MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity Jing Jing Li, MS, Hock L. Tay, PhD, Steven.

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MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity Jing Jing Li, MS, Hock L. Tay, PhD, Steven Maltby, PhD, Yang Xiang, PhD, Fiona Eyers, BSci, Luke Hatchwell, B Biomed Sci, Hong Zhou, BMed, Hamish D. Toop, BSci(Hons), Jonathan C. Morris, PhD, Parameswaran Nair, MD, PhD, Joerg Mattes, MD, PhD, Paul S. Foster, PhD, DSc, Ming Yang, PhD Journal of Allergy and Clinical Immunology Volume 136, Issue 2, Pages 462-473 (August 2015) DOI: 10.1016/j.jaci.2014.11.044 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 MiR-9 expression is induced by IFN-γ/LPS stimulation. A, MiR-9 expression levels in lung samples from mice 12 hours after exposure to LPS, IFN-γ, or IFN-γ/LPS. Data are means ± SEMs (n = 8 mice per group). B, Pulmonary macrophages stimulated with LPS, IFN-γ, or IFN-γ/LPS (γ/LPS) for 3, 6, 12, or 24 hours. Each dot represents the mean of an independent experiment performed in duplicate. C, Induced sputum samples from asthmatic patients. EOS, Eosinophilic; NEU, neutrophilic; Veh, vehicle control. Values are means ± SEMs. *P < .05 versus vehicle control. #P < .05 versus vehicle control and LPS or IFN-γ treatment at respective time points. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Inhibition of miR-9 restores DEX sensitivity, attenuating AHR induction by IFN-γ/LPS (γ/LPS). Mice were administered ant-9 or scrambled antagomir (scr), DEX, or both 12 hours before IFN-γ/LPS exposure. Twelve hours later, lung function was assessed, and lung tissues were isolated. MiR-9 levels in lung normalized to sno-202 (A), lung function measurements after exposure to IFN-γ/LPS and ant-9 or scr (B), miR-9 levels (C), and lung function measurements of mice pretreated with DEX and ant-9 or scr (D) are shown. Values are means ± SEMs (n = 8 to 10 mice per group). Veh, Vehicle control. *P < .05 versus vehicle control. #P < .05, γ/LPS+DEX+scr versus γ/LPS+DEX+ant-9. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Targeting of PPP2R5D and PPP2R2A mRNA by miR-9. A, Conserved miR-9–binding sites (highlighted) in the 3′UTR of PPP2R5D and PPP2R2A mRNAs. B, Luciferase activity in lysates of HEK293 cells transfected with constructs encoding the 3′UTR regions of PPP2R5D or PPP2R2A and miR-9 mimic or scrambled control at indicated concentrations. Values are means ± SEMs of 3 independent experiments performed in triplicate. *P < .05 and **P < .005. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Targeting miR-9 restores PPP2R5D expression and PP2A activity. Pulmonary macrophages were pretreated with ant-9 or scrambled antagomir (scr) 12 hours before IFN-γ/LPS (γ/LPS) treatment. Samples were collected 12 hours later. A and B, MiR-9 (Fig 4, A) and PPP2R5D (Fig 4, B) transcript levels were determined by using quantitative PCR. Each dot represents the mean of an independent experiment performed in triplicate. C and D, Representative Western blot images (Fig 4, C) and densitometric assessment (Fig 4, C) for PPP2R5D protein levels. Data are representative of 3 independent experiments. E and F, PP2A catalytic activity was normalized to untreated control values in pulmonary macrophages (triplicate samples from 1 experiment representative of 3 independent experiments; Fig 4, E) and lung tissue (Fig 4, F). Data are means ± SEMs (n = 6 mice per group. G and H, Western blot staining of pulmonary macrophages for phosphorylated (P-JNK) and total JNK (Fig 4, G) and P-JNK/JNK ratios (Fig 4, H) normalized to vehicle control (Veh). *P < .05. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Inhibition of miR-9 restores DEX-induced GR nuclear translocation. Pulmonary macrophages were pretreated with ant-9 or scrambled control (scr) for 12 hours before IFN-γ/LPS (γ/LPS) for 11 hours and then DEX for 1 hour. A, GR cellular localization determined by using immunofluorescence staining. B, Ratio of fluorescence intensity (nuclear/cytoplasm). Assessment was performed on 8 to 10 different slide regions on 40 to 60 cells for each treatment. C and D, MiR-9 expression levels (Fig 5, C) and PP2A activities (Fig 5, D) were also determined. Values are means ± SEMs for triplicate samples from 1 experiment and representative of 3 independent experiments. D, DEX; I, IFN-γ; L, LPS. *P < .05. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Increasing PP2A activity restores steroid sensitivity and GR nuclear translocation. Mice administered DEX and/or the PP2A activator AAL(S) after IFN-γ/LPS (γ/LPS) exposure are shown. A, Lung function was measured 12 hours later. Values are means ± SEMs (n = 8 to 12 mice per group). B and C, Isolated pulmonary macrophages treated with DEX with or without AAL(S) and GR cellular localization determined by means of immunofluorescence staining (Fig 6, B) and the ratio of fluorescence intensity (nuclear/cytoplasm; Fig 6, C). Assessment was performed on 8 to 10 different slide regions on 40 to 60 cells for each treatment. D, DEX; I, IFN-γ; L, LPS; Veh, vehicle control. *P < .05, γ/LPS+DEX, L+I+D+vehicle versus vehicle control, L+I+D+AAL(s). Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Inhibition of miR-9 restores steroid sensitivity, blocking the development of AHR in allergen-induced models of steroid-resistant AHR. A and C, Schematic representation of coexposure (Fig 7, A) and postexposure (Fig 7, C) models of LPS administration indicating timing of OVA sensitization, challenges, and DEX/ant-9 treatments. B and D, Lung function was assessed on day 17 after sensitization (Fig 7, B) and day 24 after sensitization (Fig 7, D), respectively. Values are means ± SEMs (n = 10 mice per group). Raw, Airway resistance. *P < .05 versus other groups. #P < .05, OVA/OVA versus OVA/LPS and OVA/LPS+DEX. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 Schematic representation of miR-9's effects on glucocorticoid function in pulmonary macrophages after IFN-γ/LPS exposure. MiR-9 reduces PP2A activity by reducing PPP2R5D levels, thus downregulating GR nuclear translocation and inhibiting glucocorticoid function. Ant-9 recovers PP2A activity, leading to improved glucocorticoid function. , Increased; , decreased; , phosphorylation. Red arrow, miR-9 effect; blue arrow, ant-9 effect. Journal of Allergy and Clinical Immunology 2015 136, 462-473DOI: (10.1016/j.jaci.2014.11.044) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions