Mature Mast Cells Research Articles

Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles

There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.

Abstract 4868: Tyrosine kinase-dependent modulation of tumor infiltrating immune cells in melanoma

Abstract Melanoma causes the greatest morbidity and mortality of all skin cancers and often harbors amplifications or activating mutations in KIT, a type III transmembrane receptor tyrosine kinase (RTK) involved in cancer cell growth, migration, invasion and metastasis. At present, no treatment that provide either sufficient response rates or a significant prolongation of overall survival are available for melanoma. In the last decade, several inhibitors of RTK (RTKi) have been developed for the treatment of cancer, however, a number of short-comings was observed. Therefore, novel TK inhibitors with improved selectivity were developed for the treatment of diseases associated with KIT activation. Masitinib (AB1010), a RTKi capable of blocking selectively KIT, is one such new drug. Using B16 melanoma subcutaneous graft on C57Bl6 mice, we demonstrate that Masitinib is able to limit tumor growth in vivo and increase mice survival. This anti-tumoral effect was not observed for other RTKi targeting KIT. Suprisingly, Masitinib has no effect on melanoma proliferation in vitro, suggesting instead that modulation on tumor environment takes place. Indeed, mast cells that accumulate in the tumors secrete several biologically active factors involved in the modulation of tumor growth. In vivo, Masitinib treatment completely abrogates mast cell maturation and migration resulting in decreased tumor invasion and down-regulation of several angiogenic factors. Using a mouse line deficient for mast cells (Wsh/Wsh), we confirmed that Masitinib effect is in part dependent of its inhibition on mast cell migration and maturation. Indeed, additional effect occurs on macrophage polarization leaning from “tolerogenic” toward “anti-tumoral” phenotype. Furthermore, in vivo macrophage depletion favored tumor promotion and reverses Masitinib effect on tumor growth. The results provided by this study suggest that Masitinib may be a useful therapy on melanoma by its dual role on tumor environment. It can block mast cell maturation and infiltration in the tumor site, avoiding the angiogenic effects of mast cells. It can revert tumor tolerent macrophage status leading to an increased inflammatory activity within the tumor microenvironment. These effects are not observed only on melanoma, but also on other types of cancer, suggesting the cellular mechanisms triggered by Masitinib occur in various tumoral process. For instance, a phase III clinical trial with Masitinib as GIST treatment is currently ongoing. Citation Format: Joël Babdor, Aurélie Fricot, Michaël Dussiot, Lucile Hebert, Patrice Dubreuil, Ivan Cruz Moura, Thiago Trovati Maciel, Olivier Hermine. Tyrosine kinase-dependent modulation of tumor infiltrating immune cells in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4868.

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction.

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit-independent MC-deficient (Cpa3(Cre/+)) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca(2+) desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force-Ca(2+) interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators.

Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice.

Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.

Live Staphylococcus aureus Induces Expression and Release of Vascular Endothelial Growth Factor in Terminally Differentiated Mouse Mast Cells.

Mast cells have been shown to express vascular endothelial growth factor (VEGF), thereby implicating mast cells in pro-angiogenic processes. However, the mechanism of VEGF induction in mast cells and the possible expression of VEGF in fully mature mast cells have not been extensively studied. Here, we report that terminally differentiated peritoneal cell-derived mast cells can be induced to express VEGF in response to challenge with Staphylococcus aureus, thus identifying a mast cell–bacteria axis as a novel mechanism leading to VEGF release. Whereas live bacteria produced a robust upregulation of VEGF in mast cells, heat-inactivated bacteria failed to do so, and bacteria-conditioned media did not induce VEGF expression. The induction of VEGF was not critically dependent on direct cell–cell contact between bacteria and mast cells. Hence, these findings suggest that VEGF can be induced by soluble factors released during the co-culture conditions. Neither of a panel of bacterial cell-wall products known to activate toll-like receptor (TLR) signaling promoted VEGF expression in mast cells. In agreement with the latter, VEGF induction occurred independently of Myd88, an adaptor molecule that mediates the downstream events following TLR engagement. The VEGF induction was insensitive to nuclear factor of activated T-cells inhibition but was partly dependent on the nuclear factor kappa light-chain enhancer of activated B cells signaling pathway. Together, these findings identify bacterial challenge as a novel mechanism by which VEGF is induced in mast cells.

Atopic diseases and inflammation of the brain in the pathogenesis of autism spectrum disorders.

Autism spectrum disorders (ASDs) affect as many as 1 in 45 children and are characterized by deficits in sociability and communication, as well as stereotypic movements. Many children also show severe anxiety. The lack of distinct pathogenesis and reliable biomarkers hampers the development of effective treatments. As a result, most children with ASD are prescribed psychopharmacologic agents that do not address the core symptoms of ASD. Autoantibodies against brain epitopes in mothers of children with ASD and many such children strongly correlate with allergic symptoms and indicate an aberrant immune response, as well as disruption of the blood–brain barrier (BBB). Recent epidemiological studies have shown a strong statistical correlation between risk for ASD and either maternal or infantile atopic diseases, such as asthma, eczema, food allergies and food intolerance, all of which involve activation of mast cells (MCs). These unique tissue immune cells are located perivascularly in all tissues, including the thalamus and hypothalamus, which regulate emotions. MC-derived inflammatory and vasoactive mediators increase BBB permeability. Expression of the inflammatory molecules interleukin (IL-1β), IL-6, 1 L-17 and tumor necrosis factor (TNF) is increased in the brain, cerebrospinal fluid and serum of some patients with ASD, while NF-kB is activated in brain samples and stimulated peripheral blood immune cells of other patients; however, these molecules are not specific. Instead the peptide neurotensin is uniquely elevated in the serum of children with ASD, as is corticotropin-releasing hormone, secreted from the hypothalamus under stress. Both peptides trigger MC to release IL-6 and TNF, which in turn, stimulate microglia proliferation and activation, leading to disruption of neuronal connectivity. MC-derived IL-6 and TGFβ induce maturation of Th17 cells and MCs also secrete IL-17, which is increased in ASD. Serum IL-6 and TNF may define an ASD subgroup that benefits most from treatment with the natural flavonoid luteolin. Atopic diseases may create a phenotype susceptible to ASD and formulations targeting focal inflammation of the brain could have great promise in the treatment of ASD.

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

Comparison of gDNA‐based versus mRNA‐based KIT D816V mutation analysis reveals large differences between blood and bone marrow in systemic mastocytosis

Keywords: systemic mastocytosis; KIT D816V mutation; blood; genomic DNA; messenger RNA

425 Mast cells survival and maturation in human skin are regulated and maintained by sensory nerve fibers

425 Mast cells survival and maturation in human skin are regulated and maintained by sensory nerve fibers

Down-modulation of antigen-induced activation of murine cultured mast cells sensitized with a highly cytokinergic IgE clone

Accumulating evidence suggests that several IgE clones can activate mast cells during the sensitization phase even in the absence of antigen. They were found to induce pro-inflammatory cytokine release, histamine synthesis, chemotaxis, adhesion, and accelerated maturation of mast cells, although it remains unknown whether antigen-induced responses can be affected by differences of IgE clones. We compared two IgE clones, which were different in the capacity to activate mast cells during sensitization, in terms of potentials to affect antigen-induced degranulation and cytokine releases using IL-3-dependent murine bone marrow-derived cultured mast cells (BMMCs). Antigen-induced degranulation and pro-inflammatory cytokine release were augmented, when BMMCs were sensitized with elevated concentrations of a clone IgE-3, which did not induce phosphorylation of JNK and cytokine release in the absence of antigen, whereas those were significantly rather decreased, when BMMCs were sensitized with elevated concentrations of a clone SPE-7, one of the most potent cytokinergic IgE clones, which intensively induced phosphorylation of JNK. This attenuated response with SPE-7 was accompanied by decreased tyrosine phosphorylation of the cellular proteins including Syk upon antigen stimulation. SP600125, which is known to inhibit JNK, restored the levels of antigen-induced degranulation and phosphorylation of Syk in BMMCs sensitized with higher concentrations of a clone SPE-7 when it was added before sensitization. Treatment with anisomycin, a potent activator of JNK, before IgE sensitization significantly suppressed antigen-induced degranulation. These findings suggest that differences of sensitizing IgE clones can affect antigen-induced responses and activation of JNK during sensitization might suppress antigen-induced activation of mast cells.

