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Inhibition of airways hyperresponsiveness by a chimeric STAT-6 peptide in a murine model of allergic rhinitis and asthma
S64 Abstracts
SATURDAY
Prolonged Effect of Flt3-L Plasmid to Protect Against Allergic Airway Inflammation and Airway Hyperresponsiveness J. H. Edwan1, J. E. Talmadge2, D. K. Agrawal1,3; 1Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, 2Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 3Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE. RATIONALE: We have previously reported that pUMVC3-hFLex, a plasmid, mammalian expression vector for the secretion of Flt3-Ligand (Flt3-L) reversed established allergic airway inflammation in an ovalbumin (OVA) induced mouse model of asthma. The present experiments were undertaken to examine the duration of effect of pUMVC3-hFLex treatment on a murine model of allergic airway inflammation. METHODS: Allergic airway inflammation was established in Balb/c mice by sensitization and challenge with ovalbumin (OVA), pUMVC3hFLex or pUMVC3 were administered by injection into the muscle interior tibialis. We then examined the response to therapy on airway hyperresponsiveness (AHR) to methacholine and airway inflammation 24 days after the final dose of treatment. Serum was collected to measure cytokines and immunoglobulin levels. Cytospin slides were prepared from bronchoalveolar lavage fluid (BALF) and total and differential cells were counted. RESULTS: pUMVC3-hFLex treatment completely reversed established AHR and this effect lasted for at least for 24 days after final treatment (P < 0.001). pUMVC3-hFLex treatment significantly reduced serum interleukin- (IL-) 4 levels (P < 0.001), BALF IL-10 was significantly increased (P <0.05), serum IgG2a levels were significantly attenuated (P < 0.05) . Total BALF cellularity and as well as eosinophilia were also reduced (P < 0.05). CONCLUSIONS: These results indicate that pUMVC3-hFLex treatment can reverse inflammation and AHR and maintain airway protection in experimental asthma model, and might provide a novel approach for treating asthma. Funding: NIH RO1HL070885 to DKA
254
Suppression of Established Experimental Asthma by Toll-Like Receptor 7 Activation S. Sel1, S. Sel1, M. Wegmann1, H. Garn1, G. Alber2, H. Renz1; 1Department of Clinical Chemistry and Molecular Diagnostics, Biomedical Research Center, Marburg, GERMANY, 2Institute of Immunology, University of Leipzig, Leipzig, GERMANY. RATIONALE: The aim of our study was to evaluate the impact of Tolllike receptor 7 (TLR-7) activation on established allergen triggered asthma in the mouse. METHODS: Experimental asthma was established in Balb/c mice by i.p. ovalbumin (OVA) sensitization, followed by repeated OVA aerosol challenges (primary response). Secondary response was induced two weeks later. Before and during secondary challenges mice were treated with R848 (50 g/ injection), a synthetic TLR-7 ligand. RESULTS: Subcutaneous R-848 application reversed established allergic asthma by suppressing airway eosinophilia, inflammation of the airway mucosa, OVA-specific IgE production and normalizing airway responsiveness. The same effect was achieved following intranasal R-848 application except for the presence of inflammatory cells in airway mucosa. Experiments with IL-12 -/- mice showed that the R-848 suppressing effects on airway hyperresponsiveness and mucosal inflammation were mediated via IL-12. In contrast, suppression of airway eosinophilia, IL-5 production and IgE titer were also present in IL12 -/- mice. CONCLUSIONS: These data demonstrate that the activation of TLR-7 plays an important role to interfere with established experimental asthma. Some of the observed effects are mediated via IL-12. Funding: Philipps-University of Marburg
255
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
STAT4 Deficiency Exaggerates Airway Hyperresponsiveness (AHR) After Repeated Ovalbumin (OVA) Challenge S. Matsubara, T. Kodama, K. Takeda, N. Miyahara, A. Joetham, C. Taube, A. Dakhama, J. Park, T. Koya, E. W. Gelfand; Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO. RATIONALE: In the mouse, AHR and airway allergic inflammation progressively decrease following repeated allergen challenge. It has been suggested that the Th1-promoting cytokine interleukin (IL)-12 is capable of inhibiting persistent AHR and airway inflammation. Signal transducer and activator of transcription-4 (STAT4) is phosphorylated after binding of IL-12 to its receptor. The aim of this study was to investigate the influence of STAT4 deficiency on the development of AHR and airway inflammation after repeated allergen challenge. METHODS: STAT4-deficient mice and their congenic littermates were sensitized to OVA by intraperitoneal injection (twice) and subsequently challenged (via the airways) to 1% OVA in a short term (3 times, 3 days) and long term (11 times, 5 weeks) protocol. Fourty-eight hours after the last OVA challenge, AHR and bronchoalveolar lavage (BAL) were assayed. RESULTS: STAT4 deficiency did not influence pulmonary eosinophilia in either the short- or long-term OVA-challenged mice. STAT4 deficiency significantly (p<0.05) enhanced AHR in the long-term OVA challenged mice, but not in the short-term challenged mice. Serum levels of IgE and Th2 cytokine levels in BAL fluid, IL-4, IL-5 and IL-13, were decreased following long-term OVA challenge compared with short-term challenge, but STAT4 deficiency did not alter cytokine levels in BAL fluid. CONCLUSION: These results suggest that the IL-12-STAT4 signaling pathway plays an important role in the regulation of persistent AHR without effects on Th2 cytokines or serum IgE after repeated allergen challenge. Funding: NIH grants HL-61005, HL-36577, and EPA grant R825702
