Inhibition Of Mast Cell Degranulation Research Articles

Understanding human mast cells: lesson from therapies for allergic and non-allergic diseases.

Mast cells have crucial roles in allergic and other inflammatory diseases. Preclinical approaches provide circumstantial evidence for mast cell involvement in many diseases, but these studies have major limitations - for example, there is still a lack of suitable mouse models for some mast cell-driven diseases such as urticaria. Some approaches for studying mast cells are invasive or can induce severe reactions, and very few mediators or receptors are specific for mast cells. Recently, several drugs that target human mast cells have been developed. These include monoclonal antibodies and small molecules that can specifically inhibit mast cell degranulation via key receptors (such as FcεRI), that block specific signal transduction pathways involved in mast cell activation (for example, BTK), that silence mast cells via inhibitory receptors (such as Siglec-8) or that reduce mast cell numbers and prevent their differentiation by acting on the mast/stem cell growth factor receptor KIT. In this Review, we discuss the existing and emerging therapies that target mast cells, and we consider how these treatments can help us to understand mast cell functions in disease.

Tuo-Min-Ding-Chuan Decoction Alleviate Ovalbumin-Induced Allergic Asthma by Inhibiting Mast Cell Degranulation and Down-Regulating the Differential Expression Proteins.

Allergic asthma is a stubborn chronic inflammatory disease, and is considered a co-result of various immune cells, especially mast cells, eosinophils and T lymphocytes. At present, the treatment methods of allergic asthma are limited and the side effects are obvious. Traditional Chinese medicine has been used to treat diseases for thousands of years in China. One such example is the treatment of allergic asthma, which take the characteristics of less adverse reactions and obvious curative effect. Tuo-Min-Ding-Chuan Decoction (TMDCD) is a traditional Chinese medicine compound for the treatment of allergic asthma optimized from Ma-Xing-Gan-Shi Decoction (MXGSD), which was put forward in Treatise on Febrile Diseases by Zhang Zhongjing in the Eastern Han Dynasty. The compound shows a significant clinical effect, but the mechanism of its influence on the immune system is still unclear. The purpose of this study was to observe whether TMDCD could alleviate the symptoms of ovalbumin (OVA) challenged allergic asthma mice, and to explore its immune regulatory mechanism, especially on mast cell (MC) degranulation. The results showed TMDCD could not only reduce the airway hyperresponsiveness (AHR), inflammatory cell infiltration and mucus secretion in the lung tissue of OVA challenged mice, but also decrease the levels of total IgE, OVA-specific IgE, histamine and LTC4 in serum. We found that TMDCD can downregulate the expression of Fractalkine, Tryptase ε, IL-25, CCL19, MCP-1, OX40L, Axl, CCL22, CD30, G-CSF, E-selectin, OPN, CCL5, P-selectin, Gas6, TSLP in OVA challenged mice serum by using mouse cytokines antibody array. It has been reported in some literatures that these differentially expressed proteins are related to the occurrence of allergic asthma, such as tryptase ε, MCP-1, CCL5, etc. can be released by MC. And the results of in vitro experiments showed that TMDCD inhibited the degranulation of RBL-2H3 cells stimulated by DNP-IgE/BSA. Taken together, we made the conclusion that TMDCD could reduce the infiltration of inflammatory cells in lung tissue and alleviate airway remodeling in mice with allergic asthma, showed the effects of anti-inflammatory and antiasthmatic. TMDCD could also reduce the levels of IgE, histamine, LTC4, Tryptase ε, and other MC related proteins in the serum of allergic asthma mice, and the in vitro experiments showed that TMDCD could inhibit IgE mediated degranulation and histamine release of RBL-2H3 cells, proved its anti allergic effect.

Clemastine Fumarate Attenuates Myocardial Ischemia Reperfusion Injury Through Inhibition of Mast Cell Degranulation.

Mast cell (MC) activation is associated with myocardial ischemia reperfusion injury (MIRI). Suppression of MC degranulation might be a target of anti-MIRI. This study aimed to determine whether clemastine fumarate (CLE) could attenuate MIRI by inhibiting MC degranulation. A rat ischemia and reperfusion (I/R) model was established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. Compound 48/80 (C48/80) was used to promote MC degranulation. The protective effect of CLE by inhibiting MC degranulation on I/R injury was detected by cardiac function, 2,3,5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, arrhythmia, and myocardial enzyme detection. Inflammatory factor mRNA levels, such as TNF-α, interleukin (IL)-1β, and IL-6, were detected. Cultured RBL-2H3 mast cells were pretreated with CLE and subjected to C48/80 treatment to determine whether CLE suppressed MC degranulation. Degranulation of MCs was visualized using tryptase release, Cell Counting Kit-8 (CCK-8), and cell toluidine blue (TB) staining. RBL cells were conditionally cultured with H9C2 cells to explore whether CLE could reverse the apoptosis of cardiomyocytes induced by MC degranulation. Apoptosis of H9C2 cells was detected by CCK-8, the LDH Cytotoxicity Assay Kit (LDH), TUNEL staining, and protein expression of BAX and Bcl-2. We found that CLE pretreatment further inhibited cardiac injury manifested by decreased infarct size, histopathological changes, arrhythmias, MC degranulation, and myocardial enzyme levels, improving cardiac function compared with that in the I/R group. C48/80 combined with I/R exacerbated these changes. However, pretreatment with CLE for C48/80 combined with I/R significantly reversed these injuries. In addition, CLE pretreatment improved the vitality of RBL cells and reduced tryptase release in vitro. Similarly, the supernatant of RBL cells pretreated with CLE decreased the cytotoxicity, TUNEL-positive cell rate, and BAX expression of conditioned H9C2 cells and increased the cell vitality and expression of Bcl-2. These results suggested that pretreatment with CLE confers protection against I/R injury by inhibiting MC degranulation.

Adenosine Signaling in Mast Cells and Allergic Diseases.

Adenosine is a nucleoside involved in the pathogenesis of allergic diseases. Its effects are mediated through its binding to G protein-coupled receptors: A1, A2a, A2b and A3. The receptors differ in the type of G protein they recruit, in the effect on adenylyl cyclase (AC) activity and the downstream signaling pathway triggered. Adenosine can produce both an enhancement and an inhibition of mast cell degranulation, indicating that adenosine effects on these receptors is controversial and remains to be clarified. Depending on the study model, A1, A2b, and A3 receptors have shown anti- or pro-inflammatory activity. However, most studies reported an anti-inflammatory activity of A2a receptor. The precise knowledge of the adenosine mechanism of action may allow to develop more efficient therapies for allergic diseases by using selective agonist and antagonist against specific receptor subtypes.

Hybrids between H2S-donors and betamethasone 17-valerate or triamcinolone acetonide inhibit mast cell degranulation and promote hyperpolarization of bronchial smooth muscle cells

Glucocorticoids represent the standard gold treatment of inflammation in asthmatic patients. More recently, H2S has been described to exert positive effect on this disease. Bearing in mind that an improved pharmacological activity and a reduced toxicity can be obtained through hybridization of different molecules, simultaneously modulating multiple targets, we designed and synthesized novel betamethasone 17-valerate and triamcinolone acetonide hybrids with well-known H2S-donor moieties. Synthesized compounds have been evaluated for the potential H2S-releasing profile both in cell-free environment and into the cytosol of bronchial smooth muscle cells (BSMCs). The two hybrids 4b and 5b were investigated by molecular modelling studies and results indicated that the steric accessibility of the isothiocyanate carbon atom can account for their different H2S releasing properties. Furthermore, the most promising derivatives 4b and 5b have been tested for inhibitory effect on mast cell degranulation and for the ability to induce cell membrane hyperpolarization in BSMCs. Significant inhibitory effect on mast cell degranulation was assessed, resulting to reduce β-hexosaminidase release more efficiently than the corresponding native drugs. Both compounds determined a massive membrane hyperpolarization of BSMCs and proved to be 4-fold more effective compared to reference compound NS1619. These effects represent an enrichment of the pharmacological activity of the native drugs.

