Stimulation Of Mast Cells Research Articles

286 Phase 2 proof-of-concept trial in progress—lirentelimab in adults with moderate-to-severe atopic dermatitis inadequately controlled by topical treatments

Abstract Atopic dermatitis (AD) is a chronic pruritic inflammatory dermatitis that affects approximately 16.5 million (7.3%) adults in the US, of which around 6.6 million (40%) have moderate-to-severe disease. The current standard of care includes topical treatments supplemented with corticosteroids with and without immunosuppressants. Patients with AD inadequately controlled by topical treatments and immunosuppressants are often treated with biologics and/or JAK inhibitors. Some patients do not have an adequate clinical response or are unable to tolerate these treatments prompting the need for novel therapies. Mast cells (MCs) and eosinophils (eos) are implicated in the pathogenesis of AD. Sialic acid-binding Ig-like lectin (Siglec)-8 is expressed selectively on MCs and eos; engagement with an antibody results in broad inhibition of MC activation and eos depletion. Lirentelimab (AK002) is a humanized, nonfucosylated IgG1 monoclonal antibody that binds specifically to Siglec-8. Preclinical data demonstrates that lirentelimab can broadly inhibit multiple modes of mast cell stimulation that drive AD pathogenesis. Lirentelimab, administered every 4 weeks as an infusion (IV), has been tested in over 600 healthy volunteers and patients with inflammatory and allergic diseases; in those with concomitant AD, improvements in AD were observed. Overall, lirentelimab IV has been well-tolerated; the most common AE being infusion-related reactions (IRRs) typically associated with the initial infusions. In a phase 1 study, a subcutaneous (SC) formulation of lirentelimab was well-tolerated with no IRRs. The results of these studies provide a strong rationale for conducting this phase 2 proof-of-concept, randomized, double-blind, placebo-controlled study of lirentelimab SC in adults with moderate-to-severe atopic dermatitis inadequately controlled by topical treatments (NCT05155085, ‘ATLAS’). Adults (18–80 years) are eligible for screening if they have had chronic AD presented for ≥ 3 years with moderate-to-severe symptoms defined as Eczema Area and Severity Index (EASI) score ≥ 16, involvement of ≥ 10% of the Body Surface Area (BSA) and Investigator Global Assessment (IGA) score ≥ 3, documented recent history of inadequate response to treatment with medications such as topical corticosteroids, calcineurin inhibitors, JAK inhibitors, or PDE4 inhibitors (crisaborole) and biologic-naïve or biologic-exposed (secondary loss of response, intolerance or lack of access). The complete list of inclusion/exclusion criteria will be presented. 130 patients will be randomized 1 : 1 to receive either seven SC injections of 300 mg lirentelimab or a placebo every 2 weeks and then followed for 12 weeks after the last dose. The primary endpoint is the proportion of patients who achieve EASI-75 at week 14 (2 weeks after the last dose) compared with baseline. All patients who receive study medication will be included in the safety analysis. Secondary endpoints include a percent change in EASI at week 14 from baseline and a proportion of patients with IGA of 0 or 1 and a 2-point improvement at week 14 compared to baseline. Patients who complete the study (day 99 visit of double-blind period) will be given the option to enroll in an open-label extension (OLE) for seven doses of 300 mg lirentelimab SC for 12 weeks. Qualified participants will receive medication, lab tests and medical care related to the study at no cost. This study is currently enrolling in the USA and Germany. Please visit clinicaltrials.gov (NCT05155085) or email atlas.info@allakos.com to learn more about this study.

Evaluation of You-Gui-Wan critical compounds inhibiting ALOX-5 and HDC gene expression in RBL-2H3 cells using a fractional factorial design

Ethnopharmacological relevanceThe traditional Chinese medicine (TCM) You-Gui-Wan (YGW) has been used to treat asthma for hundreds of years. Aim of the studyYGW is composed of 10 types of medicinal materials. However, the immune mechanism of YGW in asthma treatment has not been elucidated. Therefore, this study investigated asthma symptoms attenuated by YGW and the underlying immune regulatory mechanism. Materials and methodsIntratracheal (i.t.) stimulation of BALB/c mice with Dermatophagoides pteronyssinus (Der p) was performed once per week (40 μL, 2.5 μg/μL). For six consecutive weeks, different doses of YGW (0.2 g/kg and 0.5 g/kg) were orally administered 30 min before stimulation with Der p. After the last stimulation, airway hyperreactivity, lung gene expression, and total immunoglobulin E (IgE) in blood were evaluated using a whole-body plethysmograph system, real-time PCR, and ELISA, respectively. In addition, DNP-IgE/DNP-BSA was added to stimulate mast cells (RBL-2H3), and YGW or various compound compositions (Trial) were added to RBL-2H3 cells for 30 min to evaluate the effects of the drug on mast cell degranulation and on gene expression. JMP 5.1 software was used to design and analyze YGW's critical compounds by which it inhibited ALOX-5 and HDC gene expression in RBL-2H3 cells. ResultsYGW significantly decreased serum total IgE levels and airway hyperresponsiveness in asthmatic mice. YGW also reduced the gene expression of IL-6, TNF-α, IL-4, IL-13, and COX-2 in the lungs of asthmatic mice and RBL-2H3 cells. YGW and the compound (Trial 21) present in YGW inhibited the gene expression of ALOX-5 and HDC in RBL-2H3 cells. ConclusionThe experimental results indicate that YGW exhibits anti-airway hyperresponsiveness and specific immunomodulatory effects. In addition, YGW synergistically inhibits ALOX-5 and HDC gene expression in mast cells through a combination of 21 compounds, including luteolin, quercetin, and β-carotene.

PI3K/AKT-mediated autophagy inhibition facilitates mast cell activation to enhance severe inflammatory lung injury in influenza A virus- and secondary Staphylococcus aureus-infected mice

Influenza A virus infection causes considerable morbidity and mortality each year globally, and secondary bacterial infection further exacerbates the severity and fatality of the initial viral infection. Mast cells have substantial roles in protecting the respiratory tract mucosa, while their role in viral and bacterial co-infection remains unclear. The present study revealed that secondary Staphylococcus aureus infection significantly aggravated the activation of mast cells during the initial H1N1 infection both in vivo and in vitro, which was closely related to the increased inflammatory lung injury and mortality. Meanwhile, the secondary S. aureus infection suppressed autophagy and promoted inflammatory mediators released by mast cells through activating the PI3K/Akt signaling pathway. Blocking PI3K/Akt pathway by LY294002, an inhibitor of Akt phosphorylation, could rescue autophagy and inhibit the release of inflammatory mediators. Furthermore, based on the influenza A viral and secondary bacterial infected mice model, we showed that the combination of LY294002 and antiviral drug oseltamivir could effectively reduce the inflammatory damage and pro-inflammatory cytokines releasing in lungs, recovering body weight loss and improving the survival rate from the co-infections. In conclusion, secondary bacterial infection can inhibit autophagy and stimulate mast cell activation through the PI3K/Akt pathway, which might explain why secondary bacterial infection would cause severe and fatal consequences following an initial influenza A viral infection.

SARS-CoV-2 vaccine excipients polyethylene glycol and trometamol do not induce mast cell degranulation, in an in vitro model for non-IgE-mediated hypersensitivity.

The development of vaccines against SARS-CoV2 brought about several challenges, including the management of hypersensitivity reactions to these formulations. The search for underlying mechanisms involved in these adverse events initially focused on excipients which may trigger mast cell activation responses via non-IgE pathways: polyethylene glycol and trometamol. We sought to determine whether these components, in their pure form, were capable of stimulating mast cells directly. To test this hypothesis, we used an in vitro model for non-IgE-mediated activation that has previously shown degranulation responses induced via MRGPRX2 with known drug agonists of the receptor. Human LAD2 mast cells were incubated with different concentrations (1, 10, 50 mg/ml) of trometamol and of purified polyethylene glycol/Macrogol (molecular weights: 2,000, 3,350, 4,000, and 6,000). Mast cell degranulation was assessed using a beta-hexosaminidase read-out. Interestingly, degranulation responses for all reagents tested showed no significant differences from those obtained from the negative control (basal degranulation). Receptor-silencing assays were therefore not conducted. In summary, purified PEG and trometamol did not induce mast cell degranulation in this in vitro model for the study of non-IgE mechanisms of drug hypersensitivity, previously shown to be useful in the investigation of MRGPRX2 ligands. Studies using complete vaccine formulations, lipid conjugates, and receptor gene variants are needed to further clarify mechanisms of vaccine hypersensitivity.

Menthone supplementation protects from allergic inflammation in the lungs of asthmatic mice

To screen potent terpenoid compounds against allergic inflammation in vitro and in vivo, five terpenoid compounds including menthone, farnesol, oridonin, β-escin and lupeol, were first selected to compare their anti-allergic inflammation potential using mouse lung mast cells in vitro. Among five selected terpenoid compounds, just menthone treatment decreased TNF-α/IL-10 secretion ratios in lipopolysaccharide -stimulated mast cells in vitro. As a result, menthone was further chosen to treat ovalbumin (OVA)-sensitized and challenged BALB/c mice by gavage for 5 weeks. There were six groups including dietary control (DC group, 0 mg menthone/kg b.w./day), 8 (ML group), 40 (MM group) as well as 200 mg menthone/kg b.w./day (MH group) by gavage, positive control (PC group, 3 mg dexamethasone/kg b.w. by gavage before OVA challenge) and non-treatment control (NTC group, normal mice without treatment) in the experiment. Changes of inflammatory mediators, cell distribution, Th1/Th2 and pro-/anti-inflammatory cytokines secretion as well as relative gene expression amounts of six receptors related to allergic inflammation in the lungs and airways were measured. The results showed that middle menthone supplementation (40 mg menthone/kg b.w./day) in vivo decreased protein and eotaxin, but increased Th1 cytokine levels in the bronchoalveolar lavage fluid. Menthone supplementation inhibited eosinophilia, mast cell degranulation, chemokine (C–C motif) receptor 3 (CC receptor 3) and chemokine (C-X-C motif) receptor 1 (CXC receptor 1) gene expression amounts in the lungs, but restored the percentage of monocytes/macrophages. Our results suggest that menthone supplementation may alleviate allergic asthma through regulating airway allergic inflammation, protein overproduction, eosinophils infiltration, Th1/Th2 immune balance, CC receptor 3 and CXC receptor 1 gene expression amounts in the lungs but restoring the percentage of monocytes/macrophages in allergic asthmatic mice.

