Screening of Prospective Antiallergic Compound as FcεRI Inhibitors and Its Antiallergic Efficacy Through Immunoinformatics Approaches.

The occurrence of allergy, a type I hypersensitivity reaction, is rising exponentially all over the world. Sometimes, allergy proves to be fatal for atopic patients, due to the occurrence of anaphylaxis. This study is aimed to find an anti-allergic agent that can inhibit the binding of IgE to Human High Affinity IgE Receptor (FCεRI), thereby preventing the degranulation of mast cells. A considerable number of potential anti-allergic compounds were assessed for their inhibitory strength through ADMET studies. AUTODOCK was used for estimating the binding energy between anti-allergic compounds and FCεRI, along with the interacting amino acids. The docked pose showing favorable binding energy was subjected to molecular dynamics simulation study. Marrubiin, a diterpenoid lactone from Lamiaceae, and epicatechin-3-gallate appears to be effective in blocking the Human High Affinity IgE Receptor (FCεRI). This in-silico study proposes the use of marrubiin and epicatechin-3-gallate, in the downregulation of allergic responses. Due to the better inhibition constant, future direction of this study is to analyze the safety and efficacy of marrubiin in anti-allergic activities through in-vivo clinical human trials.

Cannabidiol Inhibits IgE-Mediated Mast Cell Degranulation and Anaphylaxis in Mice.

SCOPE: Cannabidiol (CBD), the most abundant non-psychoactive constituent of the plant Cannabis sativa, is known to possess immune modulatory properties. This study investigates the effects of CBD on mast cell degranulation in human and mouse primary mast cells and passive cutaneous anaphylaxis in mice. METHODS AND RESULTS: Mouse bone marrow-derived mast cells and human cord-blood derived mast cells are generated. CBD suppressed antigen-stimulated mast cell degranulation in a concentration-dependent manner. Mechanistically, CBD inhibited both the phosphorylation of FcεRI downstream signaling molecules and calcium mobilization in mast cells, while exerting no effect on FcεRI expression and IgE binding to FcεRI. These suppressive effects are preserved in the mast cells that are depleted of type 1 (CB1) and type 2 (CB2) cannabinoid receptors, as well as in the presence of CB1 agonist, CB2 agonist, CB1 inverse agonist, and CB2 inverse agonist. CBD also inhibited the development of mast cells in a long-term culture. The intraperitoneal administration of CBD suppressed passive cutaneous anaphylaxis in mice as evidenced by a reduction in ear swelling and decrease in the number of degranulated mast cells. CONCLUSION: Based on these results, the administration of CBD is a new therapeutic intervention in mast cell-associated anaphylactic diseases.

Biomarkers of Autologous Whole Blood Injection Efficacy in Patients with Chronic Spontaneous Urticaria with Autoreactivity: A Preliminary Study.

INTRODUCTION: Chronic spontaneous urticaria (CSU) with autoreactivity is often resistant to antihistamines. Autologous whole blood injection (AWBI) has shown potential efficacy in the treatment of this disease, but it is controversial. It is necessary to screen patients who are suitable for this therapy in advance. This study aimed to identify biomarkers that predict the efficacy of AWBI treatment in CSU patients with autoreactivity. METHODS: A total of 30 patients with autologous serum skin test-positive CSU treated with AWBI were included in this study; urticaria activity score (UAS7) was recorded and the treatment response was judged based on it. Levels of total serum IgE, anti-high-affinity IgE receptor (FcεRI) IgG, and basophils CD63 and FcεRI expressions, and D-dimer of all patients were determined and analyzed. RESULTS: Baseline levels of total IgE, D-dimer, basophil FcεRI and CD63 expressions showed good correlations with UAS7 variations. D-dimer, basophil FcεRI and CD63 expressions changed significantly before and after AWBI treatment in AWBI responders, and the basophil FcεRI and CD63 expressions consistently and dynamically decreased in AWBI responders during the treatment. Baseline levels of total IgE, D-dimer, basophil FcεRI and CD63 expressions showed certain predictive values for AWBI response. CONCLUSIONS: Baseline levels of total IgE, D-dimer, basophil FcεRI and CD63 expressions could be biomarkers of predicting AWBI efficacy in patients with CSU with autoreactivity.

Autoreactive IgE: Pathogenic role and therapeutic target in autoimmune diseases.

