Transcriptomic Analysis of Host Immune and Cell Death Responses Associated with the Influenza A Virus PB1-F2 Protein

Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence

Processing of Genome 5′ Termini as a Strategy of Negative-Strand RNA Viruses to Avoid RIG-I-Dependent Interferon Induction

Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5′ triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5′ triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5′ monophosphate-specific exonuclease indicates that the RIG-I-activating 5′ triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5′ end as a strategy of viruses to evade the innate immune response

Age-Dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3−/−) or the adaptor molecule Trif (TrifLps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection

Role of Position 627 of PB2 and the Multibasic Cleavage Site of the Hemagglutinin in the Virulence of H5N1 Avian Influenza Virus in Chickens and Ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens

The Critical Role of Notch Ligand Delta-like 1 in the Pathogenesis of Influenza A Virus (H1N1) Infection

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

Isolation of Bacterial Ribosomes with Monolith Chromatography

We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology

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

Inflammasome-dependent Pyroptosis and IL-18 Protect against Burkholderia pseudomallei Lung Infection while IL-1β Is Deleterious

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β

Co-ordinated Role of TLR3, RIG-I and MDA5 in the Innate Response to Rhinovirus in Bronchial Epithelium

The relative roles of the endosomal TLR3/7/8 versus the intracellular RNA helicases RIG-I and MDA5 in viral infection is much debated. We investigated the roles of each pattern recognition receptor in rhinovirus infection using primary bronchial epithelial cells. TLR3 was constitutively expressed; however, RIG-I and MDA5 were inducible by 8–12 h following rhinovirus infection. Bronchial epithelial tissue from normal volunteers challenged with rhinovirus in vivo exhibited low levels of RIG-I and MDA5 that were increased at day 4 post infection. Inhibition of TLR3, RIG-I and MDA5 by siRNA reduced innate cytokine mRNA, and increased rhinovirus replication. Inhibition of TLR3 and TRIF using siRNA reduced rhinovirus induced RNA helicases. Furthermore, IFNAR1 deficient mice exhibited RIG-I and MDA5 induction early during RV1B infection in an interferon independent manner. Hence anti-viral defense within bronchial epithelium requires co-ordinated recognition of rhinovirus infection, initially via TLR3/TRIF and later via inducible RNA helicases

Influenza Virus A Infection of Human Monocyte and Macrophage Subpopulations Reveals Increased Susceptibility Associated with Cell Differentiation

Influenza virus infection accounts for significant morbidity and mortality world-wide. Interactions of the virus with host cells, particularly those of the macrophage lineage, are thought to contribute to various pathological changes associated with poor patient outcome. Development of new strategies to treat disease therefore requires a detailed understanding of the impact of virus infection upon cellular responses. Here we report that human blood-derived monocytes could be readily infected with the H3N2 influenza virus A/Udorn/72 (Udorn), irrespective of their phenotype (CD14++/CD16−, CD14++/CD16+ or CD14dimCD16++), as determined by multi-colour flow cytometry for viral haemagglutinin (HA) expression and cell surface markers 8–16 hours post infection. Monocytes are relatively resistant to influenza-induced cell death early in infection, as approximately 20% of cells showed influenza-induced caspase-dependent apoptosis. Infection of monocytes with Udorn also induced the release of IL-6, IL-8, TNFα and IP-10, suggesting that NS1 protein of Udorn does not (effectively) inhibit this host defence response in human monocytes. Comparative analysis of human monocyte-derived macrophages (Mph) demonstrated greater susceptibility to human influenza virus than monocytes, with the majority of both pro-inflammatory Mph1 and anti-inflammatory/regulatory Mph2 cells expressing viral HA after infection with Udorn. Influenza infection of macrophages also induced cytokine and chemokine production. However, both Mph1 and Mph2 phenotypes released comparable amounts of TNFα, IL-12p40 and IP-10 after infection with H3N2, in marked contrast to differential responses to LPS-stimulation. In addition, we found that influenza virus infection augmented the capacity of poorly phagocytic Mph1 cells to phagocytose apoptotic cells by a mechanism that was independent of either IL-10 or the Mer receptor tyrosine kinase/Protein S pathway. In summary, our data reveal that influenza virus infection of human macrophages causes functional alterations that may impact on the process of resolution of inflammation, with implications for viral clearance and lung pathology