Nitric oxide inhibits IFN regulatory factor 1 and nuclear factor-κB pathways in rhinovirus- infected epithelial cells Rommy Koetzler, MD, MSc, Raza S.

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Nitric oxide inhibits IFN regulatory factor 1 and nuclear factor-κB pathways in rhinovirus- infected epithelial cells Rommy Koetzler, MD, MSc, Raza S. Zaheer, BSc, Robert Newton, PhD, David Proud, PhD Journal of Allergy and Clinical Immunology Volume 124, Issue 3, Pages 551-557 (September 2009) DOI: 10.1016/j.jaci.2009.04.041 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Selective inhibitors of IKKβ inhibit HRV-16–induced production of CXCL10 from airway epithelial cells. Both PS-1145 (A) and ML120B (B) inhibit, in a concentration-dependent manner, HRV-induced production of CXCL10 from BEAS-2B epithelial cells (n = 6). PS-1145 (C) and ML120B (D), at a concentration of 10 μmol/L, also inhibit HRV-16–induced CXCL10 production from primary cultures of HBEs (n = 3). ∗Statistical significance compared with HRV-16 alone. Journal of Allergy and Clinical Immunology 2009 124, 551-557DOI: (10.1016/j.jaci.2009.04.041) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 PAPA NONOate inhibits NF-κB DNA binding and IκBα phosphorylation. Preincubation of rh p50 for 90 minutes with 500 μmol/L of PAPA NONOate inhibited its ability to bind to either the κB1 (A) or κB2 (B) recognition sequences from the CXCL10 promoter. PAPA NONOate also prevented the HRV-16–induced phosphorylation of IκBα observed 3 hours after infection of BEAS-2B (C) or HBEs (D). All experiments are representative of n ≥ 3. Journal of Allergy and Clinical Immunology 2009 124, 551-557DOI: (10.1016/j.jaci.2009.04.041) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 PAPA NONOate inhibits IRF-1 DNA binding and HRV-16–induced IRF-1 expression. A, Preincubation of rhIRF-1 for 90 minutes with 500 μmol/L of PAPA NONOate inhibits its ability to bind to the ISRE recognition sequence in the CXCL10 promoter (representative of n = 3). B, PAPA NONOate inhibits HRV-16–induced epithelial increases in IRF-1 mRNA in BEAS-2B cells (n = 9). ∗Statistical significance compared to HRV-16 alone. C, PAPA NONOate also inhibits HRV-16–induced expression of IRF-1 protein (representative of n = 3). Journal of Allergy and Clinical Immunology 2009 124, 551-557DOI: (10.1016/j.jaci.2009.04.041) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Reduction of HRV-16–induced epithelial expression of IRF-1 by selective inhibitors of IKKβ. Both PS-1145 (A; n = 6) and ML120B (B; n = 3) inhibit HRV-16–induced increases in IRF-1 mRNA in BEAS-2B cells. Asterisk indicates statistical significance compared to HRV-16 alone. (C) PS-1145 and ML120B partially inhibit HRV-16–induced expression of IRF-1 protein (representative of n = 3). Journal of Allergy and Clinical Immunology 2009 124, 551-557DOI: (10.1016/j.jaci.2009.04.041) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Reduced nuclear translocation and binding of IRF-1 in HRV-16–infected epithelial cells treated with selective inhibitors of IKKβ. A, Both PS-1145 and ML120B inhibited HRV-16–induced nuclear translocation and binding of IRF-1 in BEAS-2B cells (representative of n = 3). B, Neither inhibitor directly affected binding of rhIRF-1 to the ISRE recognition sequence from the CXCL10 promoter (representative of n = 3). Journal of Allergy and Clinical Immunology 2009 124, 551-557DOI: (10.1016/j.jaci.2009.04.041) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions