Biomedical subjects

Roberto P Garofalo

Publications and source records attributed to Roberto P Garofalo.

At least 19 recordsLinked to original sources

PubMed2006

Retinoic acid-inducible gene I mediates early antiviral response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells.

Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respiratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I "knockdown" significantly inhibited early nuclear factor-kappaB (NF-kappaB) and interferon response factor 3 (IRF3) activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-beta), interferon-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expression levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18 h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-kappaB binding but did inhibit IFN-beta, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that TLR3 knockdown significantly reduced NF-kappaB/RelA transcription by its ability to block the activating phosphorylation of NF-kappaB/RelA at serine residue 276. We further found that TLR3 induction following RSV infection was regulated by RIG-I-dependent IFN-beta secreted from infected airway epithelial cells and was mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways controlling NF-kappaB activation.

Cell Line↗

PubMed2006

Antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung inflammation.

RATIONALE: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in children. No treatment has been shown to significantly improve the clinical outcome of patients with this infection. Recent evidence suggests that oxidative stress could play an important role in the pathogenesis of acute and chronic lung inflammatory diseases. We do not known whether RSV induces pulmonary oxidative stress and whether antioxidant treatment can modulate RSV-induced lung disease. OBJECTIVES: To investigate the effect of antioxidant administration on RSV-induced lung inflammation, clinical disease, and airway hyperreactivity (AHR). METHODS: BALB/c mice were infected with 10(7) plaque-forming units of RSV, in the presence or absence of orally administered butylated hydroxyanisole (BHA), an antioxidant. Malondialdehyde and 4-hydroxynonenal were measured in bronchoalveoar lavage (BAL) by colorimetric assay. Cytokines and chemokines were measured in BAL by Bio-Plex and leukotrienes were measured by enzyme-linked immunosorbent assay. AHR to methacholine challenge was measured by whole-body plethysmography. RESULTS: BHA treatment significantly attenuated RSV-induced lung oxidative stress, as indicated by the decrease of malondialdehyde and 4-hydroxynonenal content in BAL of RSV-infected mice. RSV-induced clinical illness and body weight loss were also reduced by BHA treatment, which inhibited neutrophil recruitment to the lung and significantly reduced pulmonary cytokine and chemokine production after RSV infection. Similarly, antioxidant treatment attenuated RSV-induced AHR. CONCLUSION: Modulation of oxidative stress represents a potential novel pharmacologic approach to ameliorate RSV-induced acute lung inflammation and potentially prevent long-term consequences associated with RSV infection, such as bronchial asthma.

Aldehydes↗

PubMed2006

Ikkepsilon regulates viral-induced interferon regulatory factor-3 activation via a redox-sensitive pathway.

Respiratory syncytial virus (RSV)-induced chemokine gene expression occurs through the activation of a subset of transcription factors, including Interferon Regulatory Factor (IRF)-3. In this study, we have investigated the signaling pathway leading to RSV-induced IRF-3 activation and whether it is mediated by intracellular reactive oxygen species (ROS) generation. Our results show that RSV infection induces expression and catalytic activity of IKKepsilon, a noncanonical IKK-like kinase. Expression of a kinase-inactive IKKepsilon blocks RSV-induced IRF-3 serine phosphorylation, nuclear translocation and DNA-binding, leading to inhibition of RANTES gene transcription, mRNA expression and protein synthesis. Treatment of alveolar epithelial cells with antioxidants or with NAD(P)H oxidase inhibitors abrogates RSV-induced chemokine secretion, IRF-3 phosphorylation and IKKepsilon induction, indicating that ROS generation plays a fundamental role in the signaling pathway leading to IRF-3 activation, therefore, identifying a novel molecular target for the development of strategies aimed to modify the inflammatory response associated with RSV infection of the lung.

Cell Line, Tumor↗

PubMed2005

Differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus.

