The amazing innate immune response to influenza A virus infection

α- and β-defensins and retrocyclins

The defensins are one major category of antimicrobial peptides found in lung fluids. The human α-defensins present in the lung are the HNPs, which are delivered to the lung by neutrophils in inflammatory states. These have strong neutralizing activity for many IAV strains.^62–64 The mechanism of antiviral activity of HNPs has not been fully elucidated. We have found that HNPs induce viral aggregation and inhibit infectivity mainly through direct interactions with the virus.^62,64–66 Salvatore et al. ⁶³ have found that HNPs also inhibit IAV through binding to epithelial cells and inhibition of protein kinase C. Unlike the collectins and other proteins that bind the viral HA, HNPs do not inhibit HA activity of IAV. ⁶⁴ Mice do not have neutrophil α-defensins, but have other antimicrobial peptides that may play a similar role.

Another class of defensins, the β-defensins, is produced by respiratory epithelial cells either constitutively or in response to inflammatory stimuli and also inhibit IAV.^62,67,68 The β-defensins are less potent as direct inhibitors of IAV than the HNPs; however, they may have important immunomodulatory roles during IAV infection as well. Ryan et al. ⁶⁸ have demonstrated that mice lacking β-defensin 1 have more severe lung inflammation when infected with IAV, although viral titers were not different compared with control mice. This article also demonstrated that IAV infection increases production of β-defensin 1 by plasmacytoid DC. Further studies of the in vivo contributions of this and other β-defensins during IAV infection will be of great interest.

Of interest, there is a third class of defensins called theta defensins or retrocylins (because of their cyclic nature) that occur in primates, but not humans, and have very strong anti-IAV activity.^62,69 The retrocyclins, like HNPs, can induce aggregation of IAV and they appear to have stronger intrinsic antiviral activity than HNPs. ⁶²

LL-37: An antiviral and immunomodulatory peptide in IAV infection

A distinct group of antimicrobial peptides are called the cathelicidins, and the one representative of this class in humans is LL-37. Recent reviews have discussed the extraordinary range of activities of LL-37, which include direct antimicrobial and antiviral activities, chemotactic activities for various immune cells and modulation of macrophage responses to inflammatory stimuli, and modulation of DC responses.^60,61 LL-37 is released from neutrophil granules, but is also produced by epithelial cells, including those in the respiratory epithelium. Leukotriene B4 has been shown to promote defense against IAV probably through its ability to stimulate release of LL-37 and β-defensins from respiratory epithelial cells. ⁷⁰ Two studies have recently established a role of LL-37 during IAV infection. Barlow et al. ⁷¹ first demonstrated that LL-37 has direct antiviral activity against IAV and contributes to host defense against the virus in vivo both by limiting viral replication and virus-induced inflammation Our laboratory then reported on the mechanism of antiviral activity of LL-37, which is distinct from that of collectins or defensins. ⁷² Unlike these proteins, LL-37 did not cause viral aggregation or alter viral uptake by epithelial cells. LL-37 did, however, cause disruption of viral membranes on electron microscopy. It is unclear whether the antiviral or immune modulatory effects of LL-37 are more important in vivo, but further studies in mice (including mice in which the homologue of human LL-37 is knocked out) will, hopefully, clarify this.

IAV recognition pathways in epithelial cells

It is now clear that there are at least three pattern recognition pathways through which cells recognize and respond to IAV infection: (i) the cytoplasmic RNA recognition protein, RIG-1; (ii) TLR3 and TLR7, which recognize viral RNA at the cell surface or in endosomal compartments; and (iii) Nucleotide oligomerization domain (NOD)-like receptors (NLR) that trigger the inflammasome pathway leading to caspase 1 activation and IL-1β and IL-18 production. RIG-1 and TLR3 are very important for epithelial cell responses,^95–98 while TLR7 is particularly important for responses of DC,^99,100 and NLR for macrophages and DC (see below). Figure 2 illustrates the various pathways of cellular response to IAV infection. OPEN IN VIEWER

Extensive data now show that RIG-1, which is an RNA helicase, is a critical mediator of response to RNA viruses, including IAV, through its ability to recognize 5’ capped single-stranded RNA in the infected cell cytoplasm. ⁹⁵ RIG-1 then signals through mitochondrial antiviral signaling protein (MAVS), and then NF-κB and IFN regulatory factor 3 (IRF3), leading to pro-inflammatory cytokine and type I IFN generation (see Takeuchi and Akira ¹⁰¹ for an excellent review). Of interest, the related RNA helicase, melanoma differentiation-associated gene 5, which is involved in poly I:C response, and response to picornaviruses and other viruses, is not involved in the response to IAV. ⁹⁶ Given the importance of RIG-1 in host defense against IAV it is not surprising that IAV has evolved to inhibit RIG-1 activation through its NS1 protein (see below).

TLR3 has been shown to be involved in the response of respiratory epithelial cells to IAV and it is up-regulated by IAV infection. ⁹⁷ Of note, activation of TLR3 appears to play mostly an adverse role in IAV infection by increasing immunopathology, in that TLR3^−/− mice had improved survival and reduced inflammatory responses despite an increase in viral titers. ⁹⁷ Although TLR4 was not found to be directly activated by IAV components, it has been shown to contribute to acute lung injury with highly pathogenic H5N1 infection. ¹⁰² Activation of TLR4 in this setting was dependent on formation of oxidized phospholipids and reduced in mice lacking a component of the NADPH oxidase. This may be a common pathway of acute lung injury, as other infectious agents and acid aspiration had similar effects.

