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IVIg Induces of Foxp3+ Regulatory T-cells in an Antigen Specific Manner in Allergic Airway Disease
AB68 Abstracts
SATURDAY
IVIg Induces of Foxp3+ Regulatory T-cells in an Antigen Specific Manner in Allergic Airway Disease A. H. Massoud1, K. Shalaby1, D. Chan1, W. M. Mourad2, C. A. Piccirillo1, B. D. Mazer1; 1McGill University, Montreal, QC, CANADA, 2 Unversity of Montreal, Montreal, QC, CANADA. RATIONALE: IVIg has been utilized to treat severe steroid-dependent asthma, but the mechanism of action remains unclear. We demonstrated that IVIg inhibits bronchial reactivity in a murine model of allergic airways disease, likely via induction of TReg. We have assessed the antigenspecificity as well as mechanism of TReg induction by IVIg. METHODS: CD4+Foxp3(GFP-)T-cells were purified from lymphoid organs of TCR-OVA-specific-Foxp3GFP mice and adoptively transferred to WT syngenic C57BL/6 animals. Recipient mice were sensitized and challenged either with OVA or Ragweed. Challenged mice received IVIg, or HSA as a control protein. Induction of CD4+Foxp3(GFP+)Treg was determined by flow cytometry in cells from lungs and lymphoid organs. A suppression assay was performed using CFSE-labled OVA-specific CD4+T-cells plus enriched Foxp3GFP+TRegs from adoptively transferred mice. The proliferative response of OVA-specific T-cells to OVA stimulation was measured by flow cytometry. Splenic dendritic cells were pretreated with IVIg, OVA or OVA+IVIg and co-cultured with CD4+T-cells from dTg mice for 5 days. Induction of Treg was assessed by flow cytometry. RESULTS: Induction of OVA-specific Foxp3GFP+TReg in lung, dLNs and spleen of OVA-IVIg-OVA mice was 4-5 fold higher compared to control groups. In Ragweed-sensitized mice adoptively transferred with TCROVA-specific-Foxp3GFP+ T-cells, IVIg induced endogenous TReg but not OVA-specific Foxp3GFP+TReg. Foxp3GFP+TReg from OVA-IVIg-OVA mice inhibited proliferation of OVAspecific T-cells 8-fold more efficiently than Foxp3GFP+TReg from other groups. Finally, OVA-IVIg pretreated DCs induced significantly more Foxp3GFP+TReg in-vitro than DCs pretreated with OVA or IVIg alone. CONCLUSIONS: IVIg induces Treg in an antigen-specific fashion. The induction of Treg appears to be mediated through conditioning of DCs.
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Implementation and Impact of a Metered Dose Inhaler Based Asthma Pathway in a Urban University Hospital M. E. Bollinger1, Z. Hasan2, D. Steacy3, K. Rafei1; 1University MD School of Medicine, Baltimore, MD, 2Lake Erie College of Osteopathic Medicine, Erie, PA, 3University of Maryland Medical System, Baltimore, MD. RATIONALE: Asthma medication delivery by metered dose inhaler (MDI) has been shown to be equivalent to nebulizers; however, their use in acute care settings is not widespread. We report on the implementation and outcomes of a metered dose inhaler based asthma pathway in an inner city academic hospital. METHODS: A multidisciplinary team developed and implemented the MDI pathway (start date 1/04 in the Emergency Department (ED) and 1/ 06 in the inpatient unit). Data were collected from the University of Maryland Medical System for all ED visits and hospitalizations for ICD9 code (493.–) between 2002 and 2009 for children 2-18 years. Demographic information, admission rates, length of stay and readmission rates were collected to include >2 years pre and post implementation of the pathway. RESULTS: Between 2002-2009, annual ED rates ranged from 1,1333,065 (mean52,616). Annual hospitalization rates ranged from 313-653 (mean5426). The demographics of the patients did not vary over the study period with mean age of 7.5 years with 90% African American, 43% female and 80% Medicaid insured. ED admission rates averaged 12.2% in the 2 years prior to the pathway and 7% in the 2 years post. Mean length of hospital stay in the 4 years prior to the pathway (‘02-‘05) was 3.51 days vs. 2.90 days in the 3 years post (’06-’09). Readmission rates were not significantly different pre and post-implementation. CONCLUSIONS: The MDI pathway has been well accepted in this inner city academic hospital with no adverse effects on admission or readmission rates or length of stay.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