Autoimmunity is the break of tolerance to self-antigens that leads to organ-specific or systemic diseases often characterized by the presence of pathogenic autoreactive antibodies (AAb) produced by plasmablast and/or plasma cells. AAb are prevalent in the general population and not systematically associated with clinical symptoms. In contrast, in some individuals, these AAb are pathogenic and drive the development of signs and symptoms of antibody-mediated autoimmune diseases (AbAID). AAb production, isotype profiles, and glycosylations are promoted by pro-inflammatory triggers linked to genetic, environmental, and hormonal parameters. Recent evidence supports a role for pathogenic AAb of the IgE isotype in a number of AbAID. Autoreactive IgE can drive the activation of mast cells, basophils, and other types of FcεRI-bearing cells and may play a role in promoting autoantibody production and other pro-inflammatory pathways. In this review, we discuss the current knowledge on the pathogenicity of autoreactive IgE in AbAID and their status as therapeutic targets. We also highlight unresolved issues including the need for assays that reproducibly quantify IgE AAbs, to validate their diagnostic and prognostic value, and to further study their pathophysiological contributions to AbAID.

Inhibiting Isoprenylation Suppresses FcεRI-Mediated Mast Cell Function and Allergic Inflammation.

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.

Fc Epsilon RI-Neuroimmune Interplay in Pruritus Triggered by Particulate Matter in Atopic Dermatitis Patients.

Atopic dermatitis (AD) is the most common chronic relapsing neuroinflammatory skin disease that is characterized by a complex and multifactorial pathophysiology. It reflects a profound interplay between genetic and environmental factors, and a recently disclosed neuroimmune dysregulation that drives skin barrier disruption, pruritus, and microbial imbalance. In terms of the key external environmental players that impact AD, air quality and itch severity linkage have been thoroughly researched. The impact of ambient air pollutants including particulate matter (PM) and AD pruritic exacerbation has been recorded despite reductions in air pollution levels in in developed countries. The developing countries have, on the contrary, experienced significant urbanization and industrialization with limited environmental protection standards in the past decades. This unprecedented construction, petrochemical industry utilization, and increment in population counts has been paired with consistent exposure to outdoor PM. This may present a key cause of AD pruritic exacerbation supported by the fact that AD prevalence has intensified globally in the past 50 years, indicating that environmental exposure may act as a trigger that could flare up itch in vulnerable persons. At the molecular level, the impact of PM on severe pruritus in AD could be interpreted by the toxic effects on the complex neuroimmune pathways that govern this disease. AD has been recently viewed as a manifestation of the disruption of both the immune and neurological systems. In light of these facts, this current review aims to introduce the basic concepts of itch sensory circuits in the neuroimmune system. In addition, it describes the impact of PM on the potential neuroimmune pathways in AD pathogenesis with a special focus on the Fc Epsilon RI pathway. Finally, the review proposes potential treatment lines that could be targeted to alleviate pruritus based on immune mediators involved in the Fc Epsilon signaling map.

MRGPRX2, drug pseudoallergies, inflammatory diseases, mechanisms and distinguishing MRGPRX2- and IgE/FcεRI-mediated events.

MRGPRX2, a novel Gaq -coupled human mast cell receptor, mediates non-immune adverse reactions without the involvement of antibody priming. Constitutively expressed by human skin mast cells, MRGPRX2 modulates cell degranulation producing pseudoallergies manifesting as itch, inflammation and pain. The term pseudoallergy is defined in relation to adverse drug reactions in general and immune/non-immune-mediated reactions in particular. A list of drugs with MRGPRX2 activity is presented, including a detailed examination of three important and widely used approved therapies: neuromuscular blockers, quinolones and opioids. For the clinician, the significance of MRGPRX2 is considered as an aid in distinguishing and ultimately identifying specific immune and non-immune inflammatory reactions. Anaphylactoid/anaphylactic reactions, neurogenic inflammation and inflammatory diseases with a clear or strongly suspected association with MRGPRX2 activation are examined. Inflammatory diseases include chronic urticaria, rosacea, atopic dermatitis, allergic contact dermatitis, mastocytosis, allergic asthma, ulcerative colitis and rheumatoid arthritis. MRGPRX2- and allergic IgE/FcεRI-mediated reactions may be clinically similar. Importantly, the usual testing procedures do not distinguish the two mechanisms. Currently, identification of MRGPRX2 activation and diagnosis of pseudoallergic reactions is generally viewed as a process of exclusion once other non-immune and immune processes, particularly IgE/FcεRI-mediated degranulation of mast cells, are ruled out. This does not take into account that MRGPRX2 signals via ß-arrestin, which can be utilized to detect MRGPRX2 activation by employing MRGPRX2 transfected cells to assess MRGPRX2 activation via two pathways, the G-protein-independent ß-arrestin pathway and the G-protein-dependent Ca2+ pathway. Testing procedures, interpretations for distinguishing mechanisms, patient diagnosis, agonist identification and drug safety evaluations are addressed.

Norisoboldine exerts antiallergic effects on IgE/ovalbumin-induced allergic asthma and attenuates FcεRI-mediated mast cell activation.

Allergic asthma is an inflammatory lung disorder, and mast cells play crucial roles in the development of this allergic disease. Norisoboldine (NOR), the major isoquinoline alkaloid present in Radix Linderae, has received considerable attention because it has anti-inflammatory effects. Herein, the aim of this study was to explore the antiallergic effects of NOR on allergic asthma in mice and mast cell activation. In a murine model of ovalbumin (OVA)-induced allergic asthma, oral administration at 5 mg/kg body weight (BW) of NOR produced strong reductions in serum OVA-specific immunoglobulin E (IgE) levels, airway hyperresponsiveness, and bronchoalveolar lavage fluid (BALF) eosinophilia, while an increase in CD4+Foxp3+ T cells of the spleen was detected. Histological studies demonstrated that NOR treatment significantly ameliorated the progression of airway inflammation including the recruitment of inflammatory cells and mucus production by decreasing levels of histamine, prostaglandin D2 (PGD2), interleukin (IL)-4, IL-5, IL-6, and IL-13 in BALF. Furthermore, our results revealed that NOR (3 âˆ¼ 30 µM) dose-dependently reduced expression of the high-affinity receptor for IgE (FcεRI) and the production of PGD2 and inflammatory cytokines (IL-4, IL-6, IL-13, and TNF-α), and also decreased degranulation of bone marrow-derived mast cells (BMMCs) activated by IgE/OVA. In addition, a similar suppressive effect on BMMC activation was observed by inhibition of the FcεRI-mediated c-Jun N-terminal kinase (JNK) signaling pathway using SP600125, a selective JNK inhibitor. Collectively, these results suggest that NOR may have therapeutic potential for allergic asthma at least in part through regulating the degranulation and the release of mediators by mast cells.

IRF3 Activation in Mast Cells Promotes FcεRI-Mediated Allergic Inflammation.

(1) Background: This study aims to elucidate a novel non-transcriptional action of IRF3 in addition to its role as a transcription factor in mast cell activation and associated allergic inflammation; (2) Methods: For in vitro experiments, mouse bone-marrow-derived mast cells (mBMMCs) and a rat basophilic leukemia cell line (RBL-2H3) were used for investigating the underlying mechanism of IRF3 in mast-cell-mediated allergic inflammation. For in vivo experiments, wild-type and Irf3 knockout mice were used for evaluating IgE-mediated local and systemic anaphylaxis; (3) Results: Passive cutaneous anaphylaxis (PCA)-induced tissues showed highly increased IRF3 activity. In addition, the activation of IRF3 was observed in DNP-HSA-treated mast cells. Phosphorylated IRF3 by DNP-HSA was spatially co-localized with tryptase according to the mast cell activation process, and FcεRI-mediated signaling pathways directly regulated that activity. The alteration of IRF3 affected the production of granule contents in the mast cells and the anaphylaxis responses, including PCA- and ovalbumin-induced active systemic anaphylaxis. Furthermore, IRF3 influenced the post-translational processing of histidine decarboxylase (HDC), which is required for granule maturation; and (4) Conclusion: Through this study, we demonstrated the novel function of IRF3 as an important factor inducing mast cell activation and as an upstream molecule for HDC activity.

Degranulation of Mast Cells as a Target for Drug Development.

Mast cells act as key effector cells of inflammatory responses through degranulation. Mast cell degranulation is induced by the activation of cell surface receptors, such as FcεRI, MRGPRX2/B2, and P2RX7. Each receptor, except FcεRI, varies in its expression pattern depending on the tissue, which contributes to their differing involvement in inflammatory responses depending on the site of occurrence. Focusing on the mechanism of allergic inflammatory responses by mast cells, this review will describe newly identified mast cell receptors in terms of their involvement in degranulation induction and patterns of tissue-specific expression. In addition, new drugs targeting mast cell degranulation for the treatment of allergy-related diseases will be introduced.