A fascinating, recently developed, concept is that the coagulation system participates in innate host defense against infections through bacterial entrapment in fibrin clots and stimulation of signaling via protease activity.^103,104 This system appears to be an ancient mechanism of early host defense. Thrombin or other proteases are known to mediate signaling in cells via activation of protease activated receptors 1 and 2 (PAR-1 and PAR-2). PAR-1 and PAR-2 expression in airway epithelium is increased during IAV infection ¹⁰⁵ and activation of these receptors can modulate signaling through TLRs. ¹⁰⁴ It is not yet clear in what settings PAR-1 or PAR-2 play beneficial or harmful roles in IAV infection. A compelling study showed that blockade of PAR-1 signaling or deletion of the PAR-1 gene in mice improved outcome of infection with pandemic H1N1 or H5N1 infection. ¹⁰⁶ These results led to the suggestion that PAR-1 inhibition might be a therapeutic strategy during severe IAV infection. In contrast, another compelling study found that PAR-1^−/− mice had improved outcome with PR-8 (mouse-adapted H1N1) infection, ¹⁰⁷ suggesting that further study is necessary before attempting therapeutic blockade of PAR-1. Both studies do indicate that PAR-1 does mediate important signaling events for recruitment of immune cells. It is too soon to try to explain the different outcomes of these studies, but differences in the viral strains used could have been important. PAR-2 appears to have different, but also important, activities in IAV infection, including stimulation of IFN-γ ¹⁰⁸ (beneficial) or activation of TLR4 ¹⁰⁴ (adverse).

The central role of type I and type III IFN response systems

The type I IFN system has long been known to have a key role in containment of IAV infection, but the diverse mechanisms through which type I IFNs mediate this effect, and how the virus counteracts these effects, are still being elucidated. ¹⁰⁹ Intracellular events triggered by type I IFNs include oligoadenylate synthetase, protein kinase R (PKR) and a host of other IFN-responsive genes. ¹¹⁰ Recently, extracellular receptors whose expression is regulated by IFNs have also been described.

Major role of IFN-inducible transmembrane protein 3, tetherin and viperin in host defense against IAV

A functional genomic screen of factors involved in restriction of IAV replication in osteosarcoma cells in vitro identified (among other proteins) the IFN-inducible transmembrane (IFITM) proteins 1, 2 and 3 as important antiviral factors. ¹¹¹ IFITM3 was particularly important in mediating the antiviral activity of IFN type I. The IFITMs were found to inhibit early replication of IAV, but also of West Nile and Dengue virus, and deletion of IFITM3 in mouse cells resulted in increased IAV replication. This was followed by another pivotal study showing a major impact of deletion of IFITM3 in mice on the course of infection with various IAV strains. ¹¹² Mice lacking IFITM3 infected with otherwise low pathogenicity IAV had markedly increased mass loss, mortality, viral loads in the lung, respiratory epithelial apoptosis, infection in the lung (as opposed to upper airways for wild type mice), neutrophil infiltration and inflammatory cytokine production. Overall, the lack of IFTIM3 converted a low pathogenicity viral infection into an infection with all the features of high pathogenicity viruses (e.g. H5N1 or 1918 H1N1). In addition, the authors found increased incidence of an uncommon allelic variant of IFTIM3 in patients hospitalized during the 2009 H1N1 pandemic. This variant was found to have a truncation of the N-terminal domain of the protein and to have reduced antiviral activity in vitro. Another recently described cell surface, type I IFN-induced antiviral protein, tetherin, has been found to inhibit IAV (and other viruses, including HIV) by tethering newly produced viral particles at the cell surface. ¹¹³ The IAV NS1 protein counteracts tetherin expression and the viral neuraminidase counteracts tetherin activity. Viperin, another recently identified IFN-inducible protein, was found to restrict budding and release of IAV particles by impeding formation of lipid raft domains in cellular membranes. ¹¹⁴ Despite confirming this in vitro effect of viperin, a further study by Tan et al. ¹¹⁵ found that viperin knockout mice did not have a different course of IAV infection in vivo than wild type mice.

Role of the Mx protein in IFN-mediated host defense against IAV

The Mx protein is an IFN-inducible protein that has long been known to mediate antiviral effects in IAV-infected cells. The mouse and human Mx proteins are referred to as Mx1 and MxA, respectively. Both confer protection, but Mx1 acts in a nuclear location, whereas MxA acts within the cytoplasm. Common laboratory mouse strains (e.g. Balb/c) lack a functional Mx1 protein, and knock in of this gene in Balb/c mice protects against lethal IAV infection. ¹¹⁶ Recently, important new insights have emerged regarding the molecular mechanisms of Mx activity. Viral strains vary in sensitivity to Mx proteins and sensitivity segregates with the NP. Avian viral strains (e.g. H5N1) are highly sensitive to Mx mediated inhibition, whereas the human pandemic H1N1 strains are not. ¹¹⁷ Zimmerman et al. ¹¹⁸ found that exchange of NP of pH1N1 with that of H5N1 resulted in loss of Mx protein sensitivity and increased lung pathology, mass loss and lethality in mice, despite the fact that the modified H5N1 strain replicated to a lower degree in vivo and in vitro than the wild type H5N1 strain. These findings strongly suggest that Mx proteins act through interacting with NP and that Mx1 is capable of down-regulating adverse pro-inflammatory responses occurring with IAV infection. Wisskirchen et al. ¹¹⁹ showed that MxA also binds to two RNA helicases (UAP56 and URH49) that are required for replication of IAV. These RNA helicases bind NP and viral ribonucleoprotein and are involved in nuclear export of viral mRNAs and prevent the accumulation of double-stranded RNA (dsRNA), thus reducing cellular type I IFN responses. The findings that Mx proteins interact with RNA helicases and viral NP indicate that interference with viral RNA replication is central to their antiviral activity. Cilloniz et al. ¹¹⁶ used global transcription profiling in lungs of wild type or Mx1^+/+ Balb/c mice infected with the highly pathogenic 1918 H1N1 IAV strains to demonstrate the molecular events associated with protection conferred by the Mx1 protein. The Mx1 protein alone improved survival from 0 to 50%, and treatment of Mx1-expressing mice with type I IFN improved survival further to 100%. This marked protective effect of IFN was only seen in the Mx1 expressing mice and was associated with specific down-regulation of pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF) and chemokines (e.g. CCL5, CXCL-10). Hence, at least in the context of highly pathogenic IAV infection, the combination of Mx1 and type I IFN is able to down-regulate damaging pro-inflammatory responses. Mx proteins therefore appear to improve the outcome of IAV infection by both antiviral and anti-inflammatory effects. They may also play a role in restricting interspecies transmission of IAV strains (e.g. avian H5N1 is highly sensitive to inhibition by MxA). ¹¹⁷

p53 as an antiviral protein

p53 has mainly been thought of as a regulator of cellular apoptosis in cells that have undergone DNA damage and as an anti-oncogene; however, it is now clear that p53 has a pivotal role in regulating antiviral responses as well. Turpin et al. ¹²⁰ demonstrated that levels of p53, phosphorylation of p53 and p53 accumulation in the nucleus in A549 cells are all increased during IAV infection. These changes occur late in the infection cycle (e.g. 8–24 h post-infection). Deletion or silencing of p53 resulted in marked reduction of apoptosis induced by IAV in respiratory epithelial cells (human or mouse). Recent studies have confirmed this increase in p53 expression and activation, and shown that IAV inhibits ubiquitination of p53 and modulates expression of p53 isoforms (which regulate expression of full length p53) resulting in the p53 increase.^121,122 An additional striking finding in the Turpin et al. ¹²⁰ study was that deletion of p53 resulted in elevation of viral titers and reduction of type I IFN transcription. These findings were recently expanded upon by Munoz-Fontela et al., ¹²³ who demonstrated that infection of p53^−/− mice results in reduced type I IFN, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α and β in the lung accompanied by increased viral replication at d 3, more mass loss and higher mortality in the mice. DC responses and CD8 cell recruitment were also impaired. Hence, lack of p53 impaired both innate and adaptive immunity to IAV. To determine if these effects result from loss of p53 in non-hematopoietic cells or hematopoietic cells chimeric mice were generated (as in the study by Shornick et al. ⁸⁶ ) in which the bone marrow expressed p53, but the other cells did not. These mice still had a defect in DC migration. This finding suggests that loss of p53 in the respiratory epithelium or other non-hematopoietic cells indirectly affects DC migration in this model. It has long been established that p53 plays numerous important roles that limit cancer development and progression (e.g. recognition of damaged DNA, promotion of apoptosis, inhibition of angiogenesis). It is remarkable to find that this extraordinary protein also has wide ranging effects in antiviral defense.

p53 directly modulates expression of the genes for MCP-1, IRF9, PKR and ISG15 through transcriptional activation and hence is central in initiation of antiviral responses through mechanisms separate from induction of apoptosis. ¹²³ Of interest, despite the ability of IAV to cause increased late accumulation and activation of p53, the viral NS1 protein has been found to associate with p53 and inhibit its transcriptional activity and pro-apoptotic effects. ¹²⁴ This finding is consistent with the various ways in which the NS1 protein inhibits the development of the IFN-mediated antiviral state in cells. IAV appears, therefore, to inhibit p53 activity early in the course of infection of epithelial cells, but to increase its activity later. This apparent dichotomy may make sense when one considers further evidence of how IAV modulates cell death pathways.

Modulation of cell death pathways during IAV infection: Apoptosis, pyroptosis, necrosis and autophagy

It is now considered that there are at least three distinct pathways of cell death, all of which involve specific signals and have distinct effects on inflammatory responses. Lamkanfi and Dixit ¹²⁵ provide an excellent recent review of these pathways in relation to microbial infection. It has become increasingly clear that pathogens (including IAV) actively modulate cell death pathways and that the host can regulate cell death pathways as a mechanism of host defense.^125,126 Although it has long been recognized that IAV induces cellular apoptosis, it has recently been shown that the virus inhibits apoptosis early in the course of infection. One important mechanism involves up-regulation of anti-apoptotic signaling through the PI3kinase/Akt pathway mediated by the viral NS1 protein^{127,128–131} (see Herold et al. for review ¹³² ). This activation of the PI3kinase pathway is observed with viral infection or recombinant expression of NS1 alone ¹²⁷ and is reduced when infecting with NS1-deleted virus. This effect was not mediated by NS1’s inhibition of type I IFN as it occurred in IFN-deficient VERO cells as well. Another mechanism through which IAV counteracts apoptosis in epithelial cells involves interaction of the viral NA with CEACAM6 leading to stimulation of pro-survival pathways, including activation of Akt. ¹³³

In the study by Zhirnov and Klenk, ¹²⁷ the activation of PI3kinase peaked at 7.5 h post-infection, whereas p53 activation and caspase 3 activation occurred later. This led to the proposal that IAV may inhibit apoptosis at earlier time points after infection to facilitate maximal viral RNA and protein production, and accelerate apoptosis at later points to facilitate later phases of the viral life cycle. It has been shown that caspase 3 activation is necessary for viral propagation^132,134 through promoting release of viral ribonucleoproteins from the nucleus of the cell. Blocking caspase 3 or NF-κB activation causes viral ribonucleoprotein retention in the nucleus and inhibits viral production.^134,135 The apoptotic phase of IAV infection involves the extrinsic apoptotic pathway mediators TRAIL and Fas ligand. ¹³⁶ The PB1-F2 protein is encoded in some viral strains through an additional open reading frame within the PB1 polymerase gene. PB1-F2 can mediate apoptosis in monocytes through the intrinsic pathway by permeabilizing mitochondrial membranes. ¹³⁷

There is an extensive literature from murine studies indicating that IAV infection stimulates oxidant generation in the lung, and that this is injurious.^88,138–140 Recent studies with isolated primary alveolar epithelial cells have demonstrated that the nuclear factor-erythroid 2 related factor 2 (Nrf2) is increased after IAV infection and protects against IAV induced apoptosis through an IFN-independent mechanism. ⁸⁸ Nrf2 was also shown to reduce IAV-induced pulmonary pathology in mice. ¹⁴¹ These findings are of interest as Nrf2 expression appears to increase ability of respiratory epithelial cells to restrict IAV replication as well. ¹⁴² Nrf2 expression is depressed by cigarette smoking and in chronic obstructive pulmonary disease, and this correlates with increased susceptibility to bacterial infection, which can be ameliorated by treatment with phytochemicals that increase Nrf2 expression.^142,143 Nrf2 was found to increase macrophage expression of phagocytic receptors and bacterial phagocytosis. ¹⁴³

Apoptosis is generally felt to be immunologically silent and not pro-inflammatory, whereas pyroptosis and necrosis are pro-inflammatory modes of cell death. Pyroptosis is the end result of inflammasome activation, but, thus far, this mode of cell death has not been reported in context of IAV infection, despite clear evidence of inflammasome activation. ¹⁴⁴ Yatim and Albert ¹²⁶ present the very interesting hypothesis that there is a integral relationship of cell death pathways and immunity, and that viruses and other microbes have driven the evolution of eukaryotic cell death pathways. Hence, we should perhaps not look at the eukaryotic cell as standing in isolation through evolution and fending off pathogen invaders, but that it has evolved through time in interaction with pathogens.

Control of cell death pathways is a fundamental to how the host deals with viral infection or infection with other intracellular pathogens. ¹²⁶ Autophagy is a constitutive process conserved from yeast to mammals that is important for recycling of cellular proteins and protein complexes, and for allowing cellular survival under stress conditions (e.g. nutrient deprivation). This process is also intimately involved in the response of cells to infection with all types of pathogens and in triggering both innate and adaptive immune responses (for excellent reviews see Dumit and Dengjel^145, and Kuballa et al. ¹⁴⁶ ). As a well researched clinical example, defects in proteins needed for autophagy have been linked to Crohn’s disease and increased inflammatory responses triggered by LPS or muramyldipeptide. We will be using the term autophagy here, although a more precise term is macro-autophagy to differentiate the process from micro-autophagy and chaperone-mediated autophagy, which occur at the level of lysosomes and do not involve the formation of the larger double-membraned autophagosome structure. In macro-autophagy this structure is formed through cooperative interaction of numerous proteins, it engulfs additional cytoplasmic proteins or ‘cargo’ and, ultimately, fuses with lysosomes to result in degradation and recycling of the cargo proteins. Viruses, in particular, have evolved ways of interfering with autophagy in different ways, often through interacting with a key autophagy-related protein called beclin-1. Beclin-1 is involved in formation of the autophagosome and also in the fusion of the autophagosome with lysosomes.

DNA viruses (e.g. herpes viruses) can inhibit autophagy formation by blocking this action of beclin-1, whereas RNA viruses (including IAV) inhibit the ability of beclin-1 to promote fusion with lysosomes. ¹⁴⁷ During IAV infection of A549 cells an accumulation of autophagosomes is observed owing to direct interaction of the M2 viral matrix protein with beclin-1. ¹⁴⁸ This results in cell death via apoptosis. This may result in blunting of innate responses to the IAV as Law et al. ¹⁴⁹ have shown that IAV-induced CXCL10 and IFN-α generation are promoted by autophagy. In addition, autophagy has been shown to be involved in degradation of viral proteins for Ag presentation via class II MHC molecules, although it appears that proteosomal processing is more important for presentation to CD4 cells in the case of IAV. ¹⁵⁰ Clearly, we have presented a simplified picture of this complex and rapidly evolving topic, as, in some instances, viruses promote autophagy (e.g. to result in death of uninfected CD4 cells mediated by the HIV envelope protein), ¹⁴⁶ and the role of autophagy in viral replication and immune responses appear to vary based on cell type and virus strains. For instance, H5N1, but not H1N1, IAV was found to promote autophagy in epithelial cells and inhibition of autophagy increased survival of mice infected with H5N1.^151,152

Autophagy is a distinct process from proteosomal degradation of ubiquitinated proteins; however, there is crosstalk between these two systems. ¹⁴⁵ Inhibition of proteosome activation reduces IAV replication by blocking viral RNA synthesis, but also by causing retention of viral NP in the cytoplasm after viral entry. ¹⁵³ In addition, in the setting of bacterial infection autophagy can limit IL-1β generation by inflammasomes providing another site of interaction between autophagy and innate immunity. ¹⁴⁶ Clearly, much has yet to be learned about the role of autophagy in response to specific pathogens, including IAV.

