Robust expression of vault RNAs induced by influenza A virus plays a critical role in suppression of PKR-mediated innate immunity

Protein kinase R (PKR) is a vital component of host innate immunity against viral infection. However, the mechanism underlying inactivation of PKR by influenza A virus (IAV) remains elusive. Here, we found that vault RNAs (vtRNAs) were greatly induced in A549 cells and mouse lungs after infection with IAV. The viral NS1 protein was shown to be the inducer triggering the upregulation of vtRNAs. Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication. Furthermore, in vivo studies showed that disrupting vtRNA expression in mice significantly decreased IAV replication in infected lungs. The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates. Interestingly, vtRNAs promoted viral replication through repressing the activation of PKR and the subsequent antiviral interferon response. In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection. Moreover, vtRNAs were also significantly upregulated by infections of several other viruses and involved in the inactivation of PKR signaling by these viruses. These results reveal a novel mechanism by which some viruses circumvent PKR-mediated innate immunity.     

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Induction of innate immunity and its perturbation by influenza viruses

Influenza A viruses (IAV) are highly contagious pathogens causing dreadful losses to human and animal, around the globe. IAVs first interact with the host through epithelial cells, and the viral RNA containing a 5′-triphosphate group is thought to be the critical trigger for activation of effective innate immunity via pattern recognition receptors-dependent signaling pathways. These induced immune responses establish the antiviral state of the host for effective suppression of viral replication and enhancing viral clearance. However, IAVs have evolved a variety of mechanisms by which they can invade host cells, circumvent the host immune responses, and use the machineries of host cells to synthesize and transport their own components, which help them to establish a successful infection and replication. In this review, we will highlight the molecular mechanisms of how IAV infection stimulates the host innate immune system and strategies by which IAV evades host responses.     

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.     

Identification of lncRNA‐155 encoded by MIR155HG as a novel regulator of innate immunity against influenza A virus infection

Long noncoding RNAs (lncRNAs) are single‐stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)‐155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA‐155. Here, we observed that expression of lncRNA‐155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA‐155 was also induced by infections with several other viruses. Disruption of lncRNA‐155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA‐155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA‐155 in human cells suppressed IAV replication, suggesting that lncRNA‐155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA‐155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA‐155 resulted in higher production of interferon‐beta (IFN‐β) and several critical interferon‐stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA‐155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B‐mediated interferon response.     

Influenza A virus interactions with macrophages: Lessons from epithelial cells

Influenza viruses are an important cause of respiratory infection worldwide. In humans, infection with seasonal influenza A virus (IAV) is generally restricted to the respiratory tract where productive infection of airway epithelial cells promotes viral amplification, dissemination, and disease. Alveolar macrophages (MΦ) are also among the first cells to detect and respond to IAV, where they play a pivotal role in mounting effective innate immune responses. In contrast to epithelial cells, IAV infection of MΦ is a “dead end” for most seasonal strains, where replication is abortive and newly synthesised virions are not released. Although the key replicative stages leading to productive IAV infection in epithelial cells are defined, there is limited knowledge about the abortive IAV life cycle in MΦ. In this review, we will explore host factors and viral elements that support the early stages (entry) through to the late stages (viral egress) of IAV replication in epithelial cells. Similarities, differences, and unknowns for each key stage of the IAV replicative cycle in MΦ will then be highlighted. Herein, we provide mechanistic insights into MΦ‐specific control of seasonal IAV replication through abortive infection, which may in turn, contribute to effective host defence.     

Fibroblast growth factor-9 expression in airway epithelial cells amplifies the type I interferon response and alters influenza A virus pathogenesis

Influenza A virus (IAV) preferentially infects conducting airway and alveolar epithelial cells in the lung. The outcome of these infections is impacted by the host response, including the production of various cytokines, chemokines, and growth factors. Fibroblast growth factor-9 (FGF9) is required for lung development, can display antiviral activity in vitro, and is upregulated in asymptomatic patients during early IAV infection. We therefore hypothesized that FGF9 would protect the lungs from respiratory virus infection and evaluated IAV pathogenesis in mice that overexpress FGF9 in club cells in the conducting airway epithelium (FGF9-OE mice). However, we found that FGF9-OE mice were highly susceptible to IAV and Sendai virus infection compared to control mice. FGF9-OE mice displayed elevated and persistent viral loads, increased expression of cytokines and chemokines, and increased numbers of infiltrating immune cells as early as 1 day post-infection (dpi). Gene expression analysis showed an elevated type I interferon (IFN) signature in the conducting airway epithelium and analysis of IAV tropism uncovered a dramatic shift in infection from the conducting airway epithelium to the alveolar epithelium in FGF9-OE lungs. These results demonstrate that FGF9 signaling primes the conducting airway epithelium to rapidly induce a localized IFN and proinflammatory cytokine response during viral infection. Although this response protects the airway epithelial cells from IAV infection, it allows for early and enhanced infection of the alveolar epithelium, ultimately leading to increased morbidity and mortality. Our study illuminates a novel role for FGF9 in regulating respiratory virus infection and pathogenesis.     