LB1030 Automated image analysis methods to detect mast cells in skin

Mast cells are a key cell in the innate immune system. These cells are tissue resident and are considered to act as sentinels for the detection of a multitude of different ligands. Activation of mast cells via stimulation of IgE receptors underlies allergic reactions to food, pollen, pet dander, and many other allergens. Stimulation of mast cells results in their activation leading to degranulation – a process in which the cell releases extracellular mediators like histamine, proteases, and cytokines. These mediators can then go on to activate other cell types nearby or can recruit more immune cells to the tissue. Some chronic pruritic diseases are thought to involve mast cells in the skin and many studies have demonstrated an increase in total mast cell number in skin from atopic dermatitis (AD) or allergic contact dermatitis (CAD). Previous methods relied on staining of skin biopsy sections with a chromogenic stain (like toulidine blue), then manual counting of the mast cells in small sections of the biopsy. More recently, the use of fluorescently-tagged avidin has been used to detect mast cells in skin biopsies, which represents a more specific detection method. A challenge still remains in the differentiation between quiescent (resting) and active (or degranulated) mast cells. Here we describe a method for the automated detection and quantification of quiescent and resting mast cells based on cell morphology, using commercially available image software analysis tools. Our method was applied to human skin biopsies that were treated to induce degranulation and to skin biopsies collected from AD patients. In contrast to earlier results, we do not observe an increase in total mast cell number nor in the number of active mast cells in the skin of AD patients. This method allows for the unbiased detection of mast cells in human skin with out the need to develop de novo proprietary algorithms.

Candidalysin, a Virulence Factor of Candida albicans, Stimulates Mast Cells by Mediating Cross-Talk Between Signaling Pathways Activated by the Dectin-1 Receptor and MAPKs.

Although mast cells (MCs) modulate the activity of effector cells during Candida albicans infection, their role in the pathogenesis of candidiasis remains unclear. Candidalysin, a C. albicans-derived peptide toxin, is a crucial factor in fungal infections. We aimed to investigate the effect of candidalysin on MC activation and the underlying molecular mechanism. Serum from candidalysin-immunized mice was used to measure candidalysin expression in patients infected with C. albicans. MC degranulation and migration were evaluated by β-hexosaminidase release assay and chemotaxis assay, respectively. EIA and ELISA were used to evaluate the production of eicosanoids and cytokines/chemokines, respectively. The production of nitric oxide (NO) was measured with a DAF-FM diacetate kit, while reactive oxygen species (ROS) production was analyzed by flow cytometry. MAPK activation was evaluated by Western blotting. We detected high candidalysin expression in the lesions of patients infected with C. albicans, and the MC number was increased in these lesions. LL-37 colocalized with MCs in the lesions of candidiasis patients. Candidalysin-enhanced MC accumulation in mice and treating LAD2 and HMC-1 cells with candidalysin induced their degranulation, migration, and production of pro- and anti-inflammatory cytokines/chemokines, eicosanoids, ROS, NO, and LL-37. Interestingly, C. albicans strains lacking candidalysin failed to induce MC activation. Moreover, candidalysin increased dectin-1 expression, and the inhibition of dectin-1 decreased MC activation. Downstream dectin-1 signaling involved the MAPK pathways. The finding that candidalysin causes cutaneous MC activation may improve our understanding of the role of MCs in the pathology of cutaneous C. albicans infection.

Enhanced presence of serotonin in nasal cavity after autologous stimulation.

