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The Anti-inflammatory Effects of IVIg: Induction and Development of Regulatory-T Cells in a Murine Model of Asthma
246
UDP Receptor P2Y6 Negatively Regulates Pulmonary Inflammation Induced by the Exposure to House Dust Mite G. Giannattasio1,2, J. R. Boyce1, J. A. Boyce1,2; 1Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA, 2Department of Medicine, Harvard Medical School, Boston, MA. RATIONALE: The G-protein-coupled receptor P2Y6 is the high-affinity receptor for the nucleotide uridine diphosphate (UDP) that is expressed in the lung on epithelial cells, vascular smooth muscle cells, monocyte/macrophages and mast cells. In this study we evaluated the role of P2Y6 in the pathogenesis of the immune response in an in vivo model of pulmonary allergic inflammation. METHODS: WT and P2Y6-/- littermates were challenged intranasally with dust mite allergen Der f 1 intranasally twice/week for 3 weeks. Bronchoalveolar lavage (BAL) fluid cell counts and lung histologic analysis were performed. Lung mRNA encoding P2Y6 receptor, proinflammatory cytokines, and goblet cell-associated proteins MUC5AC and CLCA3 were measured by qPCR. Cytokine generation by restimulated parabronchial lymph node cells was measured. RESULTS: The mRNA of P2Y6 receptor in the lung tissue was upregulated in WT mice exposed to the dust mite compared with saline-treated controls. Compared to WT controls, Der f-treated P2Y6-/- mice showed strikingly increased airway and tissue eosinophilia and goblet cell metaplasia (p < 0.01). These findings were associated with the presence of significantly higher levels of IL-13, MUC5AC and CLCA3 mRNAs within the lung tissue of P2Y6-/- mice (p < 0.05). Moreover, ex vivo restimulation of lymph node cells with the allergen induced the release of 2-fold higher amounts of IL-5 and IL-13 from P2Y6-/- compared to WT mice. CONCLUSIONS: We provide in vivo evidence that the high-affinity receptor of UDP, P2Y6, negatively regulates allergen-induced lung inflammation. These data suggest that the axis UDP/P2Y6 receptor may represent a therapeutic target in asthma and other inflammatory diseases of the lung.
247
The Effects of a Pim Kinase Inhibitor on Allergen-Induced Airway Hyperresponsiveness (AHR) and Inflammation Y. Shin1, K. Takeda1, Y. Jia1, L. Jackson2, A. D. Wright2, L. Carter2, J. Zuzack2, S. Gross2, J. Robinson2, E. Hicken2, M. Munson2, E. W. Gelfand1; 1National Jewish Health, Denver, CO, 2Array Biopharma, Boulder, CO. RATIONALE: Pim kinases are a family of 3 serine/threonine kinases whose activity is regulated by expression and can be induced by cytokines involved in allergy and asthma. Pim kinases play a role in the survival of eosinophils and basophils and their expression is increased in the BAL of asthmatic patients after allergen provocation. We investigated effects of Pim kinase inhibition on Th2 cells and following allergen sensitization and challenge. METHODS: ARRY-770 is an orally available Pim inhibitor with cellular IC50 5 110 nM. Th2 cells were generated in vitro using DO11.10 CD4+ T cells. Proliferation and cytokines were measured using FACS and ELISAs. BALB/c mice were sensitized by intraperitoneal injection of OVA (with alum) on days 0 and 14, followed by three nebulized OVA challenges on days 28-30. On each challenge day, 100mg/kg ARRY-770 was administered orally BID. AHR and BAL assessments were performed 48hrs after final airway challenge. RESULTS: ARRY-770 prevented development of Th2 effector cells and inhibited established Th2 effector cells from proliferating and producing IL-4. Following treatment with ARRY-770, development of AHR and eosinophilic airway inflammation were significantly reduced. In BAL fluid, levels of IL-4, IL-5, IL-13 and IFNg were significantly lower than in control groups. CONCLUSIONS: ARRY-770, a novel Pim kinase inhibitor, decreases Th2 responses and was effective in reducing AHR, airway inflammation and cytokine production in sensitized and challenged mice. These data identify an important role for this kinase in the development of allergen-induced lung allergic responses.
