- Email: [email protected]
Thymic stromal lymphopoietin induces cytokine production by human lung macrophages
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
316 ∗
Corresponding author. E-mail address: [email protected] (T. Victoni)
Introduction The epithelial-derived cytokine, thymic stromal lymphopoietin (TSLP), has been identified as an important cytokine in allergic inflammation. Various studies demonstrated elevate levels and expression of TSLP in airways of asthmatic. Environmental factors such as toll-like receptor ligands, viruses, microbes, allergen sources, diesel exhaust and cigarette smoke trigger TSLP production by epithelial cells. It has been described that TSLP amplifies the differentiation of peritoneal macrophages and bone marrow derived macrophages in M2-type in mice. Our aims were to investigate the role of TSLP in the M1/M2 polarization of human lung macrophages (LM). Material and methods Lung macrophages were isolated from tissues obtained after surgical resection for cancer. Macrophages were treated with LPS (10 ng/mL) or IL-4 (10 ng/mL) in presence or not of TSLP (10—30 ng/mL) for 24 h or 48 h. Production of cytokines of the M1-type (TNF-␣, CCL-2, CXCL-8 and CXCL-1) and the M2-type (CCL-13, CCL-17, CCL-18 and CCL-22) was determined by ELISA and cytokine expression by RT-qPCR. Results Exposure to TSLP did not alter the production of TNF-␣, CXCL-8 and CXCL-1 either in unstimulated LM or in LPS-stimulated LM. However, CCL-2 production was increased by TSLP at 20 ng/mL in unstimulated LM. In addition, TSLP (10 ng/mL but not 30 ng/mL) increased M2-type cytokine production (CCL-13 and CCL-17, but did not CCL-18 and CCL-22). TSLP also increased IL4-induced CCL-17 and CCL-22. Expression of CCL-17 and CCL-13 was also increased by TSLP in unstimulated LM, and expression of CCL-17 and CCL-22 was increased in IL-4-stimulantes LM. Conclusion Our results show that TSLP can activate unstimulated LM and modulate the IL-4-induced polarization of LM in the M2 subtype. Keywords Asthma; Allergic Acknowledgements CAPES/COFECUB project. Disclosure of interest The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.031 P07
Consecutive food and respiratory allergies amplify systemic and gut but not lung outcomes in mouse P. Gourbeyre 1 , G. Bouchaud 1,∗ , T. Bihouée-Roussey 2 , P. Aubert 3 , D. Lair 2 , M.A. Cheminant 2 , S. Denery-Papini 1 , M. Neunlist 3 , A. Magnan 2,4 , M. Bodinier 1 1 INRA, UR1268 BIA, rue de la géraudière, BP 71627, 44316 Nantes, France 2 Inserm UMR1087, CNRS UMR 6291, 44000 Nantes, France 3 Inserm UMR S 913, Institut des Maladies de l’Appareil Digestif (IMAD), Faculté de Médecine, 44000 Nantes, France 4 CHU DE Nantes, L’institut du thorax, Service de Pneumologie, Plate-Forme Transversale d’Allergologie, 44000 Nantes, France ∗ Corresponding author. E-mail address: [email protected] (G. Bouchaud) Aims Increasing clinical data suggest a link between food allergy and the later development of respiratory allergy. This progression may be triggered by exposures to different allergens but the mechanism implicated remains unknown. This study aimed to identify the impact of a first exposure to food allergen on the development of a new form of allergy caused by exposure to a novel allergen using a mouse model. Method In our model, mice were intraperitoneally sensitized to wheat proteins (modified gliadins) to induce a systemic response, then they were exposed orally to the same allergen and finally they were intranasally exposed to HDM (Dermatophagoides farinae
J2R (Posters) extract) a respiratory allergen without adjuvant to assess an impact on lung mucosa. Results After food and respiratory allergen exposures, mice displayed stronger amount of blood markers: IgE specific and histamine. Moreover, splenocyte secretion of IL-4 and IL-17 were increased whereas weaker levels of IFNg were observed. In parallel, Peyer patches lymphocytes secreted higher amount of IL-4 with a decreased in IFNg, IL-10 and TGF-b productions. These mice exhibited intestine damages, higher paracellular flux and modification of transcellular permeability. In contrast, airway hyperresponsiveness, inflammatory cells and cytokines in lung remained unchanged compared to the respiratory allergy model. Conclusion We show that dual exposure induces a raise in specific IgE and local Th2 and Th17 cytokines secretion before triggering phase. During the latter, gut morphology and functions were affected but not lung in dual exposed mice compared to single one underlying the organospecific impact. Altogether, our data make a step further in the elucidation of the mechanisms linking allergy history to immunological and clinical status potentially linked to atopic march development. Keywords Asthma; Allergy Disclosure of interest The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.032 P08
IL-33 is regulated by the Ubiquitin/Proteasome system C. Glineur ∗ , B. Gross , P. Marquillies , C. Duez , A. Tsicopoulos Inserm U 1019, CNRS UMR 8204, Centre d’Infection et d’Immunité de Lille, Institut Pasteur de Lille, Équipe immunité pulmonaire, 1, rue du Pr-Calmette, BP 245, 59019 Lille Cedex, France ∗ Corresponding author. E-mail address: [email protected] (C. Glineur) Introduction IL-33 is a member of the IL-1 family of cytokines produced importantly by endothelial cells. Nuclear IL-33 protein acts as a transcriptional repressor. Secreted IL-33 is involved in Th2-mediated inflammatory responses in allergic diseases such as asthma. IL-33 acts through activation of the ST2L receptor recruiting of adaptor proteins and leading to formation of an E3 ligase complex, which introduces Lys 48-linked polyubiquitin chains in ST2 leading to its degradation in the cytoplasm. The increased level of IL-33 in allergic disorders and its correlation with disease severity and the effectiveness of anti-IL-33 antibody and soluble form of ST2 in alleviating symptoms of asthma in mice support the view of IL-33 as a therapeutic target in allergic diseases. IL-33 is synthesized as a full-length form migrating at 30—34 kDa on gels. Different proteases can process full-length IL-33 to biologically active forms. Methods Cells were transfected with a construct expressing an EGFP-IL-33 protein and analyzed by cytometry in presence or absence of proteasome inhibitor or E1 enzyme inhibitor. Ubiquitinated IL-33 proteins were analyzed by western blot. Proteasome and NFB activity involved in the allergen-dependent induction of IL-33 was also evaluated. Results We show that in human and murine lung tissues, proteolytic products or products of shorter splice variants can be found in addition to higher molecular weight forms. However, in endothelial cells, the full-length and shorter forms of IL-33 are poorly detected suggesting that the IL-33 protein may be labile in proliferating cells. These experiments demonstrate that IL-33 is a target for ubiquitination but the role of this modification is unclear. Moreover, we identified high molecular weight forms of IL-33 probably corresponding to post-translational modified forms or complexes with other proteins, which could stabilize IL-33 protein. Conclusion Altogether these studies suggest that posttranslational modifications of IL-33 occur in endothelial cells
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
316 ∗
Corresponding author. E-mail address: [email protected] (T. Victoni)
Introduction The epithelial-derived cytokine, thymic stromal lymphopoietin (TSLP), has been identified as an important cytokine in allergic inflammation. Various studies demonstrated elevate levels and expression of TSLP in airways of asthmatic. Environmental factors such as toll-like receptor ligands, viruses, microbes, allergen sources, diesel exhaust and cigarette smoke trigger TSLP production by epithelial cells. It has been described that TSLP amplifies the differentiation of peritoneal macrophages and bone marrow derived macrophages in M2-type in mice. Our aims were to investigate the role of TSLP in the M1/M2 polarization of human lung macrophages (LM). Material and methods Lung macrophages were isolated from tissues obtained after surgical resection for cancer. Macrophages were treated with LPS (10 ng/mL) or IL-4 (10 ng/mL) in presence or not of TSLP (10—30 ng/mL) for 24 h or 48 h. Production of cytokines of the M1-type (TNF-␣, CCL-2, CXCL-8 and CXCL-1) and the M2-type (CCL-13, CCL-17, CCL-18 and CCL-22) was determined by ELISA and cytokine expression by RT-qPCR. Results Exposure to TSLP did not alter the production of TNF-␣, CXCL-8 and CXCL-1 either in unstimulated LM or in LPS-stimulated LM. However, CCL-2 production was increased by TSLP at 20 ng/mL in unstimulated LM. In addition, TSLP (10 ng/mL but not 30 ng/mL) increased M2-type cytokine production (CCL-13 and CCL-17, but did not CCL-18 and CCL-22). TSLP also increased IL4-induced CCL-17 and CCL-22. Expression of CCL-17 and CCL-13 was also increased by TSLP in unstimulated LM, and expression of CCL-17 and CCL-22 was increased in IL-4-stimulantes LM. Conclusion Our results show that TSLP can activate unstimulated LM and modulate the IL-4-induced polarization of LM in the M2 subtype. Keywords Asthma; Allergic Acknowledgements CAPES/COFECUB project. Disclosure of interest The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.031 P07
