Passive Cutaneous Anaphylaxis Model Research Articles

Different allergen-induced asthma and anaphylaxis show distinctive IgE pathway dependence

BackgroundThe pathogenesis of allergic asthma is classically understood to involve allergens crosslinking Immunoglobulin E (IgE) bound to mast cells, leading to the release of histamine and other inflammatory mediators. ObjectiveThis study aims to explore whether different allergens have distinct IgE-dependent mechanisms in mouse models of both asthma and anaphylaxis. MethodsWild type (WT) and IgE knockout (IgE KO) mice were used to generate asthma mouse models. Lung inflammation was assessed by histological analysis and inflammatory cells in Bronchial Alveolar Lavage Fluids (BALF). Passive Cutaneous Anaphylaxis (PCA) was conducted to quantify IgE, and Toluidine Blue staining was used to visualize mast cell recruitment. ResultsAll allergen-treated mice exhibited significant airway inflammation. Compared to WT mice, HDM-treated IgE KO mice showed an attenuated inflammatory profile, particularly with reduced eosinophils. These mice had lower serum levels of HDM-specific IgE and IgG1 as well as reduced levels of Th2 cytokines interleukin 4 (IL-4) and IL-5 in BALF. CRE-treated IgE KO mice also displayed a reduced inflammation, with significant differences only in eosinophils. Interestingly, no significant differences in airway inflammation were observed between OVA-treated IgE KO mice and WT mice. Furthermore, both HDM and CRE-induced PCA model showed significantly less Evan's blue dye leakage and mast cell recruitment in the ears of IgE KO mice compared to WT mice, with no differences for OVA-induced PCA and mast cell recruitment. ConclusionThe results highlight the variability in IgE pathway dependence among different allergens, emphasizing the need for further research to delineate the underlying mechanisms.

Ethanol Extract of Ampelopsis brevipedunculata Rhizomes Suppresses IgE-Mediated Mast Cell Activation and Anaphylaxis.

More than 20% of the world's population suffers from allergic diseases, including allergic asthma, rhinitis, and atopic dermatitis that severely reduce the patient's quality of life. The treatment of allergy has been developed, but there are still unmet needs. Ampelopsis brevipedunculata (Maxim.) Trautv. is a traditional medicinal herb with beneficial bioactivities, such as antioxidant, anti-hypertension, anti-viral, anti-mutagenic, and skin and liver (anti-hepatotoxic) protective actions. However, its anti-allergic effect has not been addressed. This study designed to investigate the pharmacological effect of an ethanol extract of A. brevipedunculata rhizomes (ABE) on mast cell and anaphylaxis models. For in vivo studies, we used ovalbumin-induced active systemic anaphylaxis (ASA) and immunoglobulin (Ig) E-mediated passive cutaneous anaphylaxis (PCA) models. In ASA model, oral administration of ABE (1, 10, and 100 mg/kg) attenuated the anaphylactic responses, such as hypothermia, serum histamine, and IgE productions. In PCA model, ABE also suppressed the plasma extravasation and swelling. The underlying mechanisms of action were identified in various mast cell types. In vitro, ABE (10, 30, and 60 µg/mL) inhibited the release of essential allergic mediators, such as histamine and β-hexosaminidase, in a concentration-dependent manner. ABE prevented the rapid increase in intracellular calcium levels induced by the DNP-HSA challenge. In addition, ABE downregulated the tumor necrosis factor-α and interleukin-4 by suppressing the activation of nuclear factor-κB. Collectively, this study is the first to identify the anti-allergic effect of ABE, suggesting that ABE is a promising candidate for treating allergic diseases.

Design of an Ara h 2 hypoallergen from conformational epitopes.

Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapysuggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Epinastine Cream: A Novel Once-Daily Therapeutic Agent for Allergic Conjunctivitis.

