Virus-infected Epithelial Cells Research Articles

RNA Sequencing of H3N2 Influenza Virus-Infected Human Nasal Epithelial Cells from Multiple Subjects Reveals Molecular Pathways Associated with Tissue Injury and Complications.

The human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. Influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. Here, we elucidated the global transcriptomic changes during H3N2 infection of human nasal epithelial cells from multiple individuals. Using RNA sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. We used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. Statistical analysis highlighted strong transcriptomic signatures significantly associated with 24 and 48 h after infection, but not at the earlier 8-h time point. In particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, B-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. These molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management.

Release of transglutaminase 2 from respiratory syncytial virus-infected airway epithelial cells and its role in disease pathogenesis

Abstract Respiratory syncytial virus (RSV) is the common cause of severe lower respiratory tract infections in young children, the elderly and the immunocompromised, for which no effective treatment or vaccine is currently available. RSV infections have also been linked to both the development and severity of asthma. Oxidative stress plays a major role in the pathogenesis of many lung inflammatory diseases. Our studies have shown that oxidative stress responses in the airways play a major role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium levels, which in turn activate calcium dependent enzymes, including transglutaminase 2 (TG2). TG2 is a transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications and has been implicated in the pathogenesis of many diseases. This study’s objective was to investigate the release of TG2 in airway epithelial cells in response to RSV infection and its potential mechanism. Studies were conducted in airway epithelial cells, a human lung carcinoma cell line and normal small alveolar epithelial cells. The changes in the TG2 levels were measured by Western blot and RT-PCR after cells were pretreated with antioxidants and infected with RSV. TG2 levels were significantly increased in the secretome with a concomitant decrease in the cell lysates, cytoplasmic and nuclear extracts in response to RSV infection. Antioxidants treatment significantly inhibited RSV-induced TG2 release. TG2 release was induced only by RSV but not RSV-induced cytokines or chemokines. These results suggest that modulation of oxidative stress represents a potential novel pharmacological approach to ameliorate virus-induced acute airway disease through TG2 release blockade.

Respiratory epithelial cells as master communicators during viral infections.

Purpose of ReviewCommunication by epithelial cells during respiratory viral infections is critical in orchestrating effective antiviral responses but also can lead to excessive inflammation. This review will evaluate studies that investigate how respiratory epithelial cells influence the behavior of immune cells and how epithelial cell/immune cell interactions contribute to antiviral responses and immunopathology outcomes.Recent FindingsPrevious studies have characterized cytokine responses of virus-infected epithelial cells. More recent studies have carefully demonstrated the effects of these cytokines on cellular behaviors within the infected lung. Infected epithelial cells release exosomes that specifically regulate responses of monocytes and neighboring epithelial cells without promoting spread of virus. In contrast, rhinovirus-infected cells induce monocytes to upregulate expression of the viral receptor, promoting spread of the virus to alternate cell types. The precise alteration of PDL expression on infected epithelial cells has been shown to switch between inhibition and activation of antiviral responses.SummaryThese studies have more precisely defined the interactions between epithelial and immune cells during viral infections. This level of understanding is critical for the development of novel therapeutic strategies that promote effective antiviral responses or epithelial repair or inhibit damaging inflammatory responses during severe respiratory viral infections.

Mouse Models Reveal Role of T-Cytotoxic and T-Reg Cells in Immune Response to Influenza: Implications for Vaccine Design.

Immunopathologic examination of the lungs of mouse models of experimental influenza virus infection provides new insights into the immune response in this disease. First, there is rapidly developing perivascular and peribronchial infiltration of the lung with T-cells. This is followed by invasion of T-cells into the bronchiolar epithelium, and separation of epithelial cells from each other and from the basement membrane leading to defoliation of the bronchial epithelium. The intraepithelial reaction may involve either CD8 or CD4 T-cytotoxic cells and is analogous to a viral exanthema of the skin, such as measles and smallpox, which occur when the immune response against these infections is activated and the infected cells are attacked by T-cytotoxic cells. Then there is formation of B-cell follicles adjacent to bronchi, i.e., induced bronchial associated lymphoid tissue (iBALT). iBALT reacts like the cortex of a lymph node and is a site for a local immune response not only to the original viral infection, but also related viral infections (heterologous immunity). Proliferation of Type II pneumocytes and/or terminal bronchial epithelial cells may extend into the adjacent lung leading to large zones filled with tumor-like epithelial cells. The effective killing of influenza virus infected epithelial cells by T-cytotoxic cells and induction of iBALT suggests that adding the induction of these components might greatly increase the efficacy of influenza vaccination.

A bacterial genotoxin causes virus reactivation and genomic instability in Epstein-Barr virus infected epithelial cells pointing to a role of co-infection in viral oncogenesis.