Serotonin, which is a vasoactive amine, is an important neurotransmitter and is involved in many behavioral and psychological phenomena, such as pain, appetite, mood, and sleep. The primary purpose of our study was to investigate the effect of high-pressure administration of sterile physiological saline isotonic solution (HpPSIS) into nasal cavity and to determine the expression of the serotonin. The study was made in two branches, the previous with 14 volunteers, the subsequent study with 40 patients with mild anxiety disorder. The middle third of the inferior turbinate epithelial cells on the right nostril was scraped using a sterile curette and indicated as (pre), then, a spray of sterilized isotonic solution at high pressure on the left nostril was delivered, and 5 minutes later a similar stimulation was delivered on the same nostril. The stimulation was made with a specific spray dispenser. The middle third of the inferior turbinate epithelial cells on the left nostril was scraped using a sterile curette and indicated as (post). Then, based on the first part of our study, we started the second part and gave a treatment on forty new patients with anxiety disorder. The results of these studies highlight the possibility of endogenous enhancement of serotonin by stimulation of mast cells. In the first part of the study, Serotonin significantly increased in protein extracts after treatment (64.35±5.33 vs. 10.97±2.17; unpaired two tailed t-test, t=9.8, df=24, p≤0.0001; F=6.035; DFn=12; DFd=12). In the second part of the study, in patients treated with HpPSIS, we observed improvement of mood, after one, two and three months, with a statistically significant reduction of DASS-21, while no reduction was observed in control patients, treated with normal pressure commercial spray. This pilot study showed that the topical treatment of HpPHIS increases serotonin levels in nasal cavity. The observation reported in this study opens the way to a new valid strategy to enhance the level of endogenous serotonin. We observed a significant improvement of ASI on patients during HpPHIS therapy.

Reduction in Allergenicity and Induction of Oral Tolerance of Glycated Tropomyosin from Crab.

Tropomyosin (TM) is an important crustacean (Scylla paramamosain) allergen. This study aimed to assess Maillard-reacted TM (TM-G) induction of allergenic responses with cell and mouse models. We analyzed the difference of sensitization and the ability to induce immune tolerance between TM and TM-G by in vitro and in vivo models, then we compared the relationship between glycation sites of TM-G and epitopes of TM. In the in vitro assay, we discovered that the sensitization of TM-G was lower than TM, and the ability to stimulate mast cell degranulation decreased from 55.07 ± 4.23% to 27.86 ± 3.21%. In the serum of sensitized Balb/c mice, the level of specific IgE produced by TM-G sensitized mice was significantly lower than TM, and the levels of interleukins 4 and interleukins 13 produced by Th2 cells in spleen lymphocytes decreased by 82.35 ± 5.88% and 83.64 ± 9.09%, respectively. In the oral tolerance model, the ratio of Th2/Th1 decreased from 4.05 ± 0.38 to 1.69 ± 0.19. Maillard reaction masked the B cell epitopes of TM and retained some T cell epitopes. Potentially, Maillard reaction products (MRPs) can be used as tolerance inducers for allergen-specific immunotherapy.

Mast cells, cortistatin, and its receptor, MRGPRX2, are linked to the pathogenesis of chronic prurigo

Chronic prurigo (CPG) is characterized by intensive itch and interactions among nerves, neuropeptides, and mast cells (MCs). The role of some neuropeptides such as cortistatin (CST) and its receptor, Mas-related G protein-coupled receptor X2 (MRGPRX2), in CPG remains poorly investigated. We evaluated first whether CST activates human skin MCs, and second whether CST and MRGPRX2 are expressed in the skin of CPG patients, and by which cells. Skin prick tests and microdialysis with CST were performed in 6 and 1 healthy volunteers, respectively. Degranulation of human skin MCs was assessed using β-hexosaminidase and histamine release assays. Skin samples from 10 patients with CPG and 10 control subjects were stained for CST, MCs, and MRGPRX2 (protein and mRNA) using immunohistochemistry, immunofluorescence, and/or in situ hybridization. Flow cytometry was used to assess CST in human skin MCs. MRGPRX2 levels were measured in serum by ELISA. CST induced concentration-dependent degranulation of human skin MCs invivo and exvivo. Skin lesions of CPG patients exhibited markedly higher numbers of CST-expressing cells, CST-expressing MCs, MRGPRX2-expressing cells, and MRGPRX2 mRNA-expressing cells than nonlesional skin. MCs were the main MRGPRX2 mRNA-expressing cells in the lesions of most CPG patients (70%). Stimulation of human skin MCs with anti-IgE led to a release of CST. The number of MRGPRX2-expressing cells correlated with disease severity (r= 0.649, P= .04). MRGPRX2 serum levels in CPG patients correlated with disease severity (r= 0.704, P= .023) and quality-of-life impairment (r= 0.687, P= .028). CST and MRGPRX2 may contribute to the pathogenesis of CPG and should be evaluated in further studies as potential biomarkers and novel therapeutic targets.

Crosslinking-mediated activation of the FcεRI: Does it need antigen for success?

Mast cells (MCs), hematopoietic cells of the myeloid lineage, are well-known for their pro-inflammatory nature contributing to the development of various allergic and autoimmune diseases. One of the characteristic receptors on MCs, the high-affinity receptor for IgE (FcεRI), is activated in its IgE-bound state via binding and crosslinking by polyvalent antigen. This results in its phosphorylation by the SRC family kinase LYN, initiating differential signaling pathways, eventually triggering immunological effector functions, such as degranulation and cytokine production. Few publications have reported on FcεRI-dependent but antigen-independent MC activation by antibody-mediated crosslinking of membrane molecules (e.g., transmembrane proteins and glycosphingolipids) that are both localized in membrane rafts and in close vicinity to the FcεRI. In this Viewpoint we will briefly introduce FcεRI-mediated MC stimulation, cite examples of FcεRI-proximal molecules, the crosslinking of which can cause FcεRI-dependent MC activation, and discuss the potential of certain viruses as well as auto-antibodies to act as indirect FcεRI-crosslinking agents. In latter cases, antigen-independent FcεRI-mediated pro-inflammatory MC activation could contribute to the development of detrimental cytokine storms.