Resveratrol preferentially inhibits IgE-dependent PGD2 biosynthesis but enhances TNF production from human skin mast cells

BackgroundResveratrol, a natural polyphenol found in the skin of red grapes, is reported to have anti-inflammatory properties including protective effects against aging. Consequently, Resveratrol is a common nutritional supplement and additive in non-prescription lotions and creams marketed as anti-aging products. Studies in mice and with mouse bone marrow-derived mast cells (BMMCs) have indicated anti-allergic effects of Resveratrol. However, the effects of Resveratrol on human primary mast cells have not been reported. MethodsHuman mast cells were isolated and purified from normal skin tissue of different donors. The effect of Resveratrol on IgE-dependent release of allergic inflammatory mediators was determined using various immunoassays, Western blotting, and quantitative real-time PCR. ResultsResveratrol at low concentrations (≤10μM) inhibited PGD2 biosynthesis but not degranulation. Accordingly, COX-2 expression was inhibited but phosphorylation of Syk, Akt, p38, and p42/44 (ERKs) remained intact. Surprisingly, TNF production was significantly enhanced with Resveratrol. At a high concentration (100μM), Resveratrol significantly inhibited all parameters analyzed except Syk phosphorylation. ConclusionsHere, we show that Resveratrol at low concentrations exerts its anti-inflammatory properties by preferentially targeting the arachidonic acid pathway. We also demonstrate a previously unrecognized pro-inflammatory effect of Resveratrol — the enhancement of TNF production from human mature mast cells following IgE-dependent activation. General significanceThese findings suggest that Resveratrol as a therapeutic agent could inhibit PGD2-mediated inflammation but would be ineffective against histamine-mediated allergic reactions. However, Resveratrol could potentially exacerbate or promote allergic inflammation by enhancing IgE-dependent TNF production from mast cells in human skin.

IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3

IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.

Expression of surface and intracellular Toll-like receptors by mature mast cells

Nowadays, more and more data indicate that mast cells play an important role in host defense against pathogens. That is why it is essential to understand the expression of Toll-like receptors (TLRs) by mast cells, because these molecules play particularly significant role in initiation host defense against microorganisms as they recognize both wide range of microbial pathogen-associated molecular patterns (PAMPs) and various endogenous damage-associated molecular patterns (DAMPs) released in response to infection. Therefore, we examined the constitutive expression of both surface and endosomal TLRs in rat native fully mature tissue mast cells. By the use of qRT-PCR we found that these cells express mRNAs for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. The expression of TLR3, TLR4, TLR5, TLR7, and TLR9 transcripts were low and comparable and only the expression of TLR2 transcript was significant. By the use of flow cytometry technique, we clearly documented that mast cells express TLR2, TLR4, and TLR5 on cell surface, while TLR3, TLR7, and TLR9 proteins are located both on the cell membrane and intracellularly. The highest expression was observed for TLR5 and the lowest for surface TLR7. These observations undoubtedly indicate that mature tissue mast cells have a broad set of TLR molecules, thus can recognize and bind bacterial, viral, and fungal PAMPs as well as various endogenous molecules generated in response to infection.

Wnt-β-Catenin Signaling Promotes the Maturation of Mast Cells.

Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs), such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs). The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.