Potential role of invariant NKT cells in the control of pulmonary inflammation and CD8+ T cell response during acute influenza A virus H3N2 pneumonia

Influenza A virus (IAV) infection results in a highly contagious respiratory illness leading to substantial morbidity and occasionally death. In this report, we assessed the in vivo physiological contribution of invariant NKT (iNKT) lymphocytes, a subset of lipid-reactive αβ T lymphocytes, on the host response and viral pathogenesis using a virulent, mouse-adapted, IAV H3N2 strain. Upon infection with a lethal dose of IAV, iNKT cells become activated in the lungs and bronchoalveolar space to become rapidly anergic to further restimulation. Relative to wild-type animals, C57BL/6 mice deficient in iNKT cells (Jα18(-/-) mice) developed a more severe bronchopneumonia and had an accelerated fatal outcome, a phenomenon reversed by the adoptive transfer of NKT cells prior to infection. The enhanced pathology in Jα18(-/-) animals was not associated with either reduced or delayed viral clearance in the lungs or with a defective local NK cell response. In marked contrast, Jα18(-/-) mice displayed a dramatically reduced IAV-specific CD8(+) T cell response in the lungs and in lung-draining mediastinal lymph nodes. We further show that this defective CD8(+) T cell response correlates with an altered accumulation and maturation of pulmonary CD103(+), but not CD11b(high), dendritic cells in the mediastinal lymph nodes. Taken together, these findings point to a role for iNKT cells in the control of pneumonia as well as in the development of the CD8(+) T cell response during the early stage of acute IAV H3N2 infection.     

Increased MDSC accumulation and Th2 biased response to influenza A virus infection in the absence of TLR7 in mice

Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune response against influenza A virus (IAV) infection; however, the role of Toll-like receptor 7 (TLR7) during the innate immune response to IAV infection and the cell types affected by the absence of TLR7 are not clearly understood. In this study, we show that myeloid derived suppressor cells (MDSC) accumulate in the lungs of TLR7 deficient mice more so than in wild-type C57Bl/6 mice, and display increased cytokine expression. Furthermore, there is an increase in production of Th2 cytokines by TLR7(-/-) compared with wildtype CD4+ T-cells in vivo, leading to a Th2 polarized humoral response. Our findings indicate that TLR7 modulates the accumulation of MDSCs during an IAV infection in mice, and that lack of TLR7 signaling leads to a Th2-biased response.     

PC61 (Anti-CD25) Treatment Inhibits Influenza A Virus-Expanded Regulatory T Cells and Severe Lung Pathology during a Subsequent Heterologous Lymphocytic Choriomeningitis Virus Infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8⁺ T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4⁺ Foxp3⁺ T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8⁺ T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8⁺ T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.     

The magnitude of the T cell response to a clinically significant dose of influenza virus is regulated by TRAIL

An immune response of appropriate magnitude should be robust enough to control pathogen spread but not simultaneously lead to immunopathology. Primary infection with influenza A virus (IAV) results in a localized pulmonary infection and inflammation and elicits an IAV-specific CD8 T cell immune response necessary for viral clearance. Clearance of IAV-infected cells, and recovery from infection, is mediated by perforin/granzyme B- and Fas/FasL-mediated mechanisms. We recently reported that TRAIL is another means by which IAV-specific CD8 T cells can kill IAV-infected cells. The current study examined the role of TRAIL in the pulmonary CD8 T cell response to a clinically significant IAV [A/PR/8/34 (PR8; H1N1)] infection (i.e., leads to observable, but limited, morbidity and mortality in wild-type [WT] mice). Compared with WT mice, IAV-infected Trail(-/-) mice experienced increased morbidity and mortality despite similar rates of viral clearance from the lungs. The increased morbidity and mortality in Trail(-/-) mice correlated with increased pulmonary pathology and inflammatory chemokine production. Analysis of lung-infiltrating lymphocytes revealed increased numbers of IAV-specific CD8 T cells in infected Trail(-/-) mice, which correlated with increased pulmonary cytotoxic activity and increased pulmonary expression of MIG and MIP-1α. In addition, there was decreased apoptosis and increased proliferation of IAV-specific CD8 T cells in the lungs of Trail(-/-) mice compared with WT mice. Together, these data suggest that TRAIL regulates the magnitude of the IAV-specific CD8 T cell response during a clinically significant IAV infection to decrease the chance for infection-induced immunopathology.     

