Roflumilast N-oxide reverses corticosteroid resistance in neutrophils from patients with chronic obstructive pulmonary disease Javier Milara, PhD, PharmD,

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Roflumilast N-oxide reverses corticosteroid resistance in neutrophils from patients with chronic obstructive pulmonary disease Javier Milara, PhD, PharmD, Javier Lluch, PhD, Patricia Almudever, PhD, PharmD, Jose Freire, PhD, Qian Xiaozhong, PhD, Julio Cortijo, PhD, PharmD Journal of Allergy and Clinical Immunology Volume 134, Issue 2, Pages 314-322.e9 (August 2014) DOI: 10.1016/j.jaci.2014.02.001 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 A-M, Basal expression of putative corticosteroid-resistant markers in neutrophils from healthy subjects and patients with COPD. Peripheral blood neutrophils from healthy subjects (n = 15) and patients with COPD (n = 15) are shown. Data are presented as a box and whisker plot with median, interquartile range, and minimum and maximum values. P values were obtained by using the Mann-Whitney test. GAPDH, Glyceradlehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A-D, Peripheral blood neutrophils were isolated from patients with COPD and incubated with NAC (1 mmol/L), dexamethasone (DEX; 1 μmol/L), LY-294002 (10 μmol/L), or combinations for 1 hour, followed by stimulation with LPS (1 μg/mL) or CSE 5%. Results are expressed as means ± SEs of 3 (3 cell population) independent experiments. Two-way ANOVA followed by post hoc Bonferroni tests: *P < .05 related to control; #P < .05 related to LPS or CSE; ♦P < .05 related to drugs alone. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Concentration-dependent inhibition of LPS- or CSE-induced IL-8 or MMP-9 release by RNO or dexamethasone (DEX) from neutrophils of healthy subjects and patients with COPD. Neutrophils were preincubated with RNO (0.1 nmol/L to 1 μmol/L) or DEX (0.1 nmol/L to 1 μmol/L) for 1 hour, followed by cell stimulation with LPS (1 μg/mL; A and C) or CSE 5% (B and D) for 6 hours. Results are expressed as means ± SEs of 3 (3 healthy subject and 3 patient with COPD cell populations) independent experiments. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 A-D, Additive/synergistic effects of RNO with dexamethasone (DEX) on IL-8 and MMP-9 secretion in neutrophils from patients with COPD. Results are expressed as means ± SEs of 3 (3 cell populations) independent experiments per condition. Two-way ANOVA, followed by post hoc Bonferroni tests: *P < .05 related to control; #P < .05 related to stimulus and single RNO or DEX exposure. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 A-H, Effects of RNO and dexamethasone (DEX) on CSE-induced corticosteroid-resistant markers. Human peripheral blood neutrophils from patients with COPD were isolated and plated on 24-well plates. Cells were incubated with RNO (1 nmol/L or 1 μmol/L), DEX (1 μmol/L), or their combination for 1 hour and stimulated with CSE. Results are expressed as means ± SEs in 3 (3 to 6 cell populations) independent experiments. Two-way ANOVA, followed by post hoc Bonferroni tests. *P < .05 related to control expression; #P < .05 related to CSE; ♦P < .05 related to 1 nmol/L RNO or 1 μmol/L DEX alone. GAPDH, Glyceradlehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 A-C, Effects of RNO and dexamethasone (DEX) on CSE-induced markers of corticosteroid resistance. Human peripheral blood neutrophils from patients with COPD were isolated and plated on 24-well plates. Cells were incubated with RNO (1 nmol/L or 1 μmol/L), DEX (1 μmol/L), or their combination for 1 hour and stimulated with CSE. mRNA levels for MKP-1 and MIF were measured, and phosphorylation of ERK1/2 was detected by means of Western blotting. Results are expressed as means ± SEs of 3 (3 to 6 cell populations) independent experiments. A representative picture for Western blots is shown in Fig 6, B. Two-way ANOVA, followed by post hoc Bonferroni tests: *P < .05 related to control; #P < .05 related to CSE; ♦P < .05 related to 2 nmol/L RNO or 1 μmol/L DEX alone. GAPDH, Glyceradlehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 IL-8 and MMP-9 release in human neutrophils from healthy subjects and patients with COPD. Peripheral blood neutrophils were isolated from healthy subjects and patients with COPD and stimulated with LPS (1 μg/mL; A and C) or CSE (5%; B and D) for 6 hours. Results are expressed as means ± SEs of 3 (3 healthy and 3 COPD cell populations) independent experiments. Two-way ANOVA, followed by post hoc Bonferroni tests: *P < .05 related to basal release in the same subject group; #P < .05 related to basal release in healthy group; ♦P < .05 related to LPS- or CSE-stimulated release in healthy group. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 A-D, Peripheral blood neutrophils were isolated from healthy subjects and incubated with 1 μmol/L RU-486, 1 μmol/L dexamethasone (DEX), or 1 μmol/L RU-486 1 hour before 1 μmol/L DEX (1 hour of incubation), followed by stimulation with LPS (1 μg/mL) or CSE 5% for 6 hours. Results are expressed as means ± SEs of 3 (3 cell population) independent experiments. Two-way ANOVA, followed by post hoc Bonferroni tests: *P < .05 related to control; #P < .05 related to LPS or CSE; ⊥P < .05 related to DEX. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 A and B, RNO inhibits ROS in neutrophils from patients with COPD. Peripheral whole blood neutrophils from patients with COPD were identified by using CD16–Alexa Fluor 645 by means of flow cytometry. Whole blood was incubated with RNO (1 nmol/L to 1 μmol/L) or the antioxidant NAC (1 mmol/L) for 1 hour and stimulated with fMLP (1 μmol/L) or CSE for 30 minutes. Intracellular ROS levels were measured in neutrophil populations by means of fluorescence H2DCF-DA dye with flow cytometry. Results are expressed as means ± SEs in 3 (4 cell populations). Two-way ANOVA, followed by post hoc Bonferroni tests: *P < .05 related to control; #P < .05 related to fMLP or CSE. RFU, Relative fluorescence units. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 RNO improves corticosteroid resistance in neutrophils from patients with COPD: mechanisms implicated. A, Cigarette smoke increases ROS levels in neutrophils from patients with COPD. The increase in ROS levels activates and increases the expression of PI3Kδ, which reduces the activity of HDAC2. Furthermore, cigarette smoke induces the NF-κB (p65) nuclear translocation switching on histone acetyltransferase, leading to histone acetylation and subsequently to transcription of genes encoding inflammatory proteins, such as IL-8 or MMP-9. In normal conditions corticosteroids reverse this by binding to GRs and recruiting HDAC2. This reverses the histone acetylation induced by NF-κB and switches off the activated inflammatory genes. However, neutrophils from patients with COPD generate oxidative stress, which impairs the activity of HDAC2. This amplifies the inflammatory response to NF-κB activation but also reduces the anti-inflammatory effect of glucocorticoids because HDAC2 is now unable to reverse histone acetylation. RNO reduces ROS formation after cigarette smoke exposure and consequently inhibits PI3Kδ activation and increases HDAC2 activity. Furthermore, RNO inhibits NF-κB (p65) nuclear translocation. Effects showed by RNO allow corticosteroids to bind HDAC2 to reverse the histone acetylation induced by NF-κB and switching off the activated inflammatory genes. B, ROS formation by cigarette smoke promotes phosphorylation of ERK1/2 and subsequently IL-8 or MMP-9 formation. Furthermore, ROS downregulates MKP1 expression (an inhibitor of ERK1/2) and increases MIF1 expression (an inhibitor of MKP1) and GRβ expression, increasing inflammation. In neutrophils from patients with COPD exposed to cigarette smoke, dexamethasone (DEX) is unable to reduce ERK1/2, MIF1, or GRβ expression or increase MKP1 expression. RNO reduces GRβ expression, allowing DEX to join GRα to translocate to the nucleus. Furthermore, RNO reduces MIF1 expression, which allows DEX to increase MKP1, reducing ERK1/2 phosphorylation and IL-8 and MMP-9 secretion. Violet lines and arrows represent the effect of cigarette smoke on neutrophils from patients with COPD. Green lines and arrows represent the effects of RNO. Journal of Allergy and Clinical Immunology 2014 134, 314-322.e9DOI: (10.1016/j.jaci.2014.02.001) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions