Interactions between cigarette smoke (CS) exposure and viral infection play an important role(s) in the pathogenesis of chronic obstructive pulmonary disease (COPD) and a variety of other disorders. A variety of lines of evidence suggest that this interaction induces exaggerated inflammatory, cytokine and tissue remodeling responses. We hypothesized that the 2′-5′OAS/RNase L system, an innate immune antiviral pathway, plays an important role in the pathogenesis of these exaggerated responses. To test this hypothesis we characterize the activation of 2′-5′ oligoadenylate synthase (OAS) in lungs from mice exposed to CS and viral PAMPs/live virus, alone and in combination. We also evaluated the inflammatory and remodeling responses induced by CS and virus/viral PAMPs in lungs from RNase L null and wild type mice. These studies demonstrate that CS and viral PAMPs/live virus interact in a synergistic manner to stimulate the production of select OAS moieties. They also demonstrate that RNase L plays a critical role in the pathogenesis of the exaggerated inflammatory, fibrotic, emphysematous, apoptotic, TGF-β1 and type I IFN responses induced by CS plus virus/viral PAMP in combination. These studies demonstrate that CS is an important regulator of antiviral innate immunity, highlight novel roles of RNase L in CS plus virus induced inflammation, tissue remodeling, apoptosis and cytokine elaboration and highlight pathways that may be operative in COPD and mechanistically-related disorders.
Rationale: Vascular endothelial growth factor (VEGF) regulates vascular, inflammatory, remodeling, and cell death responses. It plays a critical role in normal pulmonary physiology, and VEGF excess and deficiency have been implicated in the pathogenesis of asthma and chronic obstructive pulmonary disease, respectively. Although viruses are an important cause of chronic obstructive pulmonary disease exacerbations and innate responses play an important role in these exacerbations, the effects of antiviral responses on VEGF homeostasis have not been evaluated.
Objectives: We hypothesized that antiviral innate immunity regulates VEGF tissue responses.
Methods: We compared the effects of transgenic VEGF165 in mice treated with viral pathogen–associated molecular pattern polyinosinic:polycytidylic acid [poly(I:C)], mice treated with live virus, and control mice.
Measurements and Main Results: Transgenic VEGF stimulated angiogenesis, edema, inflammation, and mucin accumulation. Each of these was abrogated by poly(I:C). These inhibitory effects were dose dependent, noted when poly(I:C) was administered before and after transgene activation, and mediated by a Toll-like receptor-3–independent and RIG-like helicase (RLH)– and type I IFN receptor–dependent pathway. VEGF stimulated the expression of VEGF receptor-1 and poly(I:C) inhibited this stimulation. Poly(I:C) also inhibited the ability of VEGF to activate extracellular signal–regulated kinase-1, Akt, focal adhesion kinase, and endothelial nitric oxide synthase, and aeroallergen-induced adaptive helper T-cell type 2 inflammation. Influenza and respiratory syncytial virus also inhibited VEGF-induced angiogenesis.
Conclusions: These studies demonstrate that poly(I:C) and respiratory viruses inhibit VEGF-induced tissue responses and adaptive helper T-cell type 2 inflammation and highlight the importance of a RLH- and type I IFN receptor–dependent pathway(s) in these regulatory events. They define a novel link between VEGF and antiviral and RLH innate immune responses and a novel pathway that regulates pulmonary VEGF activity.
RIG-like helicase; mitochondrial antiviral signaling molecule; influenza virus; chronic obstructive pulmonary disease
Exacerbations of chronic obstructive pulmonary disease (COPD) are characterized by acute enhancement of airway neutrophilic inflammation under oxidative stress and can be involved in emphysema progression. However, pharmacotherapy against the neutrophilic inflammation and emphysema progression associated with exacerbation has not been established. Thioredoxin-1 has anti-oxidative and anti-inflammatory properties and it can ameliorate neutrophilic inflammation through anti-chemotactic effects and prevent cigarette smoke (CS)-induced emphysema. We aimed to determine whether thioredoxin-1 can suppress neutrophilic inflammation and emphysema progression in a mouse model of COPD exacerbation and if so, to reveal the underlying mechanisms.