Cellular stress granules and IAV infection

Another mode through which cells deal with stress is the formation of stress granules. These granules form when cellular protein translation is impaired and contain non-translating mRNAs and translation initiation components. Stress granules are felt to play a role in the ‘decision’ of whether a stressed cell should undergo apoptosis or not, in part as they sequester some apoptotic regulatory proteins. ¹⁵⁴ Viruses have been shown to induce formation of granules resembling stress granules.^155–158 Some viruses utilize stress granules to facilitate their replication.^157,159,160 This is noteworthy as stress granules can contain RIG-1 and PKR and to function in innate antiviral immunity. ¹⁶¹ Consistent with this, IAV inhibits the formation of stress granules and induction of type 1 IFN generation through actions of the NS1 protein. ¹⁶¹ NS1 was recently shown to mediate this effect through interacting with the stress granule protein, RNA-associated protein 55. ¹⁶²

microRNA changes triggered by IAV and the innate immune response

There are microRNAs (miRNAs) present in a variety of epithelial cells that can directly reduce IAV replication through acting on the PB1 gene (viral polymerase). These miRNAs (323, 491 and 654) are not perfect matches for the viral target but, nonetheless, cause degradation of viral RNA. ¹⁶³ RNA silencing is also implicated as an important antiviral mechanism for IAV ¹⁶⁴ as the NS1 protein of IAV inhibits RNA silencing in mammalian cells. IAV infection also leads to a rapid increase in miR29 in A549 cells infected with IAV in vitro or in monocytes of patients infected with IAV, and this miRNA causes up-regulation of expression of COX-2, which, in turn, leads to increased expression of IFN-λ through suppression of DNA methyltransferases. ¹⁶⁵ Clearly, our understanding of how IAV infection modulates innate immunity through induction of miRNAs is far from complete, as illustrated by a recent article by Buggele et al. ¹⁶⁶ in which miRNA microarrays were used to identify the spectrum of miRNAs induced in human lung cells by IAV strains. This study showed that at least seven miRNAs are consistently induced by IAV and that the degree of induction is greater than that induced by antiviral mediators alone. The up-regulated miRNAs are known to modify a wide array of signaling pathways. Specifically, MAPK3 and IRAK1 were shown to be targeted for mRNA degradation through the combined effect of three of the IAV-induced miRNAs. Hence, future studies will not only have to address the role of numerous miRNAs, but also their potential combinatorial effects to understand how they affect innate immunity.

AMs and recruited monocytes and macrophages

There have been numerous studies demonstrating that AMs play a key role in the initial response to IAV infection.^171,172 These cells are resident in the lung and part of the first line of defense. There is recent evidence that AM-mediated resistance to IAV can be potentiated by GM-CSF treatment. GM-CSF treatment has been found to improve survival in IAV infection, in part through acting on macrophages.^173,174 GM-CSF treated mice had increased numbers of alveolar macrophages, and the macrophages showed increased resistance to apoptotic effects of IAV. Removal of macrophages eliminated the beneficial effects of GM-CSF. As noted above alveolar epithelial cells appear to be a key source of GM-CSF in vivo and this GM-CSF also plays a critical role in recruitment of DC during IAV infection. ⁸⁷

NLR and inflammasome activation

Inflammasomes are multiprotein complexes that lead to the activation of caspase-1 and generation of active IL-1β and IL-18. These two pro-inflammatory cytokines have been shown to have independent and important roles in promoting innate and adaptive responses to IAV and many other pathogens. ¹⁷⁵ Inflammasome activation is mediated by NLR, which recognizes viral and microbial patterns, although the exact mechanism of recognition of IAV by NLRs is unknown. There are many types of inflammasomes, but thus far IAV has been shown to principally activate the NLRP3 inflammasome.^176,177 Key components of the NLRP3 inflammasome include the NLRP3 protein itself, the adaptor protein apoptosis-associated speck like protein (ASC) and caspase-1. Mice lacking NLRP3, ASC or caspase-1 were found to have increased mortality after PR-8 infection in three studies.^176–178 Of note, lack of MyD88 (adapter protein for TLR7, TLR8 and TLR9 signaling) or an alternative inflammasome molecule, NLRC4, did not affect mortality when tested in parallel studies. ¹⁷⁷ Of interest, in mice lacking the inflammasome components, cytokine, chemokine, monocyte and neutrophil responses were reduced, but lung injury was, nonetheless, increased, as evidenced by epithelial cell necrosis and collagen deposition. ¹⁷⁶ These results are similar to those reported by Schmitz et al. ¹⁷⁹ with respect to deletion of IL-1 receptors in which inflammatory responses were reduced, but survival was impaired. Viral loads were similar in NLRP3^−/− mice at d 3 after infection, but were higher by d 7, indicating a delay in viral clearance.^177,179 These results indicate that the inflammatory response was protective in this setting and that healing may be impaired in the absence of NLRP3 activation. The role of NLRP3 activation in control of adaptive responses to IAV is controversial and under active study.^177,178

