A focus in our lab has been the study of allergic diseases and mast cell proliferative disorders. Even though much is known about mast cell-derived mediators and their potential contributions to these diseases, the identification of mediators (derived or not from mast cells) and receptors that critically influence certain aspects of the allergic response and mast cell related disorders remains incompletely understood. Mast cells usually respond to an allergen via the activation of the high affinity receptor of IgE. However, some allergic-like reactions in a variety of syndromes such as urticaria are caused by largely unknown antigen-independent mechanisms. An example is vibratory urticaria (VU), a subtype of physical urticaria triggered by sustained vibration against the skin. We demonstrated a pathogenic involvement of mast cells in families with inherited vibratory urticaria and identified a missense mutation in an adhesion G-protein coupled receptor known to bind dermatan sulfate, (ADGRE2 or EMR2) associated with this condition. Human mast cells expressing mutant EMR2 sensitized them to hyperactivation in response to vibration. The findings implicate that mast cells can sense physical stimuli through a membrane receptor, a novel facet to mast cell biology and of relevance to physical urticarias. In relationship to factors influencing allergic responses (related to objective 1 of this project), it is unclear why individuals with similar levels of allergen-specific IgE may respond differently to exposure to the antigen. In collaboration with Dr. Josh Milner, we identified a role for STAT3 on anaphylaxis severity. Patients and mice with loss of function STAT3 (AD-HIES) mutations are protected against anaphylaxis to an antigen. Loss of STAT3 activity did not affect antigen-induced mediator release from mast cells, but instead reduced vascular leakage in response to mast cell derived products during anaphylaxis. This effect was mediated through stabilization of endothelial cell adherens junctions, causing defective permeability. Thus, status of STAT3 is a factor influencing anaphylactic responses to the same antigen. An implication of the study is that STAT3 inhibitors may be considered in patients with anaphylaxis. Epidemiological studies suggested more frequent anaphylactic reactions occur in adult women, although whether sex hormones can affect anaphylaxis had not been established. We used a controlled mouse model to demonstrate that anaphylactic responses were more severe and longer lasting in female than in male mice and that this was related to the female steroid estradiol. Estradiol increased tissue expression and phosphorylation of endothelial nitric oxide synthase (eNOS), whose activity producing NO is critical for blood vessel responses during anaphylactic shock. Blockage of NOS activity with the inhibitor L-NAME or genetic eNOS deficiency abolished the gender-related differences, linking increased NO production in females with vascular hyper-permeability and intensified anaphylactic responses. Our study thus provides mechanistic insights into risk factors for anaphylaxis in women. Sensitivity to an inciting allergen often requires cofactors such as non-steroidal anti-inflammatory drugs (NSAID). However, the reasons for NSAID dependency in the manifestation of anaphylaxis in some individuals but not others are unknown. Also, in relationship to objective 1 of this project, we interrogated the gene expression profiles that characterize two distinct groups of patients with food anaphylaxis: patients with documented anaphylaxis to lipid transfer protein (LTP), which is variably present in fruits, vegetables and nuts; and patients with sensitivity to LTP only after ingesting NSAIDs. Our high-throughput sequencing and gene expression analysis indicated distinct patterns in patients with food anaphylaxis without NSAID dependence compared to those with NSAID-dependent food anaphylaxis. This study presents evidence of underlying changes in gene expression that predispose to the induction of food induced anaphylaxis in the presence of co-factors such as NSAIDS, and suggests novel mechanisms that may play a role in systemic reactions to antigens. Sphingosine-1-phosphate signaling was identified as a canonical pathway exclusively downregulated in the NSAID-independent food anaphylaxis group of patients. In relationship to objective 2, since sphingosine-1-phosphate (S1P) is an important lipid mediator for immunity, sensitization to food allergens and recovery from anaphylactic shock, we are investigating the role of S1P and its receptors in mast cells and basophils, and in models of allergic inflammation. A third avenue of this project is the identification and characterization of mediators that are altered in diseases of mast cell proliferation such as mastocytosis. Neoplastic accumulation of mast cells in systemic mastocytosis (SM) associates with activating mutations in the receptor tyrosine kinase KIT. Constitutive activation of tyrosine kinase oncogenes has been linked to imbalances in oxidant/antioxidant mechanisms in other myeloproliferative disorders. However, the impact of KIT mutations on the redox status in SM and the potential therapeutic implications are not well understood. We thus examined the regulation of reactive oxygen species (ROS) and of the antioxidant protein DJ-1 (PARK-7), which increases with cancer progression and acts to lessen oxidative damage to malignant cells, in relationship with SM severity. In less advanced disease, the serum levels of DJ-1 are reduced due to activating KIT mutations in mast cells and continuous production of ROS resulting in DJ-1 oxidation and degradation. However, in patients with high mast cell burden and advanced disease, DJ-1 levels are instead increased due to escalating IL-6 which robustly induces DJ-1 transcription and overcomes DJ-1 depletion. Blockade of IL-6 receptor minimized ROS and DJ-1 dysregulation and retarded the progressive accumulation of mast cells in a mouse model of mastocytosis and thus we suggest that increased DJ-1 may favor mast cell expansion by protecting against oxidative stress. IL-6 blockade could represent an adjunctive therapy in the treatment of patients with advanced mastocytosis, as it would reduce DJ-1 levels, making mutation-positive mast cells vulnerable to oxidative damage.

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Kulinski, Joseph M; Proia, Richard L; Larson, Elisabeth M et al. (2018) S1P? Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells. Int J Mol Sci 19:
Olivera, Ana; Beaven, Michael A; Metcalfe, Dean D (2018) Mast cells signal their importance in health and disease. J Allergy Clin Immunol 142:381-393
Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli et al. (2018) Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT. Front Immunol 9:631
Yin, Yuzhi; Bai, Yun; Olivera, Ana et al. (2017) An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations. J Immunol Methods 448:105-111
Muñoz-Cano, Rosa; Pascal, Mariona; Bartra, Joan et al. (2016) Distinct transcriptome profiles differentiate nonsteroidal anti-inflammatory drug-dependent from nonsteroidal anti-inflammatory drug-independent food-induced anaphylaxis. J Allergy Clin Immunol 137:137-146
Boyden, Steven E; Metcalfe, Dean D; Komarow, Hirsh D (2016) Vibratory Urticaria and ADGRE2. N Engl J Med 375:95
Kim, Do-Kyun; Beaven, Michael A; Kulinski, Joseph M et al. (2016) Regulation of Reactive Oxygen Species and the Antioxidant Protein DJ-1 in Mastocytosis. PLoS One 11:e0162831
Taguchi, Yoshimitsu; Allende, Maria L; Mizukami, Hiroki et al. (2016) Sphingosine-1-phosphate Phosphatase 2 Regulates Pancreatic Islet ?-Cell Endoplasmic Reticulum Stress and Proliferation. J Biol Chem 291:12029-38
Kirshenbaum, Arnold S; Cruse, Glenn; Desai, Avanti et al. (2016) Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis. PLoS One 11:e0159177
Desai, Avanti; Jung, Mi-Yeon; Olivera, Ana et al. (2016) IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3. J Allergy Clin Immunol 137:1863-1871.e6

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