High FODMAP diet causes barrier loss via lipopolysaccharide-mediated mast cell activation.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) are carbohydrates thought to contribute to the symptoms of IBS. A diet in high in FODMAPs (HFM) induces gastrointestinal symptoms in patients with irritable bowel syndrome (IBS), and a diet low in FODMAPs (LFM) improves symptoms in up to 60% of patients with IBS. However, the mechanism by which FODMAPs affect IBS symptoms is unclear. We showed that mice fed on a HFM diet have mast cell activation and colonic barrier loss. Using mast cell–deficient mice with and without mast cell reconstitution, we showed that HFM-mediated colonic barrier loss is dependent on TLR4-dependent mast cell activation. In in vitro studies, we demonstrated that IBS fecal supernatant stimulates mast cells significantly more compared with fecal supernatant from healthy controls. This effect of IBS fecal supernatant on mast cell stimulation is ameliorated in the absence of the TLR4 receptor and after a LFM diet. We found that a LFM diet improves colonic barrier function and reduces mast cell activation while decreasing fecal LPS levels. Our findings indicate that a HFM diet causes mast cell activation via LPS, which in turn leads to colonic barrier loss, and a LFM diet reverses these pathophysiologic mucosal changes.

A calcium channel blocker, manoalide exerts an anti-allergic inflammatory effect through attenuating NF-κB activity

Background Many people are troubled by allergic inflammation including ocular allergic diseases, anaphylaxis, allergic rhinitis, atopic dermatitis, and eczema. Consequently, finding medications for use in allergic inflammation therapy is crucial in human health. Manoalide, a marine natural product isolated as an anti-bacterial metabolite from Luffariella variabilis, is a calcium channel blocker. However, its latent ability as an anti-allergic inflammatory agent has not yet been reported. Our research aimed to elucidate whether manoalide exerts an anti-allergic inflammatory effect in the human mast cell line, HMC-1. Methods Herein, we investigated the immunoregulatory effects and molecular mechanisms of manoalide in HMC-1 cells. Results Manoalide significantly alleviated secretion of the inflammatory cytokines interleukin (IL)-1β, thymic stromal lymphopoietin, tumor necrosis factor-α, IL-6, and IL-8 via blockage of caspase-1 without cytotoxicity in activated HMC-1 cells. Activation of nuclear factor-κB increased by mast cell stimulation was attenuated by treatment with manoalide. In addition, we demonstrated that manoalide treatment remarkably attenuated the activation of mitogen-activated protein kinases in activated-HMC-1 cells. Conclusions Taken together, our findings indicate manoalide has an anti-allergic inflammatory role, and we propose that manoalide might have potential as a novel anti-allergic inflammatory agent.

Dimerized Translationally Controlled Tumor Protein-Binding Peptide 2 Attenuates Systemic Anaphylactic Reactions Through Direct Suppression of Mast Cell Degranulation.

Dimerized translationally controlled tumor protein (dTCTP) amplifies allergic responses through activation of several types of immune cells and release of inflammatory mediators. In particular, dTCTP plays an important role in histamine release by triggering mast cells and has been proposed as a target in the treatment of allergic diseases. dTCTP-binding peptide 2 (dTBP2) is known to attenuate severe allergic rhinitis and asthma through inhibition of dTCTP activity on airway epithelial cells and T cells; however, it is unclear whether dTBP2 affects mast cell function and mast cell disease. In this study, we explored the effects of dTBP2 on mast cell degranulation and allergen-induced anaphylactic reactions. We found that bacterial product lipopolysaccharide increased the expression of dTCTP in mast cells and rapidly released dTCTP by the mast cell stimulator compound 48/80. Interestingly, the released dTCTP further promoted mast cell degranulation in an autocrine activation manner and increased calcium mobilization in mast cells, which is essential for degranulation. Furthermore, dTBP2 directly and dose-dependently inhibited in vitro mast cell degranulation enhanced by compound 48/80, suggesting a direct and potent anti-anaphylactic activity of dTBP2. dTBP2 also significantly suppressed the dTCTP-induced degranulation and histamine release through inhibition of the p38 MAPK signaling pathway and suppression of lysosomal expansion and calcium mobilization in mast cells. More importantly, in vivo administration of dTBP2 decreased mortality and significantly attenuated histamine release and inflammatory cytokine production in compound 48/80-induced systemic anaphylactic reactions. These results suggest that dTBP2 is beneficial for the control of anaphylaxis with increased dTCTP.

Ways to Address Perinatal Mast Cell Activation and Focal Brain Inflammation, including Response to SARS-CoV-2, in Autism Spectrum Disorder.

