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TLR4 is Essential for Proteinase-Mediated Allergic Lung Disease
AB58 Abstracts
SATURDAY
213
Enforced Gata3 Expression Increases Susceptibility To Allergic Airway Inflammation In Mice A. KleinJan, M. J. W. De Bruijn, Y. Levani, M. Van Nimwegen, H. C. Hoogsteden, R. W. Hendriks; Erasmus MC, Rotterdam, Netherlands. RATIONALE: Allergic asthma is a multifactorial disease with involvement of dendritic cells, T-helper-2 (Th2) cells, mast cells, eosinophils and structural cells. The inflammation is mediated by Th2 cells, which express the transcription factor Gata3. Several studies have shown that reduced gata3 is associated with reduced release of Th2 cytokines. There are, however, no studies available indicating that elevated Gata3 expression in vivo would result in higher susceptibility for Th2 diseases. We hypothesized that increased Gata3 expression in T-cells is sufficient to increase susceptibility to allergic airway inflammation. METHODS: Mice with T-cell specific transgenic Gata3 overexpression driven by the CD2 promoter (CD2-Gata3tg) and wild-type (WT) controls underwent intratracheal ovalbumin (OVA) or a house dust mite (HDM) or sham sensitizations without adjuvant. Following allergen exposures, they were analyzed for eosinophilic inflammation and T-cell cytokine production. RESULTS: In both OVA and HDM model, Gata3 overexpressing mice showed clear eosinophilic inflammation and enhanced levels for IL-5 and IL-13 in bronchoalveolar lavage (BAL) and an abundant presence of IL-4+ T-cells and IL-5+ T-cells in BAL, lung tissue and mediastinal lymph node. The WT and control animals showed no signs of allergic inflammation in the OVA model. Moreover no differences were observed in levels of IFN-g+ T-cells and IL-17+ T-cells. WT animals sensitized with HDM showed eosinophilic inflammation, which was less than in HDM sensitized CD2-Gata3tg animals. CONCLUSION: Here we prove, for the first time, that enforced expression of Gata3 in T-cells is sufficient to increase allergic airway inflammation in mice, demonstrating that Gata3 is a crucial factor in asthma.
214
TLR4 is Essential for Proteinase-Mediated Allergic Lung Disease V. K. Ongeri, S. L. Abramson, D. B. Corry; Baylor College of Medicine, Houston, TX. RATIONALE: TLR4 is essential for expression of mouse allergic lung disease in some contexts. Our laboratory showed that fungal proteinases secreted during airway infection are requisite for robust Th2 cell generation and allergic airway inflammation in mice. Furthermore, fungal proteinases cleave an endogenous ligand which in turn activates Drosophila Toll to induce anti-fungal immunity. We therefore hypothesized that fungal proteinases cleave an endogenous airway ligand leading to activation of the TLR4 pathway, involving either MyD88 or TRIF signaling intermediates, resulting in murine allergic lung disease. METHODS: Wildtype C57BL6 and TLR4-/-, MyD88-/-, and TRIF-/- mice were intranasally infected with viable fungal conidia over two weeks. Airway hyperreactivity, lung IL-4 producing cells, and eosinophilia were assessed to characterize the asthma phenotype. Alternative macrophage activation (AMA) through proteinase and TLR4 was also investigated. RESULTS: TLR4-/- mice were largely unresponsive to conidia challenge and failed to develop asthma-like disease. MyD88-/- mice developed an exaggerated response as indicated by increased sensitivity to acetylcholine challenge and increased IL-4 production and eosinophilia whereas TRIF-/- mice were similar to wildtype infected animals. Fungal proteinase induced AMA in a TLR4-dependent manner. CONCLUSIONS: Proteinase-dependent induction of the murine asthma phenotype and AMA requires proteinase exposure and TLR4 signaling through a novel pathway. These findings suggest the existence of a critical endogenous proteinase substrate that is essential for TLR4 activation and allergic disease.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
215
