Airway lipoxin A4/formyl peptide receptor 2–lipoxin receptor levels in pediatric patients with severe asthma Rosalia Gagliardo, PhD, Delphine Gras, PhD,

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Airway lipoxin A4/formyl peptide receptor 2–lipoxin receptor levels in pediatric patients with severe asthma Rosalia Gagliardo, PhD, Delphine Gras, PhD, Stefania La Grutta, MD, Pascal Chanez, MD, PhD, Caterina Di Sano, PhD, Giusy D. Albano, PhD, Isabelle Vachier, PhD, Angela M. Montalbano, PhD, Giulia Anzalone, PhD, Anna Bonanno, PhD, Loredana Riccobono, PhD, Mark Gjomarkaj, MD, Mirella Profita, PhD Journal of Allergy and Clinical Immunology Volume 137, Issue 6, Pages 1796-1806 (June 2016) DOI: 10.1016/j.jaci.2015.11.045 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Measurements of LXA4 and LTB4 concentrations in ISSs. A, Assessment of LXA4 concentrations in sputum supernatants of HC children, children with IA, and children with SA. B, Assessment of LTB4 concentrations in sputum supernatants of HC children, children with IA, and children with SA. Individual data are shown. Open squares represent HC children, open circles represent children with IA, and open triangles represent children with SA. Horizontal lines represent medians. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Immunocytochemistry analysis for FPR2/ALX in induced sputum cells from HC children, children with IA, and children with SA. A, Percentage of induced sputum cells expressing FPR2/ALXR in HC children, children with IA, and children with SA, respectively. B, Representative immunostaining of induced sputum cells for FPR2/ALXR expression (magnification ×40). NS, Not significant. *P < .01 and **P < .001. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Correlations between sputum LXA4 concentrations and sputum LTB4 concentrations in children with IA (A) and children with SA (B) and between sputum LXA4 concentrations and severe exacerbations (C) occurring in children with SA during 1-year follow-up. Open circles represent children with IA, and open triangles represent children with SA. Correlations were calculated by using the Spearman test. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Granulocyte chemotaxis assay. Assessment of PBG chemotactic activity induced by sputum supernatants obtained from HC children, children with IA, and children with SA in the presence or absence of 200 nmol/L LXA4. Results are expressed as means and SDs. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Phosphorylated GR protein expression in PBGs isolated from HC children. A, Expression of p-GR protein in PBGs. Signals corresponding to p-GR were semiquantified by using a densitometric scanner and expressed as the ratio of the band intensity of the phosphorylated protein versus the band intensity of the β-actin protein (National Institutes of Health Image/Gel Plotting analysis program). B, Representative Western blot analysis of p-GR in PBGs. Results are expressed as means and SDs. *P < .01 and **P < .001. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 DNA-binding ELISA for GR activity. Assessment of nuclear GR protein binding to GRE sequences in PBGs isolated by HC children (n = 4), children with IA (n = 4), and children with SA (n = 5) in the presence or absence of FP, LXA4, and LXA4 blocking peptide. Modulation of GR binding activity under different experimental conditions is shown. Data are expressed as means and SDs. *P < .01 and **P < .001, significantly different from baseline values. #P < .01 and ##P < .001 for the indicated comparisons. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 A and B, Flow cytometric analysis for FPR2/ALXR expression in PBGs of HC children at baseline and in the presence or absence of FP (10 nmol/L or 100 nmol/L), LXA4 (200 nmol/L), and their combination. Bars represent means ± SDs of the percentage of positive cells. *P < .01 and **P < .001, significantly different from baseline values. Representative flow cytometry of FPR2/ALXR of each experimental condition. Journal of Allergy and Clinical Immunology 2016 137, 1796-1806DOI: (10.1016/j.jaci.2015.11.045) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions