Mast cells are critical effector and regulatory cells involved in the pathogenesis of allergic inflammation. Allergic responses are generally initiated by antigen-dependent activation of the high affinity IgE receptor (Fc-epsilon-RI) expressed on the cell surface of mast cells and basophils. This causes a series of signaling events that culminate in the release mediators that cause immediate and delayed reactions. However, mast cells in particular express a variety of receptors, such as KIT (the receptor for stem cell factor, SCF) and various GPCRs whose ligands may modulate antigen-mediated mast cell activation, and thus the allergic response. Despite the importance of these events in allergic inflammation, the signaling pathways linking Fc-epsilon-RI aggregation to human mast cell activation and how other receptors modify these Fc-mediated signaling events are incompletely understood. This is partly due to the difficulty in obtaining human mast cells sufficient for analysis. We have circumvented this limitation by using primary human mast cells differentiated in culture from human blood CD34+ progenitors (Bandara et al, Methods Mol Biol, 2015) and the human mast cell line LAD2, developed in this lab and which has similar characteristics to CD34+ derived primary mast cells (Kirshenbaum et al; Int Arch Allergy Immunol, 2014). Our signaling studies have previously demonstrated that stimulation of mast cells with SCF or through Fc-epsilon-RI activates the catalytic activity of mammalian target of rapamycin (mTOR). Activation of this complex by SCF and GPCRs is important for survival, proliferation, chemotaxis and for cytokine and chemokine production in mast cells. More recently we have explored the impact of mTOR (complexes 1 and 2) on Fc-epsilon-RI-induced mast cell degranulation. Although inhibition of these complexes had no effect on degranulation, we paradoxically found that knockdown of rictor, a binding and regulatory partner of the mTORC2 complex, increased antigen-induced mast cell degranulation independently of its role on mTORC2 function. This function was specific for Fc-epsilon-RI-signaling since mast cell degranulation induced by other stimulants was unaffected by rictor knockdown. Furthermore, we detailed that rictor knockdown increased phosphorylation of LAT and PLCγ1 , calcium mobilization and the activation of PI3K activity. Thus, these studies have identified rictor as a new regulator of Fc-epsilon-RI early signaling in mast cells which could be of importance in allergic disease. The cytokine IL-33 plays a central role in type 2 immune responses and allergic inflammation. We had found that long-term exposure of human and mouse mast cells to IL-33 results in a substantial reduction of mast cell responses by attenuating signaling processes necessary for mast cell activation. Recently, we discovered that CD34+-derived and LAD human mast cells as well as murine mast cells in vivo and in vitro do not produce significant amounts of IL-33, but they are a significant source of the soluble form of the IL-33 receptor (sST2) when stimulated with antigen alone and especially in combination with IL-33 and or SCF. sST2 is known to neutralize IL-33 activity and thus our results suggest that mast cells have the ability to modulate the biologic impact of IL-33 produced locally by other cell types during allergic inflammation. These studies provide clues into the complexity of how inflammatory signals affect mast cell responses and, reciprocally, how mast cell activation modulates allergic inflammation. Our previous studies in the lab demonstrated that in human mast cells a truncated variant of the gene encoding for FcεRI (MS4A2) named t- Fc-epsilon-RI, is critical for microtubule formation, degranulation and cytokine release by facilitating trafficking of signaling proteins to the pericentrosomal and Golgi regions in response to calcium signals. Knockdown of t-Fc-epsilon-RI attenuates mast cell responses and thus t- FcεRI may represent a therapeutic target for the downregulation of allergic inflammation. Our more recent studies focused on the function in human mast cells of another member of the MS4A family, MS4A4, because of its strong link to allergy and asthma susceptibility and its known role as a regulator proliferation in other cell types. We found that silencing of MS4A4 altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes (AKT phosphorylation) while reducing it in the plasma membrane (PLCγ1 phosphorylation). As a result of the increase in AKT signaling, SCF-induced mast cell proliferation and migration were also enhanced in mast cells. Thus, our findings indicate that MS4A4 regulates mast cell function by promoting surface expression of KIT as well as signaling and imply that MS4A4 dysfunction in mast cells may result in aberrant cellular responses related to SCF function.

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2015
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Kim, Do-Kyun; Beaven, Michael A; Metcalfe, Dean D et al. (2018) Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells. J Allergy Clin Immunol 142:195-206.e8
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
Cruse, Glenn; Yin, Yuzhi; Fukuyama, Tomoki et al. (2016) Exon skipping of Fc?RI? eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy. Proc Natl Acad Sci U S A 113:14115-14120
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
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

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