The Relationship of Fractional Exhaled Nitric Oxide Levels to Allergy and Asthma Biomarkers in Young Adults M. Burnett1, G. Wegienka1, S. Havstad1, D. Ownby2, C. Cole Johnson1, E. Zoratti1; 1Henry Ford Health Systems, Detroit, MI, 2Medical College of Georgia, Augusta, GA. RATIONALE: The fractional exhaled concentration of nitric oxide (FeNO) reflects airway inflammation and is increasingly used to assess asthma control. However, FeNO levels may be impacted by factors other than asthma. We evaluated the relationship of FeNO levels to allergy and asthma biomarkers among young adult participants of a general risk birth cohort. METHODS: 18-20 year-old participants (n5137) were evaluated as part of a longitudinal study. Atopy was defined as at least one specific IgE measure >/5 0.35 kU/L to seven common allergens. FeNO levels were measured via NiOX (Aerocine, Solna, Sweden). Associations between spirometry, total IgE, and peripheral blood eosinophil levels were analyzed using Spearman’s correlations. Wilcoxon rank sum tests were used to assess differences in FeNO between groups. RESULTS: Mean FeNO in males [20.5 ppb (95% CI 17.2, 24.3)] was higher than in females [12.7 ppb (11.0, 14.7)]; p<0.001. Higher FeNO levels were also seen in atopic individuals [19.7(16.8, 23.1)] compared to those without atopy [10.7 ppb (9.5, 12.1)]; p<0.001. When excluding 34 participants that reported a prior diagnosis of asthma, atopic individuals still had higher FeNO levels [17.6 ppb (14.7, 21.0)] compared to those without atopy [11.0 ppb (9.8, 12.5)]; p<0.001. FeNO levels were positively associated with percent eosinophils (Spearman correlation r50.45; p <0.001), eosinophil count (r50.39; p <0.001) and total IgE level (r50.45; p<0.001). FeNO did not correlate with percent predicted FEV1, FVC or with FEV1/FVC. CONCLUSIONS: FeNO levels positively correlate with total IgE levels and eosinophil counts. Higher FeNO levels are also associated with male gender and atopy.
216
Variable Levels of Cytokines and Chemokines in the Proximal and Distal Asthmatic Airway J. T. Anderson1, M. Zeng1, N. Fineberg1, A. Gaggar1,2, D. D. Chaplin1, M. T. Dransfield1,2, J. S. Deshane1; 1University of Alabama at Birmingham, Birmingham, AL, 2VA Medical Center, Birmingham, AL. RATIONALE: To determine the contributions of proximal and distal airways to the formation of inflammatory mediators within asthmatic subjects, we measured cytokine and chemokine concentrations in low volume bronchial wash (BW; sampling proximal airways) compared to traditional bronchoalveolar lavage (BAL; sampling distal airways). METHODS: Asthmatic (n58) and control (n58) subjects were nonsmokers. Asthmatic subjects met GINA guidelines for mild disease and did not require corticosteroid therapy. Bronchoscopic BW was performed by instilling 10 ml of sterile saline followed by immediate aspiration (average return 3-5 ml). Without moving the bronchoscope, BAL was performed using 33 ml of saline, returning an average 10-11 ml. Concentrations of Surfactant Protein-D (ELISA) chemokines and cytokines (multiplex beadlyte assays) were measured using cell-free returned fluid from the BW and the BAL. Comparisons were made using non-parametric statistical tests. This study was approved by the UAB Institutional Review Board. RESULTS: SP-D was 11 fold higher in BAL compared to BW (p50.01) indicating that BAL sampled more distal airways. Several cytokines and chemokines (IL-4, IL-13, RANTES, IL-1RA) were present in both asthmatic BW and BAL fluid. However, GM-CSF, EGF, and Eotaxin, (p50.02) were present at detectable levels in the BW only, compared to (IFNg, IL-12(p40), FGF-2, IL-10, IL-15, IL-1b, TGF-a, TNF-b, IL-7, sIL2-Ra and IFN-a, p50.02) in the BAL only. CONCLUSIONS: BW and BAL sampled regional differences in the concentrations of cytokines and chemokines in asthmatic airways. These differences in concentrations of inflammatory mediators underscore the importance of assessing both the proximal and distal airways in studying the pathogenesis of asthma.
SATURDAY
213
Enforced Gata3 Expression Increases Susceptibility To Allergic Airway Inflammation In Mice A. KleinJan, M. J. W. De Bruijn, Y. Levani, M. Van Nimwegen, H. C. Hoogsteden, R. W. Hendriks; Erasmus MC, Rotterdam, Netherlands. RATIONALE: Allergic asthma is a multifactorial disease with involvement of dendritic cells, T-helper-2 (Th2) cells, mast cells, eosinophils and structural cells. The inflammation is mediated by Th2 cells, which express the transcription factor Gata3. Several studies have shown that reduced gata3 is associated with reduced release of Th2 cytokines. There are, however, no studies available indicating that elevated Gata3 expression in vivo would result in higher susceptibility for Th2 diseases. We hypothesized that increased Gata3 expression in T-cells is sufficient to increase susceptibility to allergic airway inflammation. METHODS: Mice with T-cell specific transgenic Gata3 overexpression driven by the CD2 promoter (CD2-Gata3tg) and wild-type (WT) controls underwent intratracheal ovalbumin (OVA) or a house dust mite (HDM) or sham sensitizations without adjuvant. Following allergen exposures, they were analyzed for eosinophilic inflammation and T-cell cytokine production. RESULTS: In both OVA and HDM model, Gata3 overexpressing mice showed clear eosinophilic inflammation and enhanced levels for IL-5 and IL-13 in bronchoalveolar lavage (BAL) and an abundant presence of IL-4+ T-cells and IL-5+ T-cells in BAL, lung tissue and mediastinal lymph node. The WT and control animals showed no signs of allergic inflammation in the OVA model. Moreover no differences were observed in levels of IFN-g+ T-cells and IL-17+ T-cells. WT animals sensitized with HDM showed eosinophilic inflammation, which was less than in HDM sensitized CD2-Gata3tg animals. CONCLUSION: Here we prove, for the first time, that enforced expression of Gata3 in T-cells is sufficient to increase allergic airway inflammation in mice, demonstrating that Gata3 is a crucial factor in asthma.
214
TLR4 is Essential for Proteinase-Mediated Allergic Lung Disease V. K. Ongeri, S. L. Abramson, D. B. Corry; Baylor College of Medicine, Houston, TX. RATIONALE: TLR4 is essential for expression of mouse allergic lung disease in some contexts. Our laboratory showed that fungal proteinases secreted during airway infection are requisite for robust Th2 cell generation and allergic airway inflammation in mice. Furthermore, fungal proteinases cleave an endogenous ligand which in turn activates Drosophila Toll to induce anti-fungal immunity. We therefore hypothesized that fungal proteinases cleave an endogenous airway ligand leading to activation of the TLR4 pathway, involving either MyD88 or TRIF signaling intermediates, resulting in murine allergic lung disease. METHODS: Wildtype C57BL6 and TLR4-/-, MyD88-/-, and TRIF-/- mice were intranasally infected with viable fungal conidia over two weeks. Airway hyperreactivity, lung IL-4 producing cells, and eosinophilia were assessed to characterize the asthma phenotype. Alternative macrophage activation (AMA) through proteinase and TLR4 was also investigated. RESULTS: TLR4-/- mice were largely unresponsive to conidia challenge and failed to develop asthma-like disease. MyD88-/- mice developed an exaggerated response as indicated by increased sensitivity to acetylcholine challenge and increased IL-4 production and eosinophilia whereas TRIF-/- mice were similar to wildtype infected animals. Fungal proteinase induced AMA in a TLR4-dependent manner. CONCLUSIONS: Proteinase-dependent induction of the murine asthma phenotype and AMA requires proteinase exposure and TLR4 signaling through a novel pathway. These findings suggest the existence of a critical endogenous proteinase substrate that is essential for TLR4 activation and allergic disease.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
215
The Relationship of Fractional Exhaled Nitric Oxide Levels to Allergy and Asthma Biomarkers in Young Adults M. Burnett1, G. Wegienka1, S. Havstad1, D. Ownby2, C. Cole Johnson1, E. Zoratti1; 1Henry Ford Health Systems, Detroit, MI, 2Medical College of Georgia, Augusta, GA. RATIONALE: The fractional exhaled concentration of nitric oxide (FeNO) reflects airway inflammation and is increasingly used to assess asthma control. However, FeNO levels may be impacted by factors other than asthma. We evaluated the relationship of FeNO levels to allergy and asthma biomarkers among young adult participants of a general risk birth cohort. METHODS: 18-20 year-old participants (n5137) were evaluated as part of a longitudinal study. Atopy was defined as at least one specific IgE measure >/5 0.35 kU/L to seven common allergens. FeNO levels were measured via NiOX (Aerocine, Solna, Sweden). Associations between spirometry, total IgE, and peripheral blood eosinophil levels were analyzed using Spearman’s correlations. Wilcoxon rank sum tests were used to assess differences in FeNO between groups. RESULTS: Mean FeNO in males [20.5 ppb (95% CI 17.2, 24.3)] was higher than in females [12.7 ppb (11.0, 14.7)]; p<0.001. Higher FeNO levels were also seen in atopic individuals [19.7(16.8, 23.1)] compared to those without atopy [10.7 ppb (9.5, 12.1)]; p<0.001. When excluding 34 participants that reported a prior diagnosis of asthma, atopic individuals still had higher FeNO levels [17.6 ppb (14.7, 21.0)] compared to those without atopy [11.0 ppb (9.8, 12.5)]; p<0.001. FeNO levels were positively associated with percent eosinophils (Spearman correlation r50.45; p <0.001), eosinophil count (r50.39; p <0.001) and total IgE level (r50.45; p<0.001). FeNO did not correlate with percent predicted FEV1, FVC or with FEV1/FVC. CONCLUSIONS: FeNO levels positively correlate with total IgE levels and eosinophil counts. Higher FeNO levels are also associated with male gender and atopy.
216
Variable Levels of Cytokines and Chemokines in the Proximal and Distal Asthmatic Airway J. T. Anderson1, M. Zeng1, N. Fineberg1, A. Gaggar1,2, D. D. Chaplin1, M. T. Dransfield1,2, J. S. Deshane1; 1University of Alabama at Birmingham, Birmingham, AL, 2VA Medical Center, Birmingham, AL. RATIONALE: To determine the contributions of proximal and distal airways to the formation of inflammatory mediators within asthmatic subjects, we measured cytokine and chemokine concentrations in low volume bronchial wash (BW; sampling proximal airways) compared to traditional bronchoalveolar lavage (BAL; sampling distal airways). METHODS: Asthmatic (n58) and control (n58) subjects were nonsmokers. Asthmatic subjects met GINA guidelines for mild disease and did not require corticosteroid therapy. Bronchoscopic BW was performed by instilling 10 ml of sterile saline followed by immediate aspiration (average return 3-5 ml). Without moving the bronchoscope, BAL was performed using 33 ml of saline, returning an average 10-11 ml. Concentrations of Surfactant Protein-D (ELISA) chemokines and cytokines (multiplex beadlyte assays) were measured using cell-free returned fluid from the BW and the BAL. Comparisons were made using non-parametric statistical tests. This study was approved by the UAB Institutional Review Board. RESULTS: SP-D was 11 fold higher in BAL compared to BW (p50.01) indicating that BAL sampled more distal airways. Several cytokines and chemokines (IL-4, IL-13, RANTES, IL-1RA) were present in both asthmatic BW and BAL fluid. However, GM-CSF, EGF, and Eotaxin, (p50.02) were present at detectable levels in the BW only, compared to (IFNg, IL-12(p40), FGF-2, IL-10, IL-15, IL-1b, TGF-a, TNF-b, IL-7, sIL2-Ra and IFN-a, p50.02) in the BAL only. CONCLUSIONS: BW and BAL sampled regional differences in the concentrations of cytokines and chemokines in asthmatic airways. These differences in concentrations of inflammatory mediators underscore the importance of assessing both the proximal and distal airways in studying the pathogenesis of asthma.