Mice were exposed to CS and then challenged with polyinosine-polycytidylic acid [poly(I:C)], an agonist for virus-induced innate immunity. Airway neutrophilic inflammation, oxidative stress and lung apoptosis were enhanced in smoke-sensitive C57Bl/6, but not in smoke-resistant NZW mice. Exposure to CS and poly(I:C) challenge accelerated emphysema progression in C57Bl/6 mice. Thioredoxin-1 suppressed neutrophilic inflammation and emphysema progression. Poly(I:C) caused early neutrophilic inflammation through keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor (GM-CSF) release in the lung exposed to CS. Late neutrophilic inflammation was caused by persistent GM-CSF release, which thioredoxin-1 ameliorated. Thioredoxin-1 enhanced pulmonary mRNA expression of MAP kinase phosphatase 1 (MKP-1), and the suppressive effects of thioredoxin-1 on prolonged GM-CSF release and late neutrophilic inflammation disappeared by inhibiting MKP-1.
Using a mouse model of COPD exacerbation, we demonstrated that thioredoxin-1 ameliorated neutrophilic inflammation by suppressing GM-CSF release, which prevented emphysema progression. Our findings deepen understanding of the mechanisms underlying the regulation of neutrophilic inflammation by thioredoxin-1 and indicate that thioredoxin-1 could have potential as a drug to counteract COPD exacerbation.
Respiratory syncytial viral (RSV) infections are a frequent cause of chronic obstructive pulmonary disease (COPD) exacerbations, which are a major factor in disease progression and mortality. RSV is able to evade antiviral defenses to persist in the lungs of COPD patients. Though RSV infection has been identified in COPD, its contribution to cigarette smoke-induced airway inflammation and lung tissue destruction has not been established. Here we examine the long-term effects of cigarette smoke exposure, in combination with monthly RSV infections, on pulmonary inflammation, protease production and remodeling in mice. RSV exposures enhanced the influx of macrophages, neutrophils and lymphocytes to the airways of cigarette smoke exposed C57BL/6J mice. This infiltration of cells was most pronounced around the vasculature and bronchial airways. By itself, RSV caused significant airspace enlargement and fibrosis in mice and these effects were accentuated with concomitant smoke exposure. Combined stimulation with both smoke and RSV synergistically induced cytokine (IL-1α, IL-17, IFN-γ, KC, IL-13, CXCL9, RANTES, MIF and GM-CSF) and protease (MMP-2, -8, -12, -13, -16 and cathepsins E, S, W and Z) expression. In addition, RSV exposure caused marked apoptosis within the airways of infected mice, which was augmented by cigarette smoke exposure. RSV and smoke exposure also reduced protein phosphatase 2A (PP2A) and protein tyrosine phosphates (PTP1B) expression and activity. This is significant as these phosphatases counter smoke-induced inflammation and protease expression. Together, these findings show for the first time that recurrent RSV infection markedly enhances inflammation, apoptosis and tissue destruction in smoke-exposed mice. Indeed, these results indicate that preventing RSV transmission and infection has the potential to significantly impact on COPD severity and progression.
Respiratory viral infections cause significant morbidity and increase the risk for chronic pulmonary graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). Our overall hypothesis is that local innate immune activation potentiates adaptive alloimmunity. In this study, we hypothesized that a viral pathogen-associated molecular pattern (PAMP) alone can potentiate pulmonary GVHD after allogeneic HCT. We, therefore, examined the effect of pulmonary exposure to polyinosinic:polycytidylic acid (poly I:C), a viral mimetic that activates innate immunity, in an established murine HCT model. Poly I:C induced a marked pulmonary T cell response in allogeneic HCT mice as compared to syngeneic HCT, with increased CD4+ cells in the lung fluid and tissue. This lymphocytic inflammation persisted at 2 weeks post poly I:C exposure in allogeneic mice and was associated with CD3+ cell infiltration into the bronchiolar epithelium and features of epithelial injury. In vitro, poly I:C enhanced allospecific proliferation in a mixed lymphocyte reaction. In vivo, poly I:C exposure was associated with an early increase in pulmonary monocyte recruitment and activation as well as a decrease in CD4+FOXP3+ regulatory T cells in allogeneic mice as compared to syngeneic. In contrast, intrapulmonary poly I:C did not alter the extent of systemic GVHD in either syngeneic or allogeneic mice. Collectively, our results suggest that local activation of pulmonary innate immunity by a viral molecular pattern represents a novel pathway that contributes to pulmonary GVHD after allogeneic HCT, through a mechanism that includes increased recruitment and maturation of intrapulmonary monocytes.
poly I:C; pulmonary graft-versus-host disease; allogeneic; lymphocytic bronchiolitis; respiratory viral infection; monocytes
Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.
Type I interferons (IFNs) constitute the first line of defense against viral infections, promote antigen presentation by dendritic cells, and play a crucial role in directly stimulating anti-viral T cell responses. However, the mechanisms underlying the diverse cellular effects of Type I IFNs are not well defined. One of the best-characterized anti-viral signaling mechanisms induced by Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. We show that requirement for cellular PKR activity could be a distinguishing feature between Type I IFN actions that mediate viral control or stimulate CD8+ T cell expansion during an acute infection with lymphocytic choriomeningitis virus (LCMV). Typically, innate immune mechanisms including Type I IFNs are considered important for viral control during a primary infection. However, we find that presence of vaccine-induced CD8+ T cell memory and accelerated generation of secondary effectors are necessary but not sufficient to provide effective protective immunity to re-infection, without the aid of innate effectors PKR and Type I IFNs. These findings have improved our understanding of virus-immune system interactions and immune correlates of anti-viral protective immunity, which might have implications in the development of effective anti-viral vaccines and immunotherapies.
Innate immune defenses are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs) 1. Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people 2. Infection is governed by hepatic immune defenses triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals IRF-3 activation to induce the expression of α/β interferon (IFN) and antiviral/interferon-stimulated genes (ISGs) that limit infection 3–10. Here we identified the poly-uridine motif of the HCV genome 3’ nontranslated region (NTR) as the PAMP substrate of RIG-I, and show that this and similar homopoly-uridine motifs present in the genome of RNA viruses is the chief feature of RIG-I recognition and immune triggering 8. 5’ terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent upon homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signaling to induce a hepatic innate immune response in vivo, and triggered IFN and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by identifying homopoly-uridine motfis present in the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and define immunogenic features of the PAMP/RIG-I interaction that could be utilized as an immune adjuvant for vaccine and immunotherapy approaches.
Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4+and CD8+T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection.
Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. Both innate and acquired immunity are essential for protection against influenza virus, and Notch and Notch ligands provide a key bridge between innate and acquired immunity. However, the role of Notch system during influenza virus infection is unknown. Here, we show that Notch ligand Delta-like 1 (Dll1) expression was up-regulated in influenza virus H1N1 challenged macrophages, and was dependent on both retinoic-acid–inducible protein I (RIG-I) and IFNα receptor (IFNαR)-mediated pathways. IFNαR-deficient mice challenged with influenza virus in vivo also display a profoundly impaired Dll1 expression with increased mortality and abrogated IFN-γ production. Treatment of WT mice during influenza infection, with either neutralizing antibodies specific for Dll1 or a γ-secretase inhibitor (GSI), which blocks Notch signaling, resulted in increased mortality, impaired viral clearance, and lower IFN-γ production. In addition, Dll1 specifically regulated IFN-γ production from both CD4+and CD8+T cells in vitro. Together, these results suggest that Notch signaling through macrophage-dependent Dll1 is critical in providing an anti-viral response during influenza infection by linking innate and acquired immunity.
Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.
Hepatitis C Virus (HCV) is a poor interferon (IFN) inducer, despite recognition of its RNA by the cytosolic RNA helicase RIG-I. This is due in part through cleavage of MAVS, a downstream adapter of RIG-I, by the HCV NS3/4A protease and through activation of the eIF2α-kinase PKR to control IFN translation. Here, we show that HCV also inhibits RIG-I activation through the ubiquitin-like protein ISG15 and that HCV triggers rapid induction of 49 genes, including ISG15, through a novel signaling pathway that precedes RIG-I and involves PKR as an adapter to recruit MAVS. Hence, we propose to divide the acute response to HCV infection into one early (PKR) and one late (RIG-I) phase, with the former controlling the latter. Furthermore, these data emphazise the need to check compounds designed as immune adjuvants for activation of the early acute phase before using them to sustain innate immunity.
Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5′-triphosphate (5′-ppp) on the viral RNA in association with a 3′ poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine “core” of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5′-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5′-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5′-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.
Pathogen recognition receptors (PRRs) are critical components of the innate immune response to viral pathogens, and function in the host to recognize pathogen-associated molecular patterns (PAMPs) in viral proteins or nucleic acids. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic PRR that senses viral RNA inside an infected cell. RIG-I recognizes hepatitis C virus (HCV) RNA as non-self through the presence of both a 5′-triphosphate (5′-ppp) and a 3′ poly-U/UC tract within the viral RNA. Here we examined the RNA structure-function attributes that define the HCV poly-U/UC tract as non-self to RIG-I, including nucleotide composition. We found that the 34 nucleotide poly-uridine “core” (U-core) within the HCV poly-U/UC tract RNA was required for non-self recognition by RIG-I, and interspersed ribocytosine nucleotides were also important to induce optimal RIG-I signaling. RIG-I/RNA binding studies revealed that RIG-I formed weaker interactions with HCV RNAs lacking poly-U sequences, and RNA interaction with multiple domains of RIG-I was required to activate signaling. Finally, RIG-I recognition of the U-core within the poly-U/UC tract activated anti-HCV responses in vitro and hepatic innate immune responses in vivo. Our studies identify long poly-uridine sequences with interspersed ribocytosines as an HCV PAMP motif that drives optimal RIG-I signaling.
DC are activated by pathogen-associated molecular patterns (PAMPs), and this is pivotal for the induction of adaptive immune responses. Thereafter, the clearance of activated DC is crucial to prevent immune pathology. While PAMPs are of major interest for vaccine science due to their adjuvant potential, it is unclear whether and how PAMPs may affect DC viability. We aimed to elucidate the possible apoptotic mechanisms that control activated DC lifespan in response to PAMPs, particularly in vivo.
We report that polyinosinic:polycytidylic acid (PolyIC, synthetic analogue of dsRNA) induces dramatic apoptosis of mouse splenic conventional DC (cDC) in vivo, predominantly affecting the CD8α subset, as shown by flow cytometry-based analysis of splenic DC subsets. Importantly, while Bim deficiency conferred only minor protection, cDC depletion was prevented in mice lacking Bim plus one of three other BH3-only proteins, either Puma, Noxa or Bid. Furthermore, we show that Type I Interferon (IFN) is necessary and sufficient for DC death both in vitro and in vivo, and that TLR3 and MAVS co-operate in IFNß production in vivo to induce DC death in response to PolyIC.
These results demonstrate for the first time in vivo that apoptosis restricts DC lifespan following activation by PolyIC, particularly affecting the CD8α cDC subset. Such DC apoptosis is mediated by the overlapping action of pro-apoptotic BH3-only proteins, including but not solely involving Bim, and is driven by Type I IFN. While Type I IFNs are important anti-viral factors, CD8α cDC are major cross-presenting cells and critical inducers of CTL. We discuss such paradoxical finding on DC death with PolyIC/Type I IFN. These results could contribute to understand immunosuppression associated with chronic infection, and to the optimization of DC-based therapies and the clinical use of PAMPs and Type I IFNs.
Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR-) and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.
Acute lung injury induced by influenza virus (IV) infection has been linked to an unbalanced release of pro-inflammatory cytokines including type I interferons (IFN) causing immune-mediated organ damage. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage-expressed IFN-β induces alveolar epithelial cell injury by autocrine induction of the pro-apoptotic TNF-related apoptosis-inducing ligand (TRAIL). Elucidating the cell-specific underlying signalling pathways revealed that IV-induced IFN-β release from alveolar macrophages (AM) strictly depended on protein kinase R- (PKR-) and NF-κB-signalling. Autocrine activation via the macrophage type I IFN receptor (IFNAR) resulted in increased expression and release of TRAIL which caused apoptosis of IV-infected and non-infected alveolar epithelial cells and promoted alveolar barrier dysfunction as demonstrated in ex vivo co-cultures and in bone marrow chimeric mouse models in vivo. Importantly, we found TRAIL highly upregulated in and released from AM of hospitalized patients with pandemic H1N1-induced lung failure. Therapeutic targeting of the macrophage IFN-β-TRAIL axis might therefore represent a promising strategy to attenuate IV-induced acute lung injury.
Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection.
Nontypeable Haemophilus influenzae (NTHi) is strongly associated with exacerbations of chronic obstructive pulmonary disease, which often coincide with viral respiratory infections. TLR2 contributes importantly to innate immunity to NTHi, but whether this pathway is affected by simultaneous antiviral responses is unknown. To analyze potential interactions, resident murine and human alveolar macrophages (AMφ) were exposed, in the presence or absence of the appropriate rIFN-β, to synthetic lipopeptides corresponding to the triacylated N-terminal fragments of three outer membrane proteins (OMP) (PCP, P4, and P6) that are highly conserved among different NTHi strains. Synthetic OMP elicited strong release of IL-6, the principal inducer of airway mucin genes, and induced CCL5 and CXCL10 from murine AMφ only when IFN-β was also present. Surprisingly, combined stimulation by OMPs and IFN-β also markedly enhanced TNF-α release by murine AMφ. Stimulation with PCP plus IFN-β induced IFN-regulatory factor 1 expression and sustained STAT1 activation, but did not alter the activation of MAPKs or NF-κB. AMφ derived from STAT1-deficient mice did not demonstrate increased production of TNF-α in response to PCP plus IFN-β. Analysis of wild-type and STAT1-deficient AMφ using real-time PCR showed that increased TNF-α production depended on transcriptional up-regulation, but not on mRNA stabilization. The synergistic effect of synthetic OMP and IFN-β was conserved between murine AMφ and human AMφ for IL-6, but not for TNF-α. Thus, IFN-β, which is produced by virally infected respiratory epithelial cells, converts normally innocuous NTHi OMP into potent inflammatory stimulants, but does so via different mechanisms in mice and humans.
PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti-T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy, accumulation the autophagy molecule LC3 around the parasite, vacuole-lysosomal fusion and killing of T. gondii in CD40-activated macrophages and microglia. Thus, our findings identified PKR as a mediator of anti-microbial activity and promoter of protection against disease caused by a non-viral pathogen, revealed that PKR is activated by CD40 via TRAF6 and TRAF2, and positioned PKR as a link between CD40-TRAF signaling and stimulation of the autophagy pathway.