The prevalence of autism spectrum disorder (ASD) continues to increase, but no distinct pathogenesis or effective treatment are known yet. The presence of many comorbidities further complicates matters, making a personalized approach necessary. An increasing number of reports indicate that inflammation of the brain leads to neurodegenerative changes, especially during perinatal life, “short-circuiting the electrical system” in the amygdala that is essential for our ability to feel emotions, but also regulates fear. Inflammation of the brain can result from the stimulation of mast cells—found in all tissues including the brain—by neuropeptides, stress, toxins, and viruses such as SARS-CoV-2, leading to the activation of microglia. These resident brain defenders then release even more inflammatory molecules and stop “pruning” nerve connections, disrupting neuronal connectivity, lowering the fear threshold, and derailing the expression of emotions, as seen in ASD. Many epidemiological studies have reported a strong association between ASD and atopic dermatitis (eczema), asthma, and food allergies/intolerance, all of which involve activated mast cells. Mast cells can be triggered by allergens, neuropeptides, stress, and toxins, leading to disruption of the blood–brain barrier (BBB) and activation of microglia. Moreover, many epidemiological studies have reported a strong association between stress and atopic dermatitis (eczema) during gestation, which involves activated mast cells. Both mast cells and microglia can also be activated by SARS-CoV-2 in affected mothers during pregnancy. We showed increased expression of the proinflammatory cytokine IL-18 and its receptor, but decreased expression of the anti-inflammatory cytokine IL-38 and its receptor IL-36R, only in the amygdala of deceased children with ASD. We further showed that the natural flavonoid luteolin is a potent inhibitor of the activation of both mast cells and microglia, but also blocks SARS-CoV-2 binding to its receptor angiotensin-converting enzyme 2 (ACE2). A treatment approach should be tailored to each individual patient and should address hyperactivity/stress, allergies, or food intolerance, with the introduction of natural molecules or drugs to inhibit mast cells and microglia, such as liposomal luteolin.

Study on degranulation of mast cells under C48/80 treatment by electroporation-assisted and ultrasound-assisted surface-enhanced Raman spectrascopy

Both electroporation-assisted and ultrasound-assisted delivery methods can rapidly deliver nanoparticles into living cells for surface-enhanced Raman scattering (SERS) detection, but these two methods have never been compared. In this study, electroporation-assisted SERS and ultrasound-assisted SERS were employed to detect the biochemical changes of degranulated mast cells induced by mast cell stimulator (C48/80). The results showed that the cell damage of electroporation based on controllable electric pulse was smaller than that of ultrasound based on cavitation. Transmission electron microscope images of cells indicated that the nanoparticles delivered by electroporation were mainly distributed in the cytoplasm, while ultrasound could transport nanoparticles to the cytoplasm and nucleus. Therefore, electroporation-assisted SERS mainly detects the biochemical information of cytoplasm, while ultrasound-assisted SERS gets more spectral signals of nucleic acid. Both methods can obtain high quality SERS signal of cells. With drug treatment, the SERS peak intensity of 733 cm−1 attributed to phosphatidylserine decreased significantly, which may be due to the activation of mast cell degranulation pathway stimulated by C48/80 agonist, resulting in a large amount of intracellular serine being used to synthesize tryptase, while the production of phosphatidylserine decreased. Further, based on principal component analysis and linear discriminant analysis (PCA-LDA approach), ultrasound-assisted SERS could achieve better sensitivity, specificity and accuracy in the discrimination and identification of drug-treated degranulated mast cells than electroporation assisted SERS. This exploratory work is helpful to realize the real-time dynamic SERS detection of intracellular biochemical components, and it also has great potential in intracellular SERS analysis, such as the cytotoxicity assay of anti-tumor drugs or cancer cell screening.

Critical Role of Protein Arginine Deiminase 4 in Mast Cell Extracellular Trap Formation Leading to Neuropathic Pain in Sickle Mice

