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The Histone Deacetylase Inhibitor Trichostatin a (TSA) Suppresses Alternaria Extract-Induced Murine Innate Allergic Inflammation By Blocking Group 2 Innate Lymphoid Cell (ILC2) Activation
Abstracts AB191
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
IL-10 Differentially Regulates IgE and IgG4 Production through Indirect Effects on Na€ıve B Cells
Adora A. Lin, MD, PhD1, Thomas B. Nutman, MD2; 1NIAID, National Institutes of Health, Bethesda, MD, 2National Institutes of Health, Bethesda, MD. RATIONALE: Allergen immunotherapy (and control of parasitic helminth-associated pathology) is associated with decreased antigen/ allergen-specific IgE and increased antigen/allergen-specific IgG4. Although T cell-derived IL-10 appears to contribute by altering the balance of antigen/allergen-specific IgE/IgG4, mechanisms mediating this alteration are largely unknown. Previous studies of IL-10’s effects on the preferential switch from IgE to IgG4 have been conducted primarily with peripheral blood mononuclear cells (PBMCs) and less commonly with purified whole B cell populations. The present study sought to explore how (and whether) IL-10 mediates preferential isotype switching to IgG4 using highly purified na€ıve B cells. METHODS: Human PBMCs and highly purified (>93%) na€ıve B cells (CD3-CD19+CD20+CD21+CD27-) were each cultured under a variety of conditions (combinations of IL-4, IL-10, and anti-CD40). Immunoglobulin (Ig) IgG and IgE isotypes were quantified from culture supernatants using ELISA or multiplexed immunoassays. RESULTS: Consistent with previous studies, in PBMC cultures, IL-4 induced the production of both IgE and IgG4 (IgG4:IgE ratio mean 2.8, range 0.4-8.7). The addition of IL-10 reduced the IL-4-induced IgE production and concurrently enhanced the induction of IgG4 such that the IgG4: IgE ratio had a mean of 42.3 (range 13.2-98.8). However, in cultures containing only na€ıve B cells, IL-10 failed to alter the IgE/IgG4 balance (IgG4: IgE ratio mean 2.5, range 0.71-4.5). CONCLUSIONS: Although IL-10 differentially regulates IgE and IgG4 production in B cells in the context of accessory cells found in PBMCs, it likely does so through indirect effects on B cell isotype switching. These effects are currently being explored at the molecular level.
623
The Histone Deacetylase Inhibitor Trichostatin a (TSA) Suppresses Alternaria Extract-Induced Murine Innate Allergic Inflammation By Blocking Group 2 Innate Lymphoid Cell (ILC2) Activation
Shinji Toki, PhD1, Kasia Goleniewska, MS1, Sara Reiss, MS1, Weisong Zhou, PhD1, Dawn C. Newcomb, PhD1,2, Melissa H. Bloodworth, BS2, Matthew T. Stier, BS2, Kelli L. Boyd, PhD2, Vasiliy V. Polosukhin, MD, PhD1, Sriram Subramaniam, MB, BS3, R. Stokes Peebles, MD1,2; 1Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, TN, 2Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 3Department of Neurology, Vanderbilt University, Nashville, TN. RATIONALE: Group 2 innate lymphoid cells (ILC2) are an important source of the type 2 cytokines IL-5 and IL-13 that are critical to allergic airway inflammation. Previous studies reported that histone deacetylase (HDAC) inhibition by trichostatin A (TSA) down-regulated adaptive allergic immune responses; however, the effect of HDAC inhibition on the early innate allergic immune response is unknown. We therefore tested the hypothesis that TSA treatment reduces Alternaria extract-induced early innate airway inflammation by blocking of ILC2 activation. METHODS: WT BALB/c mice were intranasally challenged with Alternaria extract for 4 consecutive days following TSA or vehicle treatment. Bronchoalveolar lavage (BAL) fluid and lung were harvested 24 hours after the last Alternaria extract-challenge. Protein expression of cytokines and chemokines in the lung were measured by ELISA. IL-5 and IL-13 expressing lung ILC2 were detected by flow cytometry. Eosinophilia and mucus production in the airway were evaluated by histopathological scoring. RESULTS: TSA treatment significantly decreased IL-33, a cytokine that activates lung ILC2, and the number of ILC2 expressing IL-5 and IL-13 in the lungs challenged with Alternaria extract compared to vehicle
treatment. In addition, TSA treatment significantly decreased protein expression of IL-5, IL-13, eotaxin, and eotaxin-2 in the BAL fluid and lung homogenate from Alternaria extract-challenged mice. Moreover, TSA treatment significantly decreased the number of perivascular eosinophils and mucus production in the large airways that are critical components of the asthma phenotype. CONCLUSIONS: TSA treatment reduced ILC2 activation and the early innate allergic immune responses to Alternaria extract.
624
Impact of Skin Damage on Intestinal Immune Environment and Susceptibility to Food Allergy
Ana Belen Blazquez, PhD1, Maria Cecilia Berin2; 1Ichan School of Medicine at Mount Sinai, NY, 2Associate Professor, Icahn School of Medicine at Mount Sinai, NY. RATIONALE: Sensitization can occur through inflamed skin, with subsequent oral exposure leading to manifestations of food allergy. Our aim was to determine whether skin injury could directly alter the immune environment of the gastrointestinal tract, facilitating sensitization by oral rather than epicutaneous allergen exposure METHODS: Balb/c, IL-4- and IL-13-reporter mice were tape stripped (TS) without allergen exposure weekly for 6 weeks. At week 7 mice were repeated challenged with 50mg of OVA. IgE was measured by ELISA and cell populations in intestinal tissues were quantified by flow cytometry and expressed as %CD45+ cells RESULTS: TS but not na€ıve mice developed diarrhea following repeated oral OVA exposure (0% vs 47.5% na€ıve vs TS, respectively), and elevated OVA-specific IgE. TS induced an increase in IL-13 production by mast cells in the small intestinal lamina propria. TS had a major impact on eosinophils in gastrointestinal lymph nodes. The frequency of activated eosinophils (SiglecFhigh) increased in both the mesenteric lymph nodes (MLN) (5.263% vs 20.262%, na€ıve vs TS) and Peyer’s patch (PP) (3264.6% vs 7060.8%, na€ıve vs TS); however, resting eosinophils (SiglecFint) decreased in both the MLN (9060.9% and 766 2.2%, na€ıve and TS, respectively) and in the PP (6363.8% vs 2761.1%, na€ıve vs TS). Eosinophils in the MLN and PP expressed high levels of IL-4 but not IL-13. We observed increased IL-4 expression by lineage negative CD90- cKitint cells CONCLUSIONS: We show that skin injury directly alters the intestinal immune environment. Injury expands Th2 cytokine-producing intestinal innate cell populations, and predisposes to oral sensitization to food allergens
SUNDAY
622
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
IL-10 Differentially Regulates IgE and IgG4 Production through Indirect Effects on Na€ıve B Cells
Adora A. Lin, MD, PhD1, Thomas B. Nutman, MD2; 1NIAID, National Institutes of Health, Bethesda, MD, 2National Institutes of Health, Bethesda, MD. RATIONALE: Allergen immunotherapy (and control of parasitic helminth-associated pathology) is associated with decreased antigen/ allergen-specific IgE and increased antigen/allergen-specific IgG4. Although T cell-derived IL-10 appears to contribute by altering the balance of antigen/allergen-specific IgE/IgG4, mechanisms mediating this alteration are largely unknown. Previous studies of IL-10’s effects on the preferential switch from IgE to IgG4 have been conducted primarily with peripheral blood mononuclear cells (PBMCs) and less commonly with purified whole B cell populations. The present study sought to explore how (and whether) IL-10 mediates preferential isotype switching to IgG4 using highly purified na€ıve B cells. METHODS: Human PBMCs and highly purified (>93%) na€ıve B cells (CD3-CD19+CD20+CD21+CD27-) were each cultured under a variety of conditions (combinations of IL-4, IL-10, and anti-CD40). Immunoglobulin (Ig) IgG and IgE isotypes were quantified from culture supernatants using ELISA or multiplexed immunoassays. RESULTS: Consistent with previous studies, in PBMC cultures, IL-4 induced the production of both IgE and IgG4 (IgG4:IgE ratio mean 2.8, range 0.4-8.7). The addition of IL-10 reduced the IL-4-induced IgE production and concurrently enhanced the induction of IgG4 such that the IgG4: IgE ratio had a mean of 42.3 (range 13.2-98.8). However, in cultures containing only na€ıve B cells, IL-10 failed to alter the IgE/IgG4 balance (IgG4: IgE ratio mean 2.5, range 0.71-4.5). CONCLUSIONS: Although IL-10 differentially regulates IgE and IgG4 production in B cells in the context of accessory cells found in PBMCs, it likely does so through indirect effects on B cell isotype switching. These effects are currently being explored at the molecular level.
