Bone Marrow-derived Mast Cells Research Articles

DNA Methylation Negatively Regulates Gene Expression of Key Cytokines Secreted by BMMCs Recognizing FMDV-VLPs

Virus-like particles (VLPs) have been studied and used as vaccines to control foot-and-mouth disease (FMD). Mast cells (MCs) express various pattern recognition receptors that recognize pathogens and secrete numerous cytokines to initiate and modulate immune responses. Our previous study showed that bone marrow-derived mast cells (BMMCs) can recognize foot-and-mouth disease virus-like particles (FMDV-VLPs) to differentially express various cytokines and that histone acetylation can regulate the cytokines secreted during BMMC recognition of FMDV-VLPs. To demonstrate the role of DNA methylation in this response process, BMMCs that recognize FMDV-VLPs were treated with azacytidine (5-AZA), an inhibitor of DNA methylation transferase. We prepared FMDV-VLPs as described previously and cultured the BMMCs. The transcription and expression of key cytokines and transcription factors were determined using real-time quantitative PCR (RT-qPCR) and Western blotting. Results showed that pre-treatment with AZA resulted in the increased transcription and expression of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-13, and IL-10, while the changes in IL-13 transcription and IL-6 expression were irrelevant to mannose receptors (MRs). Furthermore, analysis of the transcription factors indicated that both the transcription and expression of nuclear factor-kappa B (NF-κB) increased significantly in the AZA pre-treated group, indicating that DNA methylation may also regulate NF-κB expression to modulate TNF-α, IL-13, and IL-6. However, pre-treatment with AZA did not alter the expression of microphthalmia-associated transcription factor (MITF) or GATA-2. All the data demonstrate that DNA methylation negatively regulates the transcription and expression of TNF-α, IL-13, IL-10, and IL-6 secreted by recognizing FMDV-VLPs. These results provide new ideas for the mast cell-based design of more effective vaccine adjuvants and targeted therapies in the future.

Ligand-independent function of β2-adrenergic receptor affects IgE-mediated Ca2+ influx in mast cells

BackgroundMast cells are key effector cells that elicit immunoglobulin E (IgE)-mediated allergic inflammations. Allergen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on mast cells triggers signaling cascades that activate signal proteins and evoke extracellular Ca2+ influx, which are crucial for cytokine production. The β2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. ObjectiveAlthough β2-adrenergic-related reagents are known to influence mast cell functions, the specific intrinsic role of Adrb2 in these cells is not fully understood, potentially because of off-target effects. In this study, the additional roles of Adrb2 in mast cells were investigated, specifically the involvement of Adrb2 in FcεRI signaling, using Adrb2−/− mice. MethodsAdrb2−/− mice were used to investigate the roles of Adrb2 in mast cells by examining bone marrow-derived mast cells (BMMCs) for surface expression of mast cell markers, granule numbers, and gene expression of mast cell proteases. Cytokine production, Ca2+ influx, and nuclear factor of activated T cells (NFAT) nuclear translocation were measured in Adrb2−/− and Adrb2+/+ BMMCs upon IgE/Ag stimulation. ResultsAdrb2−/− did not affect the generation of BMMCs, their surface expression of mast cell markers, granule numbers, or gene expression of mast cell proteases, indicating that the absence of Adrb2 had no adverse effect on mast cell development. However, Adrb2−/− BMMCs exhibited reduced tumor necrosis factor α (TNFα) production and diminished Ca2⁺ influx upon IgE/Ag stimulation, which correlated with decreased NFAT translocation. Restoration of Adrb2 in Adrb2−/− BMMCs rescued cytokine production. Notably, FcεRI-mediated phosphorylation of the phospholipase PLCγ1 and mitogen-activated protein kinases (MAPKs) remained unchanged in the absence of Adrb2. ConclusionThese results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.