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Inhibition of Airways Hyperresponsiveness by a Chimeric STAT-6 Peptide in a Murine Model of Allergic Rhinitis and Asthma Y. Wang, C. McCusker, E. Fixman; Montreal Children’s Hospital Research Institute and Meakins-Chritie Laboratories, McGill University, Montreal, PQ, CANADA. RATIONALE: Allergic rhinitis and asthma are characterized by airway hyperresponsiveness (AHR) to a variety of specific and nonspecific stimuli and associated with increased production of Th2 cytokines, including IL-4 and IL-13. Activation of IL-4/IL-13 receptors containing the IL-4R alpha subunit induces tyrosine phosphorylation, dimerization and nuclear translocation of signal transducer and activator of transcription protein 6 (STAT-6). We investigated whether a chimeric STAT-6 peptide inhibited AHR in a murine model of allergic rhinitis and asthma. METHODS: Non-anesthetized 6~8 week old BALB/C mice received repeated IN application of 1%OVA, 5 microlitres in each nostril, and were then challenged, 2 weeks later, with IN OVA for 5 days. Thirty minutes before each challenges, the mice received IN chimeric STAT-6 peptide. A Change in AHR to methacholine (MCH) was used as a physiologic measure of allergen induced bronchial reactivity. RESULTS: OVA dependent AHR to MCH was significant decreased in a dose dependent manner in mice that received chimeric STAT-6 peptide compared with positive controls (P<0.01). Inflammatory cells in bronchoalveolar lavage fluid were also significantly reduced in STAT 6 peptide treated animals. Finally IL-13 expression in bronchoalveolar lavage fluid from STAT-6 peptide treated mice decreased in a dose dependent manner (P<0.05). CONCLUSIONS: These data demonstrate that IN application of a chimeric STAT-6 peptide inhibited AHR and airway inflammation in a murine model of asthma and allergic rhinitis. This chimeric peptide, delivered intranasally, thus has significant therapeutic potential for the management of asthma and allergic rhinitis. Funding: CIHR & FRSQ& Montreal Children’s Hospital Research Institute
257
SATURDAY
Prolonged Effect of Flt3-L Plasmid to Protect Against Allergic Airway Inflammation and Airway Hyperresponsiveness J. H. Edwan1, J. E. Talmadge2, D. K. Agrawal1,3; 1Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, 2Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 3Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE. RATIONALE: We have previously reported that pUMVC3-hFLex, a plasmid, mammalian expression vector for the secretion of Flt3-Ligand (Flt3-L) reversed established allergic airway inflammation in an ovalbumin (OVA) induced mouse model of asthma. The present experiments were undertaken to examine the duration of effect of pUMVC3-hFLex treatment on a murine model of allergic airway inflammation. METHODS: Allergic airway inflammation was established in Balb/c mice by sensitization and challenge with ovalbumin (OVA), pUMVC3hFLex or pUMVC3 were administered by injection into the muscle interior tibialis. We then examined the response to therapy on airway hyperresponsiveness (AHR) to methacholine and airway inflammation 24 days after the final dose of treatment. Serum was collected to measure cytokines and immunoglobulin levels. Cytospin slides were prepared from bronchoalveolar lavage fluid (BALF) and total and differential cells were counted. RESULTS: pUMVC3-hFLex treatment completely reversed established AHR and this effect lasted for at least for 24 days after final treatment (P < 0.001). pUMVC3-hFLex treatment significantly reduced serum interleukin- (IL-) 4 levels (P < 0.001), BALF IL-10 was significantly increased (P <0.05), serum IgG2a levels were significantly attenuated (P < 0.05) . Total BALF cellularity and as well as eosinophilia were also reduced (P < 0.05). CONCLUSIONS: These results indicate that pUMVC3-hFLex treatment can reverse inflammation and AHR and maintain airway protection in experimental asthma model, and might provide a novel approach for treating asthma. Funding: NIH RO1HL070885 to DKA
254
Suppression of Established Experimental Asthma by Toll-Like Receptor 7 Activation S. Sel1, S. Sel1, M. Wegmann1, H. Garn1, G. Alber2, H. Renz1; 1Department of Clinical Chemistry and Molecular Diagnostics, Biomedical Research Center, Marburg, GERMANY, 2Institute of Immunology, University of Leipzig, Leipzig, GERMANY. RATIONALE: The aim of our study was to evaluate the impact of Tolllike receptor 7 (TLR-7) activation on established allergen triggered asthma in the mouse. METHODS: Experimental asthma was established in Balb/c mice by i.p. ovalbumin (OVA) sensitization, followed by repeated OVA aerosol challenges (primary response). Secondary response was induced two weeks later. Before and during secondary challenges mice were treated with R848 (50 g/ injection), a synthetic TLR-7 ligand. RESULTS: Subcutaneous R-848 application reversed established allergic asthma by suppressing airway eosinophilia, inflammation of the airway mucosa, OVA-specific IgE production and normalizing airway responsiveness. The same effect was achieved following intranasal R-848 application except for the presence of inflammatory cells in airway mucosa. Experiments with IL-12 -/- mice showed that the R-848 suppressing effects on airway hyperresponsiveness and mucosal inflammation were mediated via IL-12. In contrast, suppression of airway eosinophilia, IL-5 production and IgE titer were also present in IL12 -/- mice. CONCLUSIONS: These data demonstrate that the activation of TLR-7 plays an important role to interfere with established experimental asthma. Some of the observed effects are mediated via IL-12. Funding: Philipps-University of Marburg
255
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
STAT4 Deficiency Exaggerates Airway Hyperresponsiveness (AHR) After Repeated Ovalbumin (OVA) Challenge S. Matsubara, T. Kodama, K. Takeda, N. Miyahara, A. Joetham, C. Taube, A. Dakhama, J. Park, T. Koya, E. W. Gelfand; Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO. RATIONALE: In the mouse, AHR and airway allergic inflammation progressively decrease following repeated allergen challenge. It has been suggested that the Th1-promoting cytokine interleukin (IL)-12 is capable of inhibiting persistent AHR and airway inflammation. Signal transducer and activator of transcription-4 (STAT4) is phosphorylated after binding of IL-12 to its receptor. The aim of this study was to investigate the influence of STAT4 deficiency on the development of AHR and airway inflammation after repeated allergen challenge. METHODS: STAT4-deficient mice and their congenic littermates were sensitized to OVA by intraperitoneal injection (twice) and subsequently challenged (via the airways) to 1% OVA in a short term (3 times, 3 days) and long term (11 times, 5 weeks) protocol. Fourty-eight hours after the last OVA challenge, AHR and bronchoalveolar lavage (BAL) were assayed. RESULTS: STAT4 deficiency did not influence pulmonary eosinophilia in either the short- or long-term OVA-challenged mice. STAT4 deficiency significantly (p<0.05) enhanced AHR in the long-term OVA challenged mice, but not in the short-term challenged mice. Serum levels of IgE and Th2 cytokine levels in BAL fluid, IL-4, IL-5 and IL-13, were decreased following long-term OVA challenge compared with short-term challenge, but STAT4 deficiency did not alter cytokine levels in BAL fluid. CONCLUSION: These results suggest that the IL-12-STAT4 signaling pathway plays an important role in the regulation of persistent AHR without effects on Th2 cytokines or serum IgE after repeated allergen challenge. Funding: NIH grants HL-61005, HL-36577, and EPA grant R825702
256
Inhibition of Airways Hyperresponsiveness by a Chimeric STAT-6 Peptide in a Murine Model of Allergic Rhinitis and Asthma Y. Wang, C. McCusker, E. Fixman; Montreal Children’s Hospital Research Institute and Meakins-Chritie Laboratories, McGill University, Montreal, PQ, CANADA. RATIONALE: Allergic rhinitis and asthma are characterized by airway hyperresponsiveness (AHR) to a variety of specific and nonspecific stimuli and associated with increased production of Th2 cytokines, including IL-4 and IL-13. Activation of IL-4/IL-13 receptors containing the IL-4R alpha subunit induces tyrosine phosphorylation, dimerization and nuclear translocation of signal transducer and activator of transcription protein 6 (STAT-6). We investigated whether a chimeric STAT-6 peptide inhibited AHR in a murine model of allergic rhinitis and asthma. METHODS: Non-anesthetized 6~8 week old BALB/C mice received repeated IN application of 1%OVA, 5 microlitres in each nostril, and were then challenged, 2 weeks later, with IN OVA for 5 days. Thirty minutes before each challenges, the mice received IN chimeric STAT-6 peptide. A Change in AHR to methacholine (MCH) was used as a physiologic measure of allergen induced bronchial reactivity. RESULTS: OVA dependent AHR to MCH was significant decreased in a dose dependent manner in mice that received chimeric STAT-6 peptide compared with positive controls (P<0.01). Inflammatory cells in bronchoalveolar lavage fluid were also significantly reduced in STAT 6 peptide treated animals. Finally IL-13 expression in bronchoalveolar lavage fluid from STAT-6 peptide treated mice decreased in a dose dependent manner (P<0.05). CONCLUSIONS: These data demonstrate that IN application of a chimeric STAT-6 peptide inhibited AHR and airway inflammation in a murine model of asthma and allergic rhinitis. This chimeric peptide, delivered intranasally, thus has significant therapeutic potential for the management of asthma and allergic rhinitis. Funding: CIHR & FRSQ& Montreal Children’s Hospital Research Institute
257