Antimicrobial activity of mucosal-associated invariant T cells.

Mucosal-associated invariant T lymphocytes (MAIT lymphocytes) are characterized by two evolutionarily conserved features: an invariant T cell antigen receptor (TCR) alpha-chain and restriction by the major histocompatibility complex (MHC)-related protein MR1. Here we show that MAIT cells were activated by cells infected with various strains of bacteria and yeast, but not cells infected with virus, in both humans and mice. This activation required cognate interaction between the invariant TCR and MR1, which can present a bacteria-derived ligand. In humans, we observed considerably fewer MAIT cells in blood from patients with bacterial infections such as tuberculosis. In the mouse, MAIT cells protected against infection by Mycobacterium abscessus or Escherichia coli. Thus, MAIT cells are evolutionarily conserved innate-like lymphocytes that sense and help fight off microbial infection.

The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus.

The Formyl Peptide Receptor 2 (FPR2) is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.

Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections.

BACKGROUND: The pathogenesis of influenza A virus (IAV) infections is a multifactorial process that includes the replication capacity of the virus and a harmful inflammatory response to infection. Formyl peptide receptor 2 (FPR2) emerges as a central receptor in inflammatory processes controlling resolution of acute inflammation. Its role in virus pathogenesis has not been investigated yet. METHODS: We used pharmacologic approaches to investigate the role of FPR2 during IAV infection in vitro and in vivo. RESULTS: In vitro, FPR2 expressed on A549 cells was activated by IAV, which harbors its ligand, annexin A1, in its envelope. FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)-dependent pathway. In vivo, activating FPR2 by administering the agonist WKYMVm-NH2 decreased survival and increased viral replication and inflammation after IAV infection. This effect was abolished by treating the mice with U0126, a specific ERK pathway inhibitor, showing that, in vivo, the deleterious role of FPR2 also occurs through an ERK-dependent pathway. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections. CONCLUSIONS: These data show that viral replication and IAV pathogenesis depend on FPR2 signaling and suggest that FPR2 may be a promising novel strategy to treat influenza.

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis.

RATIONALE: The hallmark of severe influenza virus infection is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. OBJECTIVES: To determine the role of platelets during influenza A virus infections and propose new therapeutics against influenza. METHODS: We used targeted gene deletion approaches and pharmacologic interventions to investigate the role of platelets during influenza virus infection in mice. MEASUREMENTS AND MAIN RESULTS: Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted influenza A virus pathogenesis. Activating protease-activated receptor 4, a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein IIIa protected mice from death caused by influenza viruses, and treating the mice with a specific glycoprotein IIb/IIIa antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other antiplatelet compounds (antagonists of protease-activated receptor 4, MRS 2179, and clopidogrel) were also protected from severe lung injury and lethal infections induced by several influenza strains. CONCLUSIONS: The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and antiplatelet drugs might be explored to develop new antiinflammatory treatment against influenza virus infections.

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication.

UNLABELLED: During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.

Switch from protective to adverse inflammation during influenza: viral determinants and hemostasis are caught as culprits.

Influenza viruses cause acute respiratory infections, which are highly contagious and occur as seasonal epidemic and sporadic pandemic outbreaks. Innate immune response is activated shortly after infection with influenza A viruses (IAV), affording effective protection of the host. However, this response should be tightly regulated, as insufficient inflammation may result in virus escape from immunosurveillance. In contrast, excessive inflammation may result in bystander lung tissue damage, loss of respiratory capacity, and deterioration of the clinical outcome of IAV infections. In this review, we give a comprehensive overview of the innate immune response to IAV infection and summarize the most important findings on how the host can inappropriately respond to influenza.

HLA-G1 and HLA-G5 active dimers are present in malignant cells and effusions: the influence of the tumor microenvironment.

Dimers of the nonclassical HLA-G class I molecule have recently been shown to be active structures that mediate inhibition of NK-cell cytotoxic activity through interaction with the immunoglobulin-like transcript (ILT)-2 inhibitory receptor. However, this has only been proven in trophoblasts and HLA-G transfectants. Here, we document for the first time the existence of HLA-G dimers in cancer. Indeed, we identified both surface and soluble HLA-G dimers in tumor cells and malignant ascites respectively. Interestingly, factors from the tumor microenvironment, such as interferons, enhanced the formation of HLA-G dimers and increased the protection of tumors from NK cell-mediated lysis. These data emphasize the impact of HLA-G conformation on its efficiency at inhibiting the antitumor response and thus favoring tumor progression. In view of these results, the effect of the tumor microenvironment on upregulation of HLA-G function deserves particular attention when designing cancer immunotherapy protocols.

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

Protective role for protease-activated receptor-2 against influenza virus pathogenesis via an IFN-gamma-dependent pathway.

Protease-activated receptor-2 (PAR(2)), a receptor highly expressed in the respiratory tract, can influence inflammation at mucosal surfaces. Although the effects of PAR(2) in the innate immune response to bacterial infection have been documented, knowledge of its role in the context of viral infection is lacking. We thus investigated the role of PAR(2) in influenza pathogenesis in vitro and in vivo. In vitro, stimulation of PAR(2) on epithelial cells inhibited influenza virus type A (IAV) replication through the production of IFN-gamma. In vivo, stimulation of PAR(2) using specific agonists protected mice from IAV-induced acute lung injury and death. This effect correlated with an increased clearance of IAV in the lungs associated with increased IFN- gamma production and a decreased presence of neutrophils and RANTES release in bronchoalveolar fluids. More importantly, the protective effect of the PAR(2) agonist was totally abrogated in IFN- gamma-deficient mice. Finally, compared with wild-type mice, PAR(2)-deficient mice were more susceptible to IAV infection and displayed more severe lung inflammation. In these mice higher neutrophil counts and increased RANTES concentration but decreased IFN- gamma levels were observed in the bronchoalveolar lavages. Collectively, these results showed that PAR(2) plays a protective role during IAV infection through IFN-gamma production and decreased excessive recruitment of inflammatory cells to lung alveoli.

Vav1 phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization.

The guanine nucleotide exchange factor Vav1 regulates actin polymerization and contributes to cytotoxicity by natural killer (NK) cells. An open question is how Vav1 becomes activated and what receptor can signal upstream of actin cytoskeleton rearrangement upon NK cell contact with target cells. Using transfected insect cells that express ligands of human NK cell receptors, we show that engagement of the beta2 integrin LFA-1 on NK cells by intercellular adhesion molecule (ICAM)-1 led to a tyrosine phosphorylation of Vav1 that was not sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 phosphorylation was blocked by an inhibitor of Src-family kinases, and correlated with activation of its downstream effector PAK. Binding of activation receptor 2B4 to its ligand CD48 was not sufficient for Vav1 phosphorylation. However, coengagement of 2B4 with LFA-1 resulted in an enhancement of Vav1 phosphorylation that was sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 was recruited to a detergent-resistant membrane (DRM) fraction only when 2B4 and LFA-1 were coengaged, but not after LFA-1 engagement. Therefore, binding of LFA-1 to ICAM-1 on target cells may initiate an early signaling cascade in NK cells through activation of Vav1, leading to cytoskeleton reorganization and amplification of signals from other activation receptors.

Plasminogen promotes influenza A virus replication through an annexin 2-dependent pathway in the absence of neuraminidase.

Proteolytic cleavage of haemagglutinin (HA) is essential for the infectivity of influenza A viruses (IAVs). This is usually mediated by trypsin-like proteases present in the respiratory tract. However, the ability to use plasminogen (PLG) as an alternative protease may contribute to pathogenesis of IAV infections and virus replication outside the respiratory tract. It was demonstrated previously that neuraminidase (NA) of the IAV strain A/WSN/33 can sequester PLG, allowing this virus to replicate in a PLG-dependent fashion. However, PLG also promotes replication of other IAVs, although its mode of action is poorly understood. Here, using NA-deficient viruses, we demonstrate that NA is not required for the binding of PLG and subsequent cleavage of HA. However, we demonstrate that the cellular protein annexin 2 (A2) can bind PLG and contributes to PLG-dependent cleavage of HA and subsequent IAV replication. Collectively, these results indicate that PLG promotes IAV replication in an A2-dependent fashion in the absence of NA.

Annexin II incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin.

