Ngal that might have contributed to the changes [60]. Proteomic analysis of the influenza virus–infected nasopharyngeal secretions identified several proteins involved in barrier function (such as radixin and moesin) and indicate that type III IFN may influence barrier function, further altering susceptibility to infection.

IFN-ASSOCIATED CHANGES IN THE INTENSITY OF HOST PROINFLAMMATORY SIGNALING

IFN Modulation of the Inflammasome

It is clear from several independent studies that type I IFNs are involved in the formation and activation of the inflammasome, a large multiprotein complex that mediates the activation of caspase-1 and generation of the potent proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 [73, 74]. Dysregulated activation of inflammasome signaling is involved in autoimmune disease, asthma, diabetes, obesity, and multiple sclerosis [73, 75], as well as in infection. It is not clear whether the type I IFNs positively [76–78] or negatively [30, 79] regulate the inflammasome and how this influences outcome. The answer to these questions are very much model and organism specific, with some data suggesting a positive regulatory role, and others suggesting a negative role. IFNs both regulate inflammasome activation in response to S. aureus and contribute to immunopathology. Studies using both type I and type III receptor knockout mice show that significant reductions in IL-1β production correlate with improved outcome. Neutralization of IL-1 signaling by using anakinra alleviates pulmonary pathology in response to S. aureus [80]. Levels of several other proinflammatory cytokines are also reduced in Ifnar^−/− and Il28r^−/− mice, such as KC and granulocyte-macrophage colony-stimulating factor. While IFN-λ does not directly initiate proinflammatory signaling, it can inhibit other signaling mediators. In epithelial cells, IFN-λ inhibits microRNA 21, which upregulates tumor suppressor programmed cell death protein 4, a known mediator of proinflammatory signaling [80]. Influenza decreases the production of IL-1β in response to S. aureus [81, 82], while the converse is observed for S. pneumoniae [83]. While it is apparent that IFNs are capable of influencing inflammasome activation, it does not appear to be via a conserved mechanism that influences different organisms equally.

IFN-Mediated Suppression of the Th17 Response

IFNs influence the host proinflammatory response by regulation of Th17 signaling. Th17 cells produce several cytokines, especially interleukin 17 (IL-17) and interleukin 22, that are critical in host defenses against many extracellular, intracellular, and fungal pathogens [29, 84–86]. Influenza virus infection inhibits the Th17 response by suppressing IL-23 (Figure 1). Mice lacking Ifnar do not have suppressed IL-23 or Th17 signaling and, subsequently, have improved clearance of S. aureus, compared with wild-type (WT) superinfected mice [29]. Constitutive expression of adenovirus expressing IL-23 protects mice from the influenza-induced susceptibility to infection [29], further documenting the importance of this pathway in bacterial clearance following influenza virus infection.

Multiple adoptive transfer studies have shown the capacity of type I IFN to suppress the Th17 response of γδ T cells [83, 87]. In a model of S. pneumoniae superinfection, influenza suppresses IL-17 production [83]. IL-17 production was improved in Ifnar^−/− mice, with γδ T cells shown to be the main producer. Adoptive transfer of Ifnar^−/− γδ cells to WT mice prevented the increased susceptibility to infection in the presence of influenza virus [83]. The potential mechanism whereby type I IFN influences IL-17 production by γδ cells is via interleukin 27 (IL-27). Cao et al [87] observed that type I IFNs induce IL-27, which suppresses IL-17 production by γδ cells. Mice that lack IL-27 signaling were protected from the pathology associated with both S. aureus and S. pneumoniae infection after influenza [87, 88]. Likewise, neutralizing antibody to IL-27 also prevented pneumococcal superinfection [87]. Adoptive transfer of γδ T cells lacking IL-27R to WT mice led to improved IL-17 production, myeloperoxidase production, and neutrophil recruitment [87]. Thus the ability of type I IFN to influence IL-17 signaling is one mechanism that leads to influenza increasing host susceptibility to infection.

TYPE I IFN IMPAIRS THE PHAGOCYTIC RESPONSE

During superinfection, type I IFNs hinder the neutrophil response, both in the ability of neutrophils to be recruited to the airway, as well as their microbicidal function. S. pneumoniae–superinfected mice have decreased neutrophil recruitment, which is improved in the absence of IFNAR [58, 83]. The improved neutrophil recruitment was concomitant with higher levels of the CXCR2 neutrophil chemokines KC and macrophage inflammatory protein 2 (MIP-2) [58, 89] (Figure 1). The importance of KC and MIP-2 has been shown during infection. When the chemokines are inhibited, decreased clearance of S. pneumoniae is observed, while instillation of purified KC and MIP-2 improved bacterial clearance. Functional assays have also demonstrated that type I IFN exerts negative effects on neutrophil function. In infected mice, myeloperoxidase activity is reduced when IFNAR signaling is present [58, 90]. Neutrophils isolated from influenza virus–infected mice also demonstrate a decreased capacity to kill S. aureus ex vivo, and this killing capacity is improved in the absence of IFNAR [82]. Decreased killing by superinfected neutrophils is evident in vivo after 7 days of influenza virus infection. After 3 days, the susceptibility to infection is not observed, owing to increased bactericidal activity of neutrophils and CD11c⁺ cells [82].

Another example of type I IFNs impairing phagocytic clearance of pathogens is their influence on macrophages in the upper respiratory tract. Macrophages are important for clearance of S. pneumoniae from the nasopharynx [49, 91], and their recruitment is significantly impaired during influenza virus infection. This impaired recruitment was shown to be dependent on type I IFNs. Type I IFNs exerted their effect by decreasing production of the major macrophage chemokine CCL2, an observation that was demonstrated directly in vitro with purified IFN-β, preventing production of CCL2 by macrophages in response to infection [49].

CONCLUSIONS

Secondary infections resulting from S. aureus and S. pneumoniae are a major complication of influenza virus infection. Type I and III IFN signaling is detrimental in the context of superinfection through multiple effects on inflammatory cytokines and cell recruitment, influencing the response of the inflammasome, Th17 signaling, and recruitment and function of neutrophils and macrophages, as well as affecting the nasal microbiota that influences colonization with potential pathogens. While their importance after influenza virus infection is well supported, exactly how the IFNs participate in altering host defenses to other viral illnesses remains to be explored. Of note, there are already effective strategies available, namely immunization, to decrease susceptibility to both influenza and pneumococcal infection. It is important to recognize that prevention remains an important mechanism to decrease morbidity and mortality from bacterial superinfection following influenza.

Notes

Financial support. This work was supported by the National Institutes of Health (grant R56HL125653).

Potential conflict of interest. Author certifies no potential conflicts of interest. The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.