JNK signaling during IL-3-mediated differentiation contributes to the c-kit-potentiated allergic inflammatory capacity of mast cells.

Mast cells are leukocytes that mediate various aspects of immunity and drive allergic hypersensitivity pathologies. Mast cells differentiate from hematopoietic progenitor cells in a manner that is largely IL-3 dependent. However, molecular mechanisms, including the signaling pathways that control this process, have yet to be thoroughly investigated. Here, we examine the role of the ubiquitous and critical mitogen-activated protein kinase signaling pathway due to its position downstream of the IL-3 receptor. Hematopoietic progenitor cells were harvested from the bone marrow of C57BL/6 mice and differentiated to bone marrow-derived mast cells in the presence of IL-3 and mitogen-activated protein kinase inhibitors. Inhibition of the JNK node of the mitogen-activated protein kinase pathway induced the most comprehensive changes to the mature mast cell phenotype. Bone marrow-derived mast cells differentiated during impaired JNK signaling expressed impaired c-kit levels on the mast cell surface, first detected at week 3 of differentiation. Following 1 wk of inhibitor withdrawal and subsequent stimulation of IgE-sensitized FcεRI receptors with allergen (TNP-BSA) and c-kit receptors with stem cell factor, JNK-inhibited bone marrow-derived mast cells exhibited impediments in early-phase mediator release through degranulation (80% of control), as well as late-phase secretion of CCL1, CCL2, CCL3, TNF, and IL-6. Experiments with dual stimulation conditions (TNP-BSA + stem cell factor or TNP-BSA alone) showed that impediments in mediator secretion were found to be mechanistically linked to reduced c-kit surface levels. This study is the first to implicate JNK activity in IL-3-mediated mast cell differentiation and also identifies development as a critical and functionally determinative period.

A Co-Expressed Natural Antisense RNA FCER1A-AS Controls IgE-Dependent Immunity by Promoting Expression of FcεRIα.

As the α-subunit of the high-affinity receptor for the Fc portion of immunoglobulin E (FcεRIα), FcεRIα plays a central role in IgE-mediated allergic disorders and in the immunity and immunopathology of some parasitic infections. FcεRIα is specifically expressed on basophils and mast cells, but the mechanism that controls FcεRIα expression in these cells is poorly understood. In this study, we found that the natural antisense transcript (NAT) of FcεRIα (FCER1A-AS) is co-expressed with the sense transcript (FCER1A-S) in both interleukin (IL)-3-induced FcεRIα-expressing cells and in the high FcεRIα-expressing cell line MC/9. When FCER1A-AS is selectively knocked down by the CRISPR/RfxCas13d (CasRx) approach in MC/9 cells, the expression of both FCER1A-S mRNA and proteins is markedly decreased. Furthermore, FCER1A-AS deficiency was also found to be associated with a lack of FCER1A-S expression in vivo. Correspondingly, homozygous mice deficient in FCER1A-AS demonstrated a similar phenotype to FCER1A knockout mice in Schistosoma japonicum infection and in IgE-FcεRIα-mediated cutaneous anaphylaxis. Thus, we uncovered a novel pathway for the control of FcεRIα expression by its co-expressed natural antisense transcript. IMPORTANCE FcεRIα is responsible for high-affinity binding with the Fc portion of IgE, which is critical for IgE-dependent disease responses such as allergy responses and anti-parasite immunity. FcεRIα is expressed on a few cell types, including mast cells and basophils. Although the expression of FcεRIα is known to be promoted by the IL-3-GATA-2 pathway during its differentiation, the mechanism by which FcεRIα expression is maintained remains unknown. In this study, we discovered that a natural antisense transcript, FCER1A-AS, is co-expressed with the sense transcript. The presence of FCER1A-AS is essential for sense transcript expression in mast cells and basophils, but not for the differentiation of these cells through cis-regulation. Like FcεRIα knockout mice, mice lacking FCER1A-AS also exhibit reduced survival after Schistosoma japonicum infection and a lack of IgE-mediated cutaneous anaphylaxis. Thus, a novel pathway for regulating IgE-mediated allergic diseases through noncoding RNAs has been revealed.