Inflammasome activation by IAV mainly occurs in macrophages and DCs, and in contrast to the results obtained with IFITM, hematopoietic, and not stromal, NLRP3 components were critical. ¹⁷⁸ The mechanism through which IAV triggers the inflammasomes in macrophages and DC involves viral RNA in part; however, in an elegant study by Ichinohe et al., ¹⁸⁰ an additional signal for NLRP3 activation was provided by the viral M2 ion channel. In general, inflammasomes require two signals for activation, the first causing generation of pro-forms of IL-1β, IL-18 and IL-33 and the second activating caspase-1 to cleave the pro-forms of these cytokines to the active forms. In the case of IAV, viral RNA acting through TLR7 (but not RIG-1) mediates step 1, but does not result in caspase-1 activation. The second signal involves viral replication and specifically the M2 ion channel through a mechanism that involves ion fluxes in the Golgi. Inflammasome activation also has the potential to be damaging to the host. In this regard a recent article by Lupfer et al. ¹⁸¹ demonstrated that receptor interacting protein kinase 2 (RIPK2) down-modulates inflammasome activation during IAV infection, and RIPK2 knockout mice suffer increased illness and immunopathology. ¹⁸¹ Of interest, a recent article by Stout-Delgado et al. ¹⁸² showed that elderly mice have impaired NLRP3 function in response to IAV and that this can be restored by treating the mice with nigericin, which provides another source of signal 2 for inflammasome activation through a similar mechanism as the M2 protein. These results are of great interest with regard to the impaired immune response and more adverse outcome of IAV infection in the elderly.

Spooky effects at a distance: Gut commensal bacteria modulate innate immune responses to IAV infection in the lung

Two recent studies indicate that loss of normal commensal bacteria impairs innate and adaptive immune response to IAV. Ichinohe et al. ¹⁸³ found that oral treatment of mice with neomycin alters specific subpopulations of gut flora and results in delayed viral clearance and impaired adaptive responses to IAV infection, including impaired migration of DCs to draining lymph nodes and reduced T-cell and Ab responses. This finding is of particular interest as neomycin was not absorbed through the gut lumen and did not alter airway flora. They further determined that the impaired immune responses to IAV infection resulted from diminished pro-ILβ, pro-IL18 and NLRP3 expression in the lung, and the immune deficit was seen with IAV, but not with two other lung infections that do not involve inflammasome activation (herpes simplex virus and Legionella). Hence, they concluded that the neomycin-sensitive gut bacteria provide signal 1 for NLRP3 activation at a basal level prior to IAV infection. Rectal administration of TLR agonists was able to restore IAV responses in antibiotic-treated mice. Abt et al. ¹⁸⁴ demonstrated that antibiotic treatment of mice actually results in increased mortality from IAV infection associated with increased respiratory epithelial necrosis. There was similar recruitment of neutrophils, macrophages and DCs to the lungs of antibiotic treated mice; however, macrophages isolated from these mice had defective response to type I and type II IFNs. These responses could be restored and outcome of infection in vivo improved by treating the antibiotic fed treated mice with Poly I:C. Overall, these remarkable results suggest that the dependence of immune responses to IAV on inflammasomes accounts, in part, for the ability of gut flora to modulate the course of IAV infection.

Protective role of viral uptake or phagocytosis

Phagocytosis by macrophages and neutrophils has been shown to play a protective role during IAV infection in mice.^185,186 This could, in part, reflect direct ingestion of viral particles by these cells, as demonstrated in vitro using human cells.^{25,66,187–192} Although free viral particles are internalized by endocytosis, treatment of IAV with collectins, defensins, ficolins or other innate inhibitors results in viral aggregation and ingestion of large viral aggregates by phagocytes through a process resembling phagocytosis.^16,65,193 Human AMs have recently been shown to ingest apoptotic alveolar epithelial cells. ⁸⁸ This may be the most important manner through which macrophage phagocytosis promotes recovery from IAV infection.

AMs have a variety of receptors capable of mediating viral uptake (see the excellent recent review by Londrigan et al. ¹⁹⁴ ). The primary mode of IAV entry into macrophages is through binding of the HA to appropriate sialic acids on the macrophage surface; however, macrophages can also be infected in a sialic acid-independent manner through C-type lectins expressed on the macrophage surface, including macrophage Man receptor, macrophage Gal receptor and DC-Sign.^194–196 Uptake through these receptors is calcium-dependent and dependent on glycosylation of the viral envelope proteins. ¹⁹⁵ Optimal infection of macrophages involves both sialic acid-dependent and non-sialic acid-dependent uptake. ¹⁹⁴ Scavenger receptors (SR-A and MARCO) on AMs play important roles in uptake of bacteria; however, mice lacking SR-A did not have altered infection with IAV, and MARCO^−/− mice had improved survival with IAV infection.^173,197 Of interest, the protective effects of treatment of mice with GM-CSF correlated with increased expression of the SP-A receptor SP-R210 on macrophages, and was diminished by increased expression of MARCO. ¹⁷³ In vivo, this could reflect increased SP-A-mediated clearance of IAV. ²¹ IAV infection of human AMs in vitro reduces expression of C type lectin and scavenger receptors correlating with reduced uptake of zymosan. ¹⁹⁸

AMs are major source of chemokines and cytokines

AMs exhibit a potent chemokine and cytokine response upon IAV infection.^89,198 AMs are generally not productively infected with IAV, although they express viral antigens. ¹⁹⁸ Nonetheless, viable virus is required for stimulation of cytokine and chemokine production by AMs. ¹⁹⁸ Of note, highly pathogenic H5N1 viral strains have been shown to productively infect AMs of mouse or human origin,^199,200 possibly contributing to increased pathogenicity. Human AMs produce abundant amounts of IFN-α, pro-inflammatory cytokines and chemokines, and up-regulate expression of many innate defense genes in response to viable IAV infection.^89,198 There is evidence that pathogenicity of H5N1 may result from profound stimulation of macrophage cytokine production,^201,202 although blockade of this cytokine response was not found to be protective in mice. ²⁰³ Furthermore, elimination of IL-1β production in response to severe IAV infection led to reduced lung pathology, but increased mortality. ¹⁷⁹ These findings suggest that simple blockade of IL-1β or TNF production in response to IAV is unlikely to benefit patients with severe IAV infection and may even worsen outcome.