TGF-b, IL-1b, And IL-23 Are Required For IL-13Ra1 Expression On Human Th17 Cells And IL-13 Attenuates IL-17A Production At Restimulation M. M. Huckabee, D. C. Newcomb, M. G. Boswell, K. Goleniewska, S. Reiss, R. S. Peebles, Jr, ; Vanderbilt University Medical Center, Nashville, TN. RATIONALE: IL-13 receptor (IL-13R) is comprised of IL-13Ra1 and IL-4Ra, and IL-13Ra1 is expressed on human Th17 cells but not Th1 or Th2 cells. IL-13 attenuates IL-17A production in Th17 cells, but the mechanism remains unknown. We hypothesized that one or more of the cytokines involved in Th17 polarization is responsible for IL-13Ra1 expression. Further, we hypothesized that IL-13 inhibited IL-17A production by decreasing phosphorylation of STAT3, a transcription factors required for Th17 cell differentiation. METHODS: Na€ıve human CD4+ cells were activated in the presence of anti-IL-4 and anti-IFN-g and one or more of the following cytokines: IL2, TGF-b, IL-1b, IL-6, and/or IL-23. IL-13 (10ng/ml) was given at day 0, 1, 2 or 3 of activation. T cells were also restimulated with anti-CD3 and IL-13 was added at the time of restimulation. Total RNA, total protein, and supernantants were harvested 4 days after polarization. RESULTS: TGF-b, IL-1b, and IL-23, but not IL-6, were required for IL-13Ra1 expression on human Th17 cells, and IL-13 only attenuated IL-17A production in cultures with these cytokines. STAT3 phosphorylation was decreased in the presence of IL-13. IL-13 attenuated IL-17A production when added at activation (day 0), but not later time points. Further, IL-13 attenuated IL-17A production in restimulated Th17 cells. CONCLUSIONS: IL-13Ra1 expression on CD4+ T cells requires TGF-b, IL-1b, and IL-23. IL-13 attenuates IL-17A production only at the time of polarization or at restimulation. IL-13 also decreases IL-17A production at restimulation. These results indicate that IL-13 inhibits IL-17A production early during stimulation.
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Human IL-27 Suppresses Th2 Cell Differentiation Independent of IFN-g and IL-10 S. Wang1, Y. Zhuang2, N. Erekosima2, R. Katial2, R. Alam2, H. Huang1; 1 National Jewish Health and University of Colorado Denver, Denver, CO, 2 National Jewish Health, Denver, CO. RATIONALE: Murine IL-27 is a potent suppressor of Th2 cell differentiation. However, it is unknown whether murine IL-27 suppresses Th2 cytokine production by differentiated Th2 cells and if so which signaling pathway murine IL-27 uses. Moreover, it is unclear if human IL-27 suppresses Th2 cell differentiation. We sought to further understand the mechanisms by which murine IL-27 suppresses Th2 cell differentiation and to determine whether human IL-27 suppresses Th2 cell differentiation. METHODS: Naive CD4+ T cells were isolated from C57BL/6, STAT1deficient, or T-bet-deficient mice and primed under Th2-inducing conditions in the presence or absence of murine or human IL-27. IL-4 and IFN-g expression was determined using intracellular staining and ELISA. Anti-IL-10 antibody or immunoglobulin isotype control was used to determine whether IL-27 suppresses Th2 cell differentiation dependent on IL-10. Similarly, naive human CD4+ T cells were isolated from peripheral blood and primed under Th2-inducing conditions in the presence or absence of human IL-27. The roles of IFN-g and IL-10 were assessed by using neutralizing antibodies. RESULTS: Murine IL-27 suppressed Th2 cell differentiation but failed to suppress Th2 cytokine production by previously differentiated Th2 cells. Murine IL-27 inhibited Th2 cytokine production in a STAT1-dependent but T-bet-independent manner. We found that human IL-27 suppressed IL-4 production independent of both IFN-g and IL-10. CONCLUSIONS: Murine IL-27 suppresses Th2 cell differentiation in a STAT1-dependent but T-bet-independent manner and human IL-27 suppresses Th2 cell differentiation independent of both IFN-g and IL-10.
SATURDAY
IVIg Induces of Foxp3+ Regulatory T-cells in an Antigen Specific Manner in Allergic Airway Disease A. H. Massoud1, K. Shalaby1, D. Chan1, W. M. Mourad2, C. A. Piccirillo1, B. D. Mazer1; 1McGill University, Montreal, QC, CANADA, 2 Unversity of Montreal, Montreal, QC, CANADA. RATIONALE: IVIg has been utilized to treat severe steroid-dependent asthma, but the mechanism of action remains unclear. We demonstrated that IVIg inhibits bronchial reactivity in a murine model of allergic airways disease, likely via induction of TReg. We have assessed the antigenspecificity as well as mechanism of TReg induction by IVIg. METHODS: CD4+Foxp3(GFP-)T-cells were purified from lymphoid organs of TCR-OVA-specific-Foxp3GFP mice and adoptively transferred to WT syngenic C57BL/6 animals. Recipient mice were sensitized and challenged either with OVA or Ragweed. Challenged mice received IVIg, or HSA as a control protein. Induction of CD4+Foxp3(GFP+)Treg was determined by flow cytometry in cells from lungs and lymphoid organs. A suppression assay was performed using CFSE-labled OVA-specific CD4+T-cells plus enriched Foxp3GFP+TRegs from adoptively transferred mice. The proliferative response of OVA-specific T-cells to OVA stimulation was measured by flow cytometry. Splenic dendritic cells were pretreated with IVIg, OVA or OVA+IVIg and co-cultured with CD4+T-cells from dTg mice for 5 days. Induction of Treg was assessed by flow cytometry. RESULTS: Induction of OVA-specific Foxp3GFP+TReg in lung, dLNs and spleen of OVA-IVIg-OVA mice was 4-5 fold higher compared to control groups. In Ragweed-sensitized mice adoptively transferred with TCROVA-specific-Foxp3GFP+ T-cells, IVIg induced endogenous TReg but not OVA-specific Foxp3GFP+TReg. Foxp3GFP+TReg from OVA-IVIg-OVA mice inhibited proliferation of OVAspecific T-cells 8-fold more efficiently than Foxp3GFP+TReg from other groups. Finally, OVA-IVIg pretreated DCs induced significantly more Foxp3GFP+TReg in-vitro than DCs pretreated with OVA or IVIg alone. CONCLUSIONS: IVIg induces Treg in an antigen-specific fashion. The induction of Treg appears to be mediated through conditioning of DCs.
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Implementation and Impact of a Metered Dose Inhaler Based Asthma Pathway in a Urban University Hospital M. E. Bollinger1, Z. Hasan2, D. Steacy3, K. Rafei1; 1University MD School of Medicine, Baltimore, MD, 2Lake Erie College of Osteopathic Medicine, Erie, PA, 3University of Maryland Medical System, Baltimore, MD. RATIONALE: Asthma medication delivery by metered dose inhaler (MDI) has been shown to be equivalent to nebulizers; however, their use in acute care settings is not widespread. We report on the implementation and outcomes of a metered dose inhaler based asthma pathway in an inner city academic hospital. METHODS: A multidisciplinary team developed and implemented the MDI pathway (start date 1/04 in the Emergency Department (ED) and 1/ 06 in the inpatient unit). Data were collected from the University of Maryland Medical System for all ED visits and hospitalizations for ICD9 code (493.–) between 2002 and 2009 for children 2-18 years. Demographic information, admission rates, length of stay and readmission rates were collected to include >2 years pre and post implementation of the pathway. RESULTS: Between 2002-2009, annual ED rates ranged from 1,1333,065 (mean52,616). Annual hospitalization rates ranged from 313-653 (mean5426). The demographics of the patients did not vary over the study period with mean age of 7.5 years with 90% African American, 43% female and 80% Medicaid insured. ED admission rates averaged 12.2% in the 2 years prior to the pathway and 7% in the 2 years post. Mean length of hospital stay in the 4 years prior to the pathway (‘02-‘05) was 3.51 days vs. 2.90 days in the 3 years post (’06-’09). Readmission rates were not significantly different pre and post-implementation. CONCLUSIONS: The MDI pathway has been well accepted in this inner city academic hospital with no adverse effects on admission or readmission rates or length of stay.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
TGF-b, IL-1b, And IL-23 Are Required For IL-13Ra1 Expression On Human Th17 Cells And IL-13 Attenuates IL-17A Production At Restimulation M. M. Huckabee, D. C. Newcomb, M. G. Boswell, K. Goleniewska, S. Reiss, R. S. Peebles, Jr, ; Vanderbilt University Medical Center, Nashville, TN. RATIONALE: IL-13 receptor (IL-13R) is comprised of IL-13Ra1 and IL-4Ra, and IL-13Ra1 is expressed on human Th17 cells but not Th1 or Th2 cells. IL-13 attenuates IL-17A production in Th17 cells, but the mechanism remains unknown. We hypothesized that one or more of the cytokines involved in Th17 polarization is responsible for IL-13Ra1 expression. Further, we hypothesized that IL-13 inhibited IL-17A production by decreasing phosphorylation of STAT3, a transcription factors required for Th17 cell differentiation. METHODS: Na€ıve human CD4+ cells were activated in the presence of anti-IL-4 and anti-IFN-g and one or more of the following cytokines: IL2, TGF-b, IL-1b, IL-6, and/or IL-23. IL-13 (10ng/ml) was given at day 0, 1, 2 or 3 of activation. T cells were also restimulated with anti-CD3 and IL-13 was added at the time of restimulation. Total RNA, total protein, and supernantants were harvested 4 days after polarization. RESULTS: TGF-b, IL-1b, and IL-23, but not IL-6, were required for IL-13Ra1 expression on human Th17 cells, and IL-13 only attenuated IL-17A production in cultures with these cytokines. STAT3 phosphorylation was decreased in the presence of IL-13. IL-13 attenuated IL-17A production when added at activation (day 0), but not later time points. Further, IL-13 attenuated IL-17A production in restimulated Th17 cells. CONCLUSIONS: IL-13Ra1 expression on CD4+ T cells requires TGF-b, IL-1b, and IL-23. IL-13 attenuates IL-17A production only at the time of polarization or at restimulation. IL-13 also decreases IL-17A production at restimulation. These results indicate that IL-13 inhibits IL-17A production early during stimulation.
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Human IL-27 Suppresses Th2 Cell Differentiation Independent of IFN-g and IL-10 S. Wang1, Y. Zhuang2, N. Erekosima2, R. Katial2, R. Alam2, H. Huang1; 1 National Jewish Health and University of Colorado Denver, Denver, CO, 2 National Jewish Health, Denver, CO. RATIONALE: Murine IL-27 is a potent suppressor of Th2 cell differentiation. However, it is unknown whether murine IL-27 suppresses Th2 cytokine production by differentiated Th2 cells and if so which signaling pathway murine IL-27 uses. Moreover, it is unclear if human IL-27 suppresses Th2 cell differentiation. We sought to further understand the mechanisms by which murine IL-27 suppresses Th2 cell differentiation and to determine whether human IL-27 suppresses Th2 cell differentiation. METHODS: Naive CD4+ T cells were isolated from C57BL/6, STAT1deficient, or T-bet-deficient mice and primed under Th2-inducing conditions in the presence or absence of murine or human IL-27. IL-4 and IFN-g expression was determined using intracellular staining and ELISA. Anti-IL-10 antibody or immunoglobulin isotype control was used to determine whether IL-27 suppresses Th2 cell differentiation dependent on IL-10. Similarly, naive human CD4+ T cells were isolated from peripheral blood and primed under Th2-inducing conditions in the presence or absence of human IL-27. The roles of IFN-g and IL-10 were assessed by using neutralizing antibodies. RESULTS: Murine IL-27 suppressed Th2 cell differentiation but failed to suppress Th2 cytokine production by previously differentiated Th2 cells. Murine IL-27 inhibited Th2 cytokine production in a STAT1-dependent but T-bet-independent manner. We found that human IL-27 suppressed IL-4 production independent of both IFN-g and IL-10. CONCLUSIONS: Murine IL-27 suppresses Th2 cell differentiation in a STAT1-dependent but T-bet-independent manner and human IL-27 suppresses Th2 cell differentiation independent of both IFN-g and IL-10.