Dendritic cells (DCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract. Human metapneumovirus (hMPV) is a recently identified pathogen and like its better known relative, respiratory syncytial virus (RSV), has been increasingly recognized as a major cause of respiratory morbidity in infants and in elderly persons. In the present study, we examined susceptibility as well as cellular responses of human DCs to hMPV compared with RSV. Monocyte-derived DCs (moDCs) were susceptible to infection by both viruses, but only RSV was able to induce a productive infection with release of viral progeny. Despite the fact that viral infection resulted in phenotypic maturation of moDCs, as shown by the upregulation of cell surface markers and antigen-presenting molecules (MHC I and II, CD80, CD83, CD86, CD38), RSV-infected moDCs showed a severely impaired capacity to stimulate CD4+ T cell proliferation. Compared with hMPV, RSV was a more potent inducer of inflammatory and immunomodulatory cytokines, including TNF-alpha, IL-6, IL-1beta, IL-10, and IL-12p70 in both moDCs and plasmacytoid dendritic cells (pDCs). On the other hand, hMPV, but not RSV, was able to trigger production of IFN-alpha by moDCs, while both viruses strongly induced IFN-alpha in pDCs. Finally, both viruses strikingly suppressed IFN-alpha production by moDCs or pDCs stimulated with synthetic dsRNA and CpG-ODN, respectively. The findings provide novel evidence that RSV and hMPV differentially activate human DCs and may use distinct mechanisms to interfere with the host innate and adaptive immune responses.

Antigen Presentation↗

PubMed2005

RSV-induced prostaglandin E2 production occurs via cPLA2 activation: role in viral replication.

Prostaglandins (PGs) are lipid mediators that participate in the regulation of immunological and inflammatory responses, and PG production can affect viral replication. In this study, we have investigated the mechanism of PGE2 production in airway epithelial cells, following respiratory syncytial virus (RSV) infection, and its role in viral replication. We show that RSV infection strongly induces PGE2 secretion, in a time- and replication-dependent manner, through increased cyclooxygenase-2 (COX-2) expression, which occurs independently from viral or cellular protein synthesis. RSV infection induces arachidonic acid release through induction of cytoplasmic phospholipase A2 (cPLA2) enzymatic activity and its membrane translocation. Specific inhibitors of cPLA2 significantly block RSV-induced PGE2 secretion, indicating a key role of cPLA2 in viral-induced PG production. Blocking PG secretion, through cPLA2 or COX-2 inhibition, results in impairment of RSV replication and subsequent RSV-mediated epithelial cell responses, suggesting that inhibition of PG secretion could be beneficial in RSV infection by reducing proinflammatory mediator production.

Arachidonic Acid↗

PubMed2005

A comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus.

We studied epidemiologic and immunologic factors in infants with bronchiolitis caused by influenza virus. The proportion of these infants who were male and who had an immediate family member with a history of asthma was similar to that of a control group of infants with respiratory syncytial virus (RSV) bronchiolitis. In subjects with influenza virus infection, concentrations of the beta chemokine macrophage inflammatory protein-1alpha (MIP-1alpha), but not other beta chemokines, in nasopharyngeal secretions (NPS) were greater among infants with more severe, hypoxic bronchiolitis than in subjects with mild, nonhypoxic bronchiolitis, or upper respiratory tract infection alone. Quantities of MIP-1alpha were also correlated with lower values of oxygen saturation. These findings point out epidemiologic and immunologic similarities between bronchiolitis caused by influenza and RSV, and suggest that host factors are more important than the nature of the infecting virus in the development of severe forms of bronchiolitis caused by influenza and RSV.

Bronchiolitis, Viral↗

PubMed2005

Activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the Paramyxoviridae family, Pneumovirinae subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-alpha/beta) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-alpha in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-alpha, or directly with recombinant IFN-alpha showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-alpha than hMPV. Similarly, when the ability to directly induce IFN-alpha production was assessed, RSV was clearly a weaker inducer of IFN-alpha than hMPV, as shown by both kinetics and the absolute amount of IFN-alpha secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-alpha production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-alpha production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-alpha in the lung during infection by members of the Pneumovirinae family.