Reciprocal interactions between the gut microbiome and mammary tissue microenvironment promote distant regulation of breast tumor metastasis

Abstract Establishing commensal dysbiosis, defined as an inflammatory gut microbiome with low biodiversity, prior to breast tumor initiation enhances early dissemination of hormone-receptor positive (HR+) mammary tumor cells into the circulation, distal lymph nodes, and lungs. Here, we sought to define mammary tissue mediators of dysbiosis-induced tumor dissemination. Commensal dysbiosis increased mast cell numbers in the pre-malignant mammary tissue, with numbers remaining elevated in the presence of a tumor. During early tumor progression, increased mast cell numbers correlated with increased numbers of activated fibroblasts and collagen I protein levels in mammary tissues. Fibroblast activation and tissue remodeling are associated with enhanced breast tumor metastasis. Although mast cell degranulation is known to trigger fibrosis in other disease models, it is unknown whether mammary tissue mast cells influence dissemination of tumor cells. To investigate the role of mast cells during tumor dissemination, mice were treated with mast cell stabilizers ketotifen or cromolyn. Notably, inhibition of mast cell degranulation significantly reduced fibroblast activation and dissemination of tumor cells into the blood of dysbiotic animals. Collagen levels in mammary tissues from women diagnosed with HR+ breast cancer also correlated with mast cell abundance, suggesting mast cell-mediated fibroblast activation is relevant to human disease. Together, these data demonstrate that mast cells recruited into the pre-malignant mammary tissue in response to commensal dysbiosis orchestrate early dissemination of HR+ breast tumors through activation of tissue fibroblasts.

Glaucocalyxin A Attenuates Allergic Responses by Inhibiting Mast Cell Degranulation through p38MAPK/NrF2/HO-1 and HMGB1/TLR4/NF-κB Signaling Pathways.

Glaucocalyxin A (GLA) has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. Here, in this study, the effect and mechanism of GLA on mast cell degranulation were studied. The results of the anti-DNP IgE-mediated passive cutaneous anaphylaxis (PCA) showed that GLA dramatically inhibited PCA in vivo, as evidenced by reduced Evans blue extravasation and decreased ear thickness. In addition, GLA significantly reduced the release of histamine and β-hexosaminidase, calcium influx, cytokine (IL-4, TNF-α, IL-1β, IL-13, and IL-8) production in the RBL-2H3 (rat basophilic leukemia cells), and RPMCs (peritoneal mast cells) in vitro. Moreover, we further investigated the regulatory mechanism of GLA on antigen-induced mast cells by Western blot, which showed that GLA inhibited FcεRI-mediated signal transduction and invalidated the phosphorylation of Syk, Fyn, Lyn, Gab2, and PLC-γ1. In addition, GLA inhibited the recombinant mouse high mobility group protein B1- (HMGB1-) induced mast cell degranulation through limiting nuclear translocation of NF-κBp65. Treatment of mast cells with siRNA-HMGB1 significantly inhibited HMGB1 levels, as well as MyD88 and TLR4, decreased intracellular calcium levels, and suppressed the release of β-hexosaminidase. Meanwhile, GLA increased NrF2 and HO-1 levels by activating p38MAPK phosphorylation. Consequently, these data suggest that GLA regulates the NrF2/HO-1 signaling pathway through p38MAPK phosphorylation and inhibits HMGB1/TLR4/NF-κB signaling pathway to reduce mast cell degranulation and allergic inflammation. Our findings could be used as a promising therapeutic drug against allergic inflammatory disease.

Increased Thrombin-Activatable Fibrinolysis Inhibitor in Response to Sublingual Immunotherapy for Allergic Rhinitis.

The objective of this study was to determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) as a candidate biomarker for therapeutic efficacy of sublingual immunotherapy (SLIT) and to identify the role of TAFI in the pathogenesis of allergic rhinitis (AR). Retrospective cohort study and laboratory study. Serum was collected from patients with allergies to Japanese cedar pollen before, during, and after treatment with SLIT. We measured the levels of immunoreactive TAFI, C3a, and C5a in serum by enzyme-linked immunosorbent assay (ELISA) and assessed their relative impact on a combined symptom-medication score. We also examined the impact of TAFI on mast cells and fibroblasts in experiments performed in vitro. Serum levels of TAFI increased significantly in response to SLIT. By contrast, serum C3a levels decreased significantly over time; we observed a significant negative correlation between serum levels of TAFI versus C3a and symptom-medication score. Mast cell degranulation was inhibited in response to TAFI, as it was the expression of both CCL11 and CCL5 in cultured fibroblasts. High serum levels of TAFI may be induced by SLIT. TAFI may play a critical protective role in pathogenesis of AR by inactivating C3a and by inhibiting mast cell degranulation and chemokines expression in fibroblasts. 4 Laryngoscope, 131:2413-2420, 2021.

