Coaggregation of FcεRI with FcγRIIB Inhibits Degranulation but Not Induction of Bcl-2 Family Members A1 and Bim in Mast Cells

<p/> <p>The aggregation of high-affinity immunoglobulin E (IgE) receptors (FcεRI) on mast cells is a critical event in the initiation of an allergic reaction. Coengagement of FcεRI with immunoglobulin G (IgG) low-affinity receptor FcγRIIB/CD32 inhibits degranulation and the release of inflammatory mediators from mast cells and has therefore been proposed as a new therapeutic approach for the treatment of allergies. In this study, we investigated whether FcγRIIB, besides inhibiting degranulation, negatively regulates other signalling pathways downstream of FcεRI. For this, we determined the phosphorylation and/or expression of proteins involved in the regulation of mast-cell apoptosis. Coaggregation led to an attenuation of Akt phosphorylation but did not inhibit phosphorylation of transcription factor Foxo3a or its proapoptotic target, Bim. Similarly, FcεRI-dependent expression of the prosurvival gene A1 was not affected by coaggregation. Our data demonstrate that coengagement of FcεRI and FcγRIIB inhibits degranulation but not the signalling pathways regulating Bcl-2 family members Bim and A1.</p

The impact of Bcl-2 family members on mast cell survival and apoptosis

Mast cells are long-lived effector cells of the immune system perhaps best known for their involvement in allergic diseases. There are several acute and chronic inflammations where mast cell accumulation, activation and release of mediators are important for the initiation and perpetuation of the inflammation. Better knowledge of mechanisms regulating the number of mast cells and their activity is desirable. Mast cells do not represent a homogenous population as the surrounding microenvironment will affect their effector profile and numbers. It is known that, in contrast to many inflammatory cells, activated mast cells have the capacity to recover and thereby be activated again (Fc epsilon Receptor I (FcepsilonRI) activation-induced survival). In this thesis we investigated the effect of B-cell lymphoma-2 (Bcl-2) family members on mast cell survival and apoptosis in murine and human mast cells. Murine bone marrow cells were cultured in two different ways, generating mucosal-like mast cells (MLMCs) and connective tissue-like mast cells (CTLMCs). Our in vitro-derived MLMCs and CTLMCs were found to display similar differences in chymase expression, proliferation rate and histamine content as mucosal mast cells (MMCs) and connective tissue mast cells (CTMCs) in vivo. This suggests that MLMCs and CTLMCs represent a useful in vitro model for committed mast cell lineages. Moreover, we found that CTLMCs, but not MLMCs, exhibit upregulation of the anti-apoptotic Bcl-2 family member A1 and activation-induced survival upon FcRI crosslinking. Similarly to murine mast cells, FcRI crosslinking of in vitro-derived human mast cells lead to upregulation of the human homologue of A1, bfl-1, and by the use of bfl-1 siRNA we demonstrate bfl-1 to be crucial for activation-induced human mast cell survival. Furthermore, the activation-induced survival of human mast cells is sustained in response to the inhibitors ABT-737 and roscovitine which indicate a minor role for the targeted anti-apoptotic Bcl-2 family members Bcl-XL, Bcl-2, Bcl-w and Myeloid cell leukemia-1 (Mcl-1). Taken together, we provide evidence that mast cell populations differ in their ability to survive allergic reactions and identify the Bcl-2 family member A1/Bfl-1 as a potential target for treatment of allergic diseases. The Bcl-2 homlogy 3 (BH3)-only protein Bcl-2-interacting modulator of cell death (Bim) has been found to play a role in cytokine deprivation-induced apoptosis of murine mast cells although overexpression of anti-apoptotic Bcl-2 protects mast cells more potently than loss of Bim. This indicates that other proteins, besides Bim, might be involved in this process. We describe the BH3-only protein p53 upregulated modulator of apoptosis (Puma) to be critical for the induction of mast cell apoptosis following cytokine deprivation and treatment with the DNA-damaging agent etoposide. Our data also suggest the involvement of the transcription factor Forkhead box O3A (FOXO3a) in the regulation of cytokine deprivation-induced apoptosis and the expression of Puma. Mast cells deficient for FOXO3a were markedly resistant to cytokine deprivation and overexpression of constitutively active FOXO3a caused an upregulation of Puma. We further examined the role of the two pro-apoptotic effector proteins Bcl-2-associated X protein (Bax) and Bcl-2 homologue antagonist/killer (Bak) in cytokine deprivation-induced apoptosis. Although both proteins were expressed we found a major role for Bax but not Bak in mediating mast cell apoptosis. Taken together, this identifies the pro-apoptotic Bcl-2 family members Puma and Bax to be critical for induction of apoptosis which suggest a plausible role for these pro-apoptotic proteins in the regulation of mast cell numbers in vivo

Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine). Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival

Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors

Mast cell accumulation is a hallmark of a number of diseases, including allergic asthma and systemic mastocytosis. Immunoglobulin E–mediated crosslinking of the FceRI receptors causes mast cell activation and contributes to disease pathogenesis. The mast cell lineage is one of the least studied among the hematopoietic cell lineages, and controversies remain about whether FceRI expression appears during the mast cell progenitor stage or during terminal mast cell maturation. Here, we used single-cell transcriptomics analysis to reveal a temporal association between the appearance of FceRI and the mast cell gene signature in CD341 hematopoietic progenitors in adult peripheral blood. In agreement with these data, the FceRI1 hematopoietic progenitors formed morphologically, phenotypically, and functionally mature mast cells in long-term culture assays. Single-cell transcriptomics analysis further revealed the expression patterns of prospective cytokine receptors regulating development of mast cell progenitors. Culture assays showed that interleukin-3 (IL-3) and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust FceRI downregulation. Taken together, we showed that FceRI expression appears at the progenitor stage of mast cell differentiation in peripheral blood. We also showed that external stimuli regulate FceRI expression of mast cell progenitors, providing a possible explanation for the variable FceRI expression levels during mast cell development

Single-cell analysis reveals the KIT D816V mutation in haematopoietic stem and progenitor cells in systemic mastocytosis

Background: Systemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34(+) haematopoietic progenitors can carry the mutation: however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells. Methods: Fluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and &gt;10,000 CD34(+) bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential. Findings: We found that in SM 60-99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that Fc epsilon RI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. Interpretation: The KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA

Cannabinoid non-cannabidiol site modulation of TRPV2 structure and function

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology

Divergent Effects of Acute and Prolonged Interleukin 33 Exposure on Mast Cell IgE-Mediated Functions

Background: Epithelial cytokines, including IL-33 and Thymic stromal lymphopoietin (TSLP), have attracted interest because of their roles in chronic allergic inflammation-related conditions such as asthma. Mast cells are one of the major targets of IL-33, to which they respond by secreting cytokines. Most studies performed thus far have investigated the acute effects of IL-33 on mast cells. In the current study, we investigated how acute vs. prolonged exposure of mast cells to IL-33 and TSLP affects mediator synthesis and IgE-mediated activation. Methods: Human lung mast cells (HLMCs), cord blood-derived mast cells (CBMCs), and the ROSA mast cell line were used for this study. Receptor expression and the levels of mediators were measured after treatment with IL-33 and/or TSLP. Results: IL-33 induced the release of cytokines. Prolonged exposure to IL-33 increased while TSLP reduced intracellular levels of tryptase. Acute IL-33 treatment strongly potentiated IgE-mediated activation. In contrast, 4 days of exposure to IL-33 decreased IgE-mediated activation, an effect that was accompanied by a reduction in Fc epsilon RI expression. Conclusion: We show that IL-33 plays dual roles in mast cells, in which its acute effects include cytokine release and the potentiation of IgE-mediated degranulation, whereas prolonged exposure to IL-33 reduces IgE-mediated activation. We conclude that mast cells act quickly in response to the alarmin IL-33 to initiate an acute inflammatory response, whereas extended exposure to IL-33 during prolonged inflammation reduces IgE-mediated responses. This negative feedback effect suggests the presence of a novel regulatory pathway that modulates IgE-mediated human mast cell responses

IgECL-induced survival of human mast cells.

<p>(A). CBMC upon IgECL. (B) LAD-2 cells upon IgECL. Cells were sensitized with 1 μg/mL of IgE overnight before cytokine-deprived and challenged with anti-IgE. After 24 hrs the cell viability was enumerated using trypan blue exclusion. N=3–4.</p