Animals↗

PubMed2005

Human metapneumovirus induces a profile of lung cytokines distinct from that of respiratory syncytial virus.

Lung cytokine and chemokine production by BALB/c mice infected with human metapneumovirus (hMPV) was compared to respiratory syncytial virus (RSV)-infected mice. hMPV infection induced lower levels of the inflammatory cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha but was a more potent inducer of granulocyte-macrophage colony-stimulating factor and triggered a more sustained production of the CXC chemokine KC compared to RSV. hMPV was a stronger inducer of both alpha interferon (IFN-alpha) and IFN-gamma responses than RSV. In regard to immunomodulatory cytokines, hMPV failed to induce detectable IL-10 or IL-12p70 but was a potent inducer of IL-12 p40 subunit. The implications for hMPV pathogenesis are discussed.

Cytokines↗

PubMed2005

Respiratory syncytial virus-inducible BCL-3 expression antagonizes the STAT/IRF and NF-kappaB signaling pathways by inducing histone deacetylase 1 recruitment to the interleukin-8 promoter.

Respiratory syncytial virus (RSV) is a paramyxovirus that produces airway inflammation, in part by inducing interleukin-8 (IL-8) expression, a CXC-type chemokine, via the NF-kappaB/RelA and STAT/IRF signaling pathways. In RSV-infected A549 cells, IL-8 transcription attenuates after 24 h in spite of ongoing viral replication and persistence of nuclear RelA, suggesting a mechanism for transcriptional attenuation. RSV infection induces B-cell lymphoma protein -3 (Bcl-3) expression 6 to 12 h after viral infection, at times when IL-8 transcription is inhibited. By contrast, 293 cells, deficient in inducible Bcl-3 expression, show no attenuation of IL-8 transcription. We therefore examined Bcl-3's role in terminating virus-inducible IL-8 transcription. Transient expression of Bcl-3 potently inhibited virus-inducible IL-8 transcription by disrupting both the NF-kappaB and STAT/IRF pathways. Although previously Bcl-3 was thought to capture 50-kDa NF-kappaB1 isoforms in the cytoplasm, immunoprecipitation (IP) and electrophoretic mobility shift assays indicate that nuclear Bcl-3 associates with NF-kappaB1 without affecting DNA binding. Additionally, Bcl-3 potently inhibited the STAT/IRF pathway. Nondenaturing co-IP assays indicate that nuclear Bcl-3 associates with STAT-1 and histone deacetylase 1 (HDAC-1), increasing HDAC-1 recruitment to the IL-8 promoter. Treatment with the HDAC inhibitor trichostatin A blocks attenuation of IL-8 transcription. A nuclear targeting-deficient Bcl-3 is unable to enhance HDAC-1-mediated chemokine repression. Finally, small inhibitory RNA-mediated Bcl-3 "knockdown" resulted in enhanced RSV-induced chemokine expression in A549 cells. These data indicate that Bcl-3 is a virus-inducible inhibitor of chemokine transcription by interfering with the NF-kappaB and STAT/IRF signaling pathways by complexing with them and recruiting HDAC-1 to attenuate target promoter activity.

Carrier Proteins↗

PubMed2004

Production of interferon gamma in respiratory syncytial virus infection of humans is not associated with interleukins 12 and 18.

In order to understand early events in the immune response to respiratory syncytial virus (RSV) infection, we studied the presence of various chemokines and cytokines in respiratory secretions of human infants with RSV infection. Interferon gamma (IFNgamma) was present in 30/39 (76.9%) subjects tested, but the IFNgamma-inducing cytokines interleukin (IL)12 and IL18 were detectable in 6/40 (15%) and 11/38 (28.9%) subjects, respectively. Quantities of IL12 and IL18 did not correlate with those of IFNgamma. IL18, but neither IFNgamma nor IL12 was found in significantly greater concentrations in subjects with mild, nonhypoxic forms of bronchiolitis than in those with upper respiratory illness alone or hypoxic bronchiolitis. The findings suggest that IFNgamma may be induced independently of the activities of IL12 and IL18 during RSV infection. Immune responses characterized by relatively greater release of IL18 may be associated with milder forms of bronchiolitis.