Type I interferon antagonistic properties of influenza B virus polymerase proteins

The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections. In turn, many if not all viruses have evolved various means to circumvent, resist, or counteract this host response to ensure efficient replication and propagation. Influenza viruses are no exception to this rule, and several viral proteins have been described to possess IFN‐antagonistic functions. Although the viral nonstructural protein 1 appears to be a major antagonist in influenza A and B viruses (IAV and IBV), we have previously shown that a specific motif in the IAV polymerase proteins exerts an IFN‐suppressive function very early in infection. The question remained whether a similar function would also exist in IBV polymerases. Here, we show that indeed a specific amino acid position (A523) of the PB1 protein in the IBV polymerase complex confers IFN‐antagonistic properties. Mutation of this position leads to enhanced activation of the IFN‐mediated signalling pathway after infection and subsequent reduction of virus titres. This indicates that inhibition of innate immune responses is a conserved activity shared by polymerase proteins of IAV and IBV.     

Mast Cells Expedite Control of Pulmonary Murine Cytomegalovirus Infection by Enhancing the Recruitment of Protective CD8 T Cells to the Lungs

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient Kit^W-sh/W-sh “sash” mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of “sash” mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.     

p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling

Many cellular genes and networks induced in human lung epithelial cells infected with the influenza virus remain uncharacterized. Here, we find that p21 levels are elevated in response to influenza A virus (IAV) infection, which is independent of p53. Silencing, pharmacological inhibition or deletion of p21 promotes virus replication in vitro and in vivo, indicating that p21 is an influenza restriction factor. Mechanistically, p21 binds to the C-terminus of IAV polymerase subunit PA and competes with PB1 to limit IAV polymerase activity. Besides, p21 promotes IRF3 activation by blocking K48-linked ubiquitination degradation of HO-1 to enhance type I interferons expression. Furthermore, a synthetic p21 peptide (amino acids 36 to 43) significantly inhibits IAV replication in vitro and in vivo. Collectively, our findings reveal that p21 restricts IAV by perturbing the viral polymerase complex and activating the host innate immune response, which may aid the design of desperately needed new antiviral therapeutics.     

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2′-5′-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.     

Adaptive immune cells temper initial innate responses

Toll-like receptors (TLRs) recognize conserved microbial structures called pathogen-associated molecular patterns. Signaling from TLRs leads to upregulation of co-stimulatory molecules for better priming of T cells and secretion of inflammatory cytokines by innate immune cells. Lymphocyte-deficient hosts often die of acute infection, presumably owing to their lack of an adaptive immune response to effectively clear pathogens. However, we show here that an unleashed innate immune response due to the absence of residential T cells can also be a direct cause of death. Viral infection or administration of poly(I:C), a ligand for TLR3, led to cytokine storm in T-cell- or lymphocyte-deficient mice in a fashion dependent on NK cells and tumor necrosis factor. We have further shown, through the depletion of CD4+ and CD8+ cells in wild-type mice and the transfer of T lymphocytes into Rag-1-deficient mice, respectively, that T cells are both necessary and sufficient to temper the early innate response. In addition to the effects of natural regulatory T cells, close contact of resting CD4+CD25-Foxp3- or CD8+ T cells with innate cells could also suppress the cytokine surge by various innate cells in an antigen-independent fashion. Therefore, adaptive immune cells have an unexpected role in tempering initial innate responses.     

Sialic acid‐dependent interaction of group B streptococci with influenza virus‐infected cells reveals a novel adherence and invasion mechanism

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3‐linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3‐linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co‐sedimentation assay revealed that viruses with a preference for α2,3‐linked sialic acids bind to GBS in a sialic acid‐dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination‐inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre‐infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co‐infected by influenza viruses. This co‐infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3‐linked sialic acids and human viruses binding to this linkage type.     

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia

Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3(-/-) mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3-/- animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.