Consecutive food and respiratory allergies amplify systemic and gut but not lung outcomes in mouse P. Gourbeyre 1 , G. Bouchaud 1,∗ , T. Bihouée-Roussey 2 , P. Aubert 3 , D. Lair 2 , M.A. Cheminant 2 , S. Denery-Papini 1 , M. Neunlist 3 , A. Magnan 2,4 , M. Bodinier 1 1 INRA, UR1268 BIA, rue de la géraudière, BP 71627, 44316 Nantes, France 2 Inserm UMR1087, CNRS UMR 6291, 44000 Nantes, France 3 Inserm UMR S 913, Institut des Maladies de l’Appareil Digestif (IMAD), Faculté de Médecine, 44000 Nantes, France 4 CHU DE Nantes, L’institut du thorax, Service de Pneumologie, Plate-Forme Transversale d’Allergologie, 44000 Nantes, France ∗ Corresponding author. E-mail address: [email protected] (G. Bouchaud) Aims Increasing clinical data suggest a link between food allergy and the later development of respiratory allergy. This progression may be triggered by exposures to different allergens but the mechanism implicated remains unknown. This study aimed to identify the impact of a first exposure to food allergen on the development of a new form of allergy caused by exposure to a novel allergen using a mouse model. Method In our model, mice were intraperitoneally sensitized to wheat proteins (modified gliadins) to induce a systemic response, then they were exposed orally to the same allergen and finally they were intranasally exposed to HDM (Dermatophagoides farinae
J2R (Posters) extract) a respiratory allergen without adjuvant to assess an impact on lung mucosa. Results After food and respiratory allergen exposures, mice displayed stronger amount of blood markers: IgE specific and histamine. Moreover, splenocyte secretion of IL-4 and IL-17 were increased whereas weaker levels of IFNg were observed. In parallel, Peyer patches lymphocytes secreted higher amount of IL-4 with a decreased in IFNg, IL-10 and TGF-b productions. These mice exhibited intestine damages, higher paracellular flux and modification of transcellular permeability. In contrast, airway hyperresponsiveness, inflammatory cells and cytokines in lung remained unchanged compared to the respiratory allergy model. Conclusion We show that dual exposure induces a raise in specific IgE and local Th2 and Th17 cytokines secretion before triggering phase. During the latter, gut morphology and functions were affected but not lung in dual exposed mice compared to single one underlying the organospecific impact. Altogether, our data make a step further in the elucidation of the mechanisms linking allergy history to immunological and clinical status potentially linked to atopic march development. Keywords Asthma; Allergy Disclosure of interest The authors have not supplied their declaration of conflict of interest. http://dx.doi.org/10.1016/j.rmr.2015.02.032 P08
IL-33 is regulated by the Ubiquitin/Proteasome system C. Glineur ∗ , B. Gross , P. Marquillies , C. Duez , A. Tsicopoulos Inserm U 1019, CNRS UMR 8204, Centre d’Infection et d’Immunité de Lille, Institut Pasteur de Lille, Équipe immunité pulmonaire, 1, rue du Pr-Calmette, BP 245, 59019 Lille Cedex, France ∗ Corresponding author. E-mail address: [email protected] (C. Glineur) Introduction IL-33 is a member of the IL-1 family of cytokines produced importantly by endothelial cells. Nuclear IL-33 protein acts as a transcriptional repressor. Secreted IL-33 is involved in Th2-mediated inflammatory responses in allergic diseases such as asthma. IL-33 acts through activation of the ST2L receptor recruiting of adaptor proteins and leading to formation of an E3 ligase complex, which introduces Lys 48-linked polyubiquitin chains in ST2 leading to its degradation in the cytoplasm. The increased level of IL-33 in allergic disorders and its correlation with disease severity and the effectiveness of anti-IL-33 antibody and soluble form of ST2 in alleviating symptoms of asthma in mice support the view of IL-33 as a therapeutic target in allergic diseases. IL-33 is synthesized as a full-length form migrating at 30—34 kDa on gels. Different proteases can process full-length IL-33 to biologically active forms. Methods Cells were transfected with a construct expressing an EGFP-IL-33 protein and analyzed by cytometry in presence or absence of proteasome inhibitor or E1 enzyme inhibitor. Ubiquitinated IL-33 proteins were analyzed by western blot. Proteasome and NFB activity involved in the allergen-dependent induction of IL-33 was also evaluated. Results We show that in human and murine lung tissues, proteolytic products or products of shorter splice variants can be found in addition to higher molecular weight forms. However, in endothelial cells, the full-length and shorter forms of IL-33 are poorly detected suggesting that the IL-33 protein may be labile in proliferating cells. These experiments demonstrate that IL-33 is a target for ubiquitination but the role of this modification is unclear. Moreover, we identified high molecular weight forms of IL-33 probably corresponding to post-translational modified forms or complexes with other proteins, which could stabilize IL-33 protein. Conclusion Altogether these studies suggest that posttranslational modifications of IL-33 occur in endothelial cells