We have addressed the role of bacterial co‐infection in viral oncogenesis using as model Epstein–Barr virus (EBV), a human herpesvirus that causes lymphoid malignancies and epithelial cancers. Infection of EBV carrying epithelial cells with the common oral pathogenic Gram‐negative bacterium Aggregatibacter actinomycetemcomitans (Aa) triggered reactivation of the productive virus cycle. Using isogenic Aa strains that differ in the production of the cytolethal distending toxin (CDT) and purified catalytically active or inactive toxin, we found that the CDT acts via induction of DNA double strand breaks and activation of the Ataxia Telangectasia Mutated (ATM) kinase. Exposure of EBV‐negative epithelial cells to the virus in the presence of sub‐lethal doses of CDT was accompanied by the accumulation of latently infected cells exhibiting multiple signs of genomic instability. These findings illustrate a scenario where co‐infection with certain bacterial species may favor the establishment of a microenvironment conducive to the EBV‐induced malignant transformation of epithelial cells.

Toll-Like Receptor Agonists Modulate Wound Regeneration in Airway Epithelial Cells.

Background: Impaired regeneration of airway epithelium may lead to persistence of inflammation and remodelling. Regeneration of injured epithelium is a complex phenomenon and the role of toll-like receptors (TLRs) in the stimulation of respiratory virus products in this process has not been established. Objective: This study was undertaken to test the hypothesis that the wound repair process in airway epithelium is modulated by microbial products via toll-like receptors. Methods: Injured and not-injured bronchial epithelial cells (ECs) (BEAS-2B line) were incubated with the TLR agonists poly(I:C), lipopolisacharide (LPS), allergen Der p1, and supernatants from virus-infected epithelial cells, either alone or in combination with TLR inhibitors. Regeneration and immune response in injured and not-injured cells were studied. Results: Addition of either poly(I:C) or LPS to ECs induced a marked inhibition of wound repair. Supernatants from RV1b-infected cells also decreased regeneration. Preincubation of injured and not-injured ECs with TLR inhibitors decreased LPS and poly(I:C)-induced repair inhibition. TGF-β and RANTES mRNA expression was higher in injured ECs and IFN-α, IFN-β, IL-8, and VEGF mRNA expression was lower in damaged epithelium as compared to not-injured. Stimulation with poly(I:C) increased IFN-α and IFN-β mRNA expression in injured cells, and LPS stimulation decreased interferons mRNA expression both in not-injured and injured ECs. Conclusion: Regeneration of the airway epithelium is modulated by microbial products via toll-like receptors.

Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1

The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice.IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were able to specifically neutralize HSV-1 infection in vitro via HVEM. Furthermore, we showed for the first time that HVEM-specific HSV-1 neutralizing antibodies protect mice from HSV-1 eye disease, indicating the critical role of HVEM in HSV-1 ocular infection.

Upregulation of miRNA-4776 in Influenza Virus Infected Bronchial Epithelial Cells Is Associated with Downregulation of NFKBIB and Increased Viral Survival.

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3’ UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB.

Transcriptome profiling of influenza A virus-infected lung epithelial (A549) cells with lariciresinol-4-β-D-glucopyranoside treatment.

The influenza A virus is an acute contagious pathogen that affects the human respiratory system and can cause severe lung disease and even death. Lariciresinol-4-β-D-glucopyranoside is a lignan that is extracted from Isatis indigotica, which is a medicinal herb plant that was commonly applied to treat infections, the common cold, fever and inflammatory diseases. Our previous study demonstrated that lariciresinol-4-β-D-glucopyranoside possesses anti-viral and anti-inflammatory properties. However, the comprehensive and detailed mechanisms that underlie the effect of lariciresinol-4-β-D-glucopyranoside interventions against influenza virus infection remain to be elucidated. In this study, we employed high-throughput RNA sequencing (RNA-seq) to investigate the transcriptomic responses of influenza A virus-infected lung epithelial (A549) cells with lariciresinol-4-β-D-glucopyranoside treatment. The transcriptome data show that infection with influenza A virus prompted the activation of 368 genes involved in RIG-I signalling, the inflammatory response, interferon α/β signalling and gene expression that was not affected by lariciresinol-4-β-D-glucopyranoside treatment. Lariciresinol-4-β-D-glucopyranoside exerted its pharmacological actions on the immune system, signal transduction, cell cycle and metabolism, which may be an underlying defense mechanism against influenza virus infection. In addition, 166 differentially expressed genes (DEGs) were uniquely expressed in lariciresinol-4-β-D-glucopyranoside-treated cells, which were concentrated in the cell cycle, DNA repair, chromatin organization, gene expression and biosynthesis domains. Among them, six telomere-associated genes were up-regulated by lariciresinol-4-β-D-glucopyranoside treatment, which have been implicated in telomere regulation and stability. Collectively, we employed RNA-seq analysis to provide comprehensive insight into the mechanism of lariciresinol-4-β-D-glucopyranoside against influenza virus infection.