Exudates and Transudates↗

PubMed2004

Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis.

The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic respiratory tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes in cellular gene expression. In this study, we have investigated RSV-induced changes in the nuclear proteome of A549 alveolar type II-like epithelial cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for changes in protein expression. We identified 24 different proteins by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), whose average normalized spot intensity was statistically significant and differed by +/-2-fold. Notable among the proteins identified were the cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, the TAR DNA binding protein (a protein that associates with nuclear domain 10 [ND10] structures), and heat shock protein 70- and 60-kDa isoforms (Hsp70 and Hsp60, respectively). The identification of Hsp70 was also validated by liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed that RSV induced cytoplasmic Hsp70 aggregation and nuclear accumulation. Data mining of a genomic database showed that RSV replication induced coordinate changes in Hsp family proteins, including the 70, 70-2, 90, 40, and 40-3 isoforms. Because the TAR DNA binding protein associates with ND10s, we examined the effect of RSV infection on ND10 organization. RSV induced a striking dissolution of ND10 structures with redistribution of the component promyelocytic leukemia (PML) and speckled 100-kDa (Sp100) proteins into the cytoplasm, as well as inducing their synthesis. Our findings suggest that cytoplasmic RSV replication induces a nuclear heat shock response, causes ND10 disruption, and redistributes PML and Sp100 to the cytoplasm. Thus, a high-resolution proteomics approach, combined with immunofluorescence localization and coupled with genomic response data, yielded unexpected novel insights into compensatory nuclear responses to RSV infection.

Cell Nucleus↗

PubMed2004

IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IkappaB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IkappaBalpha, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-kappaB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKbeta with the regulatory protein NEMO showed a striking reduction of lung NF-kappaB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKbeta may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.

Animals↗

PubMed2003

Reactive oxygen species mediate virus-induced STAT activation: role of tyrosine phosphatases.

Respiratory syncytial virus (RSV) is the leading cause of epidemic respiratory tract illness in children in the United States and worldwide. RSV infection of airway epithelial cells induces formation of reactive oxygen species (ROS), whose production mediates the expression of cytokines and chemokines involved the immune/inflammatory responses of the lung. In this study, we have investigated the role of ROS in RSV-induced signal transducers and activators of transcription (STAT) activation and interferon regulatory factor (IRF) gene expression in human airway epithelial cells. Our results indicate that RSV replication induces IRF-1 and -7 gene transcription, a response abrogated by antioxidants. RSV infection induces binding of STAT to the IRF-1 gamma-interferon-activated sequence (GAS) and IRF-7 interferon-stimulated responsive element (ISRE). STAT1 and STAT3 bind IRF-1 GAS, whereas STAT1, STAT2, IRF-1, and IRF-9 bind IRF-7 ISRE. Antioxidant treatment blocks RSV-induced STAT binding to both the IRF-1 GAS and IRF-7 ISRE by inhibition of inducible STAT1 and STAT3 tyrosine phosphorylation, suggesting that RSV-induced ROS formation is required for STAT activation and IRF gene expression. Although protein tyrosine phosphorylation is necessary for RSV-induced STAT activation, Janus kinase and Src kinase activation do not mediate this effect. Instead, RSV infection inhibits intracellular tyrosine phosphatase activity, which is restored by antioxidant treatment. Pharmacological inhibition of tyrosine phosphatases induces STAT activation. Together, these results suggest that modulation of phosphatases could be an important mechanism of virus-induced STAT activation. Treatment of alveolar epithelial cells with the NAD(P)H oxidase inhibitor diphenylene iodonium abolishes RSV-induced STAT activation, indicating that NAD(P)H oxidase-produced ROS are required for downstream activation of the transcription factors IRF and STAT in virus-infected airway epithelial cells.