Orf virus IL-10 reduces monocyte, dendritic cell and mast cell recruitment to inflamed skin

Orf virus (ORFV) is a zoonotic parapoxvirus that causes pustular dermatitis of sheep, and occasionally humans. Despite causing sustained infections, ORFV induces only a transient increase in pro-inflammatory signalling and the trafficking of innate immune cells within the skin seems to be impaired. An explanation for this tempered response to ORFV infection may lie in its expression of a homolog of the anti-inflammatory cytokine, interleukin (IL)-10. Using a murine model in which inflammation was induced by bacterial lipopolysaccharide, we examined the effects of the ORFV-IL-10 protein on immune cell trafficking to and from the skin. ORFV-IL-10 limited the recruitment of blood-derived Gr-1int/CD11bint monocytes, CD11c+ve/MHC-II+ve dendritic cells and c-kit+ve/FcεR1+ve mature mast cells into inflamed skin. ORFV-IL-10 also suppressed the activation of CD11c+ve/MHC-II+ve dendritic cells within the skin, reducing their trafficking to the draining lymph node. These findings suggest that expression of IL-10 by ORFV may contribute to the impaired trafficking of innate immune cells within infected skin.

A Recombinant Antibody to Siglec-8 Shows Selective ADCC Activity Against Mast Cells from Systemic Mastocytosis Patients

Background: Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by the accumulation of neoplastic mast cells (MC) in one or more extracutaneous organs. In all forms of SM, anti-mediator drugs are used to control symptoms of MC degranulation. In advanced forms of SM, organ damage is common and patients (pts) exhibit reduced life expectancy. In these individuals, cytoreductive agents such as cladribine and interferon-alpha have been used off-label, and inhibitors of KIT D816V are under investigation. A significant unmet need exists for these patients.Siglec-8 is an inhibitory receptor of the CD33-related family of sialic acid-binding, Ig-like lectins (Siglecs) that is expressed selectively on the surface of mature MC, eosinophils, and basophils. Engagement of Siglec-8 with monoclonal antibodies has been previously shown to inhibit IgE-mediated MC degranulation and to induce apoptosis of cytokine-activated eosinophils. Thus this receptor is a potential target for antibody therapy of SM with or without associated eosinophilia. Anti-Siglec-8 antibodies do not directly affect MC viability but antibodies with effector function can induce antibody cell-mediated cytotoxicity (ADCC). Here we show that a recombinant anti-Siglec-8 IgG1 monoclonal antibody can elicit ADCC activity against MC derived from SM patients ex vivo.Methods: Bone marrow (BM) aspirates from SM patients were evaluated for Siglec-8 cell-surface expression on CD117+ FcεRI+ MC or CD25+ MC by flow cytometry. For ADCC assays, BM MC enriched using CD117-targeting magnetic beads or a Siglec-8 transfected Ramos cell line were used as target cells. Peripheral blood leukocytes (PBL) or NK cells purified from peripheral blood were used as effector cells at an effector:target ratio of 10:1. Recombinant anti-Siglec-8 antibody or an isotype control antibody was added at various concentrations and the percent viable CD117+ FcεRI+ MC remaining after 48 hours of culture was determined by flow cytometry.Results: Samples from 9 pts with SM were included in the analysis (ISM, n=1; SSM, n=1; SM-CMML, n=3; SM-MDS, n=1; SM-CEL, n=1; ASM, n=1; MCL, n=1). Eight pts were KIT D816V positive. At the time of sample collection, treatments included midostaurin (n=3); cladribine (n=1); corticosteroids (n=1); and 4 pts were not receiving any biologic or cytoreductive therapy. All BM samples showed detectable CD117+ MC. Robust and selective cell-surface expression of Siglec-8 was observed in all 6 cases evaluated and 100% of CD117+ FcεRI+ MC were Siglec-8 positive by FACS, including CD25+ MC. Levels of Siglec-8 were comparable to or higher than levels on mature MC isolated from normal skin. In this limited sample size, no difference in Siglec-8 expression was observed between patients receiving different therapies or no therapy.To evaluate the ADCC activity of recombinant anti-Siglec-8 antibody on MC, enriched BM MC were incubated with anti-Siglec-8 antibody or isotype-matched control antibody at 1 μg/ mL in the presence of purified NK effector cells. In two patients evaluated, significant anti-Siglec-8-mediated ADCC activity on MC was observed using non-autologous NK cells (69% reduction, 1 pt) or autologous NK cells (76% reduction, 1 pt) indicating that anti-Siglec-8 has the potential to reduce MC burden in these patients.ADCC activity has been reported to be defective in some cancer patients. To evaluate the ability of effector cells in SM patients to mediate ADCC, an assay was developed using a Siglec-8 transfected target cell line to screen blood samples for ADCC activity induced by anti-Siglec-8 antibody. Using PBL as effector cells, ADCC was observed in all samples tested (5/5). Titration of antibody was performed on 2 samples and potent ADCC activity was observed in both, with an EC50 for target depletion of 49 and 65 ng/mL of anti-Siglec-8 antibody, respectively.Conclusion: These data provide a strong rationale for evaluating the effect of an antibody to Siglec-8 with ADCC activity in patients with SM. DisclosuresFalahati:Allakos, Inc.: Employment, Other: Options for Equity Owernship. Bright:Allakos, Inc.: Employment, Other: Options for Equity Owernship. Dorenbaum:Allakos, Inc.: Employment, Equity Ownership. Bebbington:Allakos, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tomasevic:Allakos, Inc.: Employment, Equity Ownership. George:Allakos: Research Funding; Novartis: Consultancy. Gotlib:Allakos, Inc.: Consultancy.