248
The Anti-inflammatory Effects of IVIg: Induction and Development of Regulatory-T Cells in a Murine Model of Asthma A. H. Massoud1,2, J. Guay1, S. Audusseau1, E. Bjur3, C. A. Piccirillo3, M. W. Mourad2, B. D. Mazer1; 1McGill University/Meakins Christie Laboratories, Montreal, QC, CANADA, 2Universite du Montreal, Montreal, QC, CANADA, 3McGill University, Montreal, QC, CANADA. RATIONALE: Intravenous Immunoglobulin (IVIg) has been utilized to treat severe asthma, but the mechanism of action has not been fully investigated. We have shown that administration of IVIg to OVA-sensitized and challenged mice increases CD4+-CD25+-Foxp3+ Treg cells. We investigated if IVIg induced Tregs from conventional T-cells in a model of allergic-airway disease. METHODS: 6-week-old C57BL/6 mice were sensitized and challenged intranasally with 1% OVA. IVIg or HSA was administered I.P. 24h before the first challenge. CD4+ T-cells were obtained from Tg mice harboring a knock-in allele, encoding a GFP-Foxp3 fusion protein (Foxp3GFP). Three million CD4+FOXP3- cells were transferred I.V. to syngeneic WT animals prior to sensitization. GFP expression was analyzed by flow cytometry on cell suspensions from lungs and lymphoid organs. The T-cell response to OVA in vitro was measured by [H3]Thymidine incorporation, using co-culture of splenic T-cells from HSA-treated animals with splenic DC from IVIg-treated mice and vice versa (T-cells: DC 10:1), RESULTS: There was a 3-4 fold increase in Treg in OVA-IVIg-OVA mice compared to OVA-HAS-OVA in spleens, lungs and draining LN, accompanied by increases in L-selectin induced by IVIg in an antigen-specific fashion. T-cells derived from IVIg-treated animals co-cultured with DC from HSA-treated animals remain hypo-responsive to OVA re-stimulation ex vivo. DCs from IVIg-treated mice did not reduce T-cell proliferation derived from HAS-treated mice. CONCLUSIONS: In an OVA-driven model of allergic-airways disease, IVIg induces Tregs in inflamed pulmonary tissue as well as systemic lymphoid organs. The therapeutic effects of IVIg appear to be via direct action on Treg development.
249
Education of Allied Health Personnel Key in Improving Provider Practice in a Large Health System D. VanSickle, K. Grossman, A. Sy, S. Bernstein, G. Sanders; University of Michigan Health System, Ann Arbor, MI. RATIONALE: NHLBI guidelines recommend that asthma patients receive an asthma action plan (AAP) and comprehensive disease education. Many primary care clinics are unable to fulfill that recommendation due to time and personnel constraints: provider visits are too short and most allied health personnel lack asthma management training. In December 2006, only 7% of asthma patients in our health system had an AAP documented in the electronic medical record (EMR). We assessed the effect of training allied health personnel, primarily nurses, on improving EMR documentation of AAPs. METHODS: In 2007, asthma nurse specialists educated allied health personnel at receptive primary care health centers to use our standardized asthma disease management tools, including patient education materials, documentation templates, medication device training kits, and AAPs. EMR documentation of AAPs was compared to health centers whose staff did not receive training. All physicians in our health system received email instruction on use of AAPs and patient education materials. RESULTS: Overall, the percentage of patients with a documented AAP increased from 7% in December 2006 to 41% in June 2009 (overall p < 0.0001). Health centers whose staff received training (n 5 11) improved in each annual June registry report from 15% to 22% to 43% during this period, as compared to between 5% to 9% to 26% at health centers whose staff were not trained (n 5 4) (overall p < 0.0001). CONCLUSION: On-site training of allied health personnel to provide standardized asthma patient education yielded sustained improvements in AAP use and documentation, persisting at least 2 years after the encounter.