Macrophage defects and bacterial superinfection

IAV infection impairs expression of phagocytic receptors and phagocytosis of bacteria or zymosan particles by AMs.^198,204 This could contribute to co-infection of patients with IAV and bacteria, a potentially lethal combination. ⁴ There is also extensive evidence of depressed neutrophil function and accelerated neutrophil apoptosis caused by IAV infection,^205–210 and this may contribute to bacterial superinfection.^211–213

Depression of macrophage and DC functions by IAV can also persist after the peak of IAV infection and may relate to the observation of waves of bacterial pneumonia occurring in patients seemingly in recovery from IAV infection. Didierlaurent et al. ²¹⁴ found that IAV infection in mice led to prolonged desensitization to bacterial TLR ligands, reduced chemokine generation and impaired recruitment of neutrophils after bacterial infection. Sun et al. ²¹⁵ found that production of IFN-γ by T cells after IAV infection causes depression of alveolar macrophage uptake of bacteria. In a recent study, Ghoneim et al. ²¹⁶ tracked the fate of alveolar macrophages after IAV infection and showed that the virus caused marked loss of these cells and that this loss correlated with susceptibility to lethal pneumoccal super-infection. This study showed protection with local GM-CSF treatment.

Recruited monocyte macrophages may have distinct roles compared with AMs

Monocytes and macrophages recruited to the lung during infection appear to play a more complex role than AMs in that there is evidence that they are protective, but also that they can be harmful.^{217, 218} Recently, Gong et al. ²¹⁹ showed that mice lacking SerpinB1 show increased lung inflammation and mortality after infection with seasonal IAV. ²¹⁹ There was no difference in viral clearance in this setting; however, infiltrating monocytes producing excessive TNF and IL-6 and elevated numbers of pulmonary γδ T-cells (see below) were noted in the SerpinB1 knockout mice. These results indicate that serpinB1 acts to restrain overly vigorous monocyte and γδ T-cell responses.

DC

We will not be able to do justice in this review to the extensive and fascinating role of DC in host defense against IAV and refer to reviews that deal with this topic.^1,220 Myeloid DC clearly play a key role in early detection of viral infection and in transfer of viral Antigens to draining lymph nodes to initiate an antiviral response, and plasmacytoid DC are major producers of type I IFN during IAV infection in vivo.^221,222 Of interest, myeloid and plasmacytoid DC produce distinct waves of cytokines and chemokines over time after IAV infection regulating recruitment of various immune cell types to the lung. ²²³ Of interest, the NS1 protein of IAV inhibits DC maturation and activation resulting in lowering of the ability of DC to induce T-cell responses. ²²⁴ Furthermore, different viral strains have distinct interactions with different DC subsets. ²²⁵ As with AM, DC are generally not productively infected or killed by IAV; however, H5N1 can productively infect monocyte-derived DC leading to cell death. ²²⁶ This may contribute to the lymphocyte depletion and severe pathogenicity of this viral subtype. ²²⁷ Another recent finding of interest is that recruited monocytes can rapidly differentiate into myeloid DC in vivo, and constitute a major source of type I IFNs. ²¹⁸ A number of soluble innate mediators have been found to modulate DC responses to infection, including SP-D, defensins and LL-37.^228–231

Neutrophils

Neutrophils are the first wave of recruited immune cells responding to IAV infection. Given their presence in the lung of subjects dying of severe IAV infection, there was early speculation that neutrophils might be mediators of lung injury and mortality. Neutrophil oxidants may be injurious, ²³² and neutrophil-derived proteases^233,234 and defensins ⁶⁴ can impair the functions of SP-D. IAV has recently been found to provoke formation of neutrophil extracellular traps (Tripathi S, White MR and Hartshorn KL, unpublished data) and these have been implicated in lung injury indirectly. ²³⁵ In fact, the bulk of the evidence now supports the concept that neutrophils are protective in IAV infection,^{185,236–238} even as part of the profound influx of myeloid cells caused by highly pathogenic 1918 viral strains. ³⁰ Inhibition of neutrophil extracellular trap formation by deletion of the PAD4 gene was not found to alter survival of IAV infection. ²³⁹ Neutrophils can take up IAV and various innate immune proteins, including collectins, ficolins and defensins, markedly increase neutrophil uptake of the virus. Neutrophils are also a source of defensins and LL-37. Unanticipated roles of neutrophils in facilitating the adaptive immune response to IAV have now been reported. For instance, neutrophils were shown to serve as APC ³ and to promote T-cell responses to IAV infection. ²⁴⁰ Given the abundance of these cells in the first wave of response to IAV infection in the airway, these roles in adaptive immunity could be very important.

