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Identification of CCL17 neutraligands targeting atopic diseases
J2R (Posters) 4 CHU de Bordeaux, Hôpital Pellegrin-Enfants, Pneumologie Pédiatrique, Centre d’Investigation Clinique (CIC 0005), 33076 Bordeaux, France 5 CHU de Poitiers, Pneumologie pédiatrique et EFR, 86000 Poitiers, France ∗ Corresponding author. E-mail address: [email protected] (T. Trian)
Introduction Increased bronchial smooth muscle mass is one of the key structural features of severe asthma. In adults, asthmatic airway smooth muscle cells (ASMC) demonstrate greater mitochondrial biogenesis associated with an increase in ASMC proliferation rate vs. non-asthmatic ASMC. However, to the best of our knowledge, there is no evidence that such a difference between asthmatic and non-asthmatic ASMC occurs in pre-school children. Thus, the primary aim of the study was to compare asthmatic and non-asthmatic ASMC proliferation and mitochondrial biogenesis in adults and pre-school children. The secondary aim was to assess the effect of factors released by the epithelium upon stimulation by environmental factors such as house dust mite and rhinovirus, on ASMC proliferation. Methods We cultured ASMC and bronchial epithelial cells (BEC) obtained by endobronchial biopsy from children and adults with severe asthma or undergoing bronchial endoscopy for other reasons. We then studied ASMC proliferation (cell counting and CFSE dye assay) in 10% fetal bovine serum (FBS), 0% FBS and after the addition of the BEC culture supernatant obtained after rhinovirus infection, house dust mite exposure or both. Mitochondrial mass and biogenesis were determined by western blot. Results Sixteen pre-school children with severe asthma,median aged 2.8 years, 4 control children (4.6 years), 13 adults with severe asthma (46.0 years) and 26 controls adults (65.3 years) were included. ASMC proliferation was increased in asthmatic adults, asthmatic pre-school and control children ASMC vs. control adults, with a cell doubling time of 35.0 ± 2.5 h, 24.8 ± 1.9 h, 22.6 ± 2.6 h and 47.4 ± 4.4 h, respectively (P < 0.05). This increased ASMC proliferation was associated with greater mitochondrial mass (porine rate: 50% in asthmatic adult ASMC, 52% in children ASMC vs. 20% in control adult ASMC) and biogenesis in asthmatic adult, asthmatic and control children ASMC vs. control adults (TFAM rate respectively at 44,9%, 48,1% and 62,2% vs. 18,4%). Rhinovirus and house dust mite exposure induced an increase of respectively 120,5% and 107.7% compared to the basal proliferation rate in asthmatic subjects only. Conclusion As previously described in adult asthmatics, ASMC proliferation is enhanced in both asthmatic and non-asthmatic children. This is associated with an increase in mitochondrial mass and biogenesis. ASMC proliferation is increased after a viral infection or an allergen exposure. Keywords Asthma; Allergy; Children The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.029 P05
Identification of CCL17 neutraligands targeting atopic diseases D. Abboud 1,4,∗ , F. Daubeuf 2,4 , V. Utard 1,4 , D. Bonnet 2,4 , M. Hibert 2,4 , P. Bernard 3 , J.L. Galzi 1,4 , N. Frossard 2,4 1 Biotechnologie et signalisation cellulaire, UMR 7242 CNRS, Université de Strasbourg, ESBS, Illkirch, France 2 Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France 3 GreenPharma, Orléans, France 4 LabEx Medalis, Strasbourg, France ∗ Corresponding author. E-mail address: [email protected] (D. Abboud)
315 Introduction Chemokines constitute a family of small cytokines that attract and activate leukocytes during the inflammatory response. Among them, the -chemokine CCL17 formerly known as thymus- and activation regulated chemokine (TARC), is involved in the development of atopic disorders such as asthma and atopic dermatitis. This chemokine exerts its biological effects by binding to and activating the CCR4 cell-surface receptor that belongs to the Giprotein-coupled receptor family. Enhanced expression of CCL17 as well as elevated recruitment of CCR4+ Th2 cells have been observed in asthma and atopic dermatitis. To date, most therapeutic strategies have focused on disrupting the chemokine/receptor interaction through use of chemokine receptor antagonists. We have discovered a novel class of small chemical molecules called ‘‘neutraligands’’ that bind to the chemokine itself, not to the receptor, and neutralize its biological activity. The concept has already been fully validated with the discovery of ‘‘chalcone 4’’, a small chemical that binds CXCL12, thereby preventing bronchial