Diterpenoid DGT alleviates atopic dermatitis–like responses in vitro and in vivo via targeting IL-4Rα

BackgroundAtopic dermatitis is a common chronic inflammatory skin disease characterized by relapsing eczema and intense itch. DGT is a novel synthetic heterocyclic diterpenoid derived from plants. Its therapeutic potential and mechanism(s) of action are poorly understood. ObjectivesWe investigated the potent therapeutic effect of DGT on atopic dermatitis, exploring the underlying mechanisms and determining whether DGT is a safe and well-tolerated topical treatment. MethodsWe observed anti-inflammatory effects of DGT on tumor necrosis factor-α/interferon-γ–treated human keratinocytes, and anti-allergic effects on immunoglobulin E–sensitized bone marrow–derived mast cells. In vivo, DGT was topically applied to two experimental mouse models of atopic dermatitis: oxazolone-induced sensitization and topically applied calcipotriol. Then the therapeutic effects of DGT were evaluated physiologically and morphologically. Moreover, we performed nonclinical toxicology and safety pharmacology research, including general toxicity, pharmacokinetics, and safety pharmacology on the cardiovascular, respiratory, and central nervous systems. ResultsIn keratinocytes, DGT reduced the expression of inflammatory factors, promoting the expression of barrier functional proteins and tight junctions and maintaining the steady state of barrier function. DGT also inhibited the activation and degranulation of mast cells induced by immunoglobulin E. Moreover, we found that interleukin-4 receptor-α was the possible target of DGT. Meanwhile, DGT had therapeutic effects on oxazolone/calcipotriol-treated mice. Notably, our pharmacology results demonstrated that DGT was safe and nontoxic in our studies. ConclusionDGT’s potent anti-inflammatory effects and good safety profile suggest that it is a potential candidate for the treatment of atopic dermatitis.

Unexpected Expression and Function of FcεRI in Immortalized Breast Cancer Cells: A Cautionary Null Study.

The high-affinity IgE receptor, FcεRI, is typically associated with type 2 effectors such as mast cells (MC). The relatively unique expression profile of FcεRI and accumulating evidence from pre-clinical and clinical settings, such as MC interactions with tumors, have led us to study MCs as a potential therapeutic target in breast cancer. Our work identified MCs interacting with tumor cells at primary sites using the 4T1 (BALB/c) adenocarcinoma model in vivo. However, this analysis was complicated by a surprising finding that the tumor cells intrinsically and strongly expressed FcεRI. We further studied the expression and function of FcεRI in breast cancer cells in vitro. The 4T1 cells expressed FcεRI to a level similar to mouse bone marrow-derived MC (BMMC). Additionally, two established breast cancer cultures derived from human T-47D cells, one estrogen-dependent (E3) and the other estrogen-withdrawn (EWD8), also expressed FcεRI with EWD8 cells showing the greatest abundance. Functional analyses indicated that IgE-mediated antigen stimulation did not elicit classic Ca2+ flux in breast cancer cells as seen in the respective species' MCs; however, FcεRI crosslinking could stimulate IL-6 production from the T-47D derivatives. Preliminary analysis of primary breast cancer biopsy datasets using R2: Genomics Analysis and Visualization Platform was discordant with our in vivo model and in vitro observations. Indeed, FcεRI mRNA abundance declined in metastatic breast cancers compared to non-cancerous breast tissue. Altogether, we report a previously unidentified and immunologically substantive difference between breast cancer models and human primary tumors. Investigators pursuing FcεRI-relevant therapeutics in this context should be aware of this translational barrier.

CCAAT/enhancer-binding protein α-dependent regulation of granule formation in mast cells by intestinal bacteria.

The antiallergic effects of gut microbiota have been attracting attention in recent years, but the underlying cellular and molecular mechanisms have not yet been fully understood. In this study, we aimed to investigate these mechanisms specifically focusing on mast cells. Mast cells retain intracellular granules containing various inflammatory mediators such as histamine, which are released outside the cells upon IgE and allergen stimulation. We previously reported that increased expression of the transcription factor, CCAAT/enhancer-binding protein α (C/EBPα), suppresses granule formation in mast cells and that Lacticaseibacillus casei JCM1134T (LC) upregulates C/EBPα levels. Here, granule formation in mouse bone marrow-derived mast cells was suppressed in a MyD88-dependent manner after LC treatment due to C/EBPα-dependent downregulation of the genes encoding serglycin (SRGN) and mast cell protease 4 (Mcpt4). Furthermore, C/EBPα expression was regulated by DNA methylation in the 5' region far upstream of the transcription start site. LC suppressed DNA methylation of specific CpG motifs in the 5' region of the C/EBPα gene. These results conclude that specific gut microbial components, such as those from LC, suppress granule formation in mast cells by inhibiting SRGN and Mcpt4 expression via reduced C/EBPα gene methylation.

