Defining critical roles for NF‐κB p65 and type I interferon in innate immunity to rhinovirus
2012; Springer Nature; Volume: 4; Issue: 12 Linguagem: Inglês
10.1002/emmm.201201650
ISSN1757-4684
AutoresNathan W. Bartlett, Louise Slater, Nicholas Glanville, Jennifer Haas, Gaetano Caramori, Paolo Casolari, Deborah L. Clarke, Simon D. Message, Julia Aniscenko, Tatiana Kebadze, Jie Zhu, Patrick Mallia, Joseph P. Mizgerd, Maria G. Belvisi, Alberto Papi, Sergei V. Kotenko, Sebastian L. Johnston, Michael R. Edwards,
Tópico(s)Immune Response and Inflammation
ResumoResearch Article14 November 2012Open Access Defining critical roles for NF-κB p65 and type I interferon in innate immunity to rhinovirus Nathan W. Bartlett Nathan W. Bartlett Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Louise Slater Louise Slater Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Nicholas Glanville Nicholas Glanville Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Jennifer J. Haas Jennifer J. Haas Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Gaetano Caramori Gaetano Caramori Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy Search for more papers by this author Paolo Casolari Paolo Casolari Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy Search for more papers by this author Deborah L. Clarke Deborah L. Clarke Centre for Respiratory Infections, Imperial College London, London, UK Respiratory Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK Search for more papers by this author Simon D. Message Simon D. Message Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Imperial College Healthcare National Health Service Trust, London, UK Search for more papers by this author Julia Aniscenko Julia Aniscenko Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Tatiana Kebadze Tatiana Kebadze Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Jie Zhu Jie Zhu Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Patrick Mallia Patrick Mallia Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Imperial College Healthcare National Health Service Trust, London, UK Search for more papers by this author Joseph P. Mizgerd Joseph P. Mizgerd The Pulmonary Centre, Boston University School of Medicine, Boston, Massachusetts, USA Search for more papers by this author Maria Belvisi Maria Belvisi Centre for Respiratory Infections, Imperial College London, London, UK Respiratory Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK Search for more papers by this author Alberto Papi Alberto Papi Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy Search for more papers by this author Sergei V. Kotenko Sergei V. Kotenko Department of Biochemistry and Molecular Biology, University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Sebastian L. Johnston Sebastian L. Johnston Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Imperial College Healthcare National Health Service Trust, London, UK Search for more papers by this author Michael R. Edwards Corresponding Author Michael R. Edwards [email protected] Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Nathan W. Bartlett Nathan W. Bartlett Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Louise Slater Louise Slater Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Nicholas Glanville Nicholas Glanville Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Jennifer J. Haas Jennifer J. Haas Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Gaetano Caramori Gaetano Caramori Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy Search for more papers by this author Paolo Casolari Paolo Casolari Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy Search for more papers by this author Deborah L. Clarke Deborah L. Clarke Centre for Respiratory Infections, Imperial College London, London, UK Respiratory Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK Search for more papers by this author Simon D. Message Simon D. Message Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Imperial College Healthcare National Health Service Trust, London, UK Search for more papers by this author Julia Aniscenko Julia Aniscenko Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Tatiana Kebadze Tatiana Kebadze Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Jie Zhu Jie Zhu Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Patrick Mallia Patrick Mallia Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Imperial College Healthcare National Health Service Trust, London, UK Search for more papers by this author Joseph P. Mizgerd Joseph P. Mizgerd The Pulmonary Centre, Boston University School of Medicine, Boston, Massachusetts, USA Search for more papers by this author Maria Belvisi Maria Belvisi Centre for Respiratory Infections, Imperial College London, London, UK Respiratory Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK Search for more papers by this author Alberto Papi Alberto Papi Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy Search for more papers by this author Sergei V. Kotenko Sergei V. Kotenko Department of Biochemistry and Molecular Biology, University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Sebastian L. Johnston Sebastian L. Johnston Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Imperial College Healthcare National Health Service Trust, London, UK Search for more papers by this author Michael R. Edwards Corresponding Author Michael R. Edwards [email protected] Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK Centre for Respiratory Infections, Imperial College London, London, UK Search for more papers by this author Author Information Nathan W. Bartlett1,2,3, Louise Slater1,2,3, Nicholas Glanville1,2,3, Jennifer J. Haas1,2,3, Gaetano Caramori4, Paolo Casolari4, Deborah L. Clarke3,5, Simon D. Message1,2,3,6, Julia Aniscenko1,2,3, Tatiana Kebadze1,2,3, Jie Zhu1,2,3, Patrick Mallia1,2,3,6, Joseph P. Mizgerd7, Maria Belvisi3,5, Alberto Papi4, Sergei V. Kotenko8, Sebastian L. Johnston1,2,3,6 and Michael R. Edwards *,1,2,3 1Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, London, UK 2MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK 3Centre for Respiratory Infections, Imperial College London, London, UK 4Sezione di Malattie dell'Apparato Respiratorio, Centro per lo Studio delle Malattie Infiammatorie Croniche delle Vie Aeree e Patologie Fumo Correlate dell'Apparato Respiratorio (CEMICEF), University of Ferrara, Ferrara, Italy 5Respiratory Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK 6Imperial College Healthcare National Health Service Trust, London, UK 7The Pulmonary Centre, Boston University School of Medicine, Boston, Massachusetts, USA 8Department of Biochemistry and Molecular Biology, University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA *Tel: +44 0 20 7594 3775; Fax: +44 0 20 7262 8913 EMBO Mol Med (2012)4:1244-1260https://doi.org/10.1002/emmm.201201650 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract The importance of NF-κB activation and deficient anti-viral interferon induction in the pathogenesis of rhinovirus-induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF-κB p65 nuclear expression, NF-κB p65 DNA binding in lung tissue and NF-κB-regulated airway inflammation. In vitro inhibition of NF-κB reduced rhinovirus-induced pro-inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus-infected p65-deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF-κB p65 is required for pro-inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1−/− mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF-κB p65 (to suppress inflammation but preserve anti-viral immunity) and type I IFN signalling (to enhance deficient anti-viral immunity) to treat rhinovirus-induced exacerbations of airway diseases. See accompanying article http://dx.doi.org/10.1002/emmm.201202032 The paper explained PROBLEM: Asthma is a disease affecting 300 million people worldwide. The majority of the morbidity and mortality of asthma is associated with asthma exacerbations, a form of the disease, which responds poorly to conventional asthma treatments. Respiratory infections by human rhinovirus account for most asthma exacerbations, with virus-induced lung inflammation and lung damage directly related to loss of lung function. Asthmatics are also deficient in production of type I and type III interferons, anti-viral cytokines required for defence against rhinovirus, although the role interferon plays in rhinovirus infection in vivo is incompletely elucidated. Importantly, several decades of research have suggested that NF-κB p65 is required for both virus-induced inflammation and interferon production; however, detailed investigations using virus–host combinations relevant to human disease are lacking. RESULTS: In vitro, in primary bronchial epithelial cells and cell lines, inhibition of NF-κB p65 resulted in inhibition of rhinovirus-induced pro-inflammatory cytokines, however, type I and type III interferons were not inhibited. In vivo, p65+/− mice had reduced neutrophils and pro-inflammatory cytokines but had intact anti-viral responses including interferons and intracellular anti-viral enzymes. In parallel studies, the importance of interferon in the host response to rhinovirus in vivo was revealed using interferon-α receptor 1-deficient mice (IFNAR1−/−), which had intact inflammatory responses but reduced anti-viral immunity in the form of type III interferon induction, anti-viral intracellular enzymes, lung T- and NK-cell recruitment and greater virus loads when compared to control mice. IMPACT: The data highlight the fact that in rhinovirus infection, protective anti-viral responses can be uncoupled from harmful, pro-inflammatory responses. Targeting NF-κB p65 may be a reasonable therapeutic strategy for asthma exacerbations, specifically addressing inflammation without compromising beneficial anti-viral immunity, which is sub-optimal in asthmatics. The data also question several years of research supporting the role of NF-κB p65 in type I interferon and type III interferon gene expression and that interferon is crucial to the control of rhinovirus infection in vivo. INTRODUCTION The immune response to virus infections involves a robust innate anti-viral response meditated by type I and III interferons (IFNs) and a potent inflammatory response involving both rapid immune cell recruitment and damage of infected cells and tissues. The regulation of type I IFN-β, IFN-αs and type III IFN-λs has been well studied with IFN-β being used as a model system of eukaryotic gene expression since the early 1990s (Du et al, 1993). The IFN-β promoter contains four positive regulatory domains (PRDs), which allow the DNA binding of distinct transcription factors (Du et al, 1993; Falvo et al, 2000; Thanos & Maniatis, 1995; Wathelet et al, 1998). These transcription factors include IRF-1, IRF-3 and IRF-7 as well as the heterodimeric ATF/c-Jun and members of the NF-κB or Rel family, which is composed of five related proteins, p65 (Rel A, NF-κB3), p50 (NF-κB1, precursor of which is p105), p52 (NF-κB2, precursor of which is p100), c-Rel and Rel B. Rel proteins have a central role in innate immunity with NF-κB p65 implicated in expression of type I and type III IFNs and pro-inflammatory cytokines. Numerous studies over the years including electromobility shift assays (Thanos & Maniatis, 1995), X-ray crystallography (Berkowitz et al, 2002; Panne et al, 2007) and nucleosome analysis of the IFN-β locus (Apostolou & Thanos, 2008) have overwhelmingly supported the current paradigm that all of these transcription factors are required for virus-induced IFN-β transcription; although subtle differences in different cell types have been reported. The recent use of cells from gene-deficient mice (Wang et al, 2007) have questioned the role of NF-κB p65 for IFN-β gene expression, with two recent studies from the same laboratory providing conflicting data (Wang et al, 2007, 2010). A recent editorial on this subject (Balachandran & Beg, 2011) proposes that NF-κB family members including p65 are required to maintain basal activation of the IFN-β promoter or are required very early during infection before IRF-3 activation is optimal. Furthermore, all studies to date report that p65 is also required for IFN-λ gene expression, (Onoguchi et al, 2007; Osterlund et al, 2007; Siegel et al, 2011). Importantly, the current data for IFN-β and IFN-λ gene expression is entirely based on in vitro studies mostly utilizing cell lines or gene-deficient murine embryonic fibroblasts (MEFs) with model viruses that are not important human pathogens. The role of NF-κB p65 in IFN-β and IFN-λ production has never been investigated in vivo and the wider implications of the selective targeting of NF-κB p65 in important human diseases caused by virus infections is a subject of much interest yet one poorly addressed in mouse models of human disease. Considering the lack of studies investigating the importance of p65 in IFN induction by important human viruses in vivo, we have investigated the role of NF-κB p65 and type I IFN signalling in the host defence and inflammatory response to human rhinovirus (RV) in vivo and in vitro. RVs are responsible for a range of severe human illnesses including acute exacerbations of lower airways diseases such as asthma (Johnston et al, 1995, 2005). A cardinal feature of RV infection in vitro is production of pro-inflammatory molecules, the expression of which is transcriptionally regulated by members of the NF-κB transcription factor family (Zhu et al, 1996, 1997). In asthma exacerbations, increased airway inflammation is strongly associated with clinical illness severity (Message et al, 2008; Papi et al, 2006; Wark et al, 2002) and this is thought to be mediated by NF-κB p65 activation, although studies directly demonstrating activated NF-κB p65 during RV infection or RV-induced asthma exacerbations in vivo are yet to be reported. Asthmatic subjects experience significantly increased lower respiratory tract symptoms following either natural (Corne et al, 2002) or experimental RV infection (Message et al, 2008). Impaired antiviral immunity is likely to explain this increased susceptibility to RV infection as deficient type I and type IIII IFN production by asthmatic bronchial epithelial cells (Contoli et al, 2006; Uller et al, 2010; Wark et al, 2005) and bronchoalveolar lavage (BAL) macrophages (Contoli et al, 2006) has been observed ex vivo, with the latter related to increased virus load and exacerbation severity in vivo. These relationships suggest, but are unable to definitively show, a causal relationship between IFN deficiency and disease severity. These data, along with the data reporting a requirement of p65 for IFN gene expression, suggest that approaches that inhibit p65 would suppress RV-induced IFN in the asthmatic lung, further impairing antiviral immunity, increasing virus loads, virus-induced inflammation and exacerbation severity. Therefore, understanding whether or not NF-κB p65 is involved in IFN production to RV is extremely important and this key issue is yet to be addressed as there are no data reported on the causal role of p65 or type I IFN in lung host defence against RV infection using in vivo human and mouse models of RV. Further, the requirement of p65 for RV-induced antiviral IFN expression in human bronchial epithelial cells (HBECs) in vitro is also unknown. Determining the contribution of p65 to IFN-mediated antiviral and pro-inflammatory responses is vital to identifying therapeutic targets for RV-induced lower airway diseases. Demonstrating that IFN is critical for antiviral responses to RV in vivo is necessary to justify further development of IFN-based therapies (Hayden & Gwaltney, 1984; Koltsida et al, 2011). This is the first report investigating the role of p65 in immunity to a virus in vivo and includes combined studies in human and mouse models of RV infection to demonstrate that NF-κB p65 is a central regulator of RV-induced inflammation in the airways. Furthermore, we provide evidence that suppressing p65 expression, whilst reducing airways inflammation, did not affect IFN production or antiviral immune responses, despite over 20 years of in vitro experiments that suggest the contrary. Inhibition of p65 is therefore identified as an attractive target for development of anti-inflammatory therapies that would not further impair IFN responses in virus-induced asthma exacerbations. In doing so, we have also provided clear evidence that responses mediated by type I IFN in vivo are critical for antiviral responses to RV thereby identifying IFN as another therapeutic approach likely to be beneficial. RESULTS NF-κB is activated by RV infection in vivo in the lung and in vitro in primary bronchial epithelial cells As it is not known whether RV infection leads to activation of NF-κB p65 in vivo, we initially investigated this in human and mouse models of RV infection. Fig 1A shows increased activation of NF-κB p65 as assessed by p65 nuclear stained bronchial epithelial cells in bronchial biopsies, from baseline (BL) to day4 (D4) following experimental human RV infection (Message et al, 2008). NF-κB in lung tissue was also activated in a mouse model of RV infection. Induction of binding to labelled NF-κB-containing oligonucleotides was observed in nuclear protein extracted from whole lung of RV-infected mice. No signal was observed for mice dosed with UV-inactivated virus indicating that NF-κB activation was replication-dependent. NF-κB binding was also effectively competed with 100× excess unlabelled probe demonstrating NF-κB binding specificity (Fig 1B). Activation of p65 was confirmed by nuclear p65-DNA binding experiments performed over time in Bl/6 129 mice (Fig 1B). In HBECs, RV1B caused IκBα degradation from 8 h post-infection (Fig 1C) and NF-κB-dependent reporter gene activation (Fig 1D). Transfection of HBECs with plasmids expressing constitutively active forms of the RV RNA-sensing molecules RIG-I (ΔRIG-I) (Yoneyama et al, 2004) and TRIF (ΔTRIF; Slater et al, 2010; Yamamoto et al, 2003) also activated the NF-κB-dependent reporter (Fig 1D). Figure 1. RV and RV-mediated signalling pathways activate NF-κB in vitro and in vivo. A.. Healthy human subjects (n = 5) were infected with RV16 and bronchial biopsies were taken prior to (BL) and D4 after infection and stained to quantify bronchial epithelial cell nuclear NF-κB p65. Horizontal line indicates a scale of 20 µM. Arrows indicate nuclear p65 staining. The negative control was stained with non-specific rabbit Ig rather than primary rabbit anti-p65 antibody. Graph shows each data point with mean, differences between groups were identified by t-test *p < 0.05. B.. BALB/c mice were infected intranasally with RV1B. Negative controls were dosed with UV-inactivated RV1B or PBS intranasally and NF-κB activation in lung nuclear protein extract assessed by NF-κB-DNA binding in EMSA (upper panel). Presence of p65 in RV infection in vivo was confirmed by measuring nuclear p65-DNA binding in a timecourse performed in BL/6 129 mice (lower panel). Data was analysed by one-way ANOVA (n = 1 experiment, five mice per group) **p < 0.01. C.. HBECs were infected with RV1B and IκBα degradation measured by Western blot. D.. HBECs were infected with RV1B which caused activation of a minimal NF-κB promoter at 24 h compared to medium treated cells (n = 5 independent experiments). HBECs were transfected with plasmids encoding constitutively active molecules involved in RV sensing pathways, ΔRIG-I and ΔTRIF. Both ΔRIG-I and ΔTRIF induced NF-κB reporter activation relative to empty vector controls pEF-BOS and pUNO1, respectively (n = 6 independent experiments). Reporter data is presented as fold induction versus control and analysed by t-test ***p < 0.001 unless otherwise stated, as indicated all data are expressed as mean ± SEM. Download figure Download PowerPoint Allergen challenge with RV infection increases NF-κB p65 activation and NF-κB-regulated cytokines and chemokines We have previously reported that RV infection exacerbates allergic airways inflammation (Bartlett et al, 2008). Using this model, we examined NF-κB p65 activation at early time points (8 h) observing that live RV infection during allergen challenge (RV1B-OVA) increased NF-κB p65 DNA binding in lung nuclear protein extracts when compared to live infection alone (RV1B-PBS) and allergen challenge together with inactivated RV (UV-RV1B-OVA; Fig 2A). Either RV infection alone (RV-PBS) or OVA challenge with inactivated RV1B (UV-OVA) induced more NF-κB p65 DNA binding than that observed in double-negative controls (UV-RV1B-PBS; Fig 2A). NF-κB p65 activation in the context of allergen and virus challenge was associated with markedly increased induction of IL-6, which was not induced by either stimulus alone, and clear further augmentation of IL-1β, which was induced by virus but not allergen alone (Fig 2A). The pleotropic chemokine CCL5, which is a chemoattractant for both granulocytes and lymphocytes, was highly synergistically increased in RV1B-OVA compared to all other groups (Fig 2B). Lymphocyte attracting chemokines CXCL10, CXCL11 CCL17 and CCL22 were also highest in RV1B-OVA as were the eosinophil-attracting chemokines CCL11 and CCL24 (Fig 2B). This data is direct in vivo evidence that RV infection in the allergic lung causes increased NF-κB p65 activation and expression of NF-κB-regulated pro-inflammatory cytokines and chemokines implicating this transcription factor in the pathogenesis of RV-induced exacerbation of allergic airway inflammation. Figure 2. Exacerbation of allergic airway inflammation involves enhanced NF-κB p65 activation and NF-κB p65-responsive genes. Balb/c mice were sensitized by i.p injection of OVA and challenged 10 days later on 3 consecutive days with either intranasal OVA or PBS administration; receiving intranasal RV1B or UV-RV1B on the third day. BAL and whole lung was harvested at 8 and 24 h. A.. From whole lung, nuclear NF-κB p65 DNA binding was measured at 8 h post infection. RV1B-OVA, RV1B-PBS and UV-RV1B-OVA had significantly elevated p65 binding versus UV-RV1B-PBS, and RV1B-OVA treated mice had increased p65 binding versus the other groups. At 8 h post infection, RV1B-OVA mice had increased BAL pro-inflammatory cytokines IL-6, and IL-1β compared to RV1B-PBS or UV-RV1B-OVA treated groups. B.. At 24 h post infection, RV1B-OVA mice had increased BAL CCL5, CXCL10, CXCL11, CCL17, CCL22, CCL11 and CCL24 compared to RV1B-PBS or UV-RV1B-OVA treated groups (n = 1 experiment with five mice per group or per time point). All data was analysed by one way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001 versus UV-RV1B-PBS, #p < 0.05, ##p < 0.01, ###p < 0.001 versus RV1B-OVA group. All data are expressed as mean ± SEM. Download figure Download PowerPoint Specific inhibition of NF-κB p65 does not suppress RV-induced IFNs but inhibited pro-inflammatory chemokine production in bronchial epithelial cells The major IFN subtypes induced by RV infection of bronchial epithelial cells are IFN-β and IFN-λ (Khaitov et al, 2009). Using small interfering RNA (siRNA) in HBECs, we showed that NF-κB p65 was not required for RV-induced IFN-β, IFN-λ1 or IFN-λ2/3 at 24 h (Fig 3A). In the same experiments, p65-specific siRNA significantly and almost completely prevented RV-induced expression of pro-inflammatory chemokines CCL5, CXCL8 and CXCL5 (Fig 3B). In contrast to p65, IRF3 was required for RV-induced IFN-β and IFN-λ1
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