Purpose: To investigate the in vivo efficacy of epinastine cream in type I allergic models. Methods: The dose, timing, and antiallergic effect of epinastine cream on the conjunctiva were evaluated postapplication to the eyelid skin of guinea pigs with histamine- or ovalbumin-induced allergic conjunctivitis. Additionally, we assessed its antiallergic effects on the skin postapplication to the dorsal skin of guinea pigs with ovalbumin-induced passive cutaneous anaphylaxis. Efficacy was estimated by determining the amount of dye that leaked from conjunctival or dorsal skin tissue vessels as a measure of vascular permeability, scoring the severity of allergic symptoms, and observing the scratching behaviors using clinical parameters. Results: In the histamine-induced conjunctivitis model, epinastine cream strongly inhibited conjunctival vascular permeability in a dose-dependent manner. The inhibitory effect of 0.5% epinastine cream 24 h postapplication was significantly higher than that of 0.1% epinastine hydrochloride ophthalmic solution 8 h postadministration. Additionally, the 0.5% epinastine cream inhibited conjunctival vascular permeability 15 min postapplication, and the effect was sustained over 24 h. Furthermore, the 0.5% epinastine cream effectively suppressed clinical symptom scores and exhibited ameliorated scratching bouts in conjunctival allergic reactions in the experimental allergic conjunctivitis model. Additionally, it significantly inhibited vascular permeability in skin allergic reactions in the passive cutaneous anaphylaxis model. Conclusions: The results suggest that epinastine cream is a strong, long-lasting, and skin-penetrating inhibitor of type I allergic reactions. The 0.5% epinastine cream applied once daily could be a promising, potent, and long-acting therapeutic agent for allergic conjunctivitis.

Effects and potential mechanisms of Saposhnikovia divaricata (Turcz.) Schischk. On type I allergy and pseudoallergic reactions in vitro and in vivo

Ethnopharmacological relevanceThe incidence of allergic disease is constantly increasing, but its pathogenesis is not fully understood. Saposhnikovia divaricata (SD), called ‘Fangfeng’ in China, not only can be used for antipyretic, analgesic and anti-inflammatory as a traditional Chinese medicine, but also as an active ingredient in about 8% prescriptions. However, its effects on type I allergy and pseudoallergy have not been clarified. Aim of the studyTo explore the treatment and potential mechanisms of SD and its major bioactive component Prim-O-glucosylcimifugin (POG) on type I allergy and pseudoallergy in vitro and in vivo. Materials and methodsThe inhibitory effect of SD decoction and POG on type I allergy and its possible mechanism were evaluated by using RBL-2H3 cells model in vitro and the passive cutaneous anaphylaxis (PCA) mouse model in vivo. The cell degranulation of RBL-2H3 cells induced by DNP-IgE/DNP-BSA and Compound 48/80 (C48/80) was investigated, and the molecules of degranulation related signaling pathway was further detected by qRT-PCR and Western Blot analysis. Meanwhile, therapeutic effect of SD Decoction and POG were evaluated using PCA models in vivo. The molecular docking technology was conducted to explore the potential mechanisms. ResultsIn cells model induced by DNP-IgE/DNP-BSA, the release rate of β-Hex in high dose of SD and POG groups were 43.79% and 57.01%, and the release amount of HA in high dose of SD and POG groups were 26.19 ng/mL and 24.20 ng/mL. They were significantly lower than that in the model group. Besides, SD decoction and POG could significantly inhibit intracellular Ca2+ increasing and cell apoptosis. But there is no obvious effect on cells degranulation induced by C48/80. The molecular docking results showed that 5-O-Methylvisamioside and POG could bind with FcεRI α with stronger binding ability, but weak binding ability to Mrgprx2. Moreover, qPCR and Western blot analyses indicated that SD could down-regulate Lyn/Syk/PLCγ, MAPK and PI3K/AKT/NF-κB signal pathway to inhibit IgE-dependent cell degranulation. In mice PCA model, both SD and POG could dose-dependently attenuate the Evans Blue extravasation, paw and ear swelling induced by DNP-IgE/DNP-BSA, but no significant inhibition under the PCA models induced by C48/80. ConclusionIn conclusion, SD is effective for the therapeutic of type I allergies, suggesting that SD is a potential candidate for the treatment of type I allergy, and the underlying mechanism of these effects needs to be further studied.

Silibinin attenuated pseudo-allergic reactions and mast cell degranulation via PLCγ and PI3K/Akt signaling pathway.

Anaphylaxis is a type of potentially fatal hypersensitivity reaction resulting from the activation of mast cells. Many endogenous or exogenous factors could cause this reaction. Silibinin is the main chemical component of silymarin and has been reported to have pharmacological activities. However, the anti-allergic reaction effect of silibinin has not yet been investigated. This study aimed to evaluate the effect of silibinin to attenuate pseudo-allergic reactions in vivo and to investigate the underlying mechanism in vitro. In this study, calcium imaging was used to assess Ca2+ mobilization. The levels of cytokines and chemokines, released by stimulated mast cells, were measured using enzyme immunoassay kits. The activity of silibinin was evaluated in a mouse model of passive cutaneous anaphylaxis (PCA). Western blotting was used to explore the related molecular signaling pathways. In results, silibinin markedly inhibited mast cell degranulation, calcium mobilization, and preventing the release of cytokines and chemokines in a dose-dependent manner via the PLCγ and PI3K/Akt signaling pathway. Silibinin also attenuated PCA in a dose-dependent manner. In summary, silibinin has an anti-pseudo-allergic pharmacological activity, which makes it a potential candidate for the development of a novel agent to arrest pseudo-allergic reactions.

Effects of Lactobacillus fermentation on Eucheuma spinosum polysaccharides: Characterization and mast cell membrane stabilizing activity

Eucheuma polysaccharides have varieties of biological activities. However, it is accompanied by problems like large molecular weight, high viscosity, and low utilization. Here, we first prepared fermented Eucheuma spinosum polysaccharides (F-ESP) by Lactobacillus fermentation, compared with low-temperature freeze-thaw ESP (L-ESP) prepared by the freeze-thaw method, explored the composition and structural characteristics of F-ESP and L-ESP, and evaluation of the ability of different samples to inhibit mast cell degranulation using classical mast cell model. Then, the activity of L-ESP and F-ESP in vivo was preliminarily evaluated using a passive cutaneous anaphylaxis model. Two kinds of F-ESP named F1-ESP-3 and F2-ESP-3 were obtained by fermentation of Eucheuma spinosum with the selected strains of Lactobacillus.sakei subsp.sakei and Lactobacillus.rhamnosus. Compared with the purified component L-ESP-3, the monosaccharide composition of F1-ESP-3 contains more glucuronic acid, the molecular weight reduced from >600 kDa (L-ESP-3) to 28.30 kDa (F1-ESP-3) and 33.58 kDa (F2-ESP-3), F1-ESP-3 has higher solubility and lower apparent viscosity. Fermentation did not destroy the functional groups and structure of ESP. Moreover, F1-ESP-3 significantly inhibited RBL-2H3 cell degranulation by reducing depolymerization of F-actin and Ca2+ influx. F1-ESP-3 reduced the symptoms of mast cell-mediated passive cutaneous anaphylaxis, indicating that F1-ESP-3 may have better anti-allergic activity in vivo.

Efficacy of THB001, a Potent and Selective Oral Small Molecule Inhibitor of Wild Type KIT Receptor Tyrosine Kinase, in a Rat Passive Cutaneous Anaphylaxis Model

Mast cells play a central role in the pathophysiology of allergic inflammation. Mast cell activation, migration, proliferation and survival are dependent on KIT (CD117) signaling. THB001 is a potent and selective inhibitor of wild type KIT being studied clinically as a therapy for mast cell driven diseases. The current study correlated target engagement, mast cell depletion and efficacy in an in vivo model of anaphylaxis.

