Tryptase Regulates the Epigenetic Modification of Core Histones in Mast Cell Leukemia Cells

Mast cells are immune cells that store large amounts of mast cell-restricted proteases in their secretory granules, including tryptase, chymase and carboxypeptidase A3. In mouse mast cells, it has been shown that tryptase, in addition to its canonical location in secretory granules, can be found in the nuclear compartment where it can impact on core histones. Here we asked whether tryptase can execute core histone processing in human mast cell leukemia cells, and whether tryptase thereby can affect the epigenetic modification of core histones. Our findings reveal that triggering of cell death in HMC-1 mast cell leukemia cells is associated with extensive cleavage of core histone 3 (H3) and more restricted cleavage of H2B. Tryptase inhibition caused a complete blockade of such processing. Our data also show that HMC-1 cell death was associated with a major reduction of several epigenetic histone marks, including H3 lysine-4-mono-methylation (H3K4me1), H3K9me2, H3 serine-10-phosphorylation (H3S10p) and H2B lysine-16-acetylation (H2BK16ac), and that tryptase inhibition reverses the effect of cell death on these epigenetic marks. Further, we show that tryptase is present in the nucleus of both viable and dying mast cell leukemia cells. In line with a role for tryptase in regulating nuclear events, tryptase inhibition caused increased proliferation of the mast cell leukemia cells. Altogether, the present study emphasizes a novel principle for how epigenetic modification of core histones is regulated, and provides novel insight into the biological function of human mast cell tryptase.

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host’s blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

Hypoxia modulates human mast cell adhesion to hyaluronic acid

Hypoxia is an inherent factor in the inflammatory process and is important in the regulation of some immune cell functions, including the expression of mast cell pro- and anti-inflammatory mediators. Hypoxia also influences cell adhesion to the extracellular matrix (ECM). Hyaluronic acid is one of the major components of the ECM that is involved in inflammatory and tissue regeneration processes in which mast cells play a prominent role. This prompted us to investigate the effects of hypoxia on the expression of hyaluronic acid receptors in mast cells and mast cell adhesion to this ECM component. We found that human LAD2 mast cells spontaneously adhered to hyaluronic acid in a CD44-dependent manner and that reduced oxygen concentrations inhibited or even completely abolished this adhesion process. The mechanism of hypoxia downregulation of mast cell adhesion to hyaluronic acid did not involve a decrease in CD44 expression and hyaluronidase-mediated degradation of adhesion substrates but rather conformational changes in the avidity of CD44 to hyaluronic acid. Hypoxia-mediated regulation of mast cell adhesion to extracellular matrix components might be involved in the pathogenic accumulation of mast cells observed in the course of certain diseases including rheumatoid arthritis and cancer.

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. IgE-mediated crosslinking of the FcεRI 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 there are still controversies about the identity of the mast cell progenitor, i.e., whether FcεRI expression appears during the hematopoietic progenitor stage or in maturing mast cells. Here, we used single-cell transcriptomics to reveal a temporal association between the appearance of FcεRI and the mast cell gene signature in CD34+ hematopoietic progenitors. In agreement with these data, the FcεRI+ 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 mast cell progenitor development. Culture assays showed that IL-3 and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust FcεRI downregulation. Taken together, we have demonstrated that FcεRI appears during the hematopoietic progenitor stage of mast cell differentiation and that external stimuli may regulate the FcεRI expression. Thus, the results resolve the controversy regarding the appearance of FcεRI during mast cell development.One-sentence summarySingle-cell analysis of human hematopoiesis uncovers the stage at which FcεRI appears during mast cell differentiation and reveals disparate effects of IL-3, IL-5 and IL-33 on mast cell progenitor proliferation, survival, and suppression of FcεRI expression.

Serum CD203c+ Extracellular Vesicle Serves as a Novel Diagnostic and Prognostic Biomarker for Succinylated Gelatin Induced Perioperative Hypersensitive Reaction

BackgroundPerioperative hypersensitivity reaction (HR) is an IgE-FcϵRI-mediated hypersensitivity reaction with degranulation and activation of mast cells and basophils. Several studies have focused on assessing the degranulation and activation of mast cells and basophils to diagnose and predict the prognosis of drug induced HR. However, it is challenging to isolate sufficiently pure mast cells and basophils from human sources to investigate. Effective biomarkers to assess mast cells and basophils activation in vivo could potentially have high diagnostic and prognostic values. In the present study, we investigated EVs pelleted from serum in patients with succinylated gelatin induced HR.MethodsExtracellular vesicles (EVs) were isolated using a total exosome isolation kit and ultracentrifugation, characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. Basophils were isolated from fresh peripheral blood by negative selection using Basophil Isolation Kit II. Human mast cell line was stimulated with IL4. The expression levels of proteins related to the hypersensitive response were evaluated by Western blotting and flow Cytometer. Histamine and tryptase levels were tested using a commercial ELISA kit, and gene expression of inflammatory mediators was evaluated by qRT-PCR. The receiver operating characteristic (ROC) curve was used to evaluate the specificity and sensitivity of biomarker in predicting HR.ResultsThe concentration of EVs and protein expression level of CD63, FcϵRI, CD203c and tryptase were significantly (p< 0.05) increased in HR samples. The expression level of mast cell/basophil specific CD203c were significantly increased in EVs derived from serum and basophils of HR patients, and the CD203c+-EVs production in mast cells is dramatically increased in the presence of IL4, which positively correlated with histamine, tryptase and inflammatory mediators. Moreover, the ROC curve of EVs concentration and CD203c expression indicated that CD203c+-EVs had a strong diagnostic ability for HR.ConclusionSerum CD203c+-EVs serves as a novel diagnostic and prognostic biomarker for HR.