Sesquiterpene-Loaded Co-Polymer Hybrid Nanoparticle Effects on Human Mast Cell Surface Receptor Expression, Granule Contents, and Degranulation.

Biodegradable polymeric nanoparticles (NPs) such as poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) have been used as drug delivery systems for natural and synthetic compounds and are designed to control the loading and release of biodegradable materials to target cells, tissues, and organs. Eremophilane-type sesquiterpenes have anti-inflammatory properties but are lipophilic, cytotoxic, and not biocompatible with many cells. To determine whether biodegradable PLGA/PVA could improve the biocompatibility of sesquiterpenes, sesquiterpene-loaded NPs were synthesized and their effects on human mast cells (LAD2), the major effector cells of allergic inflammation, were determined. NPs composed of PLGA/PVA and two types of sesquiterpenes (fukinone, PLGA/PVA-21 and 10βH-8α,12-epidioxyeremophil-7(11)-en-8β-ol, PLGA/PVA-22) were produced using a microfluidic synthesis method. The NPs’ size distribution and morphology were evaluated by dynamic light scattering and cryogenic transmission electron microscopy (TEM). PLGA/PVA-21 and PLGA/PVA-22 were 60 to 70 nm and were readily internalized by LAD2 as shown by flow cytometry, fluorescence microscopy, and TEM. While unencapsulated sesquiterpenes decreased LAD2 cell viability by 20%, PLGA/PVA-21 and PLGA/PVA-22 did not alter LAD2 viability, showing that encapsulation improved the biocompatibility of the sesquiterpenes. PLGA/PVA-21 and PLGA/PVA-22 decreased the expression of genes encoding the subunits of the high affinity immunoglobulin E receptor (FcεR1α, FcεR1β, FcεR1γ) and the stem cell factor receptor (Kit,), suggesting that hybrid NPs could alter mast cell responses to antigens and shift their maturation. Similarly, PLGA/PVA-21 and PLGA/PVA-22 inhibited tryptase expression but had no effect on chymase expression, thereby promoting a shift to the tryptase-positive phenotype (MCT). Lastly, PLGA/PVA-21 and PLGA/PVA-22 inhibited mast cell degranulation when the LAD2 cells were activated by IgE crosslinking and FcεRI. Overall, our results suggest that PLGA/PVA-21 and PLGA/PVA-22 alter human mast cell phenotype and activation without modifying viability, making them a more biocompatible approach than treating cells with sesquiterpenes alone.

Human Umbilical Cord Mesenchymal Stem Cell Therapy Mitigates Interstitial Cystitis by Inhibiting Mast Cells.

BackgroundInterstitial cystitis (IC) is a recurrent and chronic inflammatory disease that compromises patients’ quality of life. Effective treatments for IC are limited. This study aimed to evaluate the therapeutic potency of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) in an IC-induced rat model and investigate the potential molecular mechanism in a mast cell model (rat basophilic leukemia cells, RBL-2H3) in treating IC in a coculture system.Material/MethodsThe rat model of IC was induced by cyclophosphamide (CYP). Rats were randomly divided into 3 groups: sham, IC+PBS, and IC+MSC. In the coculture system, RBL-2H3 cells were sensitized overnight to Compound 48/80 (C48/80), cocultured with UC-MSCs for 3 days, and collected for subsequent experiments. RBL-2H3 cells were randomly divided into 3 groups: sham, C48, and UC-MSCs (C48+MSC).ResultsThe UC-MSCs marked by thymidine analog 5-ethynyl-2-deoxyuridine (EdU) were transplanted in the treatment group, and were densely distributed in the bladder. Accordingly, the conscious cystometry was measured and the bladder tissues were harvested. Compared with the sham group, the treated IC rats exhibited shorter bladder voiding intervals (307±35 vs 217±37 s; P<0.01), more integral epithelia, and less collagen fiber aggregation, infiltration and degranulation of mast cells, and inflammatory cytokines in the bladder tissue. In the coculture system, compared with the C48 group, the UC-MSC-treated RBL-2H3 cells had suppressed degranulation.ConclusionsUC-MSCs treatment showed a promising therapeutic effect on treating IC in vivo and in vitro. UC-MSCs inhibit mast cell degranulation in IC and could be a potential therapeutic target to ameliorate inflammation in IC.