SATURDAY
Abstracts AB63
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
UDP Receptor P2Y6 Negatively Regulates Pulmonary Inflammation Induced by the Exposure to House Dust Mite G. Giannattasio1,2, J. R. Boyce1, J. A. Boyce1,2; 1Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA, 2Department of Medicine, Harvard Medical School, Boston, MA. RATIONALE: The G-protein-coupled receptor P2Y6 is the high-affinity receptor for the nucleotide uridine diphosphate (UDP) that is expressed in the lung on epithelial cells, vascular smooth muscle cells, monocyte/macrophages and mast cells. In this study we evaluated the role of P2Y6 in the pathogenesis of the immune response in an in vivo model of pulmonary allergic inflammation. METHODS: WT and P2Y6-/- littermates were challenged intranasally with dust mite allergen Der f 1 intranasally twice/week for 3 weeks. Bronchoalveolar lavage (BAL) fluid cell counts and lung histologic analysis were performed. Lung mRNA encoding P2Y6 receptor, proinflammatory cytokines, and goblet cell-associated proteins MUC5AC and CLCA3 were measured by qPCR. Cytokine generation by restimulated parabronchial lymph node cells was measured. RESULTS: The mRNA of P2Y6 receptor in the lung tissue was upregulated in WT mice exposed to the dust mite compared with saline-treated controls. Compared to WT controls, Der f-treated P2Y6-/- mice showed strikingly increased airway and tissue eosinophilia and goblet cell metaplasia (p < 0.01). These findings were associated with the presence of significantly higher levels of IL-13, MUC5AC and CLCA3 mRNAs within the lung tissue of P2Y6-/- mice (p < 0.05). Moreover, ex vivo restimulation of lymph node cells with the allergen induced the release of 2-fold higher amounts of IL-5 and IL-13 from P2Y6-/- compared to WT mice. CONCLUSIONS: We provide in vivo evidence that the high-affinity receptor of UDP, P2Y6, negatively regulates allergen-induced lung inflammation. These data suggest that the axis UDP/P2Y6 receptor may represent a therapeutic target in asthma and other inflammatory diseases of the lung.
247
The Effects of a Pim Kinase Inhibitor on Allergen-Induced Airway Hyperresponsiveness (AHR) and Inflammation Y. Shin1, K. Takeda1, Y. Jia1, L. Jackson2, A. D. Wright2, L. Carter2, J. Zuzack2, S. Gross2, J. Robinson2, E. Hicken2, M. Munson2, E. W. Gelfand1; 1National Jewish Health, Denver, CO, 2Array Biopharma, Boulder, CO. RATIONALE: Pim kinases are a family of 3 serine/threonine kinases whose activity is regulated by expression and can be induced by cytokines involved in allergy and asthma. Pim kinases play a role in the survival of eosinophils and basophils and their expression is increased in the BAL of asthmatic patients after allergen provocation. We investigated effects of Pim kinase inhibition on Th2 cells and following allergen sensitization and challenge. METHODS: ARRY-770 is an orally available Pim inhibitor with cellular IC50 5 110 nM. Th2 cells were generated in vitro using DO11.10 CD4+ T cells. Proliferation and cytokines were measured using FACS and ELISAs. BALB/c mice were sensitized by intraperitoneal injection of OVA (with alum) on days 0 and 14, followed by three nebulized OVA challenges on days 28-30. On each challenge day, 100mg/kg ARRY-770 was administered orally BID. AHR and BAL assessments were performed 48hrs after final airway challenge. RESULTS: ARRY-770 prevented development of Th2 effector cells and inhibited established Th2 effector cells from proliferating and producing IL-4. Following treatment with ARRY-770, development of AHR and eosinophilic airway inflammation were significantly reduced. In BAL fluid, levels of IL-4, IL-5, IL-13 and IFNg were significantly lower than in control groups. CONCLUSIONS: ARRY-770, a novel Pim kinase inhibitor, decreases Th2 responses and was effective in reducing AHR, airway inflammation and cytokine production in sensitized and challenged mice. These data identify an important role for this kinase in the development of allergen-induced lung allergic responses.
248
The Anti-inflammatory Effects of IVIg: Induction and Development of Regulatory-T Cells in a Murine Model of Asthma A. H. Massoud1,2, J. Guay1, S. Audusseau1, E. Bjur3, C. A. Piccirillo3, M. W. Mourad2, B. D. Mazer1; 1McGill University/Meakins Christie Laboratories, Montreal, QC, CANADA, 2Universite du Montreal, Montreal, QC, CANADA, 3McGill University, Montreal, QC, CANADA. RATIONALE: Intravenous Immunoglobulin (IVIg) has been utilized to treat severe asthma, but the mechanism of action has not been fully investigated. We have shown that administration of IVIg to OVA-sensitized and challenged mice increases CD4+-CD25+-Foxp3+ Treg cells. We investigated if IVIg induced Tregs from conventional T-cells in a model of allergic-airway disease. METHODS: 6-week-old C57BL/6 mice were sensitized and challenged intranasally with 1% OVA. IVIg or HSA was administered I.P. 24h before the first challenge. CD4+ T-cells were obtained from Tg mice harboring a knock-in allele, encoding a GFP-Foxp3 fusion protein (Foxp3GFP). Three million CD4+FOXP3- cells were transferred I.V. to syngeneic WT animals prior to sensitization. GFP expression was analyzed by flow cytometry on cell suspensions from lungs and lymphoid organs. The T-cell response to OVA in vitro was measured by [H3]Thymidine incorporation, using co-culture of splenic T-cells from HSA-treated animals with splenic DC from IVIg-treated mice and vice versa (T-cells: DC 10:1), RESULTS: There was a 3-4 fold increase in Treg in OVA-IVIg-OVA mice compared to OVA-HAS-OVA in spleens, lungs and draining LN, accompanied by increases in L-selectin induced by IVIg in an antigen-specific fashion. T-cells derived from IVIg-treated animals co-cultured with DC from HSA-treated animals remain hypo-responsive to OVA re-stimulation ex vivo. DCs from IVIg-treated mice did not reduce T-cell proliferation derived from HAS-treated mice. CONCLUSIONS: In an OVA-driven model of allergic-airways disease, IVIg induces Tregs in inflamed pulmonary tissue as well as systemic lymphoid organs. The therapeutic effects of IVIg appear to be via direct action on Treg development.
249
Education of Allied Health Personnel Key in Improving Provider Practice in a Large Health System D. VanSickle, K. Grossman, A. Sy, S. Bernstein, G. Sanders; University of Michigan Health System, Ann Arbor, MI. RATIONALE: NHLBI guidelines recommend that asthma patients receive an asthma action plan (AAP) and comprehensive disease education. Many primary care clinics are unable to fulfill that recommendation due to time and personnel constraints: provider visits are too short and most allied health personnel lack asthma management training. In December 2006, only 7% of asthma patients in our health system had an AAP documented in the electronic medical record (EMR). We assessed the effect of training allied health personnel, primarily nurses, on improving EMR documentation of AAPs. METHODS: In 2007, asthma nurse specialists educated allied health personnel at receptive primary care health centers to use our standardized asthma disease management tools, including patient education materials, documentation templates, medication device training kits, and AAPs. EMR documentation of AAPs was compared to health centers whose staff did not receive training. All physicians in our health system received email instruction on use of AAPs and patient education materials. RESULTS: Overall, the percentage of patients with a documented AAP increased from 7% in December 2006 to 41% in June 2009 (overall p < 0.0001). Health centers whose staff received training (n 5 11) improved in each annual June registry report from 15% to 22% to 43% during this period, as compared to between 5% to 9% to 26% at health centers whose staff were not trained (n 5 4) (overall p < 0.0001). CONCLUSION: On-site training of allied health personnel to provide standardized asthma patient education yielded sustained improvements in AAP use and documentation, persisting at least 2 years after the encounter.
SATURDAY
Abstracts AB63
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2