Annexin A1 Mimetic Peptide Ac2-26 Modulates the Function of Murine Colonic and Human Mast Cells

Mast cells (MCs) are main effector cells in allergic inflammation and after activation, they release stored (histamine, heparin, proteases) and newly synthesized (lipid mediators and cytokines) substances. In the gastrointestinal tract the largest MC population is located in the lamina propria and submucosa whereas several signals such as the cytokine IL-4, seem to increase the granule content and to stimulate a remarkable expansion of intestinal MCs. The broad range of MC-derived bioactive molecules may explain their involvement in many different allergic disorders of the gastrointestinal tract. Annexin A1 (AnxA1) is a 37 KDa glucocorticoid induced monomeric protein selectively distributed in certain tissues. Its activity can be reproduced by mimetic peptides of the N-terminal portion, such as Ac2-26, that share the same receptor FPR-L1. Although previous reports demonstrated that AnxA1 inhibits MC degranulation in murine models, the effects of exogenous peptide Ac2-26 on intestinal MCs or the biological functions of the Ac2-26/FPR2 system in human MCs have been poorly studied. To determine the effects of Ac2-26 on the function of MCs toward the possibility of AnxA1-based therapeutics, we treated WT and IL-4 knockout mice with peptide Ac2-26, and we examined the spontaneous and compound 48/80 stimulated colonic MC degranulation and cytokine production. Moreover, in vitro, using human mast cell line HMC-1 we demonstrated that exogenous AnxA1 peptide is capable of interfering with the HMC-1 degranulation in a direct pathway through formyl peptide receptors (FPRs). We envisage that our results can provide therapeutic strategies to reduce the release of MC mediators in inflammatory allergic processes.

Roles of IgE and Histamine in Mast Cell Maturation

Mast cells are activated upon immunoglobulin E (IgE)-mediated antigen stimulation, and release a wide variety of mediators, including histamine to trigger inflammatory responses. The surface expression levels of Fcε receptor I (FcεRI), a high affinity receptor of IgE, were found to be positively regulated by IgE. IgE could protect murine cultured mast cells from apoptotic cell death induced by the deprivation of interleukin-3 and a certain kind of IgE could activate immature mast cells in the absence of antigens, leading to the release of pro-inflammatory cytokines and a transient increase in histamine synthesis. Histamine synthesis in mast cells was found to be required for the maturation of murine connective tissue-type mast cells, raising the possibility that IgE indirectly modulates local mast cell maturation. Although it remains controversial to what extent this concept of “monomeric IgE effects” could have relevance in the modulation of human mast cell functions, the therapeutic effects of anti-IgE antibodies might be accounted for in terms of the decreased serum IgE concentrations. Because drastic increases in serum IgE concentrations are often observed in patients with atopic dermatitis and chronic urticaria, a close investigation of the roles of IgE in mast cell maturation should contribute to development of novel therapeutic approaches for these inflammatory diseases.

Lignans from Machilus thunbergii as Thymic Stromal Lymphopoietin Inhibitors

Thymic stromal lymphopoietin (TSLP) plays an important role in the pathophysiology of various allergic diseases that are mediated by T helper cell type-2 (Th2) responses, including asthma and atopic dermatitis. The primary focus of this study was the identification of potent inhibitors of the TSLP signaling pathway for potential therapeutic use. The 80% methanol extract of Machilus thunbergii bark significantly inhibited the signal transducer and activator of transcription 5 (STAT5) phosphorylation in human mast cell (HMC)-1 cells. Through activity-guided isolation, three lignans (1–3) were obtained and identified as (+)-galbelgin (1), meso-dihydroguaiaretic acid (2), and machilin A (3). Among them, two lignans (1 and 2) significantly inhibited STAT5 phosphorylation and TSLP/TSLPR interaction, as determined by ELISA. Our results indicated that lignans isolated from M. thunbergii are a promising resource for the treatment of allergic diseases.

YKL-40 Enhanced the Permeability of HDMECs with Histamine Through Activating Akt and p38 Pathways

Background, YKL-40 is currently considered as an important marker of endothelial dysfunction. Chronic spontaneous urticaria (CSU) is a common vascular skin disease. The increased vascular permeability play an important role in the occurrence and pathogenesis of CSU.Objective, the aim of this study is to explore the role of YKL-40 on the permeability of HDMECs.Methods, in this study, the mRNA level of YKL-40 in human mast cell line (HMC-1) were detected by RT-PCR. The effects of YKL-40 on vascular permeability, VE-cadherin release, VE-cadherin disruption in human dermal microvascular endothelial cells (HDMECs) were investigated by transwell, ELISA or immunofluorescence. The phosphorylation of VE-cadherin, p38 and Akt, in histamine plus YKL-40 treated HDMECs were detected by Western Blot.Results, we found that YKL-40 significantly promoted the permeability changes and leaded to the released, disruption of VE-cadherin in HDMECs induced by histamine. Furthermore, YKL-40 also enhanced the Akt and p38 pathways. Conclusion, we suggest that YKL-40 may serve as pro-permeability cytokines, and play a role in the pathogenesis of CSU. This study will help to further elucidate the pathogenesis of CSU and provide a new target for the development of anti-histamine resistance drugs for CSU.