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Roflumilast Ameliorates Ovalbumin-Induced Neutrophilic Airway Inflammation in Mice: Role of CD4+ CD25+ FOXP3+ Regulatory T-Cells
Abstracts AB379
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
L13
Differential Role of Foxp3+ Regulatory T Cells during Allergen Sensitization and Challenge
Prof Booki Min, DVM, PhD; Lerner Research Institute/NB30, Cleveland, OH. RATIONALE: The regulatory functions of Foxp3+ regulatory T (Treg) cells, a central regulator in inflammation, during allergic inflammation remain unclear. METHODS: Cockroach antigen is chosen as a model allergen. B6 Foxp3DTR mice that express the diphtheria toxin (DT) receptor under the Foxp3 promoter were sensitized with intraperitoneal injection of CA in Alum. Two weeks later, the animals were intranasally challenged with CA in PBS for 4 consecutive days. Mice are sacrificed 24 hours after the last CA challenge. Treg cells were depleted by administering DT at various time points (at sensitization, prior to challenge, and post challenge). Inflammatory CD4 T cell responses were evaluated. RESULTS: Following allergen challenge, 30;40% of CD4 T cells infiltrating the inflamed sites expressed Foxp3, indication of Treg cells. The level peaked around one day after the last antigen challenge and gradually diminished thereafter. When Treg cells were depleted prior to or post allergen challenge, the lung inflammation was severely exacerbated. Infiltrating CD4 T cell levels were markedly increased, and the proportions of T cells producing inflammatory cytokines (IL-4, IL-10, IL-13, and IL17) were substantially elevated. Interestingly, depleting Treg cells at the time of allergen sensitization resulted in significant increase of CD4 T cell infiltration. While Th2 type cytokine producing CD4 T cells were greatly increased, we found that T cells producing IL-17 were rather decreased. CONCLUSIONS: The current study demonstrates that Treg cells may play a critical role in regulating allergic inflammatory responses not only during antigen challenge but also during antigen sensitization.
L14
Roflumilast Ameliorates Ovalbumin-Induced Neutrophilic Airway Inflammation in Mice: Role of CD4+ CD25+ FOXP3+ Regulatory T-Cells
Prof Mohamed G. Elsakkar, Sr1, Prof Olla A. Sharaki2, Dr Dina M. Abdallah2, Dr Dalia K. Mostafa2, and Dr Fadia T. Shekondali3; 1Alexandria University, Alexandria, Egypt, Alexandria, Egypt, 2Faculty of Medicine, Alexandria University, Alexandria, Egypt, Alexandria, Egypt, 3Faculty of Medicine, EL-Zawia University, Libya, EL-Zawia, Libya. RATIONALE: More severe forms of asthma seem to have neutrophilic inflammation with less reversible airway obstruction and poor response to treatment. Regulatory T cells (Treg) are characterized by their ability to suppress effector T cells. Prevention of cAMP degradation strongly increased suppressive potency of Treg cells. We hypothesized that the use of roflumilast, a selective PDE4 inhibitor may augment the Treg cells suppression on neutrophilic airway inflammatory responses. METHODS: Male CD1 mice were divided into 4 groups, 12 mice each. Normal control group, ovalbumin (OVA) sensitized and challenged group which served as positive control, and roflumilast-treated OVA-sensitized and challenged group. Bronchoalveolar lavage (BAL) fluid were assessed for IL-4, IFN-g and IL-10 cytokines, the cell pellets were cytologically examined. The lungs were excised for histo-pathological examination using H&E, periodic acid Schiff and Masson’s trichrome stains. Splenocytes were separation and analyzed for identification of CD4+ CD25+ FOXP3+ Treg cells. ANOVA one-way test was used for the statistical analysis of the data. A level of P < 0.05 was considered statistically significant. RESULTS: Treatment with roflumilast significantly attenuated OVAinduced increase in inflammatory cytokines (IL-4 and IFN-g), suppressed recruitment of inflammatory cells in BAL fluids and lungs, and inhibited BAL fluid neutrophilia (P < 0.05). It reduces goblet cell metaplasia and bronchial fibrosis. Roflumilast significantly increased percentage of CD4+
CD25+ FOXP3+ Treg cells, associated with increased IL-10 in BAL fluid (P < 0.05). CONCLUSIONS: Treatment with roflumilast ameliorates airway neutrophilic inflammation associated with significant increase in Treg cell number and function.
L15
Covalent Heterbivalent Allergies
Inhibitors
for
Food
Mr Peter E. Deak1, and Dr Basar Bilgicer, PhD2; 1University of Notre Dame, Notre Dame, IN, 2University of Notre Dame, Notre Dame. RATIONALE: Food allergies are a common problem in the developing world and frequently cause severe life-threatening anaphylaxis reactions and peanut allergies are the most common severe food allergy. We believe that by identifying and inhibiting the allergen specific immunoglobulin E (sIgE) molecules which are responsible for multivalent allergen binding and subsequent triggering of mast cell and basophil degranulation, these allergic reactions can be prevented. METHODS: In order to identify the sIgEs of interest in peanut allergies, we used a nanoparticle based IgE binding epitope evaluation technique we developed called nanoallergens. By presenting relevant IgE binding peptides taken from the primary sequence of the major peanut allergens and presenting them on a liposomal surface, we can identify which epitope trigger degranulation to rat basophil leukemia (RBL) cells sensitized with peanut allergic patient serum. Next, using the sIgE binding sequences, we designed covalent heterobivalent inhibitors (cHBIs) which can specifically bind sIgEs for peanuts and form covalent bonds with IgEs of interest, permanently inhibiting them. Combinations of these inhibitors were incubated with four different allergy patient sera and then their sIgE binding and inhibitory effects were observed using ELISA and RBL cell assays. RESULTS: Using only a two cHBI combination, we showed selective binding to allergy patient sera and a complete prevention of RBL cell degranulation over a wide range of crude peanut extract concentration. CONCLUSIONS: We demonstrate the clinical potential of cHBIs to inhibit allergic reactions and the potential for the nanoallergen-cHBI combination to provide personalized treatments for any allergies from foods to drugs.
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
L13
Differential Role of Foxp3+ Regulatory T Cells during Allergen Sensitization and Challenge
Prof Booki Min, DVM, PhD; Lerner Research Institute/NB30, Cleveland, OH. RATIONALE: The regulatory functions of Foxp3+ regulatory T (Treg) cells, a central regulator in inflammation, during allergic inflammation remain unclear. METHODS: Cockroach antigen is chosen as a model allergen. B6 Foxp3DTR mice that express the diphtheria toxin (DT) receptor under the Foxp3 promoter were sensitized with intraperitoneal injection of CA in Alum. Two weeks later, the animals were intranasally challenged with CA in PBS for 4 consecutive days. Mice are sacrificed 24 hours after the last CA challenge. Treg cells were depleted by administering DT at various time points (at sensitization, prior to challenge, and post challenge). Inflammatory CD4 T cell responses were evaluated. RESULTS: Following allergen challenge, 30;40% of CD4 T cells infiltrating the inflamed sites expressed Foxp3, indication of Treg cells. The level peaked around one day after the last antigen challenge and gradually diminished thereafter. When Treg cells were depleted prior to or post allergen challenge, the lung inflammation was severely exacerbated. Infiltrating CD4 T cell levels were markedly increased, and the proportions of T cells producing inflammatory cytokines (IL-4, IL-10, IL-13, and IL17) were substantially elevated. Interestingly, depleting Treg cells at the time of allergen sensitization resulted in significant increase of CD4 T cell infiltration. While Th2 type cytokine producing CD4 T cells were greatly increased, we found that T cells producing IL-17 were rather decreased. CONCLUSIONS: The current study demonstrates that Treg cells may play a critical role in regulating allergic inflammatory responses not only during antigen challenge but also during antigen sensitization.
