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Background: Immunoglobulin E (IgE) belongs to a class of immunoglobulins involved in immune response to specific allergens. However, the roles of IgE and IgE receptor (FcεR1) in pathological cardiac remodeling and heart failure are unknown. Methods: Serum IgE levels and cardiac FcεR1 expression were assessed in diseased hearts from human and mouse. The role of FcεR1 signaling in pathological cardiac remodeling was explored in vivo by FcεR1 genetic depletion, anti-IgE antibodies, and bone marrow transplantation. The roles of the IgE-FcεR1 pathway were further evaluated in vitro in primary cultured rat cardiomyocytes and cardiac fibroblasts (CFs). RNA sequencing and bioinformatic analyses were used to identify biochemical changes and signaling pathways that are regulated by IgE/FcεR1. Results: Serum IgE levels were significantly elevated in patients with heart failure as well as in 2 mouse cardiac disease models induced by chronic pressure overload via transverse aortic constriction and chronic angiotensin II infusion. Interestingly, FcεR1 expression levels were also significantly upregulated in failing hearts from human and mouse. Blockade of the IgE-FcεR1 pathway by FcεR1 knockout alleviated transverse aortic constriction– or angiotensin II–induced pathological cardiac remodeling or dysfunction. Anti-IgE antibodies (including the clinical drug omalizumab) also significantly alleviated angiotensin II–induced cardiac remodeling. Bone marrow transplantation experiments indicated that IgE-induced cardiac remodeling was mediated through non–bone marrow–derived cells. FcεR1 was found to be expressed in both cardiomyocytes and CFs. In cultured rat cardiomyocytes, IgE-induced cardiomyocyte hypertrophy and hypertrophic marker expression were abolished by depleting FcεR1. In cultured rat CFs, IgE-induced CF activation and matrix protein production were also blocked by FcεR1 deficiency. RNA sequencing and signaling pathway analyses revealed that transforming growth factor-β may be a critical mediator, and blocking transforming growth factor-β indeed alleviated IgE-induced cardiomyocyte hypertrophy and cardiac fibroblast activation in vitro. Conclusions: Our findings suggest that IgE induction plays a causative role in pathological cardiac remodeling, at least partially via the activation of IgE-FcεR1 signaling in cardiomyocytes and CFs. Therapeutic strategies targeting the IgE-FcεR1 axis may be effective for managing IgE-mediated cardiac remodeling.

Purpose Neutrophils play a critical role in defending against threats such as microbial infection, yet their activation during innate immune response incites collateral damage to healthy tissues. We have previously shown that corneal injury induces mast cells to express the neutrophil chemoattractant CXCL2. Here we delineate the mechanism of injury-induced, non-IgE-mediated mast cell activation at the ocular surface. Methods Corneal injury was induced by mechanical removal of the epithelium and anterior stroma in mast cell deficient (cKitW-sh) and C57BL/6 mice using Algerbrush II. Corneas were analyzed for frequencies of total CD45+ inflammatory cells, CD11b+Ly6G+ neutrophils, and cKit+FceR1+ mast cells using flow cytometry. Mast cells were stimulated with different inflammatory factors known to increase during corneal injury (IL-33, IL-1β, IL-36γ, IL-6, SDF1α and Substance P) and assessed for the secretion of β-hexosaminidase, tryptase and CXCL2 using ELISA. IL-33 neutralizing antibody (1 mg/ml) was administered locally for mast cell inhibition in vivo. Results Mast cell deficient mice failed to recruit early neutrophils to the injured corneas. IL-33 stimulation upregulated CXCL2 secretion by mast cells. Corneal injury resulted in amplified expression of IL-33 at the cornea and epithelium was identified as its primary source. Topical neutralization of IL-33 at the ocular surface inhibited mast cell activation, limited neutrophil infiltration, and reduced corneal inflammatory haze, normalizing tissue architecture following ocular injury. Conclusions These data implicate IL-33 in mast cell activation and early neutrophil recruitment in non-allergic inflammation, suggesting IL-33 as a potential therapeutic target in inflammatory disorders of the ocular surface.

The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB–dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow–derived mast cells from MALT1 knockout (MALT1−/−) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1−/− and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell–derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.