Interferon-Related Gene Expression in Respiratory Syncytial Virus (RSV) Infected Pediatric Bronchial Epithelial Cells (BECs) Is Inversely Correlated with Lung Function

Interferon-Related Gene Expression in Respiratory Syncytial Virus (RSV) Infected Pediatric Bronchial Epithelial Cells (BECs) Is Inversely Correlated with Lung Function

Inducible GBP5 Mediates the Antiviral Response via Interferon-Related Pathways during Influenza A Virus Infection

Guanylate binding protein (GBP) 5 belongs to the GBP family, which is involved in important cellular processes, including signal transduction, translation, vesicle trafficking, and exocytosis. Structurally, GBPs display a high degree of homology and share highly conserved GTP-binding or hydrolysis domains. GBP5 was reported to be a critical cellular factor in inflammasome assembly. However, little is known about its role in the host antiviral innate immune response. In this study, we found that GBP5 expression was significantly elevated in influenza patients and influenza A virus-infected A549 human lung epithelial cells. The overexpression of GBP5 inhibited virus replication by enhancing the expression of virus-induced interferon (IFN) and IFN-related effectors. Knockdown of GBP5 had the opposite effect. Moreover, GBP5 enhanced endogenous IFN expression by interacting with the NF-κB-essential modulator complex and stimulating NF-κB signaling. Additionally, the expression of proinflammatory factors, such as IL-6, IL-8, tumor necrosis factor-α, cyclooxygenase-2, and inducible nitric oxide synthase, was also activated by GBP5. Taken together, our results reveal that GBP5 inhibited virus replication through the activation of IFN signaling and proinflammatory factors.

Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8–10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

Caspase-mediated degradation of host cortactin that promotes influenza A virus infection in epithelial cells

Influenza A virus (IAV) is well-known to exploit host factors to its advantage. Here, we report that IAV exploits host cortactin, an actin filament-stabilising protein for infection in epithelial cells. By using RNA interference-mediated knockdown and overexpression approach, we demonstrate that cortactin promotes IAV infection. However, cortactin polypeptide undergoes the degradation during late IAV infection. By perturbing the lysosome and proteasome, two main compartments governing the degradation of mammalian proteins, we demonstrate that a lysosome-associated apoptotic pathway mediates the degradation of cortactin in IAV-infected cells. However, we could not detect cleaved cortactin fragments by western blotting using the antibodies recognising either N-terminal/Central or C-terminal cortactin regions, which suggested the presence of multiple caspase cleavage sites. Indeed, CaspDB, a recently-described database predicted up to 35 caspase cleavage motifs present across cortactin polypeptide. The data presented indicate that host cortactin potentially has a dual but contrasting role during IAV infection.

Oxidative stress in Nipah virus-infected human small airway epithelial cells.

Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development.

Regulation of ADAM10 and ADAM17 by Sorafenib Inhibits Epithelial-to-Mesenchymal Transition in Epstein-Barr Virus-Infected Retinal Pigment Epithelial Cells.

The a-disintegrin-and-metalloprotease (ADAM) family proteins are widely expressed in the different layers of the retina throughout development. The effect of ADAM proteins on the epithelial-to-mesenchymal transition (EMT) in proliferative vitreoretinopathy (PVR) or AMD is yet to be elucidated. In this study we used Epstein-Barr virus (EBV)-transformed adult retinal pigment epithelial (ARPE) cells to investigate how sorafenib, a multikinase inhibitor, modulates ADAM proteins to control EMT. Epithelial to mesenchymal transition and related mechanisms in EBV-infected ARPE cells were determined by RT-PCR, Western blot, invasion assay, ELISA assay, and gene silencing with siRNA. Mesenchymal-like ARPE/EBV cells exhibited considerably increased cellular migration and invasion compared with ARPE cells and produced EMT-related cytokines. Sorafenib significantly inhibited production of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α and blocked the activation of migration-related signaling molecules, such as HIF-1α, p-STAT3, MMP2, and Ang-1. The expression of mature ADAM10, ADAM17, and cleaved Notch 1 proteins in ARPE/EBV cells was downregulated after treatment with sorafenib through the regulatory activity of nardilysin (NRD-1). Gene silencing of NRD-1 in ARPE/EBV cells attenuated secretion of EMT-related cytokines and expression of ADAM10 and 17 and upregulated epithelial markers. Sorafenib controls the mesenchymal characteristics of EBV-infected ARPE cells. Nardilysin and ADAM family proteins might be new targets for the prevention or control of EMT in retinal diseases.