Basophils beyond allergic and parasitic diseases.

Basophils bind IgE via FcεRI-αßγ2, which they uniquely share only with mast cells. In doing so, they can rapidly release mediators that are hallmark of allergic disease. This fundamental similarity, along with some morphological features shared by the two cell types, has long brought into question the biological significance that basophils mediate beyond that of mast cells. Unlike mast cells, which mature and reside in tissues, basophils are released into circulation from the bone marrow (constituting 1% of leukocytes), only to infiltrate tissues under specific inflammatory conditions. Evidence is emerging that basophils mediate non-redundant roles in allergic disease and, unsuspectingly, are implicated in a variety of other pathologies [e.g., myocardial infarction, autoimmunity, chronic obstructive pulmonary disease, fibrosis, cancer, etc.]. Recent findings strengthen the notion that these cells mediate protection from parasitic infections, whereas related studies implicate basophils promoting wound healing. Central to these functions is the substantial evidence that human and mouse basophils are increasingly implicated as important sources of IL-4 and IL-13. Nonetheless, much remains unclear regarding the role of basophils in pathology vs. homeostasis. In this review, we discuss the dichotomous (protective and/or harmful) roles of basophils in a wide spectrum of non-allergic disorders.

Ephedrine Alkaloid-Independent High-Affinity Immunoglobulin-E Receptor (FcεRI) Internalization Results in CCL2 Production without Inducing Mast Cell Degranulation.

Mast cells (MCs) play an important role in allergies, leading to the development of MC-targeted therapies. Ephedra herb (Mao) has potent anti-allergic activity, but contains ephedrine alkaloids (EAs); therefore, its hazardous effects are taken into consideration during its clinical use. We previously reported that Mao attenuates robust MC degranulation by an allergen through high-affinity immunoglobulin E (IgE) receptor (FcεRI) internalization, in which an EA-independent mechanism was suggested to be at play. This study aimed to deepen our understanding of the potential of Mao against FcεRI internalization using two strains with different EA contents. Mao extracts were administered to bone marrow-derived MCs (BMMCs), and their cellular responses, including FcεRI internalization, were analyzed. In addition, physiological events were evaluated using a passive cutaneous anaphylactic (PCA) reaction mouse model. BMMCs mediate the production of diverse inflammatory mediators. Among these, the potent chemokine CCL2 is thought to be differentially regulated from other pro-inflammatory mediators. We found that Mao significantly induces CCL2 expression in BMMCs despite suppressing robust degranulation through FcεRI internalization. Importantly, this was a distinctly EAs-independent response. In the PCA reaction, local MC activation following allergen challenge was suppressed by Mao treatment, which strengthened the view that Mao sufficiently decreased the rapid activation of MCs and promoted CCL2 secretion. Collectively, these observations provide additional insights into the mechanism of Mao-induced silent FcεRI internalization in MCs and the complex and heterogeneous secretory responses operating in MCs.

Urticaria and basophils.

Chronic spontaneous urticaria (CSU) is a common skin disease without an etiology in the vast majority of cases. The similarity of symptoms and pathology to allergen-induced skin reactions supports that skin mast cell IgE receptor activation is also involved in CSU. Accumulating evidence also supports a role for blood basophils in disease expression. Blood basopenia is noted in active CSU disease with the recruitment of blood basophils to skin lesion sites. Blood basophils further display altered IgE receptor mediated degranulation patterns in two phenotypes that improve in remission. In active CSU subjects, changes in IgE receptor signaling molecule expression levels accompany the altered degranulation function in blood basophils. The success of therapies targeting IgE in CSU patients have also shown that altered blood basophil phenotypes and enumeration have potential use as a disease biomarker.

Endocannabinoid modulation of allergic responses: Focus on the control of FcεRI-mediated mast cell activation.