Synergistic anti-allergy activity using a combination of Enterococcus faecalis IC-1 and luteolin

Allergies are a global issue. However, medical intervention for allergy treatment is limited. Recent studies have focused on allergy prevention with food components. Both the lactic acid bacteria Enterococcus faecalis IC-1 (IC-1) and the flavonoid luteolin have been shown to have an anti-allergic effect. Following the concept of diet diversity and synergistic effects, the purpose of this study was to explore the anti-allergic activity of a combination of IC-1 and luteolin. A combination of IC-1 and luteolin had anti-allergic effects with the passive cutaneous anaphylaxis reaction and strongly inhibited mast cell degranulation in a Caco-2/RBL-2H3 cells co-culture system. Oral administration with a combination of IC-1 and luteolin significantly decreased blood IgE content in ovalbumin (OVA)-induced allergic mice and alleviated symptoms of an allergy attack. These might have been done through correcting Th2 shifting with the allergy state since a combination of IC-1 and luteolin increased the IFN-γ content in blood. Furthermore, in vitro differentiation of the Th2 subset confirmed that a combination of IC-1 and luteolin inhibited naïve T cell from differentiating into a Th2 subset through inhibition of Th2 transcription factor GATA-3 expression. The combination also promoted the gene expression level of IFN-γ in mice bone marrow-derived dendritic cells. These results suggested a synergistic anti-allergic effect using a combination of IC-1 and luteolin both in reducing IgE content and in inhibiting mast cell degranulation during the allergy state.

Discovery, Function, and Therapeutic Targeting of Siglec-8.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass cell surface receptors that have inhibitory activities on immune cells. Among these, Siglec-8 is a CD33-related family member selectively expressed on human mast cells and eosinophils, and at low levels on basophils. These cells can participate in inflammatory responses by releasing mediators that attract or activate other cells, contributing to the pathogenesis of allergic and non-allergic diseases. Since its discovery in 2000, initial in vitro studies have found that the engagement of Siglec-8 with a monoclonal antibody or with selective polyvalent sialoglycan ligands induced the cell death of eosinophils and inhibited mast cell degranulation. Anti-Siglec-8 antibody administration in vivo to humanized and transgenic mice selectively expressing Siglec-8 on mouse eosinophils and mast cells confirmed the in vitro findings, and identified additional anti-inflammatory effects. AK002 (lirentelimab) is a humanized non-fucosylated IgG1 antibody against Siglec-8 in clinical development for mast cell- and eosinophil-mediated diseases. AK002 administration has safely demonstrated the inhibition of mast cell activity and the depletion of eosinophils in several phase 1 and phase 2 trials. This article reviews the discovery and functions of Siglec-8, and strategies for its therapeutic targeting for the treatment of eosinophil- and mast cell-associated diseases.

Mechanisms of Drug Desensitization: Not Only Mast Cells.

Drug desensitization (DD) allows transient clinical tolerance to the drug in reactive patients and it is frequently and successfully used in the management of both IgE and non IgE-mediated hypersensitivity reactions (HRs). The underlying mechanisms behind this process is not well understood. The desensitization procedure is associated with the inhibition of mast cells degranulation and cytokine production, that, is attributable, at least partially, to the abrogation of Ca2+ mobilization; in vitro findings and in vivo mouse models of rapid desensitization show that the organization and spatial distribution of actin is critical for Ca2+ mobilization. Some clinical observations may suggest the induction of a longer memory of tolerance by DD and they raise the suspicion that other cells and mechanisms are involved in DD. Some data are emerging about the modifications of immune responses during DD in patients with previous immediate HRs. In particular, an increase of regulatory cytokines, mainly represented by IL-10, has been shown, and more importantly, the appearance of IL-35 producing T regulatory cells has been described during DD. The release of controlled cellular mediators by mast cells over time and the development of the antigen-specific regulation of adaptive response allow to safely and successfully reach the target dose of a first line drug during DD.