Nano-silica particles synergistically IgE-mediated mast cell activation exacerbating allergic inflammation in mice.

BackgroundAllergic respiratory diseases have increased dramatically due to air pollution over the past few decades. However, studies are limited on the effects of inorganic components and particulate matter with different particle sizes in smog on allergic diseases, and the possible molecular mechanism of inducing allergies has not been thoroughly studied.MethodsFour common mineral elements with different particle sizes in smog particles were selected, including Al2O3, TiO2, Fe2O3, and SiO2. We studied the relationship and molecular mechanism of smog particle composition, particle size, and allergic reactions using mast cells, immunoglobulin E (IgE)-mediated passive cutaneous anaphylaxis (PCA) model, and an ovalbumin (OVA)-induced asthmatic mouse model in vitro and in vivo, combined with transmission electron microscopy, scanning transmission X-ray microscopy analysis, and transcriptome sequencing.ResultsOnly 20 nm SiO2 particles significantly increased β-hexosaminidase release, based on dinitrophenol (DNP)-human serum albumin (HSA) stimulation, from IgE-sensitized mast cells, while other particles did not. Meanwhile, the PCA model showed that Evan’s blue extravasation in mice was increased after treatment with nano-SiO2 particles. Nano-SiO2 particles exposure in the asthmatic mouse model caused an enhancement of allergic airway inflammation as manifested by OVA-specific serum IgE, airway hyperresponsiveness, lung inflammation injury, mucous cell metaplasia, cytokine expression, mast cell activation, and histamine secretion, which were significantly increased. Nano-SiO2 particles exposure did not affect the expression of FcϵRI or the ability of mast cells to bind IgE but synergistically activated mast cells by enhancing the mitogen-activated protein kinase (MAPK) signaling pathway, especially the phosphorylation levels of the extracellular signal-regulated kinase (ERK)1/2. The ERK inhibitors showed a significant inhibitory effect in reducing β-hexosaminidase release.ConclusionOur results indicated that nano-SiO2 particles stimulation might synergistically activate IgE-sensitized mast cells by enhancing the MAPK signaling pathway and that nano-SiO2 particles exposure could exacerbate allergic inflammation. Our experimental results provide useful information for preventing and treating allergic diseases.

Efficacy of Anti-Allergen Therapeutics in Preclinical Allergy Model Systems Correlate with Symptomatic Relief in a Clinical Setting

The passive cutaneous anaphylaxis (PCA) mouse model measures local mast cell activation-induced vascular permeability and has been used to study IgE-mediated hypersensitivity. The PCA model was utilized to preclinically select allergen-specific monoclonal antibody (mAb) cocktails subsequently shown to provide symptomatic relief in individuals with birch or cat allergy as measured by significant reductions in total nasal symptom score (TNSS) following nasal allergen challenge (NAC) and reduction in allergen-specific skin prick tests (SPT) relative to baseline.

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα^−/−) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα^−/− MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.

The influence of acetylsalicylic acid and alcohol on absorption kinetics of hen´s egg white in a human passive cutaneous anaphylaxis model.

BackgroundDespite the well-known fact that acetylsalicylic acid (ASA) can induce anaphylaxis in patients susceptible to wheat-dependent exercise-induced anaphylaxis, few studies have sought to investigate the effects of cofactors on type-1 food allergy and none with ASA and hen’s egg and hen’s egg and alcohol combined.Methods and resultsWe applied the experimental model of ‘passive cutaneous anaphylaxis’ in humans to study whether the absorption kinetics of egg white is altered while being treated with ASA or under the influence of alcohol. Donor sera from four egg allergic patients with specific immunoglobulin E (s-IgE) to ovalbumin (0.1–8.87–19.5–170 kUA/L) were injected intracutaneously into the forearm of 12 healthy volunteers who were then challenged separately to: 1) egg white 2) egg white + ASA and 3) egg white + alcohol. ‘Time to wheal’ and ‘wheal size’ were compared among the three experiments.We saw that ‘time to wheal’ with both ASA (P = 0.001) and alcohol (P = 0.019) added as cofactor significantly decreased compared with baseline.ConclusionIn this passive cutaneous anaphylaxis model, ASA and alcohol affected both reaction time and size of reactions elicited after egg ingestion. This suggests that patients with egg allergy could have faster and more severe reactions during ASA treatment or under alcohol influence.

