Mast cells are effector and regulatory cells involved in the pathogenesis of allergic inflammation, generally through the activation of the high affinity IgE receptor (Fc-epsilon-RI). Binding of an antigen to IgE/Fc-epsilon-RI complexes initiates intracellular signaling events that drive mast cell responses. In addition, signaling by other cell surface receptors can activate mast cells or modify their responses to the IgE receptor. A deeper understanding of the signaling mechanisms initiated by receptors expressed in mast cells and their crosstalk can give clues on the regulation of allergic reactions and treatment options for human disease. Recently, we found that an adhesion G-protein coupled receptor, ADGRE2 (EMR2) is abundantly expressed in human mast cells and that a missense variant (p.C492Y) of ADGRE2 identified in patients with autosomal dominant vibratory urticaria associates with disease presentation. Patients with this variant exhibit mast cell hyperreactivity upon mechanical stimuli of repetitive nature resulting in localized hives, increased histamine levels in serum and increased extracellular tryptase staining in the dermis compared to normal subjects. The p.C492Y variant destabilizes a non-covalent interaction between the alpha and beta subunits of ADGRE2, rendering the receptor more prone to dissociating the alpha subunit after vibration and resulting in its activation. In fiscal year (FY) 2019, in collaboration with Drs. Schwartz, Lyons, and Milner we have unveiled another mechanism for the activation of ADGRE2 by proteolytic cleavage of the alpha ADGRE2 subunit mediated by an uncommon heterotretramer of mast cell tryptase. Heterotetramers of alpha and beta tryptase are present in patients with hereditary alpha-tryptasemia, but not in other individuals that usually have beta homotetramers. This mechanism links their genotype with the higher responsiveness of these patients to vibratory stimuli in the skin. Another focus in our lab has also been the study of mast cell proliferative disorders. In systemic mastocytosis (SM), neoplastic accumulation of mast cells associates with genetic variants, particularly the missense mutation D816V, in the tyrosine kinase receptor KIT, rendering it constitutively active. Normal KIT signaling is important for the differentiation, proliferation and survival of mast cells and also enhances mast cell responses to allergic stimulation. However, oncogenic signaling due to D816V or other KIT variants results in abnormal mast cell responses. We have previously implicated the sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) axis as a critical pathway in oncogenic KIT signaling leading to abnormal mast cell growth, a knowledge that can be helpful when considering alternatives approaches to the treatment of aggressive SM, where tyrosine kinase inhibitors have shown limited long-term improvement. In FY 2019, we have demonstrated a role for aberrant signaling of mast cells with D816V-KIT in the persistent IL-6 production in SM. Patients with SM have increased serum levels of IL-6 in association with disease severity and progression. However, the cell source and mechanisms involved are not well understood. We found that intracellular IL-6 staining by flow cytometry and immunofluorescence was primarily associated with mast cells and suggested a higher percentage of IL-6 positive mast cells in patients with higher D816V allelic burden. Among the aberrant signals from D816V-KIT, we identified increased JAK2 activity and MEK/ERK- and PI3K-derived signals as drivers of IL-6 transcription and consequent IL-6 release, the former two by regulating the activation/expression of STAT5. The involvement of STAT5 in persistent IL-6 production in D816V-KI mast cells contrasts with mechanisms described in other malignant cells where autocrine feed-forward loops involving IL-6 and STAT3 are common. The study provides the first clues into mechanisms leading to persistent IL-6 production in mastocytosis and potential target molecules for therapeutic intervention. During FY 2019 we have also completed studies on the identification of other vehicles of intercellular communication released by mast cells that may contribute to the pathology of mast cell proliferative diseases. We demonstrated that serum of patients with SM contains elevated concentrations of extracellular vesicles (EVs) with a mast cell signature and which associate with markers of disease severity. Based on the associations of the number of EVs in SM with hepatomegaly and alkaline phosphatase levels, a marker of liver function, we asked whether these EVs could contribute to the abnormal liver pathology associated with mastocytosis. In FY 2019, we reported that KIT contained in these EVs in macrocytosis was transferred into hepatic stellate cells eliciting proliferation, cytokine production, and differentiation, processes known to occur in hepatic fibrosis pathologies. Confirming these conclusions, the effects were reduced by KIT inhibition or neutralization and recapitulated by enforced expression of KIT or constitutively active D816V-KIT. Our data also indicated that stellate cells were activated in vivo in livers from mice injected with SM-EVs. This study suggests that EVs in SM have the potential to induce a fibrotic phenotype in stellate cells, a characteristic change associated with liver pathology, by introducing functionally active KIT into stellate cells. The identification of EVs from neoplastic cells in mastocytosis has opened the possibility that their specific cargos can be shuttled into cells other than stellate cells, dysregulating their function and thus contributing to some pathological aspects associated with mastocytosis. Studies of SM-EVs on other cell targets is a subject of further investigation during the current FY2019.