Inhibitory activity of narirutin on RBL-2H3 cells degranulation

Context: It is an efficient strategy to apply inhibition of mast cell degranulation for evaluating anti-allergic effects of compounds. Previous works confirmed that narirutin had anti–allergic activity in OVA induced allergic asthma murine model. However, the mechanism is not clear. Objective: Here, inhibitory mechanism of narirutin on RBL-2H3 cells degranulation was investigated. Materials and methods: Cell viability was analyzed by CCK-8 kits, cell degranulation was analyzed by ELISA methods, morphology and ultrastructure of cells was observed by atomic force microscopy, intracellular Ca 2+ concentration was measured by fluorescence microscopre, mRNA expression were measured by PCR, and signaling pathways were measured by WB. Results: The results showed that narirutin have no direct effects on mRNA expression of FcεRI subunit. However, it inhibited Ca2+ influx by suppressing the phosphorylation of Syk, LAT and PLCγ1 signaling pathway transduction. Subsequently, the inhibition of Ca2+ influx directly leads to NF-κB signaling pathway transduction decreased. Narirutin can also suppress the phosphorylation of MAPK signaling pathways by decreasing the expression of P-p38, P-ERK and P-JNK, inhibit the synergistic effect for Ca2+ influx, and then reduce the release of IL-4, TNF-α, histamine and β-HEX. Conclusion: Our study suggested that the inhibitory mechanism of narirutin on RBL-2H3 cells degranulation could be related to regulate MAPK, NF-κB and Tyrosine kinase signaling pathway.

Evaluation of Anti-asthmatic Activity of Salvadora oleoids Leaves

Salvadora oleoids (Family: Salvadoraceae) is being used in the traditional medicine for the treatment of severe bronchitis and asthma. So, the study aims to evaluate anti-asthmatic activity of S. oleoids leaves. Leaves are reported to contain flavonoids, saponins, steroids, alkaloids, and tannins, known to possess similar effects. Flavonoids have been shown to smooth muscle relaxant, bronchodilator, antioxidant, and anti-inflammatory activity. The flavonoids are also reported to inhibit mast cell degranulation. Saponins are reported to mast cell stabilizing, anti-allergic, anti-inflammatory and antihistaminic activity. Tannins are reported to inhibit cyclooxygenase (COX) enzyme, decrease vascular permeability and antioxidant activity. Steroids are reported to an extraordinarily strong anti-inflammatory activity by inhibiting cytokine release IL-1, 2 and 6. The present investigation was undertaken to evaluate the anti-asthmatic activity of ethanolic extract of S. oleoids leaves in experimental animals. Asthma is a chronic inflammatory disorder of the airways. Limitations of currently available therapies and associated side effects have created an urge to search new and better treatment options. Thus, present study was designed to evaluate anti-asthmatic activity of S. oleoids leaves. Ethanolic extract of S. oleoides was prepared and used in the dose of 200 mg/kg (p.o.). Various in-vivo models like histamine induced bronchoconstriction in guinea pigs and milk induced leukocytosis in mice were used for evaluating anti-asthmatic activity of the drugs. Ethanolic extract of S. oleoides showed a significant broncho-dilatory, anti-histaminic and anti-inflammatory activity. Thus, ethanolic extract of S. oleoides have significant anti-asthmatic activity. Thus, ethanolic extract of S. oleoides leaves have proved to be an effective drug in prevention of asthma.

Nuclear factor E2 related factor (NRF2) inhibits mast cell- mediated allergic inflammation via SIRT4-mediated mitochondrial metabolism.