FAK inhibitor PF-431396 suppresses IgE-mediated mast cell activation and allergic inflammation in mice

Mast cells (MCs) initiate and maintain allergic inflammation. Upon being stimulated with immunoglobulin (Ig)E and antigen (Ag), MCs exhibit FcεRI (high-affinity IgE) receptor-mediated degranulation, cytokine secretion, and increased focal adhesion kinase (FAK) activity. The aims of this study were to examine mechanisms of FAK regulation in IgE-mediated MC activation and the effects of FAK inhibition on MC-mediated allergic responses. FAK activity was manipulated with short hairpin RNA (shRNA) knockdown, FAK overexpression, and the FAK inhibitor PF-431396 (PF). Gene expression and kinase activation were analyzed with quantitative molecular biology assays. PF effects were tested in the passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and allergic conjunctivitis (AC) mouse models. Our results showed that FAK overexpression increased IgE-mediated degranulation and reduced the dexamethasone inhibitory effect on MCs activation. The FAK inhibitor PF diminished MC release of β-hexosaminidase (β-hex), histamine, and inflammatory cytokines, via a mechanism that involves MAPK and NF-κB signaling pathways. CaMKII was identified as a robust FAK-associating protein. Inhibition of CaMKII activation by KN-93 suppressed FAK activity and its downstream pathway. PF attenuated inflammatory responses in our PCA and ASA models, and relieved signs of allergic disease in AC model mice. In conclusions, MC degranulation and production of inflammatory mediators in allergic disease may be consequent to FcεRI crosslinking inducing CaMKII-mediated activation of FAK activity. FAK inhibition may represent a new MC-suppressing treatment strategy for the treatment of allergic diseases.

EGR3-HDAC6-IL-27 Axis Mediates Allergic Inflammation and Is Necessary for Tumorigenic Potential of Cancer Cells Enhanced by Allergic Inflammation-Promoted Cellular Interactions.

The objective of this study was to investigate mechanisms of allergic inflammation both in vitro and in vivo in details. For this, RNA sequencing was performed. Early growth response 3 gene (Egr3) was one of the most highly upregulated genes in rat basophilic leukemia (RBL2H3) cells stimulated by antigen. The role of Egr3 in allergic inflammation has not been studied extensively. Egr3 was necessary for passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Egr3 promoter sequences contained potential binding site for NF-κB p65. NF-κB p65 directly regulated Egr3 expression and mediated allergic inflammation in vitro. Histone deacetylases (HDACs) is known to be involved in allergic airway inflammation. HDAC6 promoter sequences contained potential binding site for EGR3. EGR3 showed binding to promoter sequences of HDAC6. EGR3 was necessary for increased expression of histone deacetylase 6 (HDAC6) in antigen-stimulated RBL2H3 cells. HDAC6 mediated allergic inflammation in vitro and PSA. TargetScan analysis predicted that miR-182-5p was a negative regulator of EGR3. Luciferase activity assay confirmed that miR-182-5p was a direct regulator of EGR3. MiR-182-5p mimic inhibited allergic inflammation both in vitro and in vivo. Cytokine array showed that HDAC6 was necessary for increased interleukin-27 (IL-27) expression in BALB/C mouse model of PSA. Antigen stimulation did not affect expression of EBI3, another subunit of IL-27 in RBL2H3 cells or BALB/C mouse model of PCA or PSA. IL-27 receptor alpha was shown to be able to bind to HDAC6. IL-27 p28 mediated allergic inflammation in vitro, PCA, and PSA. Mouse recombinant IL-27 protein promoted features of allergic inflammation in an antigen-independent manner. HDAC6 was necessary for tumorigenic and metastatic potential enhanced by PSA. PSA enhanced the metastatic potential of mouse melanoma B16F1 cells in an IL-27-dependent manner. Experiments employing culture medium and mouse recombinant IL-27 protein showed that IL-27 mediated and promoted cellular interactions involving B16F1 cells, lung macrophages, and mast cells during allergic inflammation. IL-27 was present in exosomes of antigen-stimulated RBL2H3 cells. Exosomes from antigen-stimulated RBL2H3 cells enhanced invasion of B16F1 melanoma cells in an IL-27-dependemt manner. These results present evidence that EGR3-HDAC6-IL-27 axis can regulate allergic inflammation by mediating cellular interactions.