The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate.

Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.

Aging promotes mast cell activation and antigen presenting capacity in atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Aging is an independent and dominant risk factor for atherosclerosis and is associated with a low-grade chronic inflammation termed inflammaging. This age-induced pro-inflammatory environment may have great impact on plaque-residing immune cells, including mast cells. Mast cells have been found to accumulate in the human atherosclerotic plaque upon disease progression and have been associated with plaque instability. However, it is currently unknown whether aging drives the pro-atherogenic effects of mast cells in atherosclerosis. To assess the effects of aging on mast cell phenotype, we examined mast cell populations in young and old male Ldlr-/- mice fed either a chow (CD) or Western-type (WD) diet. We observed a 6-fold increase in the number of mast cells in the aged atherosclerotic aorta compared to young (Y-CD: 4.5±0.7 vs. O-CD: 279.4±9 cells, p<0.01; Y-WD: 45.3±15.2 vs. O-CD: 279.4±9 cells, p<0.01). Furthermore, we detected a marked increase in activation status with age as shown by the significantly augmented number of CD63+ mast cells in the atherosclerotic aorta (Y-CD: 1.8±0.6 vs. O-CD: 259.7±85.4 cells, p<0.01; Y-WD: 41.3±13.6 vs. O-CD: 259.7±85.4 cells, p<0.01). In line, we identified that aged bone-marrow derived mast cells displayed elevated basal CD63 expression compared to young cells, indicative of intrinsic low-grade activation. Moreover, we demonstrated that aging increases the amount of MHCII+ mast cells in atherosclerosis, allowing them to act as antigen-presenting cells, which correspondingly led to increased CD4+ T cell proliferation in vitro (young: 35.2±1.2% vs. old: 45.8±1.2%, p<0.001). Finally, single-cell RNA sequencing of human atherosclerotic plaques confirmed mast cell-specific expression of MHC-II orthologs. To conclude, we established that aging induces phenotypic changes of mast cells in atherosclerosis, regarding both activation status and antigen presenting capacity, which can contribute to disease progression.

Aspalathin, a Primary Rooibos Flavonoid, Alleviates Mast Cell-Mediated Allergic Inflammation by the Inhibition of FcεRI Signaling Pathway.

Mast cells are primary cells initiating allergic inflammation by the release of various allergic mediators, such as histamine and pro-inflammatory cytokines. Aspalathin (ASP) is the predominant flavonoid found exclusively in rooibos, an herb that has been traditionally used in allergy relief therapy. In the present study, we investigated the beneficial effects of ASP on mast cell-mediated allergic inflammation. For in vivo study, two well-known mast cell-mediated local and systemic allergic inflammation mouse models were used: passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis mouse models (ASA). Oral administration of ASP dose-dependently suppressed immunoglobulin (Ig)E-mediated PCA responses evidenced by Evans blue extravasation, ear thickening, and mast cell degranulation. ASP also significantly mitigated ovalbumin-induced ASA responses, including hypothermia, histamine secretion, and the production of IgE and interleukin-4. Notably, ASP was more effective in suppressing allergic inflammation than nothofagin, another prominent flavonoid known as an anti-allergic component of rooibos. The regulatory mechanism of mast cell activation by ASP was clarified using mast cell line and primary cultured mast cells (RBL-2H3 and mouse bone marrow-derived mast cells). ASP reduced IgE-stimulated mast cells degranulation and intracellular calcium influx by the inhibition of FcεRI signaling pathway (Lyn, Fyn, and Syk). Moreover, ASP reduced pro-inflammatory cytokine expressions by inhibiting two major transcription factors, nuclear factor of activated T cells and nuclear factor-κB. Collectively, we proposed that ASP could be a potential therapeutic candidate for the treatment of mast cell-mediated allergic inflammatory diseases.

SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool.

The high-affinity IgE receptor FcεRI is the mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the FcεRI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE resulting in degranulation and proinflammatory cytokine production. In comparison to the T- and B-cell receptor complexes, for which several co-receptors orchestrating the initial signaling events have been described, information is scarce about FcεRI-associated proteins. Additionally, it is unclear how FcεRI signaling synergizes with input from other receptors and how regulators affect this synergistic response. We found that the HDL receptor SR-BI (gene name: Scarb1/SCARB1) is expressed in MCs, functionally associates with FcεRI, and regulates the plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains. This impacted the activation of MCs upon co-stimulation of the FcεRI with receptors known to synergize with FcεRI signaling. Amongst them, we investigated the co-activation of the FcεRI with the receptor tyrosine kinase KIT, the IL-33 receptor, and GPCRs activated by adenosine or PGE2. Scarb1-deficient bone marrow-derived MCs showed reduced cytokine secretion upon co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating respective MC activation. Mimicking Scarb1 deficiency by cholesterol depletion employing MβCD, we identified PKB and PLCγ1 as cholesterol-sensitive proteins downstream of FcεRI activation in bone marrow-derived MCs. When MCs were co-stimulated with stem cell factor (SCF) and Ag, PLCγ1 activation was boosted, which could be mitigated by cholesterol depletion and SR-BI inhibition. Similarly, SR-BI inhibition attenuated the synergistic response to PGE2 and anti-IgE in the human ROSAKIT WT MC line, suggesting that SR-BI is a crucial regulator of synergistic MC activation.

Identification of pyruvic and maleic acid as potential markers for disease activity and prognosis in chronic urticaria

BackgroundPopulation-based studies have highlighted the link between chronic urticaria (CU) and metabolic syndrome, and metabolic alterations have been revealed in CU. However, to our knowledge, a comprehensive metabolomics study on a large cohort of CU patients has not been reported. ObjectiveTo explore the underlying metabolic subtypes and novel metabolite biomarkers for CU diagnosis and therapy. MethodsPlasma samples from 80 CU patients and 82 healthy controls (HCs) were collected for metabolomics quantification and performed bioinformatics analysis. Another independent cohort consisting of 144 CU patients was studied to validate the findings. Bone marrow-derived mast cells (BMMCs) and IgE-induced passive cutaneous anaphylaxis (PCA) mice were utilized for in vitro and in vivo experiments, respectively. ResultsWe observed clear metabolomics difference between CU and HC. Meanwhile, differential metabolites N6-acetyl-l-lysine, L-aspartate, maleic acid and pyruvic acid were used to respectively construct random forest classifiers, and achieved AUCs greater than 0.85, suggesting their potential as diagnostic biomarkers of CU. More importantly, by exploring the underlying metabolic subtypes of CU, we found that the low abundance of pyruvic acid and maleic acid was significantly related to the activity of CU, poor efficacy of second-generation H1-antihistamines (sgAHs), and short relapse-free time. The results were validated in the independent cohort. Moreover, supplementation with pyruvate or maleate could significantly attenuate IgE-mediated mast cells activation in vitro and in vivo. ConclusionsThe combination of plasma pyruvic acid and maleic acid may be effective biomarkers for predicting the disease activity, therapeutic efficacy as well as prognosis for CU patients.

Nur77 Mediates Anaphylaxis by Regulating miR-21a.

