Identification and Characterization of Signaling Pathways and Mediators Regulating Mast Cell-Related Disorders

National Institute Of Allergy And Infectious Diseases

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Abstract

1-One of the main aims of this project is to identify mediators or vehicles of intercellular communication, including extracellular vesicles (EV’s), that contribute to the pathogenesis and/or the presentation of allergic diseases and mast cell-related diseases. EVs are membrane-bound, nano-sized vesicular structures secreted by most cell types into body fluids. EVs contain and shuttle distinct biological molecules that can be taken up by recipient cells and alter their cellular functions, and thus are considered vehicles for cell-to-cell communication. Previously, we have shown that EVs released by neoplastic mast cells and those found in the serum of patients with systemic mastocytosis, a mast-cell related disorder, can alter pathways in non-hematopoietic recipient cells. For instance, EVs from these patients, when taken up by hepatic stellate cells, induced an abnormal fiber-producing phenotype in these cells; and when taken up by osteoblasts, they inhibited their ability to produce bone. These findings linked the altered composition of circulating EVs in mastocytosis with their potential to contribute to liver fibrosis and osteoporosis - two common presentations in this disorder, and provided the rationale for pursuing similar avenues in other mast cell-related diseases, such as allergic disorders. In FY2025, we have been investigating the composition of circulating EVs in a cohort of pediatric patients with high levels of total IgE and food-allergen-specific IgE before and after challenge with these foods. The findings will provide insight into the mechanisms behind the presentation of allergic reactions and sensitivity of these patients to the food allergens. For example, the data indicate that increased markers of activated neutrophils distinguish subpopulations of children with high and low sensitivity to peanut. Furthermore, current data also suggest that the composition of EVs in these children with food allergies differs from that in children without allergies, providing clues into the altered immunological environment of these individuals. This may allow us to identify markers or factors that impact the positive or negative reactions of these patients to food allergen challenges. Related to EVs in mast-cell related disorders, in FY2024 we completed a study that supported the concept that EVs released from a cell in an altered state or activated by a stimulus have a cascading effect in vivo, causing changes in receptor availability and signaling processes in other cells. Specifically, we found that ligand-induced activation of the tyrosine kinase receptor KIT in mast cells or constitutively active KIT due to gain-of-function mutations in KIT commonly found in patients with mastocytosis, results in the secretion of more numerous and unique EVs containing KIT, which was reversed by inhibition of KIT signaling (PMID: 38938682). These EVs transferred oncogenic KIT to hepatic stellate cells, linking the dissemination via EVs of oncogenic KIT from a cell with a somatic mutation to another cell type and thus contributing to a disease presentation such as liver fibrosis. Along these lines, in FY2024 and 2025 we found that human mast cells activated via the high affinity receptor for IgE (FceRI) also secrete more EVs than resting mast cells and have a distinct cargo. FceRI is characteristically and abundantly expressed in mast cells and basophils. After exposure to a cognate antigen (or allergen), the IgE bound to the receptor is crosslinked, activating the receptor and resulting in the release of allergic and inflammatory mediators that mediate allergic reactions. However, the function of EVs released after activation of FceRI is currently unknown. During FY2025 we have been characterizing EVs secreted by mast cells after exposure to antigen-specific IgE (sensitization) and after challenge with the specific antigen. Surprisingly, we observed IgE sensitization increases the release of EVs by human and mouse mast cells in vitro and in mice, and that the lipid and protein composition of these EVs is also distinct from the composition of EVs released by FceRI-activated or resting mast cells. Of note, IgE sensitization of mast cells not only increased the number of EVs released but also the amount of IgE bound to FceRI on the EV surface per particle. Such IgE-containing EVs were able to transfer their IgE/FceRI complexes to naïve mast cells conferring them the ability to degranulate in response to the specific antigen. Given that allergic individuals typically have IgE levels above the normal range, these data raise the possibility that circulating EVs from allergic patients may also confer activation potential to other immune cells with an impact on allergic inflammation. A summary of several scientific efforts related to mast cell-related diseases, including some of those described above, was recently published in a review envisioned during a Symposium honoring Dr. Metcalfe’s 40 years of service in the Federal Government (PMID: 40633596). 2- In a separate project in FY2025, we analyzed data collected during the COVID-19 pandemic to evaluate the value of basophil activation tests (BAT) in predicting allergic reactions to the mRNA COVID-19 vaccine (BNT162b2). Basophils, as like mast cells, are major effector cells in allergy. Allergic reactions to the COVID-19 mRNA vaccine contribute to vaccine hesitancy and vaccine avoidance. Thus, identifying a test that accurately predicts allergic reactions to mRNA vaccines is critical. Our analysis, unfortunately, indicated that BAT, which is used commonly used as a predictor of other allergic conditions, cannot predict allergic reactions to the BNT162b2 vaccine. Instead, we found that the number of previous vaccinations received increased basophil reactivity, thus representing a confounding factor for interpreting BAT as a predictive test for reactions. Further studies are necessary to understand how repeated vaccination increases basophil activation and to find a test that can accurately predict allergic reactions to the mRNA vaccine (PMID: 40497015). 3- During previous fiscal years, we studied the potential contributions of the receptor 4 for the lipid mediator sphingosine -1-phosphate (S1P), S1PR4, on the innate and adaptive responses. S1P is an important lipid mediator for immunity, sensitization to food allergens and recovery from anaphylactic shock, and it acts partly by binding to its receptors (S1PRs). Several studies have indicated an association between S1P and allergic disorders. One of the five members of the S1PR receptor family, S1PR4, is confined to hematopoietic cells and yet its role in immune regulation remains ill-understood. By the end of FY2024 we published a study showing that loss of S1PR4 results in smaller draining lymph nodes after footpad immunization in mice, with reduced cell numbers and activation status of effector B and T cells in germinal centers (GC), and faulty maturation of the lymph node vascular network. These deficiencies were a consequence of reduced infiltration of circulating lymphocytes into the lymph node - a vital process for antigen surveillance meant to establish productive GC. Surprisingly, we revealed that at the very early stages of acute inflammation, S1PR4 in neutrophils, but not in lymphocytes, was important for local vascular changes in the tissue and optimal redirection of lymphocytes to lymph nodes. Our manuscript provides insight into novel roles for S1PR4 in immunity and highlights an underappreciated link between the innate and adaptive immune systems (PMID: 39188715).