623
The Histone Deacetylase Inhibitor Trichostatin a (TSA) Suppresses Alternaria Extract-Induced Murine Innate Allergic Inflammation By Blocking Group 2 Innate Lymphoid Cell (ILC2) Activation
Shinji Toki, PhD1, Kasia Goleniewska, MS1, Sara Reiss, MS1, Weisong Zhou, PhD1, Dawn C. Newcomb, PhD1,2, Melissa H. Bloodworth, BS2, Matthew T. Stier, BS2, Kelli L. Boyd, PhD2, Vasiliy V. Polosukhin, MD, PhD1, Sriram Subramaniam, MB, BS3, R. Stokes Peebles, MD1,2; 1Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, TN, 2Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 3Department of Neurology, Vanderbilt University, Nashville, TN. RATIONALE: Group 2 innate lymphoid cells (ILC2) are an important source of the type 2 cytokines IL-5 and IL-13 that are critical to allergic airway inflammation. Previous studies reported that histone deacetylase (HDAC) inhibition by trichostatin A (TSA) down-regulated adaptive allergic immune responses; however, the effect of HDAC inhibition on the early innate allergic immune response is unknown. We therefore tested the hypothesis that TSA treatment reduces Alternaria extract-induced early innate airway inflammation by blocking of ILC2 activation. METHODS: WT BALB/c mice were intranasally challenged with Alternaria extract for 4 consecutive days following TSA or vehicle treatment. Bronchoalveolar lavage (BAL) fluid and lung were harvested 24 hours after the last Alternaria extract-challenge. Protein expression of cytokines and chemokines in the lung were measured by ELISA. IL-5 and IL-13 expressing lung ILC2 were detected by flow cytometry. Eosinophilia and mucus production in the airway were evaluated by histopathological scoring. RESULTS: TSA treatment significantly decreased IL-33, a cytokine that activates lung ILC2, and the number of ILC2 expressing IL-5 and IL-13 in the lungs challenged with Alternaria extract compared to vehicle
treatment. In addition, TSA treatment significantly decreased protein expression of IL-5, IL-13, eotaxin, and eotaxin-2 in the BAL fluid and lung homogenate from Alternaria extract-challenged mice. Moreover, TSA treatment significantly decreased the number of perivascular eosinophils and mucus production in the large airways that are critical components of the asthma phenotype. CONCLUSIONS: TSA treatment reduced ILC2 activation and the early innate allergic immune responses to Alternaria extract.
624
Impact of Skin Damage on Intestinal Immune Environment and Susceptibility to Food Allergy
Ana Belen Blazquez, PhD1, Maria Cecilia Berin2; 1Ichan School of Medicine at Mount Sinai, NY, 2Associate Professor, Icahn School of Medicine at Mount Sinai, NY. RATIONALE: Sensitization can occur through inflamed skin, with subsequent oral exposure leading to manifestations of food allergy. Our aim was to determine whether skin injury could directly alter the immune environment of the gastrointestinal tract, facilitating sensitization by oral rather than epicutaneous allergen exposure METHODS: Balb/c, IL-4- and IL-13-reporter mice were tape stripped (TS) without allergen exposure weekly for 6 weeks. At week 7 mice were repeated challenged with 50mg of OVA. IgE was measured by ELISA and cell populations in intestinal tissues were quantified by flow cytometry and expressed as %CD45+ cells RESULTS: TS but not na€ıve mice developed diarrhea following repeated oral OVA exposure (0% vs 47.5% na€ıve vs TS, respectively), and elevated OVA-specific IgE. TS induced an increase in IL-13 production by mast cells in the small intestinal lamina propria. TS had a major impact on eosinophils in gastrointestinal lymph nodes. The frequency of activated eosinophils (SiglecFhigh) increased in both the mesenteric lymph nodes (MLN) (5.263% vs 20.262%, na€ıve vs TS) and Peyer’s patch (PP) (3264.6% vs 7060.8%, na€ıve vs TS); however, resting eosinophils (SiglecFint) decreased in both the MLN (9060.9% and 766 2.2%, na€ıve and TS, respectively) and in the PP (6363.8% vs 2761.1%, na€ıve vs TS). Eosinophils in the MLN and PP expressed high levels of IL-4 but not IL-13. We observed increased IL-4 expression by lineage negative CD90- cKitint cells CONCLUSIONS: We show that skin injury directly alters the intestinal immune environment. Injury expands Th2 cytokine-producing intestinal innate cell populations, and predisposes to oral sensitization to food allergens
SUNDAY
622