Search Results (19)

Background/Objectives: This study evaluated the immunomodulatory effects of multiple A. bisporus strains, applying both in vitro and in vivo murine models and aiming to identify specific strains with immune modulatory properties. Methods: Sixteen A. bisporus strains were screened in a dendritic cell (DC)–CD4⁺ T cell co-culture system, using cells from female C57BL/6 mice, for their capacity to induce Th1- and Th17-associated cytokine production. Based on these cytokine profiles, three potent strains (MES01856, MES01706, MES01637), one non-responder strain (MES01515), and the commercially available A15 strain were selected for investigation in a murine model, an ovalbumin (OVA)-induced allergic food allergy. In these sensitized female C3H/HeOuJ mice, ear swelling, anaphylactic shock scores, OVA-specific IgE, and serum mouse mast cell protease-1 (mMCP-1) were measured following an allergen challenge. Additionally, IL-6, IL-10, and TNF-α secretion from bone marrow-derived dendritic cells (BMDCs) was measured after stimulation with these A. bisporus fresh ground extracts (FGEs). Results: In vitro, increases in cytokine production were obtained, with MES01856, MES01706, and MES01637 identified as the most potent inducers. In vivo, OVA sensitization elicited significant ear swelling and elevated shock scores in the mice. Dietary supplementation with MES01706 or MES01515 significantly reduced ear swelling, while the other strains resulted in non-significant changes. No significant changes were observed in shock scores, OVA-specific IgE, or mMCP-1. Conclusions: Multiple different A. bisporus strains elicit specific immunomodulating effects both in vitro and in in vivo murine models, identifying the MES01706 strain as one with the highest potential to beneficially affect immune function. Full article

The cochlea is susceptible to damage from ototoxic agents such as cisplatin, yet the mechanisms underlying cochlear injury remain incompletely understood. Mast cells (MCs), key immune players in allergic and inflammatory responses, have recently been identified in the rodent cochlea and implicated in cisplatin-induced ototoxicity. Our study investigated the role of MC degranulation in cochlear damage and evaluated the activation capacity of cochlear-resident MCs. Bone marrow-derived MCs (BMMCs) were cultured and induced to degranulate via IgE-anti-DNP/DNP stimulation, and the supernatants were applied to cochlear explants. Cochlear explants were also treated with Compound 48/80 (CP48/80) or cisplatin to assess MC activation. Morphological changes were assessed and hair cells (HC) quantified via phalloidin staining, while ELISA measured mediator release. Supernatants from degranulated BMMC induced a dose-dependent HC loss and tissue damage. A significant chymase and tryptase release was triggered by CP48/80 from cochlear MCs, with chymase elevation detected even at low concentrations. Cochlear MCs were rapidly activated by cisplatin exposure, elevating chymase and histamine levels, and the effects were attenuated by the MC stabilizer sodium cromolyn. Notably, tryptase remained undetectable post-cisplatin treatment, suggesting tissue-specific MC responses. These findings establish MC degranulation as an early event in cisplatin-induced cochlear injury, mediated by chymase and histamine. Our study highlights MCs as potential therapeutic targets for mitigating ototoxicity and underscores the need to explore MC-driven pathways in hearing loss. Full article

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. Full article

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. Full article

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. Full article

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. Full article

Mast cells can recognize foot-and-mouth disease virus-like particles (FMDV-VLPs) via mannose receptors (MRs) to produce differentially expressed cytokines. The regulatory role of chromatin accessibility in this process is unclear. Bone marrow-derived mast cells (BMMCs) were cultured, and an assay of transposase-accessible chromatin sequencing (ATAC-seq) was applied to demonstrate the regulation of chromatin accessibility in response to the BMMCs’ recognition of FMDV-VLPs. A pathway enrichment analysis showed that peaks associated with the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), and other signaling pathways, especially the NF-κB pathway, were involved in the BMMCs’ recognition of VLPs. Moreover, transcription factors including SP1, NRF1, AP1, GATA3, microphthalmia-associated transcription factor (MITF), and NF-κB-p65 may bind to the motifs with altered chromatin accessibility to regulate gene transcription. Furthermore, the expression of NF-κB, interleukin (IL)-9, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the BMMCs of the VLP group increased compared with that of the BMMCs in the control group, whereas the expression of IL-10 did not differ significantly between groups. After inhibiting the MRs, the expression of NF-κB, IL-9, TNF-α, and IFN-γ decreased significantly, whereas the expression of IL-10 increased. The expression of MAPK and IL-6 showed no significant change after MR inhibition. This study demonstrated that MRs expressed on BMMCs can affect the NF-κB pathway by changing chromatin accessibility to regulate the transcription of specific cytokines, ultimately leading to the differential expression of cytokines. These data provide a theoretical basis and new ideas for the development of a novel vaccine for FMD. Full article