Nur77 belongs to the NR4A subfamily of orphan nuclear hormone receptors. It has been shown to play important roles in metabolism, cancer progression, cellular differentiation, and the regulation of immune process. However, there has yet to be research reporting on the role of Nur77 in allergic inflammations such as anaphylaxis. This study aimed to identify molecules that could mediate allergic inflammations. To this end, we performed RNA sequencing analysis employing bone marrow-derived mast cells (BMMCs). Antigen (DNP-HSA) stimulation increased the expression levels of transcription factors such as Nr4a3 (NOR1), Nr4a1 (Nur77), and Nr4a2 (Nurr1). We focused our study on Nur77. Antigen stimulation increased the expression of Nur77 in a time- and dose-dependent manner in rat basophilic leukemia cells (RBL2H3). The downregulation of Nur77 prevented both antigen-induced increase in β-hexosaminidase activity as well as hallmarks of allergic reactions such as HDAC3, COX2, and MCP1 in RBL2H3 cells. Nur77 was necessary for both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). TargetScan analysis predicted that miR-21a would be a negative regulator of Nur77. miR-21a mimic negatively regulated PCA and PSA by inhibiting the hallmarks of allergic reactions. ChIP assays showed that c-JUN could bind to the promoter sequences of Nur77. Antigen stimulation increased the expression of c-JUN in RBL2H3 cells. Altogether, our findings demonstrate the regulatory role played by Nur77-miR-21a loop in allergic inflammations such as anaphylaxis, making this the first report to present the role played by Nur77 in an allergic inflammation. Our results suggest that Nur77 and miR-21 might serve as targets for developing anti-allergy drugs.

Abstract 7469: Inflammation plays an important role in the progression of chronic myeloid leukemia

Abstract The development of Tyrosine Kinase Inhibitors (TKIs) resulted in a significant increase in the life expectancy of patients with chronic myeloid leukemia (CML). Nevertheless, TKIs still fail in a significant proportion of therapy-naïve patients, or their effectiveness is hampered by secondary resistance. It is therefore urgent to explore the underlying mechanisms in order to develop new therapeutic approaches to cure CML instead of controlling the disease with a lifelong TKI therapy. Using the ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML we show that BCR::ABL1 leads to the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression. This is supported by our data from CML patients, where we also observe higher levels of pro-inflammatory cytokines in the serum compared to healthy individuals. Furthermore, we demonstrate that BCR::ABL1 drives the expansion of bone marrow derived mast cells (BMMCs) and sensitizes them for FcεRI triggered degranulation. Interestingly, these BMMCs show differences in their lipid metabolism. Hence, sphingomyelin and lysophosphatidylcholine, both known to be involved in inflammation and cancer progression, are upregulated in BCR::ABL1-positive mast cells compared to their negative counterparts. By crossing in the Cpa3Cre mediated model for mast cell deficiency, we demonstrate for the first time that the lack of mast cells prevents the BCR::ABL1 induced upregulation of pro-inflammatory cytokines and also the development of splenomegaly. In addition, we were able to show that splenomegaly in CML patients is associated with high bone marrow mast cell counts and that an upregulation of pro-inflammatory cytokines in patient serum samples correlated with tryptase levels. In summary, our study identifies mast cells and their inflammatory cytokines as important factors in disease progression and suggests considering them as additional targets in CML therapy. Citation Format: Melanie Langhammer, Julia Schöpf, Timo Jaquet, Katharina Horn, Moritz Angel, Daniel Mohl, Simon Lagies, Corinna Spohr, Daniel Christen, Franziska Maria Uhl, Tiago Maié, Henrike Jacobi, Thorsten B. Feyerabend, Julia Huber, Marcus Panning, Cassian Sitaru, Ivan Costa, Robert Zeiser, Konrad Aumann, Heiko Becker, Till Braunschweig, Steffen Koschmieder, Bernd Kammerer, Khalid Shoumariyeh, Michael Huber, Mirle Schemionek-Reinders, Tilman Brummer, Sebastian Halbach. Inflammation plays an important role in the progression of chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7469.

Staphylococcal superantigen-like protein 3 triggers murine mast cell adhesion by binding to CD43 and augments mast cell activation.

