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Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells  Rommy Koetzler, MD, MSc, Raza S. Zaheer,

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Presentation on theme: "Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells  Rommy Koetzler, MD, MSc, Raza S. Zaheer,"— Presentation transcript:

1 Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells  Rommy Koetzler, MD, MSc, Raza S. Zaheer, BSc, Shahina Wiehler, MSc, Neil S. Holden, PhD, Mark A. Giembycz, PhD, David Proud, PhD  Journal of Allergy and Clinical Immunology  Volume 123, Issue 1, Pages e9 (January 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 PAPA NONOate inhibits induction of CXCL10 mRNA and protein in HRV-16–infected epithelial cells. A and B, In BEAS-2B cells (n = 7) induction of CXCL10 mRNA and protein was inhibited in a concentration-dependent manner (P < .05, ANOVA). Asterisks indicate significant inhibition compared with HRV-16 alone by means of appropriate post hoc tests. C and D, PAPA NONOate (500 μmol/L) also inhibits CXCL10 production in HBEs (n = 9). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Inhibition of HRV-16–induced CXCL10 in epithelial cells is not mediated through cGMP. A, The cell-permeable analog 8-bromo-cGMP did not mimic the effects of PAPA NONOate (500 μmol/L) in BEAS-2B cells (n = 4). B, Similarly, an inhibitor of soluble guanylyl cyclase (ODQ) did not reverse the effects of PAPA NONOate (n = 4). Asterisks indicate significant inhibition compared to HRV-16 alone. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 PAPA NONOate inhibits HRV-16–induced activation of CXCL10 promoter–luciferase constructs. A, Diagrammatic representations of full-length (972-bp) and truncated (376-bp) promoters. B, PAPA NONOate (solid bars) inhibits activation of full-length and truncated constructs to a similar degree. Asterisks indicate significant inhibition compared with HRV-16 alone (open bars, n = 7). C, Mutation of individual NF-κB and ISRE, but not AP-1, recognition sequences in the truncated promoter reduces HRV-16–induced promoter activation. Asterisks indicate significant inhibition by PAPA NONOate (solid bars) compared with virus alone (open bars, n = 6). C/EBP, CCAAT/enhancer binding protein. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 PAPA NONOate inhibits HRV-16–induced activation of reporter constructs. A, PAPA NONOate significantly inhibits HRV-16–induced activation of constructs containing 5 copies of either the NF-κB1 or NF-κB2 recognition sequences (n = 6). B, PAPA NONOate significantly inhibits HRV-16–induced activation of constructs containing 2 sequential copies of ISRE–NF-κB1 or ISRE–NF-κB2 sequences (n = 6). C, PAPA NONOate significantly inhibits HRV-16–induced activation of the truncated promoter containing mutations in both the AP-1 and NF-κB2 recognition sequences (n = 6). In each case asterisks indicate significant inhibition by PAPA NONOate (solid bars) compared with virus alone (open bars). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Effects of PAPA NONOate on nuclear translocation, binding, or both of NF-κB in HRV-16–infected BEAS-2B cells. A, HRV-16 infection induced 2 bands on incubation of nuclear extracts with the NF-κB1 oligonucleotide. Induction of both bands was inhibited by PAPA NONOate. B, Supershift experiments demonstrate that antibody to p65 shifts only the upper band, whereas antibody to p50 shifts both bands. Antibodies to p52 and cRel were without effect. Data are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Effects of PAPA NONOate on nuclear translocation, binding, or both of IRFs in HRV-16–infected BEAS-2B cells. A, HRV-16 infection induced 3 bands on incubation of nuclear extracts with the ISRE oligonucleotide. Induction of all 3 bands was inhibited by PAPA NONOate. B, Supershift experiments demonstrate that antibody to IRF-1 completely shifts the upper band. Antibodies to IRF-2, IRF-3, IRF-7, or ISGF-3 were without effect on any band. Data are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Sodium nitroprusside (SNP) inhibits HRV-16–induced epithelial production of CXCL10. A, BEAS-2B cells were exposed to HRV-16 in the presence or absence of 500 μmol/L SNP, and CXCL10 protein was measured after 24 hours (n = 5). B, SNP also inhibited HRV-16–induced CXCL10 production from primary HBEs (n = 5). Asterisks indicate significant inhibition compared to HRV-16 alone. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 PAPA NONOate inhibits dsRNA-mediated induction of CXCL10 mRNA and protein in epithelial cells. A and B, In BEAS-2B cells (n = 6) induction of CXCL10 mRNA and protein was inhibited in a concentration-dependent manner (P < .05, ANOVA). Asterisks indicate significant inhibition compared with dsRNA alone by using appropriate post hoc tests. C and D, PAPA NONOate (500 μmol/L) also inhibits CXCL10 production in HBEs (n = 8). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 The inhibitor of soluble guanylyl cyclase, ODQ, dose-dependently inhibits PAPA NONOate–induced accumulation of cGMP in BEAS-2B cells. Cells pretreated with an inhibitor of phosphodiesterase V were exposed to PAPA NONOate (500 μmol/L) in the presence or absence of ODQ (3-30 μmol/L). Accumulation of cGMP was measured after 2 hours (n = 4). Asterisks indicate significant inhibition compared to HRV-16 + NONOate alone. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Inhibition of HRV-16–induced CXCL10 in HBEs is not mediated through cGMP. A, Exposure of primary HBEs to the cell-permeable analog 8-bromo-cGMP ( μmol/L) did not mimic the ability of PAPA NONOate (500 μmol/L) to inhibit HRV-16–induced CXCL10 production (n = 3). B, Similarly, preincubation of primary HBEs with ODQ (3-30 μmol/L), an inhibitor of soluble guanylyl cyclase, did not reverse the PAPA NONOate–mediated inhibition of HRV-16–induced CXCL10 production (n = 3). Asterisks indicate significant inhibition compared to HRV-16 alone. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 PAPA NONOate inhibits dsRNA-induced activation of CXCL10 promoter–luciferase constructs. PAPA NONOate inhibits dsRNA-induced activation of the 376-bp truncated construct. Mutation of individual NF-κB and ISRE, but not AP-1, recognition sequences in the truncated promoter reduces dsRNA-induced promoter activation. Asterisks indicate significant inhibition by PAPA NONOate (solid bars) compared with dsRNA alone (open bars, n = 6). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 EMSA time-course experiments
EMSA time-course experiments. Time courses of HRV-16–induced binding of nuclear proteins to oligonucleotides containing NF-κB1 oligonucleotide (A), NF-κB2 oligonucleotide (B), or ISRE oligonucleotide (C). Specific bands are shown by arrows. Data are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Effects of PAPA NONOate on nuclear translocation, binding, or both of NF-κB in HRV-16–infected BEAS-2B cells. A, HRV-16 infection induced 2 bands on incubation of nuclear extracts with the NF-κB2 recognition sequence. Induction of both bands was inhibited with PAPA NONOate. B, Supershift experiments demonstrate that antibody to p65 shifts only the upper band, whereas antibody to p50 shifts both bands. Antibodies to p52 and cRel were without effect. Data are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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