Mast cells: a novel therapeutic avenue for cardiovascular diseases?

Mast cells are tissue-resident immune cells strategically located in different compartments of the normal human heart (the myocardium, pericardium, aortic valve and close to nerves) as well as in atherosclerotic plaques. Cardiac mast cells produce a broad spectrum of vasoactive and proinflammatory mediators, which have potential roles in inflammation, angiogenesis, lymphangiogenesis, tissue remodeling and fibrosis. Mast cells release preformed mediators (e.g., histamine, tryptase, chymase) and de novo synthesized mediators [e.g., cysteinyl leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)], as well as cytokines and chemokines, which can activate different resident immune cells (e.g., macrophages) and structural cells (e.g., fibroblasts, endothelial cells) in the human heart and aorta. The transcriptional profiles of various mast cell populations highlight their potential heterogeneity and distinct gene and proteome expression. Mast cell plasticity and/or heterogeneity enable these cells the potential for performing different, even opposite, functions in response to changing tissue contexts. Human cardiac mast cells display significant differences compared to mast cells isolated from other organs. These characteristics make cardiac mast cells intriguing, given their dichotomous potential roles of inducing or protecting against cardiovascular diseases. Identification of cardiac mast cell subpopulations represents a prerequisite for understanding their potential multifaceted roles in health and disease. Several new drugs specifically targeting human mast cell activation are under development or in clinical trials. Mast cells and/or their subpopulations can potentially represent novel therapeutic targets for cardiovascular disorders.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

Nomenclature of allergic diseases and hypersensitivity reactions: Adapted to modern needs: An EAACI position paper.

The exponential growth of precision diagnostic tools, including omic technologies, molecular diagnostics, sophisticated genetic and epigenetic editing, imaging and nano-technologies and patient access to extensive health care, has resulted in vast amounts of unbiased data enabling in-depth disease characterization. New disease endotypes have been identified for various allergic diseases and triggered the gradual transition from a disease description focused on symptoms to identifying biomarkers and intricate pathogenetic and metabolic pathways. Consequently, the current disease taxonomy has to be revised for better categorization. This European Academy of Allergy and Clinical Immunology Position Paper responds to this challenge and provides a modern nomenclature for allergic diseases, which respects the earlier classifications back to the early 20th century. Hypersensitivity reactions originally described by Gell and Coombs have been extended into nine different types comprising antibody- (I-III), cell-mediated (IVa-c), tissue-driven mechanisms (V-VI) and direct response to chemicals (VII). Types I-III are linked to classical and newly described clinical conditions. Type IVa-c are specified and detailed according to the current understanding of T1, T2 and T3 responses. Types V-VI involve epithelial barrier defects and metabolic-induced immune dysregulation, while direct cellular and inflammatory responses to chemicals are covered in type VII. It is notable that several combinations of mixed types may appear in the clinical setting. The clinical relevance of the current approach for allergy practice will be conferred in another article that will follow this year, aiming at showing the relevance in clinical practice where various endotypes can overlap and evolve over the lifetime.

The Normal Range of Baseline Tryptase Should Be 1 to 15 ng/mL and Covers Healthy Individuals With HαT.

Physiological levels of basal serum tryptase vary among healthy individuals, depending on the numbers of mast cells, basal secretion rate, copy numbers of the TPSAB1 gene encoding alpha tryptase, and renal function. Recently, there has been a growing debate about the normal range of tryptase because individuals with the hereditary alpha tryptasemia (HαT) trait may or may not be symptomatic, and if symptomatic, uncertainty exists as to whether this trait directly causes clinical phenotypes or aggravates certain conditions. In fact, most HαT-positive cases are regarded as asymptomatic concerning mast cell activation. To address this point, experts of the European Competence Network on Mastocytosis (ECNM) and the American Initiative in Mast Cell Diseases met at the 2022 Annual ECNM meeting and discussed the physiological tryptase range. Based on this discussion, our faculty concluded that the normal serum tryptase range should be defined in asymptomatic controls, inclusive of individuals with HαT, and based on 2 SDs covering the 95% confidence interval. By applying this definition in a literature screen, the normal basal tryptase in asymptomatic controls (HαT-positive persons included) ranges between 1 and 15 ng/mL. This definition should avoid overinterpretation, unnecessary referrals, and unnecessary anxiety or anticipatory fear of illness in healthy individuals.

Landscape of mast cell populations across organs in mice and humans.

Mast cells (MCs) are tissue-resident immune cells that exhibit homeostatic and neuron-associated functions. Here, we combined whole-tissue imaging and single-cell RNA sequencing datasets to generate a pan-organ analysis of MCs in mice and humans at steady state. In mice, we identify two mutually exclusive MC populations, MrgprB2+ connective tissue-type MCs and MrgprB2neg mucosal-type MCs, with specific transcriptomic core signatures. While MrgprB2+ MCs develop in utero independently of the bone marrow, MrgprB2neg MCs develop after birth and are renewed by bone marrow progenitors. In humans, we unbiasedly identify seven MC subsets (MC1-7) distributed across 12 organs with different transcriptomic core signatures. MC1 are preferentially enriched in the bladder, MC2 in the lungs, and MC4, MC6, and MC7 in the skin. Conversely, MC3 and MC5 are shared by most organs but not skin. This comprehensive analysis offers valuable insights into the natural diversity of MC subtypes in both mice and humans.

Allergy: Mechanistic insights into new methods of prevention and therapy.

In the past few decades, the prevalence of allergic diseases has increased worldwide. Here, we review the etiology and pathophysiology of allergic diseases, including the role of the epithelial barrier, the immune system, climate change, and pollutants. Our current understanding of the roles of early life and infancy; diverse diet; skin, respiratory, and gut barriers; and microbiome in building immune tolerance to common environmental allergens has led to changes in prevention guidelines. Recent developments on the mechanisms involved in allergic diseases have been translated to effective treatments, particularly in the past 5 years, with additional treatments now in advanced clinical trials.

New Insights into the Pathogenesis of Mastocytosis: Emerging Concepts in Diagnosis and Therapy.

Mastocytosis is a heterogeneous group of neoplasms defined by a numerical increase and accumulation of clonal mast cells (MCs) in various organ systems. The disease may present as cutaneous mastocytosis or systemic mastocytosis (SM). On the basis of histopathological and molecular features, clinical variables, and organ involvement, SM is divided into indolent SM, smoldering SM, SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia. Each variant is defined by unique diagnostic criteria and a unique spectrum of clinical presentations. A key driver of MC expansion and disease evolution is the oncogenic machinery triggered by mutant forms of KIT. The genetic background, additional somatic mutations, and comorbidities also contribute to the course and prognosis. Patients with SM may also suffer from mediator-related symptoms or even an MC activation syndrome. This article provides an update of concepts on the genetics, etiology, and pathology of mastocytosis, with emphasis on diagnostic criteria and new treatment concepts.

CD8+ T cell differentiation status correlates with the feasibility of sustained unresponsiveness following oral immunotherapy.

While food allergy oral immunotherapy (OIT) can provide safe and effective desensitization (DS), the immune mechanisms underlying development of sustained unresponsiveness (SU) following a period of avoidance are largely unknown. Here, we compare high dimensional phenotypes of innate and adaptive immune cell subsets of participants in a previously reported, phase 2 randomized, controlled, peanut OIT trial who achieved SU vs. DS (no vs. with allergic reactions upon food challenge after a withdrawal period; n = 21 vs. 30 respectively among total 120 intent-to-treat participants). Lower frequencies of naïve CD8+ T cells and terminally differentiated CD57+CD8+ T cell subsets at baseline (pre-OIT) are associated with SU. Frequency of naïve CD8+ T cells shows a significant positive correlation with peanut-specific and Ara h 2-specific IgE levels at baseline. Higher frequencies of IL-4+ and IFNγ+ CD4+ T cells post-OIT are negatively correlated with SU. Our findings provide evidence that an immune signature consisting of certain CD8+ T cell subset frequencies is potentially predictive of SU following OIT.

Dynamin-related protein 1 differentially regulates FcεRI- and substance P-induced mast cell activation.

BACKGROUND: The mitochondrial fission protein dynamin-related protein 1 (Drp1) has been suggested to regulate mast cell (MC) activation by certain stimuli in vitro, but its functions in MCs activated by various stimuli in vivo have not yet been examined. OBJECTIVE: We sought to analyze Drp1 function in both mouse and human MCs. METHODS: We used human peripheral blood-derived cultured MCs and 2 genetic mouse models in which MCs were depleted of Drp1: Drp1fl/flMcpt5cre+/- mice and Drp1fl/flCpa3cre+/- mice. RESULTS: In mice, Drp1 depletion enhanced FcεRI-induced MC activation while suppressing substance P-stimulated MC activation in vitro and in vivo. This was also true in human peripheral blood-derived cultured MCs in vitro after pharmacologic inhibition of Drp1. CONCLUSION: Drp1 differentially regulates MC activation by various stimuli. Promoting Drp1 activation might therefore represent a novel therapy for suppressing IgE-dependent MC activation. Further, inhibiting Drp1 activation might mitigate other MC-dependent responses, such as those induced by substance P.

An optimized protocol for phenotyping human granulocytes by mass cytometry.

Granulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3). Granulocyte integrity and detection of protein markers were optimized so that rare granulocyte populations could be deeply characterized by single cell mass cytometry. For complete details on the use and execution of this protocol, please refer to Vivanco Gonzalez et al. (2020).

Exponential magnetophoretic gradient for the direct isolation of basophils from whole blood in a microfluidic system.