Staphylococcus aureus is a noteworthy pathogen in allergic diseases, as four staphylococcal exotoxins activate mast cells, a significant contributor to inflammation, in an IgE-independent manner. Although the adhesion of mast cells is an essential process for their immune responses, only a small number of exotoxins have been reported to affect the process. Here, we demonstrated that staphylococcal superantigen-like (SSL) 3, previously identified as a toll-like receptor 2 agonist, induced the adhesion of murine bone marrow-derived mast cells to culture substratum. SSL3-induced adhesion was mediated by fibronectin in an Arg-Gly-Asp (RGD) sequence-dependent manner, suggesting the integrins were involved in the process. Additionally, SSL3 was found to bind to an anti-adhesive surface protein CD43. SSL3 induced the adhesion of HEK293 cells expressing exogenous CD43, suggesting that CD43 is the target molecule for adhesion induced by SSL3. Evaluation of SSL3-derived mutants showed that the C-terminal region (253-326), specifically T285 and H307, are necessary to induce adhesion. SSL3 augmented the IL-13 production of mast cells in response to immunocomplex and SSL12. These findings reveal a novel function of SSL3, triggering cell adhesion and enhancing mast cell activation. This study would clarify the correlation between S. aureus and allergic diseases such as atopic dermatitis.

IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells.

Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates μ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express μ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of μ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between μ-opioid and TLR4 receptors.

Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

Butyrate, Valerate, and Niacin Ameliorate Anaphylaxis by Suppressing IgE-Dependent Mast Cell Activation: Roles of GPR109A, PGE2, and Epigenetic Regulation.

Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs invitro and invivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs invivo and invitro involving GPR109A, PGE2, and epigenetic regulation.

Effect of stearoyl-coenzyme a desaturase 1 (SCD1) on the function of mast cells

Background: The prevalence of childhood asthma and obesity is increasing, while obesity increases the risk and severity of asthma. Lipid metabolism has been considered as an important factor in the pathogenesis of obesity-associated asthma. Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that catalyzes the production of monounsaturated fatty acids (MUFA).Methods: In the present study, the microarray data retrieved from the Gene Expression Comprehensive Database (GEO) was analyzed to further clarify the impact of SCD1 on Mast cell activation related lipid mediators and the correlation between SCD1 and obesity asthma in the population.Results: SCD1 was highly expressed in IgE-activated bone marrow-derived mast cells (BMMCs). Meanwhile, SCD1 was also verified expressed highly in dinitrophenyl human serum albumin (DNP-HAS) stimulated RBL-2H3 cells. The expression of SCD1 was up-regulated in peripheral blood leukocytes of asthmatic children, and was positively correlated with skinfold thickness of upper arm, abdominal skinfold and body mass index (BMI). Inhibition of SCD1 expression significantly suppressed the degranulation, lipid mediator production, as well as the migration ability in DNP-HAS-stimulated RBL-2H3 cells.Conclusion: SCD1 is involved in obese-related asthma through regulating mast cells.

Akebia saponin D attenuates allergic airway inflammation through AMPK activation.

Akebia saponin D (ASD) is a bioactive triterpenoid saponin extracted from Dipsacus asper Wall. ex DC.. This study aimed to investigate the effects of ASD on allergic airway inflammation. Human lung epithelial BEAS-2B cells and bone marrow-derived mast cells (BMMCs) were pretreated with ASD (50, 100 and 200μΜ) and AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) (1mM), and then stimulated with lipopolysaccharide (LPS) or IL-33. Pretreatment with ASD and AICAR significantly inhibited TNF-α and IL-6 production from BEAS-2B cells, and IL-13 production from BMMCs. Moreover, pretreatment with ASD and AICAR significantly increased p-AMPK expression in BEAS-2B cells. Inhibition of AMPK by siRNA and compound C partly abrogated the suppression effect of ASD on TNF-α, IL-6, and IL-13 production. Asthma murine model was induced by ovalbumin (OVA) challenge and treated with ASD (150 and 300mg/kg) or AICAR (100mg/kg). Infiltration of eosinophils, neutrophils, monocytes, and lymphocytes, and production of TNF-α, IL-6, IL-4, and IL-13 were attenuated in ASD and AICAR treated mice. Lung histopathological changes were also ameliorated after ASD and AICAR treatment. Additionally, it showed that treatment with ASD and AICAR increased p-AMPK expression in the lung tissues. In conclusion, ASD exhibited protective effects on allergic airway inflammation through the induction of AMPK activation.

Cultures of Human Skin Mast Cells, an Attractive In Vitro Model for Studies of Human Mast Cell Biology.

Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.