Extracellular trap (ET) formation primarily meant to ensnare the pathogens, is also involved in sterile inflammation, platelet activation, impaired wound healing, oxidative stress, organ damage, etc (Jorch and Kubes, reviewed in Nat Med 2017). We have identified a novel role of mast cell ETs (MCETs) in contributing to neuropathic pain via Protein Arginine Deiminase 4 (PAD4), an enzyme that catalyzes chromatin decondensation in the nucleus by converting arginine to citrulline on histones. We used homozygous BERK sickle (HbSS) mice expressing human sickle hemoglobin (Hb) and control BERK (HbAA) mice expressing normal human HbA, and mast cells isolated from their dorsal skin. MCETs were visualized by laser scanning confocal microscopy in 100 μm thick dorsal skin sections and in mast cells in vitro with SYTO13 and SYTOX Orange (Invitrogen) and citrullinated histone antibodies (Abcam); and co-localized in the skin using mast cell-specific c-kit, FcεR1 and tryptase, nerve-fiber-specific anti-NF200 and anti-PGP9.5 and anti-CD31 for vasculature.We observed vibrant clusters of trap-shooting mast cells surrounding the vasculature and nerve fibers in the skin of sickle mice. The exuberant spread of MCETs and tryptase permeated the vessel lumen and may contribute to observed increase in circulating tryptase, DNA and endothelial activation in sickle mice. Notably, large aggregates of MCETs and mast cells appeared occluding blood vessels, and tightly surrounded the axonal nerve fibers demonstrating signs of injury and axonal sprouting, features underlying neuropathic pain. We stimulated mast cells in vitro with 40µM hemin for 2h after 2-hour priming with 1ng/ml TNFα to create a sickle microenvironment in vitro . SYTO13 and citrullinated histone staining showed that nearly all the cells from sickle but fewer cells from control mice showed MCET formation with hemin after priming with TNFα. Tubulin staining showed contraction and extension into long fibers in hemin/TNFα treated sickle mast cells, suggesting that ongoing hemolysis releasing free-heme in an inflammatory mileau in SCD may contribute to MCETosis observed in the skin of sickle mice. Transcripts of TLR4 and FcεR1 are significantly elevated in sickle compared to control mast cells (Vincent et al., Blood 2013). Silencing of TLR4 and/or FcεR1 did not inhibit hemin/TNFα-induced MCET formation completely. However, inhibition of PAD4 with 10µM GSK484 (Cayman) completely abrogated hemin/TNFα-induced MCET formation. Several signaling pathways including PKB/Akt, MAPK/ERK, p38MAPK and Syk were constitutively activated in sickle mast cells. Collectively, these in vitro observations suggest that multiple receptors and pathways converge at the downstream activation of PAD4 leading to MCETosis.Since MCETs incited direct peripheral nerve injury, we examined if inhibition of MCETs in vivo using PAD4 inhibitor would attenuate pain. Sickle and control mice were treated subcutaneously with PAD4 inhibitor GSK484 (20 mg/kg/day) or 0.9% saline for 30 days. GSK484 significantly reduced cold and heat hyperalgesia after 48h (p=.0066) and 72h (p=.0002), respectively, but reduced mechanical hyperalgesia significantly after 1 week (p=.0036) of treatment Vs baseline before treatment. Attenuation of hyperalgesia remained effective until 30 days, last day of treatment and observation, suggesting that mice do not develop analgesic tolerance. Mast cell degranulation was decreased by 90% in the skin (p=.0004); white blood cells and serum GM-CSF were significantly reduced (p= .010 and <.0001, respectively), but the hematocrit was significantly increased (p=.035) in sickle mice treated with GSK484 Vs saline. Skin secretagogue of GSK484 treated sickle mice showed about 50% reduction in inflammatory cytokines CCL2/MCP-1, TNFα and RANTES (p=.0229, .0154 and .0290, respectively Vs saline treated mice).Together, these data demonstrate an anti-inflammatory effect of GSK484 in the periphery and attenuation of neuropathic pain. Thus, mast cell degranulation and MCETosis contribute to inflammation and neurovascular injury leading to inflammatory as well as neuropathic pain. We speculate that MCETosis may also contribute to vascular dysfunction and organ damage in SCD. Thus, PAD4 inhibition provides a potential therapeutic target to attenuate the onset and progression of pain and perhaps other pathological consequences of SCD. DisclosuresGupta:Tautona Group: Consultancy, Honoraria; Fera Pharmaceuticals LLC: Consultancy, Honoraria.

Differential Use of the Signaling Molecules BTK and PLC-γ in Mast Cells Stimulated Via the Receptors FcεRI and KIT

Mast cells belong to the myeloid lineage of the hemopoietic system, are localized to the tissue-environment interphase, and execute regulatory and protective roles in both innate and adaptive immunity. However, mast cells also are involved in pathophysiological processes ranging from allergic diseases to autoimmune diseases and tumors. Two receptors expressed on the surface of mast cells and prominently involved in physiology and pathophysiology of mast cell biology are the high-affinity receptor for IgE (FcεRI) and the receptor tyrosine kinase KIT (a.k.a. CD117), the receptor for stem cell factor (SCF). Whereas antigen (Ag)-triggered activation of the IgE-bound FcεRI results in immediate release of preformed mediators (degranulation) and production of arachidonic acid metabolites and cytokines/chemokines, SCF-induced mast cell activation is only causing the latter. On the other side, SCF is inducing mast cell proliferation and chemotaxis, two responses that are not caused by FcεRI activation. Intriguingly, the signal transduction downstream of these receptors involves activation of the same signaling pathways and engagement of comparable sets of signaling proteins, raising the question, how differential mast cell effector functions are controlled by these receptors. Therefore, it is essential to learn more about the molecular pathways leading to FcεRI and KIT-mediated activation of mast cells. In the presented work, primary murine mast cells were stimulated either with Ag via the IgE-bound FcεRI or with SCF via its receptor KIT and differential signal transduction was analyzed. We put our focus on two central signaling elements in mast cells, the cytosolic tyrosine kinase BTK and its target, phospholipase C-γ, and also addressed differences between using suboptimal and optimal ligand concentrations. Cell responses like degranulation, proinflammatory cytokine production, activating protein phosphorylation, and intracellular calcium mobilization were analyzed making use of mast cells from wild-type and transgenic (knock-out) mice as well as selective pharmacological inhibitors. Interestingly, marked differences were found with respect to pathway activation, importance of particular signaling proteins, and induction of effector responses. Ag-triggered degranulation is thought to be dependent on BTK/PLC-γ-mediated calcium mobilization and thus it was unexpected to observe only a weak effect of PLC-γ inhibition. On the other side, SCF-induced calcium mobilization was strictly dependent on PLC-γ, however, only a minor involvement of BTK could be observed. In combination with the additional results obtained in our study, future analyses of differential pathway organization by multiprotein assemblies as well as kinetic control mechanisms appear to be important. Detailed knowledge of these mechanisms will help to precisely promote beneficial and strictly control detrimental mast cell responses. DisclosuresNo relevant conflicts of interest to declare.