For influenza viruses to become infectious, the proteolytic cleavage of hemagglutinin (HA) is essential. This usually is mediated by trypsin-like proteases in the respiratory tract. The binding of plasminogen to influenza virus A/WSN/33 leads to the cleavage of HA, a feature determining its pathogenicity and neurotropism in mice. Here, we demonstrate that plasminogen also promotes the replication of other influenza virus strains. The inhibition of the conversion of plasminogen into plasmin blocked influenza virus replication. Evidence is provided that the activation of plasminogen is mediated by the host cellular protein annexin II, which is incorporated into the virus particles. Indeed, the inhibition of plasminogen binding to annexin II by using a competitive inhibitor inhibits plasminogen activation into plasmin. Collectively, these results indicate that the annexin II-mediated activation of plasminogen supports the replication of influenza viruses, which may contribute to their pathogenicity.

Protease-activated receptor 1 inhibition protects mice against thrombin-dependent respiratory syncytial virus and human metapneumovirus infections.

BACKGROUND AND PURPOSE: Protease-activated receptor 1 (PAR1) has been demonstrated to be involved in the pathogenesis of viral diseases. However, its role remains controversial. The goal of our study was to investigate the contribution of PAR1 to respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections. EXPERIMENTAL APPROACH: Pharmacological approaches were used to investigate the role of PAR1 during RSV and hMPV infection, in vitro using epithelial A549 cells and in vivo using a mouse model of virus infection. KEY RESULTS: In vitro, the PAR1 antagonist RWJ-56110 reduced the replication of RSV and hMPV in A549 cells. In agreement with these results, RWJ-56110-treated mice were protected against RSV and hMPV infections, as indicated by less weight loss and mortality. This protective effect in mice correlated with decreased lung viral replication and inflammation. In contrast, hMPV-infected mice treated with the PAR1 agonist TFLLR-NH2 showed increased mortality, as compared to infected mice, which were left untreated. Thrombin generation was shown to occur downstream of PAR1 activation in infected mice via tissue factor exposure as part of the inflammatory response, and thrombin inhibition by argatroban reduced the pathogenicity of the infection with no additive effect to that induced by PAR1 inhibition. CONCLUSION AND IMPLICATIONS: These data show that PAR1 plays a detrimental role during RSV and hMPV infections in mice via, at least, a thrombin-dependent mechanism. Thus, the use of PAR1 antagonists and thrombin inhibitors may have potential as a novel approach for the treatment of RSV and hMPV infections.

From Naproxen Repurposing to Naproxen Analogues and Their Antiviral Activity against Influenza A Virus.

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.

FPR2: A Novel Promising Target for the Treatment of Influenza.

The Formyl-peptide receptor-2 (FPR2) is a seven transmembrane G protein-coupled receptor, which plays an important role in sensing of bacteria and modulation of immune responses. FPR2 is also used by viruses for their own profit. Annexin A1, one of the multiple ligands of FPR2, is incorporated in the budding virus membrane of influenza A viruses (IAV). Thereby, once IAV infect a host cell, FPR2 is activated. FPR2-signaling leads to an increase in viral replication, a dysregulation of the host immune response and a severe disease. Conversely, experiments using FPR2 antagonists in a preclinical model of IAV infections in mice showed that blocking FPR2 protects animals from lethal infections. Thus, FPR2 represents a very attractive host target against influenza. In this review we will give an overview on the pathogenesis of influenza with a focus on the role of FPR2 and we will discuss the advantages of using FPR2 antagonists to treat the flu.

Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.

Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza.

Exosomes bearing HLA-G are released by melanoma cells.

Tumor cells release membrane vesicles, named exosomes, capable of specific cytotoxic T-lymphocyte activation by transferring tumor antigens to dendritic cells. By contrast, the nonclassical human leucocyte antigen (HLA)-G class I molecule displays immunotolerant properties and can be ectopically expressed by tumor cells, thereby allowing their escape from immunosurveillance. We describe here that a melanoma cell line, named Fon, established from an HLA-G-positive melanoma biopsy, spontaneously expressed high levels of the HLA-G1 membrane-bound isoform. Exosomes released by Fon cells were purified and analyzed both for their density on sucrose gradient and their protein composition by Western blotting and flow cytometry. Besides the expression of well-described proteins such as Lamp-2, notably, these melanoma-derived exosomes bore HLA-G1. In addition, exosomes harboring HLA-G1 were secreted by the HLA-G-negative M8 melanoma cells transfected with the HLA-G1 cDNA. Thus, the presence of tolerogenic HLA-G molecules on melanoma-derived exosomes may provide a novel way for tumors to modulate host's immune response.

PAR1 contributes to influenza A virus pathogenicity in mice.

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.