inflammation and hyperresponsiveness in animal models of asthma (Hachet-Haas et al., JBC 2008; Galzi et al., Pharmacol Ther 2010; Gasparik et al., ACS Med Chem Lett 2012; Daubeuf et al., JBC 2013). Our aim was to identify new CCL17 neutraligands, and evaluate their in vivo activity in mouse models of cutaneous and respiratory Th2driven allergic inflammation. Methods We have set up a cell-based high throughput assay to identify new CCL17 neutraligands. HEK293 cells were transfected with the CCL17 receptor, CCR4, together with the G protein G␣qi5 that will increase the intracellular Ca2+ response (FlexStation3) upon activation with CCL17. A library of pure natural substances, as well as a subset of the academic library of Strasbourg (1000 compounds) was screened. The hits were tested in two Th2-driven murine models: — an 8-day model of allergic hypereosinophilia to ovalbumin; — a model of allergic dermatitis to MC903 (calcipotriol). Results Two hit molecules were selected from the HTS assay, compounds A and B, with CCL17-neutralizing activity: when preincubated with the chemokine, they blocked CCL17-induced Ca2+ responses (2 g/mL; IC50s = 5 and 8 M, respectively); by contrast, when preincubated with the cells, i.e. with the CCR4 receptor, the Ca2+ responses were not affected, indicating the compounds were CCL17 neutraligands and not CCR4 receptor antagonists. We show that compounds A and B (350 mol/kg, I.P.) significantly reduced the number of cells collected in broncho-alveolar lavage fluid, in particular eosinophils (40 and 60% inhibition, respectively) in the model of allergic hypereosinophilia. In addition, compounds A and B (350 mol/kg) administered topically prevented MC903induced ear redness and thickness, and decreased plasma IgE levels (by 90%). Conclusion Our results show successful identification of CCL17 neutraligands that efficiently control Th2 inflammation, and will help understand the role of CCL17 in asthma and atopic dermatitis. Disclosure of interest The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.030 P06
Thymic stromal lymphopoietin induces cytokine production by human lung macrophages T. Victoni 1,2,3,∗ , C. Abrial 1 , H. Salvator 1 , M. Brollo 1 , S. Grassin Delyle 1 , V. Lagente 2 , E. Naline 1 , P. devillier 1 1 UPRES EA220-Université de Versailles Saint-Quentin, Hôpital Foch, France 2 UMR991 Inserm, Faculté de Pharmacie, Université de Rennes 1, Rennes, France 3 Laboratório de Reparo Tecidual, DHE/IBRAG/UERJ, Rio de Janeiro, Brazil
Introduction Increased bronchial smooth muscle mass is one of the key structural features of severe asthma. In adults, asthmatic airway smooth muscle cells (ASMC) demonstrate greater mitochondrial biogenesis associated with an increase in ASMC proliferation rate vs. non-asthmatic ASMC. However, to the best of our knowledge, there is no evidence that such a difference between asthmatic and non-asthmatic ASMC occurs in pre-school children. Thus, the primary aim of the study was to compare asthmatic and non-asthmatic ASMC proliferation and mitochondrial biogenesis in adults and pre-school children. The secondary aim was to assess the effect of factors released by the epithelium upon stimulation by environmental factors such as house dust mite and rhinovirus, on ASMC proliferation. Methods We cultured ASMC and bronchial epithelial cells (BEC) obtained by endobronchial biopsy from children and adults with severe asthma or undergoing bronchial endoscopy for other reasons. We then studied ASMC proliferation (cell counting and CFSE dye assay) in 10% fetal bovine serum (FBS), 0% FBS and after the addition of the BEC culture supernatant obtained after rhinovirus infection, house dust mite exposure or both. Mitochondrial mass and biogenesis were determined by western blot. Results Sixteen pre-school children with severe asthma,median aged 2.8 years, 4 control children (4.6 years), 13 adults with severe asthma (46.0 years) and 26 controls adults (65.3 years) were included. ASMC proliferation was increased in asthmatic adults, asthmatic pre-school and control children ASMC vs. control adults, with a cell doubling time of 35.0 ± 2.5 h, 24.8 ± 1.9 h, 22.6 ± 2.6 h and 47.4 ± 4.4 h, respectively (P < 0.05). This increased ASMC proliferation was associated with greater mitochondrial mass (porine rate: 50% in asthmatic adult ASMC, 52% in children ASMC vs. 20% in control adult ASMC) and biogenesis in asthmatic adult, asthmatic and control children ASMC vs. control adults (TFAM rate respectively at 44,9%, 48,1% and 62,2% vs. 18,4%). Rhinovirus and house dust mite exposure induced an increase of respectively 120,5% and 107.7% compared to the basal proliferation rate in asthmatic subjects only. Conclusion As previously described in adult asthmatics, ASMC proliferation is enhanced in both asthmatic and non-asthmatic children. This is associated with an increase in mitochondrial mass and biogenesis. ASMC proliferation is increased after a viral infection or an allergen exposure. Keywords Asthma; Allergy; Children The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.029 P05