Despite their rarity in peripheral blood, basophils play important roles in allergic disorders and other diseases including sepsis and COVID-19. Existing basophil isolation methods require many manual steps and suffer from significant variability in purity and recovery. We report an integrated basophil isolation device (i-BID) in microfluidics for negative immunomagnetic selection of basophils directly from 100 µL of whole blood within 10 minutes. We use a simulation-driven pipeline to design a magnetic separation module to apply an exponentially increasing magnetic force to capture magnetically tagged non-basophils flowing through a microtubing sandwiched between magnetic flux concentrators sweeping across a Halbach array. The exponential profile captures non-basophils effectively while preventing their excessive initial buildup causing clogging. The i-BID isolates basophils with a mean purity of 93.9% ± 3.6% and recovery of 95.6% ± 3.4% without causing basophil degradation or unintentional activation. Our i-BID has the potential to enable basophil-based point-of-care diagnostics such as rapid allergy assessment.

KIT as a master regulator of the mast cell lineage.

The discovery in 1987/1988 and 1990 of the cell surface receptor KIT and its ligand, stem cell factor (SCF), was a critical achievement in efforts to understand the development and function of multiple distinct cell lineages. These include hematopoietic progenitors, melanocytes, germ cells, and mast cells, which all are significantly affected by loss-of-function mutations of KIT or SCF. Such mutations also influence the development and/or function of additional cells, including those in parts of the central nervous system and the interstitial cells of Cajal (which control gut motility). Many other cells can express KIT constitutively or during immune responses, including dendritic cells, eosinophils, type 2 innate lymphoid cells, and taste cells. Yet the biological importance of KIT in many of these cell types largely remains to be determined. We here review the history of work investigating mice with mutations affecting the white spotting locus (which encodes KIT) or the steel locus (which encodes SCF), focusing especially on the influence of such mutations on mast cells. We also briefly review efforts to target the KIT/SCF pathway with anti-SCF or anti-Kit antibodies in mouse models of allergic disorders, parasite immunity, or fibrosis in which mast cells are thought to play significant roles.

Drug-induced mast cell eradication: A novel approach to treat mast cell activation disorders?

Mast cell (MC) activation is a key event in allergic reactions, other inflammatory states, and MC activation syndromes. MC-stabilizing agents, mediator-targeting drugs, and drugs interfering with mediator effects are often prescribed for these patients. However, the clinical efficacy of these drugs varies depending on the numbers of involved MCs and the underlying pathology. One straightforward approach would be to eradicate the primary target cell. To date however, no MC-eradicating treatment approach has been developed for patients with MC activation disorders. Nevertheless, recent data suggest that long-term treatment with agents effectively inhibiting KIT function results in the virtual eradication of tissue MCs and a sustained decrease in serum tryptase levels. In many of these patients, MC depletion is associated with a substantial improvement in mediator-induced symptoms. In patients with an underlying KIT D816V-positive mastocytosis, such MC eradication requires an effective inhibitor of KIT D816V, such as avapritinib. However, the use of KIT inhibitors must be balanced against their potential side effects. Here we discuss MC-eradicating strategies in various disease models, the feasibility of this approach, available clinical data, and future prospects for the use of KIT-targeting drugs in MC activation disorders.

IgE antibodies increase honeybee venom responsiveness and detoxification efficiency of mast cells.

BACKGROUND: In contrast to their clearly defined roles in allergic diseases, the physiologic functions of Immunoglobulin E antibodies (IgEs) and mast cells (MCs) remain enigmatic. Recent research supports the toxin hypothesis, showing that MCs and IgE-related type 2 immune responses can enhance host defense against certain noxious substances, including honeybee venom (BV). However, the mechanisms by which MCs can interfere with BV toxicity are unknown. In this study, we assessed the role of IgE and certain MC products in MC-mediated BV detoxification. METHODS: We applied in vitro and in vivo fluorescence microscopyimaging, and flow cytometry, fibroblast-based toxicity assays and mass spectrometry to investigate IgE-mediated detoxification of BV cytotoxicity by mouse and human MCs in vitro. Pharmacologic strategies to interfere with MC-derived heparin and proteases helped to define the importance of specific detoxification mechanisms. RESULTS: Venom-specific IgE increased the degranulation and cytokine responses of MCs to BV in vitro. Passive serum sensitization enhanced MC degranulation in vivo. IgE-activated mouse or human MCs exhibited enhanced potential for detoxifying BV by both proteolytic degradation and heparin-related interference with toxicity. Mediators released by IgE-activated human MCs efficiently degraded multiple BV toxins. CONCLUSIONS: Our results both reveal that IgE sensitization enhances the MC's ability to detoxify BV and also assign efficient toxin-neutralizing activity to MC-derived heparin and proteases. Our study thus highlights the potential importance of IgE, MCs, and particular MC products in defense against BV.

Proposed Diagnostic Criteria and Classification of Canine Mast Cell Neoplasms: A Consensus Proposal.

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.