Inhibition of c-Fos expression attenuates IgE-mediated mast cell activation and allergic inflammation by counteracting an inhibitory AP1/Egr1/IL-4 axis

BackgroundActivator protein-1 (AP1), a c-Fos–JUN transcription factor complex, mediates many cytobiological processes. c-Fos has been implicated in immunoglobulin (Ig)E activation of mast cells (MCs) via high-affinity IgE Fc receptor (FcεRI) binding. This study examined c-Fos involvement in MC activation and tested the effects of the c-Fos/AP1 inhibitor T-5224 on MCs activation and allergic responses.MethodsIn vitro studies were conducted with two MC model systems: rat basophilic leukemia cells (RBLs) and mouse bone marrow derived mast cells (BMMCs). MC degranulation and effector functions were examined with β-hexosaminidase release and cytokine secretion assays. c-Fos/AP1 was inhibited with T-5224. c-Fos activity was suppressed with short hairpin RNA targeting c-Fos (shFos). In vivo immune responses were evaluated in passive cutaneous anaphylaxis (PCA) and ovalbumin-induced active systemic anaphylaxis (ASA) models, as well as in an oxazolone (OXA)-induced model of atopic dermatitis, a common allergic disease.Resultsc-Fos expression was elevated transcriptionally and translationally in IgE-stimulated MCs. c-Fos binding of the Egr1 (early growth response 1) promoter upregulated Egr1 transcription, leading to production of interleukin (IL)4. T-5224 reduced FcεRI-mediated MC degranulation (evidenced by β-hexosaminidase activity and histamine levels) and diminished EGR1 and IL4 expression. T-5224 attenuated IgE-mediated allergic responses in PCA and ASA models, and it suppressed MC-mediated atopic dermatitis in mice.ConclusionIgE binding can activate MCs via a c-Fos/Egr1/IL-4 axis. T-5224 suppresses MC activation in vitro and in vivo and thus represents a promising potential strategy for targeting MC activation to treat allergic diseases.

GlcNAc is a mast-cell chromatin-remodeling oncometabolite that promotes systemic mastocytosis aggressiveness