L14
Roflumilast Ameliorates Ovalbumin-Induced Neutrophilic Airway Inflammation in Mice: Role of CD4+ CD25+ FOXP3+ Regulatory T-Cells
Prof Mohamed G. Elsakkar, Sr1, Prof Olla A. Sharaki2, Dr Dina M. Abdallah2, Dr Dalia K. Mostafa2, and Dr Fadia T. Shekondali3; 1Alexandria University, Alexandria, Egypt, Alexandria, Egypt, 2Faculty of Medicine, Alexandria University, Alexandria, Egypt, Alexandria, Egypt, 3Faculty of Medicine, EL-Zawia University, Libya, EL-Zawia, Libya. RATIONALE: More severe forms of asthma seem to have neutrophilic inflammation with less reversible airway obstruction and poor response to treatment. Regulatory T cells (Treg) are characterized by their ability to suppress effector T cells. Prevention of cAMP degradation strongly increased suppressive potency of Treg cells. We hypothesized that the use of roflumilast, a selective PDE4 inhibitor may augment the Treg cells suppression on neutrophilic airway inflammatory responses. METHODS: Male CD1 mice were divided into 4 groups, 12 mice each. Normal control group, ovalbumin (OVA) sensitized and challenged group which served as positive control, and roflumilast-treated OVA-sensitized and challenged group. Bronchoalveolar lavage (BAL) fluid were assessed for IL-4, IFN-g and IL-10 cytokines, the cell pellets were cytologically examined. The lungs were excised for histo-pathological examination using H&E, periodic acid Schiff and Masson’s trichrome stains. Splenocytes were separation and analyzed for identification of CD4+ CD25+ FOXP3+ Treg cells. ANOVA one-way test was used for the statistical analysis of the data. A level of P < 0.05 was considered statistically significant. RESULTS: Treatment with roflumilast significantly attenuated OVAinduced increase in inflammatory cytokines (IL-4 and IFN-g), suppressed recruitment of inflammatory cells in BAL fluids and lungs, and inhibited BAL fluid neutrophilia (P < 0.05). It reduces goblet cell metaplasia and bronchial fibrosis. Roflumilast significantly increased percentage of CD4+
CD25+ FOXP3+ Treg cells, associated with increased IL-10 in BAL fluid (P < 0.05). CONCLUSIONS: Treatment with roflumilast ameliorates airway neutrophilic inflammation associated with significant increase in Treg cell number and function.
L15
Covalent Heterbivalent Allergies
Inhibitors
for
Food
Mr Peter E. Deak1, and Dr Basar Bilgicer, PhD2; 1University of Notre Dame, Notre Dame, IN, 2University of Notre Dame, Notre Dame. RATIONALE: Food allergies are a common problem in the developing world and frequently cause severe life-threatening anaphylaxis reactions and peanut allergies are the most common severe food allergy. We believe that by identifying and inhibiting the allergen specific immunoglobulin E (sIgE) molecules which are responsible for multivalent allergen binding and subsequent triggering of mast cell and basophil degranulation, these allergic reactions can be prevented. METHODS: In order to identify the sIgEs of interest in peanut allergies, we used a nanoparticle based IgE binding epitope evaluation technique we developed called nanoallergens. By presenting relevant IgE binding peptides taken from the primary sequence of the major peanut allergens and presenting them on a liposomal surface, we can identify which epitope trigger degranulation to rat basophil leukemia (RBL) cells sensitized with peanut allergic patient serum. Next, using the sIgE binding sequences, we designed covalent heterobivalent inhibitors (cHBIs) which can specifically bind sIgEs for peanuts and form covalent bonds with IgEs of interest, permanently inhibiting them. Combinations of these inhibitors were incubated with four different allergy patient sera and then their sIgE binding and inhibitory effects were observed using ELISA and RBL cell assays. RESULTS: Using only a two cHBI combination, we showed selective binding to allergy patient sera and a complete prevention of RBL cell degranulation over a wide range of crude peanut extract concentration. CONCLUSIONS: We demonstrate the clinical potential of cHBIs to inhibit allergic reactions and the potential for the nanoallergen-cHBI combination to provide personalized treatments for any allergies from foods to drugs.