Cell Line↗

PubMed2003

Ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells.

Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. RSV replication is a potent activator of the epithelial-cell genomic response, influencing the expression of a spectrum of cellular pathways, including proinflammatory chemokines of the CC, CXC, and CX(3)C subclasses. Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nontoxic antiviral agent currently licensed for the treatment of severe RSV lower respiratory tract infections. Because ribavirin treatment reduces the cytopathic effect in infected cells, we used high-density microarrays to investigate the hypothesis that ribavirin modifies the virus-induced epithelial genomic response to replicating virus. Ribavirin treatment administered in concentrations of 10 to 100 micro g/ml potently inhibited RSV transcription, thereby reducing the level of RSV N transcripts to approximately 13% of levels in nontreated cells. We observed that in both the absence and the presence of ribavirin, RSV infection induced global alterations in the host epithelial cell, affecting approximately 49% of the approximately 6,650 expressed genes detectable by the microarray. Ribavirin influences the expression of only 7.5% of the RSV-inducible genes (total number of genes, 272), suggesting that the epithelial-cell genetic program initiated by viral infection is independent of high-level RSV replication. Hierarchical clustering of the ribavirin-regulated genes identified four expression patterns. In one group, ribavirin inhibited the expression of the RSV-inducible CC chemokines MIP-1 alpha and -1 beta, which are important in RSV-induced pulmonary pathology, and interferon (IFN), a cytokine important in the mucosal immune response. In a second group, ribavirin further up-regulated a set of RSV- and IFN-stimulated response genes (ISGs) encoding antiviral proteins (MxA and p56), complement products, acute-phase response factors, and the STAT and IRF transcription factors. Because IFN-beta expression itself was reduced in the ribavirin-treated cells, we further investigated the mechanism for up-regulation of the IFN-signaling pathway. Enhanced expression of IFI 6-16, IFI 9-27, MxA/p78, STAT-1 alpha, STAT-1 beta, IRF-7B, and TAP-1-LMP2 transcripts were independently reproduced by Northern blot analysis. Ribavirin-enhanced TAP-1-LMP2 expression was a transcriptional event where site mutations of the IFN-stimulated response element (ISRE) blocked RSV and ribavirin-inducible promoter activity. Furthermore, ribavirin up-regulated the transcriptional activity of a reporter gene selectively driven by the ISRE. In specific DNA pull-down assays, we observed that ribavirin enhanced RSV-induced STAT-1 binding to the ISRE. We conclude that ribavirin potentiates virus-induced ISRE signaling to enhance the expression of antiviral ISGs, suggesting a mechanism for the efficacy of combined treatment with ribavirin and IFN in other chronic viral diseases.

Antiviral Agents↗

PubMed2002

Metapneumovirus and acute wheezing in children.

A new respiratory virus, human metapneumovirus, was recently identified. We detected this virus by PCR in ten (8%) of 132 consecutive children admitted to Turku Hospital, Finland, for acute expiratory wheezing (median age 7 months, range 4-25). The mean duration of hospital stay was 2.5 days (SD 1.6) and mean duration of respiratory symptoms was 19 days (8). The white blood cell count, C-reactive protein, and regulated upon activation, normal T-cell-expressed and T-cell-secreted (RANTES) concentrations in nasal secretion remained low, whereas interleukin 8 concentrations in nasal secretion were high. Human metapneumovirus is a clinically important causative agent of acute wheezing in young children.

Adolescent↗

PubMed2002

Respiratory syncytial virus-induced activation of nuclear factor-kappaB in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways.

The transcription factor nuclear factor (NF)-kappaB controls the expression of numerous respiratory syncytial virus (RSV)-inducible inflammatory and immunomodulatory genes. Using a BALB/c mouse model, the present article shows that RSV potently and specifically activates NF-kappaB in vivo, a process that involves nuclear translocation of the subunits RelA, p50, and c-Rel in the lung. By depletion of alveolar macrophages (AMs) in BALB/c mice and use of C3H/HeJ mice lacking a functional Toll-like receptor (TLR)-4 signaling pathway, we demonstrate the existence of distinct but sequentially integrated RSV-inducible early NF-kappaB responses in the lung. The first response occurs early after RSV inoculation, is AM and TLR4 dependent, and is viral replication independent, whereas the second response involves epithelial cells and/or inflammatory cells, is TLR4 independent, and requires viral replication. NF-kappaB may be considered a central activator of not only inflammatory but also innate immune responses to RSV.

Animals↗

PubMed2002

Regulation of RANTES promoter activation in alveolar epithelial cells after cytokine stimulation.

Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine family of proteins implicated in a variety of diseases characterized by lung eosinophilia and inflammation, strongly produced by stimulated airway epithelial cells. Because such cytokines as tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN-gamma) have been shown to enhance RANTES induction in airway epithelial cells and RANTES gene expression appears to be differentially regulated depending on the cell type and the stimulus applied, in this study we have elucidated mechanisms that operate to control RANTES induction on exposure to TNF-alpha and/or IFN-gamma. Our results indicate that TNF-alpha and IFN-gamma synergistically induce RANTES protein secretion and mRNA expression. RANTES transcription is activated only after stimulation with TNF-alpha, but not IFN-gamma, which affects RANTES mRNA stabilization. Promoter deletion and mutagenesis experiments indicate that the nuclear factor (NF)-kappaB site is the most important cis-regulatory element controlling TNF-induced RANTES transcription, although NF-interleukin-6 binding site, cAMP responsive element (CRE), and interferon-stimulated responsive element (ISRE) also play a significant role. TNF-alpha stimulation induces nuclear translocation of interferon regulatory factor (IRF)-3, which in viral infection binds the RANTES ISRE and is necessary for activation of RANTES transcription. However, TNF-induced IRF-3 translocation does not result in IRF-3 binding to the RANTES ISRE. Although viral infection can activate an ISRE-driven promoter, TNF cannot, indicating that RANTES gene enhancers are controlled in a stimulus-specific fashion. Identification of molecular mechanisms involved in RANTES gene expression is fundamental for developing strategies to modulate lung inflammatory responses.

Cells, Cultured↗

PubMed2002

Interleukin-8 gene regulation in intestinal epithelial cells infected with rotavirus: role of viral-induced IkappaB kinase activation.

Rotavirus is the major etiologic agent of diarrhea in children and the most common cause of severe pediatric gastroenteritis. Rotavirus infection is limited to mature enterocytes that line the villi of the small intestine. Gut epithelial cells, upon infection and cytokine stimulation, are able to produce chemokines, a family of small chemotactic cytokines that regulate the migration and activation of leukocytes. We have previously shown that rotavirus infection of the intestinal epithelial cell line HT-29 induces increased expression of the CXC chemokine interleukin- (IL) 8. Mechanisms responsible for the transcriptional regulation of the IL-8 gene in intestinal epithelial cells during viral infections have not been fully elucidated. Therefore, the purpose of this study was to define the molecular mechanisms of IL-8 gene expression in HT-29 cells infected with rotavirus. Transient transfection analysis of 5' deletions and mutations of the IL-8 promoter driving expression of luciferase reporter gene indicates that the activating protein- (AP) 1 and nuclear factor- (NF) kappaB elements are necessary for IL-8 promoter activation during rotavirus infection. The importance of NF-kappaB activation for IL-8 gene expression was further demonstrated by the inhibition of rotavirus-induced IL-8 gene transcription and protein synthesis following blockade of degradation of the NF-kappaB cytoplasmic inhibitor IkappaB-alpha. Rotavirus infection of HT-29-induced IkappaB kinase (IKK) activation and overexpression of a dominant negative mutant of IKK-beta greatly reduced rotavirus-induced IL-8 promoter activation and NF-kappaB-driven transcription, indicating that IKK is involved in rotavirus-induced IL-8 gene expression and NF-kappaB activation.

Epithelial Cells↗