Allergic reactions are highly prevalent pathologies initiated by the production of IgE antibodies against harmless antigens (allergens) and the activation of the high-affinity IgE receptor (FcεRI) expressed in the surface of basophils and mast cells (MCs). Research on the mechanisms of negative control of those exacerbated inflammatory reactions has been intense in recent years. Endocannabinoids (eCBs) show important regulatory effects on MC-mediated immune responses, mainly inhibiting the production of pro-inflammatory mediators. However, the description of the molecular mechanisms involved in eCB control of MC activation is far from complete. In this review, we aim to summarize the available information regarding the role of eCBs in the modulation of FcεRI-dependent activation of that cell type, emphasizing the description of the eCB system and the existence of some of its elements in MCs. Unique characteristics of the eCB system and cannabinoid receptors (CBRs) localization and signaling in MCs are mentioned. The described and putative points of cross-talk between CBRs and FcεRI signaling cascades are also presented. Finally, we discuss some important considerations in the study of the effects of eCBs in MCs and the perspectives in the field.

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

Kaempferol Suppresses the Activation of Mast Cells by Modulating the Expression of FcεRI and SHIP1.

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcεRI on BMMCs, but the mRNA levels of FcεRIα, ß, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcεRI on BMMCs was still observed when protein synthesis or protein transporter was inhibited. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NF-E2-related factor 2 (NRF2)-a master transcription factor of antioxidant stress-in BMMCs, the inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we found that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. The kaempferol-induced upregulation of SHIP1 was also observed in peritoneal MCs. The knockdown of SHIP1 by siRNA significantly enhanced IgE-induced degranulation of BMMCs. A Western blotting analysis showed that IgE-induced phosphorylation of PLCγ was suppressed in kaempferol-treated BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcεRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

Mast Cell Proteases Cleave Prion Proteins and a Recombinant Ig against PrP Can Activate Human Mast Cells.

IgE Abs, best known for their role in allergic reactions, have only rarely been used in immunotherapies. Nevertheless, they offer a potential alternative to the more commonly used IgGs. The affinity of IgE Ag binding influences the type of response from mast cells, so any immunotherapies using IgEs must balance Ag affinity with desired therapeutic effect. One potential way to harness differential binding affinities of IgE is in protein aggregation diseases, where low-affinity binding of endogenous proteins is preferred, but enhanced binding of clusters of disease-associated aggregated proteins could target responses to the sites of disease. For this reason, we sought to create a low-affinity IgE against the prion protein (PrP), which exists in an endogenous monomeric state but can misfold into aggregated states during the development of prion disease. First, we determined that mast cell proteases tryptase and cathepsin G were capable of degrading PrP. Then we engineered a recombinant IgE Ab directed against PrP from the V region of a PrP-specific IgG and tested its activation of the human mast cell line LAD2. The αPrP IgE bound LAD2 through Fc receptors. Crosslinking receptor-bound αPrP IgE activated SYK and ERK phosphorylation, caused Fc receptor internalization, and resulted in degranulation. This work shows that a recombinant αPrP IgE can activate LAD2 cells to release enzymes that can degrade PrP, suggesting that IgE may be useful in targeting diseases that involve protein aggregation.

Commensal Bacteria and the Lung Environment Are Responsible for Th2-Mediated Memory Yielding Natural IgE in MyD88-Deficient Mice.

IgE Abs are a common mediator of allergic responses and are generally produced in type 2 immune responses to allergens. Allergen stimulation of IgE-bound FcεRI on mast cells or basophils induces the production of chemical mediators and cytokines. In addition, IgE binding to FcεRI without allergen promotes the survival or proliferation of these and other cells. Thus, spontaneously produced natural IgE can increase an individual's susceptibility to allergic diseases. Mice deficient in MyD88, a major TLR signaling molecule, have high serum levels of natural IgE, the mechanism for which remains unknown. In this study, we demonstrated that the high serum IgE levels were maintained from weaning by memory B cells (MBCs). IgE from plasma cells and sera from most Myd88-/- mice, but none of the Myd88+/- mice, recognized Streptococcus azizii, a commensal bacterium overrepresented in the lungs of Myd88-/- mice. IgG1+ MBCs from the spleen also recognized S. azizii. The serum IgE levels declined with the administration of antibiotics and were boosted by challenge with S. azizii in Myd88-/- mice, indicating the contribution of S. azizii-specific IgG1+ MBCs to the natural IgE production. Th2 cells were selectively increased in the lungs of Myd88-/- mice and were activated upon addition of S. azizii in the lung cells ex vivo. Finally, lung nonhematopoietic cells, and CSF1 overproduced therefrom, were responsible for natural IgE production in Myd88-/- mice. Thus, some commensal bacteria may prime the Th2 response and natural IgE production in the MyD88-defective lung environment in general.