Formononetin (FNT) is a plant-derived isoflavone natural product with anti-inflammatory, antioxidant, and anti-allergic properties. We showed previously that FNT inhibits immunoglobulin E (IgE)-dependent mast cell (MC) activation, but the effect of FNT on IgE-independent MC activation is yet unknown. Our aim was to investigate the effects and possible mechanisms of action of FNT on IgE-independent MC activation and pseudoallergic inflammation. We studied the effects of FNT on MC degranulation in vitro with a cell culture model using compound C48/80 to stimulate either mouse bone marrow-derived mast cells (BMMCs) or RBL-2H3 cells. We subsequently measured β-hexosaminase and histamine release, the expression of inflammatory factors, cell morphological changes, and changes in NF-κB signaling. We also studied the effects of FNT in several in vivo murine models of allergic reaction: C48/80-mediated passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and 2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD). The results showed that FNT inhibited IgE-independent degranulation of MCs, evaluated by a decrease in the release of β-hexosaminase and histamine and a decreased expression of inflammatory factors. Additionally, FNT reduced cytomorphological elongation and F-actin reorganization and attenuated NF-κB p65 phosphorylation and NF-κB-dependent promoter activity. Moreover, the administration of FNT alleviated pseudoallergic responses in vivo in mouse models of C48/80-stimulated PCA and ASA, and DNCB-induced AD. In conclusion, we suggest that FNT may be a novel anti-allergic drug with great potential to alleviate pseudoallergic responses via the inhibition of IgE-independent MC degranulation and NF-κB signaling. Full article

Ethyl caffeate (EC) is a natural phenolic compound that is present in several medicinal plants used to treat inflammatory disorders. However, its anti-inflammatory mechanisms are not fully understood. Here, we report that EC inhibits aryl hydrocarbon receptor (AhR) signaling and that this is associated with its anti-allergic activity. EC inhibited AhR activation, induced by the AhR ligands FICZ and DHNA in AhR signaling-reporter cells and mouse bone marrow-derived mast cells (BMMCs), as assessed by AhR target gene expressions such as CYP1A1. EC also inhibited the FICZ-induced downregulation of AhR expression and DHNA-induced IL-6 production in BMMCs. Furthermore, the pretreatment of mice with orally administered EC inhibited DHNA-induced CYP1A1 expression in the intestine. Notably, both EC and CH-223191, a well-established AhR antagonist, inhibited IgE-mediated degranulation in BMMCs grown in a cell culture medium containing significant amounts of AhR ligands. Furthermore, oral administration of EC or CH-223191 to mice inhibited the PCA reaction associated with the suppression of constitutive CYP1A1 expression within the skin. Collectively, EC inhibited AhR signaling and AhR-mediated potentiation of mast cell activation due to the intrinsic AhR activity in both the culture medium and normal mouse skin. Given the AhR control of inflammation, these findings suggest a novel mechanism for the anti-inflammatory activity of EC. Full article

Mast cells may contribute to osteoporosis development, because patients with age-related or post-menopausal osteoporosis exhibit more mast cells in the bone marrow, and mastocytosis patients frequently suffer from osteopenia. We previously showed that mast cells crucially regulated osteoclastogenesis and bone loss in ovariectomized, estrogen-depleted mice in a preclinical model for post-menopausal osteoporosis and found that granular mast cell mediators were responsible for these estrogen-dependent effects. However, the role of the key regulator of osteoclastogenesis, namely, receptor activator of NFκB ligand (RANKL), which is secreted by mast cells, in osteoporosis development has, to date, not been defined. Here, we investigated whether mast-cell-derived RANKL participates in ovariectomy (OVX)-induced bone loss by using female mice with a conditional Rankl deletion. We found that this deletion in mast cells did not influence physiological bone turnover and failed to protect against OVX-induced bone resorption in vivo, although we demonstrated that RANKL secretion was significantly reduced in estrogen-treated mast cell cultures. Furthermore, Rankl deletion in mast cells did not influence the immune phenotype in non-ovariectomized or ovariectomized mice. Therefore, other osteoclastogenic factors released by mast cells might be responsible for the onset of OVX-induced bone loss. Full article