Identification of CCL17 neutraligands targeting atopic diseases D. Abboud 1,4,∗ , F. Daubeuf 2,4 , V. Utard 1,4 , D. Bonnet 2,4 , M. Hibert 2,4 , P. Bernard 3 , J.L. Galzi 1,4 , N. Frossard 2,4 1 Biotechnologie et signalisation cellulaire, UMR 7242 CNRS, Université de Strasbourg, ESBS, Illkirch, France 2 Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France 3 GreenPharma, Orléans, France 4 LabEx Medalis, Strasbourg, France ∗ Corresponding author. E-mail address: [email protected] (D. Abboud)
315 Introduction Chemokines constitute a family of small cytokines that attract and activate leukocytes during the inflammatory response. Among them, the -chemokine CCL17 formerly known as thymus- and activation regulated chemokine (TARC), is involved in the development of atopic disorders such as asthma and atopic dermatitis. This chemokine exerts its biological effects by binding to and activating the CCR4 cell-surface receptor that belongs to the Giprotein-coupled receptor family. Enhanced expression of CCL17 as well as elevated recruitment of CCR4+ Th2 cells have been observed in asthma and atopic dermatitis. To date, most therapeutic strategies have focused on disrupting the chemokine/receptor interaction through use of chemokine receptor antagonists. We have discovered a novel class of small chemical molecules called ‘‘neutraligands’’ that bind to the chemokine itself, not to the receptor, and neutralize its biological activity. The concept has already been fully validated with the discovery of ‘‘chalcone 4’’, a small chemical that binds CXCL12, thereby preventing bronchial inflammation and hyperresponsiveness in animal models of asthma (Hachet-Haas et al., JBC 2008; Galzi et al., Pharmacol Ther 2010; Gasparik et al., ACS Med Chem Lett 2012; Daubeuf et al., JBC 2013). Our aim was to identify new CCL17 neutraligands, and evaluate their in vivo activity in mouse models of cutaneous and respiratory Th2driven allergic inflammation. Methods We have set up a cell-based high throughput assay to identify new CCL17 neutraligands. HEK293 cells were transfected with the CCL17 receptor, CCR4, together with the G protein G␣qi5 that will increase the intracellular Ca2+ response (FlexStation3) upon activation with CCL17. A library of pure natural substances, as well as a subset of the academic library of Strasbourg (1000 compounds) was screened. The hits were tested in two Th2-driven murine models: — an 8-day model of allergic hypereosinophilia to ovalbumin; — a model of allergic dermatitis to MC903 (calcipotriol). Results Two hit molecules were selected from the HTS assay, compounds A and B, with CCL17-neutralizing activity: when preincubated with the chemokine, they blocked CCL17-induced Ca2+ responses (2 g/mL; IC50s = 5 and 8 M, respectively); by contrast, when preincubated with the cells, i.e. with the CCR4 receptor, the Ca2+ responses were not affected, indicating the compounds were CCL17 neutraligands and not CCR4 receptor antagonists. We show that compounds A and B (350 mol/kg, I.P.) significantly reduced the number of cells collected in broncho-alveolar lavage fluid, in particular eosinophils (40 and 60% inhibition, respectively) in the model of allergic hypereosinophilia. In addition, compounds A and B (350 mol/kg) administered topically prevented MC903induced ear redness and thickness, and decreased plasma IgE levels (by 90%). Conclusion Our results show successful identification of CCL17 neutraligands that efficiently control Th2 inflammation, and will help understand the role of CCL17 in asthma and atopic dermatitis. Disclosure of interest The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.030 P06
Thymic stromal lymphopoietin induces cytokine production by human lung macrophages T. Victoni 1,2,3,∗ , C. Abrial 1 , H. Salvator 1 , M. Brollo 1 , S. Grassin Delyle 1 , V. Lagente 2 , E. Naline 1 , P. devillier 1 1 UPRES EA220-Université de Versailles Saint-Quentin, Hôpital Foch, France 2 UMR991 Inserm, Faculté de Pharmacie, Université de Rennes 1, Rennes, France 3 Laboratório de Reparo Tecidual, DHE/IBRAG/UERJ, Rio de Janeiro, Brazil