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Exogenous IFN-β has antiviral and anti-inflammatory properties in primary bronchial epithelial cells from asthmatic subjects exposed to rhinovirus Julie A. Cakebread, PhD, Yunhe Xu, PhD, Chris Grainge, PhD, Valia Kehagia, MD, Peter H. Howarth, DM, Stephen T. Holgate, DSc, Donna E. Davies, PhD Journal of Allergy and Clinical Immunology Volume 127, Issue 5, Pages e9 (May 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 1 Induction of antiviral genes by IFN-β. PBECs from nonasthmatic (open circles, n = 10) and asthmatic (solid circles, n = 10) donors were incubated in serum free medium (SFM) or challenged with 100 IU/mL IFN-β for 6 hours. After RNA extraction, expression of IRF-7 and MDA-5 was assessed by means of RT-qPCR and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ubiquitin C (UBC) by using the ΔΔCT method. Statistical analysis was performed with the Student t test or Mann-Whitney U test. ∗∗P < .001. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 2 IFN-β attenuates replication of RV1B in PBECs. PBECs (n = 6) were infected for 72 hours with RV1B (1 × 103 TCID50 units/105 cells) without or with exogenous IFN-β (100 IU/mL) or were left uninfected in serum free medium (SFM). Photomicrographs illustrate cell morphology under the stated conditions. Expression of viral RNA was quantified by using RT-qPCR and viral titers by using TCID50. Statistical analysis was performed with the Wilcoxon signed-rank test. ∗∗P = .008. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 3 Induction of inflammatory genes by IFN-β. PBECs from nonasthmatic (open circles, n = 10) and asthmatic (solid circles, n = 10) donors were incubated in serum free medium (SFM) or challenged with 100 IU/mL IFN-β for 6 hours. After RNA extraction, IP-10 and RANTES mRNA was assessed by means of RT-qPCR and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ubiquitin C (UBC) mRNA by using the ΔΔCT method. Statistical analysis was performed with the Student t test or Mann-Whitney U test. ∗P < .02. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 4 Upregulation of IP-10 in response to RV1B infection. PBECs from asthmatic subjects (n = 8) were infected with RV1B for up to 72 hours. IP-10 mRNA expression was quantified with RT-qPCR and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ubiquitin C (UBC) by using the ΔΔCT method. Protein secretion in cell supernatants was measured with the BD Cytometric Bead Array (CBA). Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Fig 5 Exogenous IFN-β significantly suppresses IP-10 and RANTES protein production in response to RV1B infection. PBECs from asthmatic subjects (n = 8) were infected with RV1B without or with exogenous IFN-β (A and B). In panels C and D PBECs were also treated with serum free medium (SFM), IFN-β alone, or UV-irradiated viral controls. Protein secretion in cell supernatants was measured with the BD Cytometric Bead Array. Statistical analysis was performed with the Student t test or Wilcoxon signed-rank test. ∗P = .05 and ∗∗P < .02. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Analysis of STAT1 and STAT2 activation by means of Western blotting
Analysis of STAT1 and STAT2 activation by means of Western blotting. PBECs from 3 nonasthmatic (open bars) and 3 asthmatic (solid bars) donors were incubated in serum free medium alone or treated with IFN-β at 100 IU/mL for 1 hour, and then cells were lysed into SDS sample buffer. Phosphorylation of STAT1 and STAT2 was analyzed by means of Western blotting and quantified relative to total STAT protein loaded by means of densitometry. There was no significant phosphorylation of STAT1 or STAT2 in the absence of IFN-β (not shown), and there was no difference between IFN-β–induced phosphorylation of STAT1 or STAT2 between the 2 groups. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Induction of antiviral genes in PBECS from nonasthmatic or asthmatic donors by IFN-β. PBECs from nonasthmatic (open circles, n = 10) and asthmatic (solid circles, n = 10) donors were exposed to serum free medium (SFM) alone or challenged with 100 IU/mL IFN-β for 6 hours. After RNA extraction, expression of RIG-I, TLR3, 2′-5′-oligoadenylate synthetase (2′5′OAS), and PKR was assessed by means of RT-qPCR and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ubiquitin C (UBC) by using the ΔΔCT method. Statistical analysis was performed with the Student t test or Mann-Whitney U test. ∗∗P < There were no significant differences between the groups. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Viral replication and cytopathic effect after infection with a low dose of RV1B. PBECs from 8 asthmatic subjects were infected with RV1B (1 × 103 TCID50 units/105 cells) for up to 72 hours. Photomicrographs show cytopathic effects of RV1B at 24, 48, and 72 hours compared with noninfected controls cultured in serum free medium (SFM) alone. Expression of viral RNA was quantified by using RT-qPCR and viral titers by using TCID50. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Upregulation of RANTES in response to RV1B infection
Upregulation of RANTES in response to RV1B infection. PBECs from asthmatic subjects (n = 8) were infected with RV1B for up to 72 hours. RANTES mRNA expression was quantified by using RT-qPCR and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ubiquitin C (UBC) by using the ΔΔCT method. Protein secretion in cell supernatants was measured with the BD Cytometric Bead Array (CBA). Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Exogenous IFN-β significantly suppresses IP-10 and RANTES mRNA production in response to RV1B infection. PBECs from asthmatic subjects (n = 8) were infected with RV1B without or with exogenous IFN-β (100 IU/mL). IP-10 and RANTES mRNA was quantified at 72 hours relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ubiquitin C (UBC) by using the ΔΔCT method. Data were analyzed with the Wilcoxon signed-rank test. ∗∗P < .01. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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Induction of IL-6 and IL-8 protein release from PBECs from asthmatic donors after RV1B infection. PBECs from asthmatic subjects (n = 7) were infected with RV1B for up to 72 hours or with inactive UV-irradiated RV1B as a control (tested at 72 hours); IL-6 and IL-8 protein release into the culture supernatants was measured by means of ELISA (upper panels). In the lower panels the PBECs were challenged with RV1B without or with exogenous IFN-β. Protein secretion in cell supernatants was measured by using ELISA. Statistical analysis used the Wilcoxon signed-rank test. ∗∗P < .01. RV1B had no significant effect on IL-8 release compared with UV-irradiated controls. IFN-β did not affect IL-8 release in the presence of RV1B. Journal of Allergy and Clinical Immunology , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions
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