IL-10 in Mast Cell-Mediated Immune Responses: Anti-Inflammatory and Proinflammatory Roles.

Mast cells (MCs) play critical roles in Th2 immune responses, including the defense against parasitic infections and the initiation of type I allergic reactions. In addition, MCs are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, cancers, allograft rejections, and lifestyle diseases. Whereas antigen-specific IgE is a well-known activator of MCs, which express FcεRI on the cell surface, other receptors for cytokines, growth factors, pathogen-associated molecular patterns, and damage-associated molecular patterns also function as triggers of MC stimulation, resulting in the release of chemical mediators, eicosanoids, and various cytokines. In this review, we focus on the role of interleukin (IL)-10, an anti-inflammatory cytokine, in MC-mediated immune responses, in which MCs play roles not only as initiators of the immune response but also as suppressors of excessive inflammation. IL-10 exhibits diverse effects on the proliferation, differentiation, survival, and activation of MCs in vivo and in vitro. Furthermore, IL-10 derived from MCs exerts beneficial and detrimental effects on the maintenance of tissue homeostasis and in several immune-related diseases including contact hypersensitivity, auto-immune diseases, and infections. This review introduces the effects of IL-10 on various events in MCs, and the roles of MCs in IL-10-related immune responses and as a source of IL-10.

Immunomodulatory effects of Brominated Flame Retardants on Cytokine Production in Mast cells

Polybrominated diphenyl ethers (PBDEs), extensively used as flame-retardants have been classified as persistent environmental pollutants. PBDEs have also been shown to cause neurotoxicity, immunotoxicity, and endocrine disruption; however, the exact mechanisms are largely unknown. In this study, we propose to investigate PBDE-induced immunotoxicity in mast cells. We examined the effects of two of the most commonly occurring brominated diphenyl ether (BDE) congeners, BDE-47 and -99, and a metabolite of BDE-47, 6-OH-BDE-47 on MC/9 mast cells. MC/9 cells treated with BDE-47, -99, and 6-OH-BDE-47 (0.25, 0.5, 5, 20 and 40μM, 24h) were stimulated with IgE/antigen (1μg/ml), and levels of IL-6 and TNF-α were measured by ELISA. Our results indicate that PBDEs inhibit the IgE-mediated cytokine release in MC/9 cells in a concentration-dependent manner. Interestingly, exposure to BDE-47 and 6-OH-BDE47 decreased IL-6 release in a concentration-dependent manner; however, BDE-99 decreased IL-6 release only at concentrations above 20 μM. These results demonstrate that tetra-brominated BDE-47 and its 6-OH metabolite can suppress cytokine release with increasing concentrations, while penta-brominated BDE-99 congener inhibits IL-6 release only at higher concentrations. We observed a similar decrease in TNF-α release when tge MC/9 cells were exposed to the BDE-47, -99, and 6-OH-BDE-47. Mast cell degranulation measured by β-hexoaminidase also showed that BDE congeners decreased degranulation in a concentration-dependent manner in IgE stimulated mast cells. In order to test effects on cell viability, we exposed the MC/9 cells to increasing concentrations of the BDE congeners and measured cytotoxicity at 24 and 48h by MTS assay. We found that tetra-brominated BDE-47 exhibited more potent cytotoxicity (EC50 µM =42.38 at 24h and 32.33 at 48h) in comparison to the penta-brominated BDE-99 (EC50 µM =48.41 and 35.04 at 48h) in the MC/9 cells. However, the 6-OH metabolite of BDE-47 was more cytotoxic in comparison to its parent compound BDE-47 as well as penta-brominated BDE-99 (EC50 µM =25.82 at 24h and 20.16 at 48h). We are further characterizing the development of apoptosis in the MC9 cells in the presence of BDE congeners. Our results suggest that PBDE congeners can modulate cytokine release in mast cells, and the extent of inhibition is dependent on the bromination of PBDEs. These results address the need to better understand the molecular and cellular mechanisms by which PBDE exposure can mediate immunotoxicity.