Asthma, which is a chronic inflammatory disease of the airways, is usually caused by allergens in which various structures and immune cells are involved. Ephedra sinica, the most commonly used Chinese medicine, has significant clinical effects on asthma, but its components are complex and the mechanism of action has not been fully elucidated. Among its components, we identified an amide alkaloid (EB-A) and investigated its anti-asthmatic activity and the underlying mechanisms. In this study, we replicated an OVA-sensitized/challenged allergic asthma mouse model, and divided the mice into a model (OVA) group, positive drug (Y, 0.5 mg/kg/day) group, and EB-A treatment with low (Low, 10 mg/kg/day) and high dose (High, 20 mg/kg/day) groups. Asthma-related features were analyzed through the airway hyperresponsiveness (AHR), cough and wheeze indexes, allergen-specific IgE, prostaglandin D2 (PDG2), and lung histology in mice. The levels of apoptosis and reactive oxygen species (ROS) in the primary lung cells, cytokines in the serum and broncho-alveolar lavage fluid (BALF), and proteinase-activated receptor-2 (PAR2) pathway activation in the lung tissue were measured to evaluate the inflammatory injury and lung epithelial barrier damage in the mice. Dendritic cell (DC) maturation and mast cell (MC) activation were verified in vitro and in vivo. Furthermore, the effect of a PAR2 activation in lung epithelial cells on the maturation of DCs was evaluated by the co-culture system of (human bronchial epithelial cell lines) 16HBE and bone marrow-derived dendritic cells (BMDCs). The results showed that EB-A inhibited the typical asthmatic phenotypes, as well as lung injury and inflammation, MC activation and degranulation, and DC maturation in the OVA-sensitized/challenged BALB/c mice. In addition, EB-A inhibited the expression of PAR2 in the lung epithelial cells and significantly interfered with the maturation of DCs after inhibiting PAR2. Taken together, our study firstly demonstrated that EB-A could ameliorate OVA-induced allergic asthma by inhibiting MC activation and DC maturation, and the molecular mechanism of EB-A’s anti-asthmatic activity might be mediated by inhibiting PAR2. Our data provide a molecular justification for the use of EB-A in the treatment of allergic asthma. Full article

Bone-marrow-derived mast cells are matured from bone marrow cells in medium containing 20% fetal calf serum (FCS), interleukin (IL)-3 and stem-cell factor (SCF) and are used as in vitro models to study mast cells (MC) and their role in health and disease. In vivo, however, BM-derived hematopoietic stem cells account for only a fraction of MC; the majority of MC in vivo are and remain tissue resident. In this study we established a side-by-side culture with BMMC, fetal skin MC (FSMC) or fetal liver MC (FLMC) for comparative studies to identify the best surrogates for mature connective tissue MC (CTMC). All three MC types showed comparable morphology by histology and MC phenotype by flow cytometry. Heterogeneity was detected in the transcriptome with the most differentially expressed genes in FSMC compared to BMMC being Hdc and Tpsb2. Expression of ST2 was highly expressed in BMMC and FSMC and reduced in FLMC, diminishing their secretion of type 2 cytokines. Higher granule content, stronger response to FcεRI activation and significantly higher release of histamine from FSMC compared to FLMC and BMMC indicated differences in MC development in vitro dependent on the tissue of origin. Thus, tissues of origin imprint MC precursor cells to acquire distinct phenotypes and signatures despite identical culture conditions. Fetal-derived MC resemble mature CTMC, with FSMC being the most developed. Full article

In this study, we investigated the anti-allergic effects of 3,4-dihydroxybenzaldehyde (DHB) isolated from the marine red alga, Polysiphonia morrowii, in mouse bone-marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-sensitized mice. DHB inhibited IgE/bovine serum albumin (BSA)-induced BMCMCs degranulation by reducing the release of β-hexosaminidase without inducing cytotoxicity. Further, DHB dose-dependently decreased the IgE binding and high-affinity IgE receptor (FcεRI) expression and FcεRI-IgE binding on the surface of BMCMCs. Moreover, DHB suppressed the secretion and/or the expression of the allergic cytokines, interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF)-α, and the chemokine, thymus activation-regulated chemokine (TARC), by regulating the phosphorylation of IκBα and the translocation of cytoplasmic NF-κB into the nucleus. Furthermore, DHB attenuated the passive cutaneous anaphylactic (PCA) reaction reducing the exuded Evans blue amount in the mouse ear stimulated by IgE/BSA. These results suggest that DHB is a potential therapeutic candidate for the prevention and treatment of type I allergic disorders. Full article

Sargassum horneri (S. horneri), an edible brown alga, has been proposed as a functional food with an improvement effect on abnormal skin immune responses. The present study investigates the anti-allergic effect of an ethanol extract from S. horneri (SHE) on immunoglobulin E (IgE)/bovine serum albumin (BSA)-mediated activation in bone marrow-derived cultured-mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) reaction in mice. SHE markedly and dose-dependently suppressed the degranulation of BMCMCs by reducing the β-hexosaminidase and histamine release without cytotoxicity. In addition, SHE significantly decreased the FcεRI expression on the surface of BMCMCs and its IgE binding. Moreover, SHE reduced the mRNA expression and the production of allergic cytokines; interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-13; interferon (IFN)-γ and/or tumor necrosis factor (TNF)-α; and a chemokine, thymus and activation-regulated chemokine (TARC), by suppressing the activation of Src-family kinases and nuclear factor (NF)-κB signaling. In further study, the application of SHE reduced the PCA reaction in an IgE/BSA-induced type I allergic mice model. Taken together, we suggest that SHE has an anti-allergic effect in type I allergic responses. Full article

Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs. Full article