Mast cell (MC)-mediated inflammation is essential for allergic rhinitis, and nuclear factor E2 related factor (NRF2) is found to inhibit inflammation. This study investigated whether NRF2 could inhibit MC inflammation and its molecular mechanisms concerning SIRT4. Real-time quantitative PCR (RT-qPCR) and western blot were used to detect gene expression, and Elisa kit was used to detect the content of histamine and inflammatory cytokines in the medium of MCs, and Seahorse XF instrument was used to measure the mitochondrial metabolism of MCs. Knockdown SIRT4 and establish SIRT4 overexpression of HMC-1 cells to study the function of SIRT4. As an activator of NRF2, 4-Octyl Itaconate increases not only NRF2 expression but also increases SIRT4 expression. Although 4-Octyl Itaconate could reduce the histamine release and degranulation of MCs, which was induced by compound 48/80, SIRT4 knockdown decreased the inhibition of 4-Octyl Itaconate. Similarly,4-Octyl Itaconate inhibited the secretion of inflammatory cytokines (TNF-α, IL-1β, IL6, and IL-8) by MCs, which was induced by LPS, but SIRT4 knockdown decreases the inhibition of 4-Octyl Itaconate. Also, the up-regulation of SIRT4 significantly inhibited mitochondrial metabolism in MCs and inhibited SIRT1 and P-p65 protein expression after inducing by 100 ng/mL LPS for 1 hour. NRF2 inhibits MC degranulation and MC-mediated inflammation by promoting SIRT4, and SIRT4 overexpression inhibits the mitochondrial metabolism of MCs.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8

The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Paeoniflorin inhibits IgE-mediated allergic reactions by suppressing the degranulation of mast cells though binding with FcϵRI alpha subunits

Paeoniflorin (PF), a monoterpene glycoside isolated from the aqueous extract of the Chinese herb Radix Paeoniae Alba, has been used for treating various inflammatory diseases. In this study, we aimed to investigate the anti-allergic activities of PF. The anti-anaphylactic activity of PF was investigated using mast cell (MC) degranulation assay as well as Ca2+ influx in vitro and skin swelling and extravasation assays in vivo. The results showed that PF inhibited MC degranulation (histamine release from 74.5 ± 4.95 ng/ml to 58.7 ± 6.06 ng/ml) and Ca2+ influx challenged by DNP-BSA in vitro. In addition, PF reduced the degree of swelling and Evans blue exudation in mice paws. Furthermore, PF dose-dependently reduced serum inflammatory mediator release in mice sensitized with ovalbumin for 48 h by inhibiting MC degranulation. Molecular docking study revealed that PF bound better with the α subunit of FcϵRI than with the β subunit. SPR revealed that PF had a strong affinity interaction with FcϵRI α subunit and the KD value was (7.08 ± 0.97) e−6 M. Our findings revealed that PF inhibited anaphylactic responses in vivo and in vitro, and it can be considered a novel FcϵRI inhibitor for preventing MC-related allergic diseases.

Two Japanese pepper (Zanthoxylum piperitum) fruit-derived compounds attenuate IgE-mediated allergic response in vitro and in vivo via inhibition of mast cell degranulation

Zanthoxylum piperitum (ZP, ‘Japanese pepper’) is a traditional medicine and pepper used in Asian countries such as Japan. Hydroxy-α-sanshool, a pungent-tasting substance contained within ZP, has been reported to slightly suppress immunoglobulin E (IgE)-mediated mast cell degranulation. The current study aims to newly identify anti-allergic compounds derived from ZP. We examine the inhibitory mechanisms behind IgE-mediated mast cell degranulation. By inhibitory effect-guided isolation, we identified degranulation inhibitory compounds derived from ZP fruit: 1-acetoxy-7-hydroxy-3, 7-dimethylocta-2E, 5E-diene (ZP1) and 8-hydroxygeranyl acetate (ZP2). ZP1 and ZP2 inhibited IgE-mediated degranulation and A23187-mediated degranulation in RBL-2H3 mast cells. Our findings suggest the inhibition of degranulation by ZP1 and ZP2 was by inhibition of Lyn phosphorylation, followed by inhibition of intracellular Ca2+ mobilization, protein kinase C alpha phosphorylation, membrane ruffling, and granule-to-plasma membrane fusion. Oral administration of ZP1 or ZP2 attenuated an IgE-mediated passive cutaneous anaphylactic reaction in mice. Histological observation suggests that this effect occurred via inhibition of mast cell degranulation. These findings indicate that ZP1 and ZP2 attenuate allergic reaction via inhibition of IgE-mediated mast cell degranulation.