As noted above potentially adverse effects of neutrophils include production of oxidants as mice lacking functional NADPH oxidase have improved outcome with IAV, ²³² and many studies now show that anti-oxidants are protective in the context of IAV infection. IAV directly stimulates oxidant production by neutrophils, and this response is linked to neutrophil apoptosis and impaired responses of neutrophils to subsequent bacterial challenges. Of interest, pre-incubation of IAV with collectins protects the cells against this IAV induced impairment.^25,205

NK cells

Once again, we do not propose to do justice to the major and complex role of NK cells in influenza viral infection, but we refer to a recent review. ²⁴¹ Recent studies have demonstrated that mice lacking NK cells have worse outcome with IAV. ²⁴² NK cells contribute to control of viral replication and produce cytokines that modulate the immune response to IAV. Production of IL-22 by NK cells and NKT cells has an important role in repair of epithelial damage caused by IAV.^243,244 IL-15 contributes to host defense against IAV by recruiting NK cells to the lung. ²⁴⁵ NK cells have also been shown to contribute to lung pathology in models of IAV infection.^246,247 Of interest, NKT cells have been found to suppress immuno-pathology caused by IAV, in part through suppressing accumulation of inflammatory monocytes.^248,249 Impaired function of NK cells resulting from IAV infection is also reported,^250,251 and this may also contribute to bacterial super-infection. ²⁵² Notably, the response of NK cells to IAV infection is mediated through binding of the viral HA to sialic acids present on the NK receptor NKp46.^253,254 This appears to be another example of what, at first, appears to be a non-specific, carbohydrate-dependent binding event, leading to a directed innate immune response. As with many other topics we have reviewed, NK cells also play important roles in bridging the innate and adaptive responses to IAV. ²⁵⁵

Discovery of innate lymphocyte cells and their role in IAV infection

It was previously assumed that lymphocytes (other than NK cells) do not participate in the initial innate response to infection with a novel IAV strain, but that they come into play during the adaptive immune response. It is now clear, however, that there are several populations of innate lymphoid cells that exist and that some of these participate in the initial response to IAV infection. Neill et al. ²⁵⁶ used mice engineered to express GFP under the IL-13 promoter and identified a novel subset of IL-13 producing lymphoid cells, which they termed ‘nuocytes’ (as nu is the thirteenth letter of the Greek alphabet). These cells are rare in the resting state, but expand greatly in response to IL-25 and IL-33. They could not be assigned to any known hematopoetic cell lineage and they were shown to be critically important in mediating protective type-2 immunity to helminth infection. ²⁵⁶ IAV infection induces IL-33 generation, ²⁵⁷ raising the question of whether innate lymphoid cells are generated during IAV infection. Chang et al. ²⁵⁸ demonstrated that a H3N1 strain of IAV induces rapid development of airway hyper-reactivity (AHR) in mice, even in the absence of adaptive immunity (i.e. in Rag2^-/-, mice which lack T- and B-cells) or in NKT cells (i.e. in CD1d-deficient mice). This AHR response was shown to depend on the presence of a subset of cells the authors referred to as ‘natural helper cells’, which are not present in mice lacking the IL-33 receptor ST2. Of note, mice lacking ST2 could still develop AHR through the adaptive immune pathway triggered by ovalbumin sensitization. Hence, the AHR pathway triggered by the natural helper cells is a fully innate response and distinguishable from classic adaptive type-2 responses. These extraordinary findings may explain, in part, the ability to IAV to trigger asthma exacerbations which is a major source of morbidity. ²⁵⁹

An article by Monticelli et al. ²⁶⁰ demonstrated another, more beneficial, aspect of innate lymphoid cells in response to IAV. First, they demonstrated that a similar population of cells (lineage-negative, with similar markers and producing IL-13 and IL-5 in response to IL-33) exists in human lungs. Then, they showed in a mouse model using PR-8 H1N1 that these innate lymphoid cells accumulate in response to IAV infection and that they are a major source of amphiregulin, which mediates their ability to promote epithelial tissue repair after infection. Amphiregulin alone was found to promote tissue repair and improve oxygenation in mice depleted of the innate lymphoid cells. Again, this process occurred in Rag2^−/− mice as well. The subject of innate lymphoid cells is rapidly evolving and an excellent recent review is provided by Spits and Cupedo. ²⁶¹ It is clear that these cells, IL-33 and amphiregulin may play roles in both host recovery and tolerance of infection and immunopathogenesis of IAV and hopefully many new insights will emerge in the near future. A potential adverse effect of amphiregulin is that it is also involved in development of fibrotic responses in the lung; however, this aspect may not be important in the context of an acute, self-limited infection like IAV.

γδ T-cells and IL-17

A surprising, and important, recent finding is that IL-17 plays an important role in the immune pathology induced by severe IAV infection (in this case the PR-8 infection model in mice). ²⁶² IL-17 is an important trigger of neutrophil recruitment that can be generated as part of an adaptive immune response; however, IL-17 is also produced by a subset of innate T-cells (γδ T-cells). These cells led to a rapid and sustained generation of IL-17 in PR-8 infected mice (2–7 d post-infection) that reached high levels prior to the recruitment of Ag-specific lymphocytes to the lung. Of note, deletion of the IL-17 response by use of mice lacking the IL-17 receptor resulted in improved survival and reduced mass loss in the mice without changing the viral load. This effect correlated with reduction in neutrophil influx and reduction in generation of oxidized phospholipids, which have been shown to mediate acute lung injury related to severe influenza infection (e.g. by H5N1) or acid aspiration through activation of TLR4 signaling.^102,262 Of note, the adverse effect of IL-17 was not mediated through IL-6 or TNF. A common point between the IL-17 and TLR3 studies is that in both settings deletion of these innate responses improves outcome of infection without affecting viral load. It is important to make a distinction between the innate γδ T-cells and the protective subset of CD4 T-cells that produce IL-17 as part of the adaptive immune response (Th17 cells). For instance, McKinstry et al. ²⁶³ showed that IL-10 deficiency promotes a Th17 response leading to improved survival in mice challenged with high dose IAV infection.