AbstractSystemic mastocytosis (SM) is a KIT-driven hematopoietic neoplasm characterized by the excessive accumulation of neoplastic mast cells (MCs) in various organs and, mainly, the bone marrow (BM). Multiple genetic and epigenetic mechanisms contribute to the onset and severity of SM. However, little is known to date about the metabolic underpinnings underlying SM aggressiveness, which has thus far impeded the development of strategies to leverage metabolic dependencies when existing KIT-targeted treatments fail. Here, we show that plasma metabolomic profiles were able to discriminate indolent from advanced forms of the disease. We identified N-acetyl-d-glucosamine (GlcNAc) as the most predictive metabolite of SM severity. High plasma levels of GlcNAc in patients with advanced SM correlated with the activation of the GlcNAc-fed hexosamine biosynthesis pathway in patients BM aspirates and purified BM MCs. At the functional level, GlcNAc enhanced human neoplastic MCs proliferation and promoted rapid health deterioration in a humanized mouse model of SM. In addition, in the presence of GlcNAc, immunoglobulin E-stimulated MCs triggered enhanced release of proinflammatory cytokines and a stronger acute response in a mouse model of passive cutaneous anaphylaxis. Mechanistically, elevated GlcNAc levels promoted the transcriptional accessibility of chromatin regions that contain genes encoding mediators of receptor tyrosine kinases cascades and inflammatory responses, thus leading to a more aggressive phenotype. Therefore, GlcNAc is an oncometabolite driver of SM aggressiveness. This study suggests the therapeutic potential for targeting metabolic pathways in MC-related diseases to manipulate MCs effector functions.

Neurokinin A blocks the ABCA1 channel to promote IL-10-dependent mast cell regulation

Abstract Cutaneous mast cells (MCs) reside in close proximity to peripheral sensory nerve fibers and are susceptible to regulation by neuropeptides. The tachykinin family member, Neurokinin A (NKA) binds with highest affinity to the Neurokinin 2 Receptor, expressed on MCs. MCs are poised to respond to NKA, but its role in the MC biology is, to our knowledge, under-explored. The objective of this study was to evaluate the effects of NKA on bone marrow (BM) MCs and peritoneal (P)MCs in vitro and in the murine model of passive cutaneous anaphylaxis (PCA) in vivo. Neurokinin A inhibited FceRI-initiated phosphorylation and nuclear localization of STAT5, and release of TNF and IL-13 in an IL-10-dependent manner. NKA affected the MC secretome, abrogating ABCA1-dependent release of the cysteine protease, calpain. Extracellular calpain, in turn, degraded IL-10. In vivo, NKA administration reduced PCA, inhibiting edema, and induction of inflammatory cytokines and in a manner that relied on MC derived-IL-10. Likewise, direct inhibition of the ABCA1 phenocopied the effects of NKA, reducing IgE-initiated MC responses in vitro and in vivo. In conclusion, NKA minimizes IgE-initiated inflammation though an IL-10-dependent mechanism and inhibits ABCA1-dependent release of the IL-10 degrading enzyme, calpain. This study illuminates a novel tier of neuropeptide mediated MC regulation through the ABCA1. Strategies interfering with the ABCA1 may be promising therapeutics for targeting MCs in cutaneous inflammatory diseases.

Immunological validation of the pharmacological and anti‐allergic action of glycyrrhetinic acid from Glycyrrhizae Radix

ABSTRACTAimGlycyrrhetinic acid (GA) is an important and active metabolite derived from glycyrrhizin (GL) in Glycyrrhizae Radix (GR) that induces anti‐allergic, anti‐inflammatory, anti‐oxidant, anti‐viral, and anti‐microbial effects in vivo. To validate these beneficial actions of GA as an active compound of GR in vivo, we prepared a monoclonal antibody against GA.MethodsThe monoclonal antibody, mAb6D6, was prepared and evaluated for cross‐reactivity with GA and its analogs using competitive enzyme‐linked immunosorbent assay. Subsequently, mAb6D6 was used to validate the anti‐allergic action of GA, which was orally administered as GL or hot water extracts of GR, in the IgE‐mediated passive cutaneous anaphylaxis model for immediate hypersensitivity and contact hypersensitivity model.ResultsMAb6D6 specifically identified GA and had no reactivity against other oleanane‐type compounds and endogenous steroids. This antibody could neutralise the anti‐allergic action of GA in both models in which mice were orally treated with GL or extracts of GR after intravenous injection of the antibody.ConclusionMAb6D6 neutralised the activity of GA in vivo. Therefore, it is essential to analyse the role of GA from GR in Kampo medicine and elucidate its mechanism of action.