PKR was identified more than 30 years ago as an inhibitor of viral replication. It is unknown if PKR promotes protection against disease caused by non-viral pathogens. We addressed this question using Toxoplasma gondii, a major parasitic pathogen. T. gondii can cause cerebral and/or eye disease primarily in immunosuppressed patients and newborns. After infection with T. gondii, PKR-deficient mice exhibited high parasite loads in the eye and brain and were more susceptible to ocular and cerebral toxoplasmosis. Macrophages and microglia are important effectors of protection against T. gondii. These cells required PKR signaling to kill the parasite in response to stimulation via CD40, a molecule that promotes protection against ocular and cerebral toxoplasmosis. CD40 functioned only through its TRAF6 binding site to activate PKR, but this process was also dependent on TRAF2 where this molecule likely acted as an intermediary that promoted TRAF6-PKR association and PKR activation. PKR linked CD40-TRAF signaling to stimulation of the autophagy pathway and T. gondii killing. Our studies identified a previously unappreciated role of PKR as mediator of anti-microbial activity and promoter of resistance against disease caused by a non-viral pathogen, as well as provided new insight on the molecular link between CD40 and PKR.
Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.
Pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs), such as viral RNA, drives innate immune responses against West Nile virus (WNV), an emerging neurotropic pathogen. Here we demonstrate that WNV PAMPs orchestrate endothelial responses to WNV via competing innate immune cytokine signals at the blood-brain barrier (BBB), a multicellular interface with highly specialized brain endothelial cells that normally prevents pathogen entry. While Th1 cytokines increase the permeability of endothelial barriers, type I interferon (IFN) promoted and stabilized BBB function. Induction of innate cytokines by pattern recognition pathways directly regulated BBB permeability and tight junction formation via balanced activation of the small GTPases Rac1 and RhoA, which in turn regulated the transendothelial trafficking of WNV. In vivo, mice with attenuated type I IFN signaling or IFN induction (Ifnar−/−
Irf7−/−) exhibited enhanced BBB permeability and tight junction dysregulation after WNV infection. Together, these data provide new insight into host-pathogen interactions at the BBB during neurotropic viral infection.
West Nile virus (WNV) is an emerging pathogen capable of infecting the central nervous system (CNS), causing fatal encephalitis. However, the mechanisms that control the ability of WNV to cross the blood-brain barrier (BBB) and access the CNS are unclear. In this study, we show that detection of WNV by host tissues induces innate immune cytokine expression at the BBB, regulating BBB structure and function and impacting transendothelial trafficking of WNV. This regulatory effect is shown to happen rapidly following exposure to virus, to occur independently of viral replication within BBB cells, and to require the signaling of cytoskeletal regulatory Rho GTPases. These results provide new understanding of host-pathogen interactions at the BBB during viral encephalitis.
The innate immune response must be mobilized promptly yet judiciously via Toll-like receptors (TLRs) to protect the lungs against pathogens. Stimulation of murine peritoneal macrophage (PMø) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I interferon β (IFN-β), leading to autocrine activation of the transcription factor STAT1. Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar Mø (AMø) or control PMø from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (poly I:C, specific for TLR3) or bacteria (Pam3Cys, specific for TLR2, and re-purified LPS, specific for TLR4). AMø did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-β. This unique AMø response was not due to altered TLR expression, or defective immediate early gene response, as measured by expression of TNF-α and three beta chemokines. Instead, AMø differed from PMø in not producing bioactive IFN-β, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMø to induce STAT1 phosphorylation in PMø. Consequently, AMø did not produce the microbicidal effector molecule nitric oxide following TLR4 or TLR3 stimulation unless exogenous IFN-β was also added. Thus, murine AMø respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-β secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense.
Macrophages; Lung; Signal Transduction; Cytokines; Nitric Oxide
Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.
Japanese encephalitis (JE) is major emerging encephalitis, and more than 60% of global population inhabits JE endemic areas. The etiological virus is currently spreading to previously unaffected regions due to rapid changes in climate and demography. However, the impact of TLR molecules on JE progression has not been addressed to date. We found that the distinct outcomes of JE progression occurred in TLR3 and TLR4-dependent manner, i.e. TLR3−/− mice were highly susceptible, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation manifested by early CD11b+Ly-6Chigh monocyte infiltration, high expression of proinflammatory cytokines, as well as increased BBB permeability. In contrast, TLR4 ablation provided potent type I IFN innate response in infected mice, as well as in myeloid-derived cells closely associated with strong induction of antiviral ISG genes, and also resulted in enhanced humoral, CD4+, and CD8+ T cell responses along with altered plasmacytoid DC and CD4+Foxp3+ Treg number. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were coupled with reduced JE lethality. Our studies provide an insight into the role of each TLR molecule on the modulation of JE, as well as its mechanism of neuroinflammation control during JE progression.
Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.
While viruses that target the liver often cause lengthy infections with considerable morbidity, there is limited understanding of how they evade host responses. We have studied hepatitis A virus (HAV), an important cause of acute hepatitis in humans. Although HAV infection typically results in hepatic inflammation, there is no disease in the liver during the first weeks of infection despite robust virus replication. This suggests that HAV either fails to stimulate or efficiently evades recognition by host innate immune sensors. Our prior work showed HAV disrupts RIG-I/MDA5 signaling by targeting MAVS, an essential adaptor protein, for degradation by 3ABC, a precursor of the only HAV protease, 3Cpro. Here, we show here that a distinct viral processing intermediate, the 3CD protease-polymerase, disrupts TLR3 signaling by degrading its adaptor protein, TRIF. HAV has evolved a novel strategy to target two different host adaptor proteins with a single protease, using its 3Dpol RNA polymerase to modify the substrate specificity of its 3Cpro protease when fused to it in the 3CD precursor, thus allowing it to target non-canonical 3Cpro recognition sequences in TRIF. This remarkable example of viral adaptation allows the virus to target two different host adaptor proteins with a single viral protease.
Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.
Hepatitis C Virus (HCV) is the most common bloodborne pathogen for which no vaccine is available. Infection with the virus often leads to persistent (or chronic) infection. Patients with chronic HCV infection can develop progressive liver disease and liver failure, leading to the need for a transplant. It is not fully understood why some people clear the virus and others develop persistent infection. Understanding differences in how patients respond to the virus in the early phases of infection may lead to better treatment of HCV. Here, we use a highly conserved region of the HCV genome to examine innate immunological responses to HCV. We found that plasmacytoid dendritic cells, innate cells keyed to respond with anti-viral interferon proteins, recognize the virus. Additionally, we show that pDCs use RIG-I in the recognition of this virus, which was previously thought to be dispensable in pDCs. The proteins secreted by these cells can control viral replication in a cell-based laboratory system. In cells isolated from healthy donors, we found that fresh human cells can respond in the same manner to the virus as the laboratory strain of cells, and there was a correlation with genetic differences. Our study offers novel insight to how the body recognizes HCV during early infection and host-virus interactions that mediate viral control of this common infection.
The interferon (IFN)-inducible antiviral state is mediated in part by the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-β by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-β by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-β production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-β in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-β induction that could affect tissue protection and survival during viral infections.
Type I interferons (IFNs) such as IFN-β are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-β production in mouse embryonic fibroblasts and in virus-infected mice. Here we report that high basal levels of OAS/RNase L in macrophages reduce, rather than increase, virus induction of IFN-β. RNA damage and apoptosis caused by RNase L were the likely reasons for the decreased IFN-β production in virus-infected macrophages. Our studies suggest that during viral infections, the OAS/RNase L pathway can either enhance or suppress IFN production, depending on the cell type. IFN regulation by RNase L is suggested to contribute to tissue protection and survival during viral infections.
•We report interactions of Toll-like receptors (TLRs) with COX enzymes in vivo.•COX-2 was broadly induced by LPS (TLR4) but more locally by poly(I:C) (TLR3).•COX-1/2 deletion modified the response to TLR activation in a TLR-specific manner.•COX-2 deletion enhanced interferon responses to viral-type TLR3/7/9 ligands.•COX-2 inhibition could provide a novel anti-viral therapeutic strategy.