Leptin Is Oversecreted by Respiratory Syncytial Virus-Infected Bronchial Epithelial Cells and Regulates Th2 and Th17 Cell Differentiation

Background: Infection of human bronchial epithelial cells (hBECs) with respiratory syncytial virus (RSV) has been shown to induce a Th lymphocyte subset drift, e.g. enhanced differentiation of Th2 and Th17 subsets, which is a classic characteristic of asthma. However, the molecules responsible for the drift in Th subsets remain unknown. This study aims to determine the expression of leptin in RSV-infected hBECs, and its role in Th2 and Th17 cell differentiation and extracellular regulated kinase (ERK) 1/2 phosphorylation. Methods: Cultured hBECs were infected with RSV. mRNA expression of the LEP gene in cells was measured by real-time PCR while LEP protein secretion in culture medium was measured by ELISA. Th differentiation was investigated in cultured human peripheral blood mononuclear cells following stimulation with recombinant human leptin. Th2 and Th17 subsets were examined by flow cytometry. Phosphorylation of the ERK1/2 protein in lymphocytes was detected by Western blot and immunofluorescence. Results: LEP mRNA expression was significantly upregulated in RSV-infected hBECs while the leptin protein level in the supernatants of RSV-infected hBECs was significantly increased. Stimulation of lymphocytes with leptin increased the differentiation of the Th17 subset and ERK1/2 phosphorylation, but suppressed Th2 subset differentiation. Conclusion: Leptin was oversecreted by RSV-infected hBECs, which promoted Th17 subset differentiation but suppressed Th2 subset differentiation possibly via regulating ERK1/2 phosphorylation.

Modulation of sterol biosynthesis regulates viral replication and cytokine production in influenza A virus infected human alveolar epithelial cells

Highly pathogenic H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals remain the primary choice for treating H5N1 diseases and have their limitations. Encouraging findings highlight the beneficial effects of combined treatment of host targeting agents with immune-modulatory activities. This study evaluated the undefined roles of sterol metabolic pathway in viral replication and cytokine induction by H5N1 virus in human alveolar epithelial cells. The suppression of the sterol biosynthesis by Simvastatin in human alveolar epithelial cells led to reduction of virus replication and cytokine production by H5N1 virus. We further dissected the antiviral role of different regulators of the sterol metabolism, we showed that Zometa, FPT inhibitor III, but not GGTI-2133 had anti-viral activities against both H5N1 and H1N1 viruses. More importantly, FPT inhibitor III treatment significantly suppressed cytokine production by H5N1 virus infected alveolar epithelial cells. Since both viral replication itself and the effects of viral hyper-induction of cytokines contribute to the immunopathology of severe H5N1 disease, our findings highlights the therapeutic potential of FPT inhibitor III for severe human H5N1 diseases. Furthermore, our study is the first to dissect the roles of different steps in the sterol metabolic pathway in H5N1 virus replication and cytokine production.

Clustered microRNAs of the Epstein-Barr virus cooperatively downregulate an epithelial cell-specific metastasis suppressor.

The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo. EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.

Manipulated Microenvironment in Human Papilloma Virus–Infected Epithelial Cells: Is the CD40–CD154 Pathway Beneficial for Host or Virus?

In this issue, Tummers et al. (2014) demonstrate that high-risk human papilloma viruses (hrHPVs) attenuate the magnitude of responses to CD40 ligation and the epithelial cells' (ECs) capacity to attract leukocytes. These results suggest that hrHPVs can escape from host immune surveillance by modulating pro-inflammatory responses in infected ECs, resulting in persistent infections and potential carcinogenesis.

Phosphoinositide-3-kinase p110δ isoform promotes influenza pathogenicity (MUC5P.858)

Abstract We have found that mice deficient in a tissue specific isoform of the signaling enzyme phosphoinositide-3 kinase (PI3K) p110δ are protected from PR8 influenza virus infection and have reduced weight loss and lung viral loads post-infection. Lung inflammation and immune cell infiltration is also decreased after viral challenge. In addition, p110δ KO mice had fewer influenza virus infected alveolar epithelial cells when infected with a GFP-flu virus and GFP expression was measured in lung epithelial cells. Cytokine measurements in bronchoalveolar lavages suggest p110δ KO mice exhibit increased IFN-gamma and MIG post PR8 infection. Slightly increased numbers of NK cells are detected in the lungs of p110δ KO mice as early as day 3 post-infection however activation was decreased within this population as compared to controls, suggesting they are not the primary source of resistance to influenza. In order to determine which cell type was causing the decreased pathogenicity we used two different tissue specific knockout mouse lines. Mice with a myeloid specific KO of p110δ showed similar weight loss and viral loads to C57BL/6 controls, suggesting alveolar macrophages are not primarily responsible for the observed protection in p110δ KO mice. However, mice with alveolar epithelial cell (AEC) specific deletion of p110δ were protected from influenza virus challenge. These data suggest PI3K signaling cascade is important for influenza virus protection within AEC.