IL-17 enhances the migration of B cells during asthma by inducing CXCL13 chemokine production in structural lung cells Roua Al-Kufaidy, MS, Alejandro.

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IL-17 enhances the migration of B cells during asthma by inducing CXCL13 chemokine production in structural lung cells Roua Al-Kufaidy, MS, Alejandro Vazquez-Tello, PhD, Ahmed S. BaHammam, MD, Saleh Al-Muhsen, MD, Qutayba Hamid, MD, PhD, Rabih Halwani, PhD Journal of Allergy and Clinical Immunology Volume 139, Issue 2, Pages 696-699.e5 (February 2017) DOI: 10.1016/j.jaci.2016.07.037 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 B-cell infiltration in bronchial tissue increased in asthma and correlated with IL-17 cytokines stimulation and elevated CXCL13 expression. A, B cells in bronchial tissues were immunolabeled with anti-CD20 antibodies and expressed as number/mm2. B, Abundant B cells in an airway follicle, stained with anti-CD20 antibodies. C, Representative CXCL13 immunoreactivity in biopsies from those with severe asthma (i, ii) and control subjects (iii). D, Quantification of CXCL13+ cells in biopsies. E, CXCL13 mRNA expression levels in bronchial tissues of subjects with asthma and healthy control subjects. F, Fold increase in IL-17–stimulated CXCL13 mRNA expression levels in fibroblasts and endothelial cells by RT-PCR. G, Flow cytometric analysis of fibroblasts stimulated with or without IL-17A+17F and immunostained with anti-CXCL13 antibodies. H, Fold increase in IL-17–stimulated CXCL13+ fibroblast and endothelial cell numbers. NS, Nonsignificant. *P < .05. Journal of Allergy and Clinical Immunology 2017 139, 696-699.e5DOI: (10.1016/j.jaci.2016.07.037) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IL-17–induced CXCL13 expression is required for B-cell migration and regulated by NF-κB pathway activation. A, Percentage of asthmatic B cells migrated toward culture medium from fibroblasts stimulated with IL-17 cytokines, in the presence/absence of either anti–IL-8 or anti-CXCL13 antibodies. B, Similar as (Fig 2, A) with medium from endothelial cells. C, Blockage of the NF-κB pathway in fibroblasts stimulated with IL-17 cytokines abolished B-cell migration. D, ELISA determination of CXCL13 proteins released into the medium by fibroblasts, stimulated with or without IL-17 cytokines and NF-κB inhibitor added. DMSO, Dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; NS, nonstimulated. *P < .05. **P < .01. Journal of Allergy and Clinical Immunology 2017 139, 696-699.e5DOI: (10.1016/j.jaci.2016.07.037) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 A, Representative figure of cell follicles (H&E staining) formed in an asthmatic bronchial tissue. B, Number of B-cell follicles/mm2 in bronchial tissue of subjects with asthma and healthy subjects. C, IL-17A+ cells/mm2 in bronchial tissues of subjects with mild/moderate and severe asthma and healthy control subjects. D, IL-17 mRNA expression levels in bronchial biopsies from subjects with mild/moderate or severe asthma and healthy control subjects. E, Fold increase in secreted CXCL13 protein levels from fibroblasts stimulated with or without IL-17. F, Fold increase in CXCL13 mRNA in epithelial cells (healthy) stimulated with or without IL-17. G, Same as Fig E1, F, except in asthmatic epithelial cells. H, Same as Fig E1, F, except in asthmatic ASM cells. I, Western analysis indicating the absence of CXCL13 protein expression following stimulation of asthmatic primary epithelial cells with IL-17 cytokines. (Fig E1, D-H, n = 10). ASM, Airway smooth muscle; H&E, hematoxylin and eosin; SE, standard error. Data are expressed as means ± SE. *P < .05. Journal of Allergy and Clinical Immunology 2017 139, 696-699.e5DOI: (10.1016/j.jaci.2016.07.037) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 CXCR5, VLA-4, and LFA-1 receptors are upregulated on B cells following stimulation with IL-17. A, Relative expression of CXCR5 mRNA in B cells from subjects with asthma and healthy control subjects following stimulation with IL-17. B, Representative flow cytometric analysis of asthmatic B cells immunostained with anti-CXCR5 antibodies, stimulated with or without IL-17. C, % of CXCR5+ cells following stimulation with IL-17 from healthy and severe asthmatic B cells. D, Fold increase in VLA-4 and LFA-1 mRNA expression levels in asthmatic B cells following stimulation with IL-17 cytokines normalized to baseline. LFA-1, Lymphocyte function-associated antigen 1; SE, standard error; VLA-4, very late antigen-4. Data are expressed as means ± SE. *P < .05 compared with the nonstimulated (NS) condition. **P < .05 compared with the control condition. Journal of Allergy and Clinical Immunology 2017 139, 696-699.e5DOI: (10.1016/j.jaci.2016.07.037) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions