- Email: [email protected]
Airway NK Cells are Involved in Innate Th2-like Inflammation in Response to an Environmental Antigen, Alternaria
Abstracts S255
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Airway NK Cells are Involved in Innate Th2-like Inflammation in Response to an Environmental Antigen, Alternaria K. Iijima, T. Kobayashi, K. R. Bartemes, H. Kita; Mayo Clinic, Rochester, MN. RATIONALE: The molecular mechanisms underlying the initiation of Th2-type immune responses are not well understood. Exposure to environmental antigens, such as Alternaria, cockroach, and mite, are implicated in the development and exacerbation of allergic diseases and asthma in humans. The goal of this project was to investigate the immunological outcomes of Alternaria exposure and to dissect the molecular mechanism. METHODS: Non-sensitized naive mice were exposed intranasally to Alternaria alternata extract every 3 days and monitored for 8 days. Bronchoalveolar lavage fluids and lungs were analyzed by ELISA, RT-PCR, and FACS. RESULTS: Airway exposure of naive mice to Alternaria induced airway eosinophilia within 12 hours, and eosinophilia increased exponentially up to 8 days. IL-5 protein was detected in the airways as early as 4 hours after the initial Alternaria exposure; increased mRNA expression for IL-5, IL-13, IFN-g, eotaxin-2, but not IL-4, was verified by RT-PCR. The early production of IL-5 (i.e. 12 hours after initial Alternaria exposure) was abolished in Beige mice, but was unaffected in Rag1-knockout (KO), nude, mast cell-deficient or NKT-deficient mice. In addition, IL-5 production and eosinophilia were inhibited in mice treated with anti-NK cell antibody (anti-asialo-GM1) or NK-cell deficient Ly49A transgenic mice. By FACS analysis, lung NK-like cells (CD3e-, NK1.1-, CD16/321) showed robust intracellular IL-5 in both wild-type and Rag1-KO mice. CONCLUSIONS: Exposure to Alternaria triggers prompt Th2-like innate immune responses in the airways. Airway NK cells may play a pivotal role as an interface between innate and adaptive Th2-type airway inflammation. Funding: Mayo Foundation and NIH grant AI34486
986
Sputum Protease/Anti-Protease Imbalance Distinguishes Progressive Airflow Limitation in Adolescents with Asthma: CAMP Continuation Study R. A. Covar, P. Szefler, J. Murphy, A. Liu, J. D. Spahn, R. Harbeck, B. J. Efaw, D. Sundstrom, S. J. Szefler; National Jewish Center, Denver, CO. RATIONALE: To evaluate physiological and biological markers of inflammation associated with airflow limitation in adolescents and young adults with a long-term history of persistent asthma. METHODS: In the NHLBI Childhood Asthma Management Program (CAMP), four patterns of airflow limitation, determined by abnormal FEV1/FVC ratio (5 percentile of NHANES III reference values) prior to randomization and 9 years later, were identified: current limitation (Persistent and Late Obstruction Groups) and no current limitation (Stable and Early Obstruction Groups). Exhaled nitric oxide, body box plethysmography, and biomarkers from induced sputum were measured eight years after ending the 5-year treatment phase. RESULTS: Participants with current limitation (20 Persistent and 10 Late Obstruction) differed from those with no limitation (9 Early Obstruction and 16 Stable Groups) by measures of large and small airway function (FEV1/FVC and FEF25-75), but not lung volume measurements. Those with current airflow limitation, as compared to those with no limitation, had lower mean 6 SD levels of sputum IL8 (log 2.8 6 0.4 vs 3.2 6 0.5 pg/ml, p 5 0.01), TIMP1 (log 1.5 6 0.6 vs. 1.9 6 0.5 ng/ml, p 5 0.03), and elastase/alpha-1-proteinase inhibitor complex (log 3.8 6 0.5 vs 4.1 6 0.4 ng/ml, p 5 0.03), without differences in MMP9 levels or activity, free elastase activity, or neutrophil count. The Late Obstruction Group had the lowest levels of these biomarkers: log TIMP1 1.4 6 0.6 ng/ml; IL8 2.8 6 0.5 pg/ml; and elastase/alpha-1-proteinase inhibitor complex 5 3.5 6 0.6 ng/ml. CONCLUSIONS: Reduced levels of TIMP1 and elastase/alpha-1-proteinase inhibitor complex, not elevated free elastase activity, MMP9 or neutrophil count, suggest an underlying protease/anti-protease imbalance that may distinguish progressive airflow limitation in asthma. Funding: NIH HR16048; HL081335; HL075416; M01 RR00051
987
Improvements in Airway Hyperresponsiveness and Inflammation in an Asthma Model via Upregulation of Anti-Oxidant Responses with 2-Cyano-3,12-Dioxooleana-1,9 (11)-Diene-28-Oic Acid Trifluoroethyl Amide (CDDO-TFEA) E. G. Brooks1, D. Nanyez1, S. Shulz1, C. Meyer2, M. B. Sporn3, D. J. Hochman1; 1The University of Texas Medical Branch, Galveston, TX, 2 Reata Pharmaceuticals Inc., Dallas, TX, 3Dartmouth Medical School, Hanover, NH. RATIONALE: Elevated levels of reactive oxygen species (ROS) induce cellular injury and promote airway inflammation and hyperresponsiveness (AHR) in animal models of asthma. CDDO-TFEA, a compound that upregulates transcriptional anti-oxidant response elements (ARE) through the Nrf2 pathway, may be useful in protecting against oxidative injury in asthma. METHODS: BALB/c mice were sensitized with ovalbumin 14 days prior to ovalbumin (OVA) challenge. Mice were treated daily with 3 to 60 mg/kg CDDO-TFEA just prior to 1% OVA aerosol challenge on 6 successive days. Responses were assessed by whole body plethysmography, enumeration of inflammatory cells and cytokines in broncho-alveolar lavage (BAL) and assessment of serum IgE levels by OVA-specific ELISA. RESULTS: CDDO-TFEA treated mice demonstrated reduced AHR (80% reduction, p < .05)., reduced BAL eosinophil numbers (50% reduction, p < .05) and reduced serum IgE levels after treatment with doses ranging from 3 to 30 mg/kg. No toxicity occurred at therapeutic doses. However, toxicity was noted at 60 mg/kg and was associated with a return of AHR and inflammation. CONCLUSIONS: Administration of CDDO-TFEA in a murine model of asthma was associated with a reduction in AHR, airway inflammation and IgE formation. These results provide evidence that oxidative stress is an important feature of asthma and a potential target for therapeutic intervention. Funding: Reata Pharmaceuticals Inc.
988
Esophageal Remodeling Develops as a Consequence of Tissue Specific Interleukin (IL)-5 Induced Eosinophilia M. Wang, M. Collins, P. Putnam, M. E. Rothenberg, A. Mishra; Cincinnati Children Hospital Medical Center, cincinnati, OH. RATIONALE: Eosinophilic esophagitis (EE) has been associated with esophageal remodeling, but the mechanisms involved are poorly understood. We examined the mechanism of the induction of esophageal remodeling in EE. METHODS: Esophageal remodeling was assessed by histology in patient biopsies and in mouse esophageal tissue sections following the induction of experimental EE in wild type, IL-5 deficient and IL-5 transgenic mice. The semi-quantitative analysis of basal layer and lamina propria collagen thickness was measured by morphometric analysis. TGF-b, MUC5AC and IL-5 mRNA were measured by real time PCR. RESULTS: An impressive accumulation of collagen in the epithelial mucosa and lamina propria, and basal layer thickening was observed in the esophageal biopsies from EE patients (P < 0.001) compared to normal individuals as well as in mice with experimental EE (P < 0.001). Significantly reduced lamina propria collagen and basal layer thickness were observed in IL-5 deficient mice (P < 0.001, P < 0.01 respectively) compared to wild type mice following the induction of experimental EE. Furthermore, the esophagus of CD2-IL-5 transgenic mice showed increased thickness of basal layer and collagen accumulation compared to non-transgenic mice. IL-5 intestine transgenic (iIL-5) mice did not have EE-like esophageal changes, although both CD2-IL-5 and iIL-5 mice showed eosinophils in the esophagus. In addition, TGF-b, MUC5AC and IL-5 mRNA expression are increased in the esophageal biopsies of EE patients (P < 0.05) and in the mouse model of EE (P < 0.05) compared to normal controls. CONCLUSIONS: These findings provide evidence that IL-5-mediated eosinophilia is essential in the induction of esophageal remodeling. Funding: NIH
TUESDAY
985
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Airway NK Cells are Involved in Innate Th2-like Inflammation in Response to an Environmental Antigen, Alternaria K. Iijima, T. Kobayashi, K. R. Bartemes, H. Kita; Mayo Clinic, Rochester, MN. RATIONALE: The molecular mechanisms underlying the initiation of Th2-type immune responses are not well understood. Exposure to environmental antigens, such as Alternaria, cockroach, and mite, are implicated in the development and exacerbation of allergic diseases and asthma in humans. The goal of this project was to investigate the immunological outcomes of Alternaria exposure and to dissect the molecular mechanism. METHODS: Non-sensitized naive mice were exposed intranasally to Alternaria alternata extract every 3 days and monitored for 8 days. Bronchoalveolar lavage fluids and lungs were analyzed by ELISA, RT-PCR, and FACS. RESULTS: Airway exposure of naive mice to Alternaria induced airway eosinophilia within 12 hours, and eosinophilia increased exponentially up to 8 days. IL-5 protein was detected in the airways as early as 4 hours after the initial Alternaria exposure; increased mRNA expression for IL-5, IL-13, IFN-g, eotaxin-2, but not IL-4, was verified by RT-PCR. The early production of IL-5 (i.e. 12 hours after initial Alternaria exposure) was abolished in Beige mice, but was unaffected in Rag1-knockout (KO), nude, mast cell-deficient or NKT-deficient mice. In addition, IL-5 production and eosinophilia were inhibited in mice treated with anti-NK cell antibody (anti-asialo-GM1) or NK-cell deficient Ly49A transgenic mice. By FACS analysis, lung NK-like cells (CD3e-, NK1.1-, CD16/321) showed robust intracellular IL-5 in both wild-type and Rag1-KO mice. CONCLUSIONS: Exposure to Alternaria triggers prompt Th2-like innate immune responses in the airways. Airway NK cells may play a pivotal role as an interface between innate and adaptive Th2-type airway inflammation. Funding: Mayo Foundation and NIH grant AI34486
986
Sputum Protease/Anti-Protease Imbalance Distinguishes Progressive Airflow Limitation in Adolescents with Asthma: CAMP Continuation Study R. A. Covar, P. Szefler, J. Murphy, A. Liu, J. D. Spahn, R. Harbeck, B. J. Efaw, D. Sundstrom, S. J. Szefler; National Jewish Center, Denver, CO. RATIONALE: To evaluate physiological and biological markers of inflammation associated with airflow limitation in adolescents and young adults with a long-term history of persistent asthma. METHODS: In the NHLBI Childhood Asthma Management Program (CAMP), four patterns of airflow limitation, determined by abnormal FEV1/FVC ratio (5 percentile of NHANES III reference values) prior to randomization and 9 years later, were identified: current limitation (Persistent and Late Obstruction Groups) and no current limitation (Stable and Early Obstruction Groups). Exhaled nitric oxide, body box plethysmography, and biomarkers from induced sputum were measured eight years after ending the 5-year treatment phase. RESULTS: Participants with current limitation (20 Persistent and 10 Late Obstruction) differed from those with no limitation (9 Early Obstruction and 16 Stable Groups) by measures of large and small airway function (FEV1/FVC and FEF25-75), but not lung volume measurements. Those with current airflow limitation, as compared to those with no limitation, had lower mean 6 SD levels of sputum IL8 (log 2.8 6 0.4 vs 3.2 6 0.5 pg/ml, p 5 0.01), TIMP1 (log 1.5 6 0.6 vs. 1.9 6 0.5 ng/ml, p 5 0.03), and elastase/alpha-1-proteinase inhibitor complex (log 3.8 6 0.5 vs 4.1 6 0.4 ng/ml, p 5 0.03), without differences in MMP9 levels or activity, free elastase activity, or neutrophil count. The Late Obstruction Group had the lowest levels of these biomarkers: log TIMP1 1.4 6 0.6 ng/ml; IL8 2.8 6 0.5 pg/ml; and elastase/alpha-1-proteinase inhibitor complex 5 3.5 6 0.6 ng/ml. CONCLUSIONS: Reduced levels of TIMP1 and elastase/alpha-1-proteinase inhibitor complex, not elevated free elastase activity, MMP9 or neutrophil count, suggest an underlying protease/anti-protease imbalance that may distinguish progressive airflow limitation in asthma. Funding: NIH HR16048; HL081335; HL075416; M01 RR00051
987
Improvements in Airway Hyperresponsiveness and Inflammation in an Asthma Model via Upregulation of Anti-Oxidant Responses with 2-Cyano-3,12-Dioxooleana-1,9 (11)-Diene-28-Oic Acid Trifluoroethyl Amide (CDDO-TFEA) E. G. Brooks1, D. Nanyez1, S. Shulz1, C. Meyer2, M. B. Sporn3, D. J. Hochman1; 1The University of Texas Medical Branch, Galveston, TX, 2 Reata Pharmaceuticals Inc., Dallas, TX, 3Dartmouth Medical School, Hanover, NH. RATIONALE: Elevated levels of reactive oxygen species (ROS) induce cellular injury and promote airway inflammation and hyperresponsiveness (AHR) in animal models of asthma. CDDO-TFEA, a compound that upregulates transcriptional anti-oxidant response elements (ARE) through the Nrf2 pathway, may be useful in protecting against oxidative injury in asthma. METHODS: BALB/c mice were sensitized with ovalbumin 14 days prior to ovalbumin (OVA) challenge. Mice were treated daily with 3 to 60 mg/kg CDDO-TFEA just prior to 1% OVA aerosol challenge on 6 successive days. Responses were assessed by whole body plethysmography, enumeration of inflammatory cells and cytokines in broncho-alveolar lavage (BAL) and assessment of serum IgE levels by OVA-specific ELISA. RESULTS: CDDO-TFEA treated mice demonstrated reduced AHR (80% reduction, p < .05)., reduced BAL eosinophil numbers (50% reduction, p < .05) and reduced serum IgE levels after treatment with doses ranging from 3 to 30 mg/kg. No toxicity occurred at therapeutic doses. However, toxicity was noted at 60 mg/kg and was associated with a return of AHR and inflammation. CONCLUSIONS: Administration of CDDO-TFEA in a murine model of asthma was associated with a reduction in AHR, airway inflammation and IgE formation. These results provide evidence that oxidative stress is an important feature of asthma and a potential target for therapeutic intervention. Funding: Reata Pharmaceuticals Inc.
988
Esophageal Remodeling Develops as a Consequence of Tissue Specific Interleukin (IL)-5 Induced Eosinophilia M. Wang, M. Collins, P. Putnam, M. E. Rothenberg, A. Mishra; Cincinnati Children Hospital Medical Center, cincinnati, OH. RATIONALE: Eosinophilic esophagitis (EE) has been associated with esophageal remodeling, but the mechanisms involved are poorly understood. We examined the mechanism of the induction of esophageal remodeling in EE. METHODS: Esophageal remodeling was assessed by histology in patient biopsies and in mouse esophageal tissue sections following the induction of experimental EE in wild type, IL-5 deficient and IL-5 transgenic mice. The semi-quantitative analysis of basal layer and lamina propria collagen thickness was measured by morphometric analysis. TGF-b, MUC5AC and IL-5 mRNA were measured by real time PCR. RESULTS: An impressive accumulation of collagen in the epithelial mucosa and lamina propria, and basal layer thickening was observed in the esophageal biopsies from EE patients (P < 0.001) compared to normal individuals as well as in mice with experimental EE (P < 0.001). Significantly reduced lamina propria collagen and basal layer thickness were observed in IL-5 deficient mice (P < 0.001, P < 0.01 respectively) compared to wild type mice following the induction of experimental EE. Furthermore, the esophagus of CD2-IL-5 transgenic mice showed increased thickness of basal layer and collagen accumulation compared to non-transgenic mice. IL-5 intestine transgenic (iIL-5) mice did not have EE-like esophageal changes, although both CD2-IL-5 and iIL-5 mice showed eosinophils in the esophagus. In addition, TGF-b, MUC5AC and IL-5 mRNA expression are increased in the esophageal biopsies of EE patients (P < 0.05) and in the mouse model of EE (P < 0.05) compared to normal controls. CONCLUSIONS: These findings provide evidence that IL-5-mediated eosinophilia is essential in the induction of esophageal remodeling. Funding: NIH
TUESDAY
985