Cyclooxygenase 2 (COX)-2 is induced by bacterial and viral infections and has complex, poorly understood roles in anti-pathogen immunity. Here, we use a knock-in luciferase reporter model to image Cox2 expression across a range of tissues in mice following treatment with the either the prototypical bacterial pathogen-associated molecular pattern (PAMP), LPS, which activates Toll-like receptor (TLR)4, or with poly(I:C), a viral PAMP, which activates TLR3. LPS induced Cox2 expression in all tissues examined. In contrast, poly(I:C) elicited a milder response, limited to a subset of tissues. A panel of cytokines and interferons was measured in plasma of wild-type, Cox1−/− and Cox2−/− mice treated with LPS, poly(I:C), MALP2 (TLR2/6), Pam3CSK4 (TLR2/1), R-848 (TLR7/8) or CpG ODN (TLR9), to establish whether/how each COX isoform modulates specific PAMP/TLR responses. Only LPS induced notable loss of condition in mice (inactivity, hunching, piloerection). However, all TLR agonists produced cytokine responses, many of which were modulated in specific fashions by Cox1 or Cox2 gene deletion. Notably we observed opposing effects of Cox2 gene deletion on the responses to the bacterial PAMP, LPS, and the viral PAMP, poly(I:C), consistent with the differing abilities of the PAMPs to induce Cox2 expression. Cox2 gene deletion limited the plasma IL-1β and interferon-γ responses and hypothermia produced by LPS. In contrast, in response to poly(I:C), Cox2−/− mice exhibited enhanced plasma interferon (IFNα,β,γ,λ) and related cytokine responses (IP-10, IL-12). These observations suggest that a COX-2 selective inhibitor, given early in infection, may enhance and/or prolong endogenous interferon responses, and thereby increase anti-viral immunity.
Cyclooxygenase; LPS; Poly(I:C); Toll-like receptor; TLR3; TLR4
Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called “priming loop”, we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3−/−IRF7−/− double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1−/−IL-28Rα−/−). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.
The response of cells to virus infection depends on Interferons (IFNs), a group of cytokines which activate the expression of hundreds of genes that help control viral replication inside infected cells. While type I IFN was discovered in 1957, type III IFN (IFNλ, IL-28/29) was characterized recently and is known for its role in the response to hepatitis C virus. Airway epithelia are the primary target of influenza virus, and we studied how infection induces IFNs and which IFN is most important for the epithelial anti-influenza response. We found that infected epithelia detect virus through the cytoplasmic RIG-I/MAVS recognition system, leading to activation of the transcription factor IRF7 and subsequent induction of both type I and III IFNs. All ensuing cellular responses to infection are dependent on the production and secretion of IFNs, as responses are lost in epithelia lacking receptors for both type I and III IFNs. Finally, gene induction is indistinguishable in single receptor-deficient and wild-type cells, indicating that the two IFN systems are completely redundant in epithelia. Thus, influenza infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two overlapping and redundant amplification loops to upregulate antiviral genes.
Increasing evidence indicates that chronic inflammatory and immune responses play key roles in the development and progression of COPD. Recent data provide evidence for a role in the NLRP3 inflammasome in the airway inflammation observed in COPD. Cigarette smoke activates innate immune cells by triggering pattern recognition receptors (PRRs) to release “danger signal”. These signals act as ligands to Toll-like receptors (TLRs), triggering the production of cytokines and inducing innate inflammation. In smokers who develop COPD there appears to be a specific pattern of inflammation in the airways and parenchyma as a result of both innate and adaptive immune responses, with the predominance of CD8+ and CD4+ cells, and in the more severe disease, with the presence of lymphoid follicles containing B lymphocytes and T cells. Furthermore, viral and bacterial infections interfere with the chronic inflammation seen in stable COPD and exacerbations via pathogen-associated molecular patterns (PAMPs). Finally, autoimmunity is another novel aspect that may play a critical role in the pathogenesis of COPD. This review is un update of the currently discussed roles of inflammatory and immune responses in the pathogenesis of COPD.