Fine particulate matter (PM2.5) promotes IgE-mediated mast cell activation through ROS/Gadd45b/JNK axis

BackgroundMast cells play an important role in allergic responses and persistently exposure to environmental fine particulate matter (PM2.5) exacerbates allergic diseases,but the details remained elucidative. ObjectivesTo investigate the effect of PM2.5 on IgE-mediated mast cell responses through an IgE-mediated mouse model and mast cell activation. MethodsThe β-hexosaminidase release and a BALB/c model of passive cutaneous anaphylaxis (PCA) was used to test IgE-mediated mast cells activation in vitro and in vivo. RNA-Seq technique was conducted to study the gene expression profile. Reactive oxygen species (ROS) production was measured by flow-cytometry. RT-PCR,WB and ELISA were performed to examine targeting molecules expression. ResultsPM2.5 facilitated IgE-mediated degranulation and increased cytokines expression in mast cells. Meanwhile, the Evan’s blue extravasation as well as serum cytokines in mice was increased after treatment with PM2.5. Furthermore, PM2.5 treatment dramatically increased the expression of Gadd45b which is an oxidative stress molecule that directly activates down-stream pathway, such as MEKK4/JNK. PM2.5 treatment activated MEKK4, JNK1/2 but not ERK1/2 and p38. Meanwhile, Knockdown of Gadd45b significantly attenuated PM2.5-mediated JNK1/2 activation and expression of cytokines. In addition, a JNK1/2-specific inhibitor SP600125 blocked IgE-mediated mast cell activation and cytokine release in PCA model mice. Moreover, PM2.5 treatment increased the ROS level and ROS inhibitor dramatically blocked the PM2.5-induced ROS production and reversed the PM2.5-mediated gene expression in the mitochondrial respiratory chain. ConclusionsPM2.5 regulates ROS production through Gadd45b/MEKK4/JNK pathway, facilitating IgE-mediated mast cell activation.

Anti-type I allergic effects of Jing-Fang powder extracts via PI3K/Akt pathway in vitro and in vivo

Jing-Fang powder (Schizonepeta tenuifolia Briq. and Saposhnikovia divaricata (Turcz.) Schischk.) was used to treat chronic bronchitis, asthma and chronic urticaria. Based on the preliminary results of screening research on the antiallergic effective parts of Jing-Fang powder, its ethyl acetate extract fractions (JFEE) and isolate D (JFEE-D) showed the best anti-allergic effect. RBL-2H3 cell activation degranulation model and mice passive cutaneous anaphylaxis (PCA) reaction model were used to investigate the effects and mechanisms of JFEE and JFEE-D on IgE-mediated type I allergic reactions. LC–MS was utilized to determine the composition of JFEE and JFEE-D. We found that JFEE and JFEE-D significantly reduced β-HEX, histamine, IL-4, IL-6 levels in cell supernatants, and improved the degree and morphology of cell degranulation. JFEE and JFEE-D significantly inhibited the increase of ear vascular permeability and abnormal increase of serum IgE, TNF-α, IL-6 levels. JFEE and JFEE-D inhibited mRNA expression of PI3K and Akt and down-regulated protein expression of PI3K, Akt, p-Akt, and PLCγ1 in sensitized RBL-2H3 cells. The combined use of JFEE and JFEE-D with pathway inhibitor Wortmannin revealed synergistic down-regulation of PI3K, Akt, and p-Akt protein expression. The combined use of pathway agonist IGF-1, JFEE and JFEE-D down-regulated increase of p-Akt/Akt protein expression. Moreover, JFEE and JFEE-D significantly inhibited protein expression of PI3K, p-Akt and PLCγ1 in PCA model mice. These results show that JFEE and JFEE-D inhibit type I allergic reactions by inhibiting PI3K/Akt signaling pathway.