Novel Functional Roles for Ten-Eleven-Translocation 2 (Tet2) in Normal and Leukemic Growth of Mast Cells

KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. DisclosuresNo relevant conflicts of interest to declare.

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

Interleukin-33 and Mast Cells Bridge Innate and Adaptive Immunity: From the Allergologist's Perspective.

Interleukin (IL) 33, a member of the IL-1 superfamily, is an “alarmin” protein and is secreted in its active form from damaged cells undergoing necrotic cell death. Mast cells are one of the main effector cell types in allergic disorders. They secrete a variety of mediators, including T helper 2 cytokines. As mast cells have high-affinity IgE receptors (FcεRI) on their surface, they can capture circulating IgE. IgE-bound mast cells degranulate large amounts of histamine, heparin, and proteases when they encounter antigens. As IL-33 is an important mediator of innate immunity and mast cells play an important role in adaptive immune responses, interactions between the two could link innate and adaptive immunity. IL-33 promotes the adhesion of mast cells to laminin, fibronectin, and vitronectin. IL-33 increases the expression of adhesion molecules, such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in endothelial cells, thus enhancing mast cell adhesion to blood vessel walls. IL-33 stimulates mast cell proliferation by activating the ST2/Myd88 pathway; increases mast cell survival by the activation of survival proteins such as Bcl-XL; and promotes the growth, development, and maturation of mast cell progenitors. IL-33 is also involved in the activation of mature mast cells and production of different proinflammatory cytokines. The interaction of IL-33 and mast cells could have important clinical implications in the field of clinical urology. Epithelial dysfunction and mast cells could play an important role in the pathogenesis of interstitial cystitis. Urinary levels of IL-33 significantly increase in patients with interstitial cystitis. In addition, the number of mast cells significantly increase in the urinary bladders of patients with interstitial cystitis. Therefore, inhibition of mast cell activation and degranulation in response to increase in IL-33 is a potential therapeutic target in the treatment of interstitial cystitis.

Murine and human mast cell progenitors

The development of mature mast cells (MCs) from hematopoietic progenitor cells as well as the identification and characterization of committed progenitor cells are a current focus of mast cell research. Most published reports in this area are on the origin and differentiation of MCs in mice. Evidence for the human system, i.e. derived from primary human MCs, is widely lacking. Based on the published data, MCs develop either from a committed progenitor or from a common basophil/mast cell precursor. This review summarizes the current knowledge on MC development and MC differentiation.