Diabetic kidney disease (DKD) is a key microvascular complication of diabetes, with few therapies for targeting renal disease pathogenesis and progression. We performed transcriptional and protein studies on 103 unique blood and kidney tissue samples from patients with and without diabetes to understand the pathophysiology of DKD injury and its progression. The study was based on the use of 3 unique patient cohorts: peripheral blood mononuclear cell (PBMC) transcriptional studies were conducted on 30 patients with DKD with advancing kidney injury; Gene Expression Omnibus (GEO) data was downloaded, containing transcriptional measures from 51 microdissected glomerulous from patients with DKD. Additionally, 12 independent kidney tissue sections from patients with or without DKD were used for validation of target genes in diabetic kidney injury by kidney tissue immunohistochemistry and immunofluorescence. PBMC DKD transcriptional analysis, identified 853 genes (p < 0.05) with increasing expression with progression of albuminuria and kidney injury in patients with diabetes. GEO data was downloaded, normalized, and analyzed for significantly changed genes. Of the 325 significantly up regulated genes in DKD glomerulous (p < 0.05), 28 overlapped in PBMC and diabetic kidney, with perturbed FcER1 signaling as a significantly enriched canonical pathway. FcER1 was validated to be significantly increased in advanced DKD, where it was also seen to be specifically co-expressed in the kidney biopsy with tissue mast cells. In conclusion, we demonstrate how leveraging public and private human transcriptional datasets can discover and validate innate immunity and inflammation as key mechanistic pathways in DKD progression, and uncover FcER1 as a putative new DKD target for rational drug design.

Hua Cao,1 Chungang Xiao,1 Zhangxiu He,2 Hong Huang,3,4 Huifang Tang1,4 1The First Affiliated Hospital, Department of Cardiology, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, People’s Republic of China; 2The First Affiliated Hospital, Department of Nephrology, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, People’s Republic of China; 3The First Affiliated Hospital, Institute of Clinical Medicine, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, People’s Republic of China; 4The First Affiliated Hospital, Institute of Cardiovascular Disease, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, People’s Republic of ChinaCorrespondence: Hong Huang; Huifang TangThe First Affiliated Hospital, Institute of Cardiovascular Disease, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, People’s Republic of ChinaEmail trave1@126.com; tanghuifang999@163.comAbstract: Cardiac remodeling is accompanied by cardiac hypertrophy, fibrosis, and dysfunction, eventually leading to heart failure (HF). However, the molecular mechanisms involved in cardiac remodeling are complicated, especially the association with immune. Immunoglobulin E (IgE) is a class of immunoglobulins involved in immune response to specific allergens. Recently, Zhao et al characterized a novel specific role of IgE and its high affinity receptor (Fc&epsiv;R1) in directly promoting pathological myocardial remodeling and cardiac dysfunction. Additionally, upon blocking IgE-Fc&epsiv;R1 signaling using Fc&epsiv;R1 genetic depletion or by administrating the anti-IgE monoclonal antibody omalizumab (Oma) in mice, they observed that cardiac hypertrophy and cardiac interstitial fibrosis induced by angiotensin II (Ang II) or transverse aortic constriction (TAC) were significantly suppressed. In contrast, IgE administration alone can aggravate pathological cardiac remodeling and dysfunction. RNA-seq and downstream analysis indicated that TGF-β was the common pathway and the most pivotal mediator in IgE-Fc&epsiv;R1-induced cardiac remodeling and dysfunction. Furthermore, the administration of a TGF-β inhibitor could ameliorate cardiac remodeling and improve cardiac function. Therefore, these findings suggest that IgE-Fc&epsiv;R1 maybe promising therapeutic targets for cardiac remodeling and provide an experimental basis for the use of omalizumab for HF patients combined with high serum IgE levels or allergic diseases.Keywords: cardiac remodeling, IgE- Fc&epsiv;R1, immunoglobulins, TGF-&#x03B2

Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcϵRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three β-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcϵR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both β and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcϵRI.

Background Food-induced anaphylaxis is a serious allergic reaction caused by Fce-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis. Methods Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1-/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FceR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined. Results Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1-/- mice were completely whereas female C5ar1-/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1-/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1-/- BMMCs of both sexes were significantly reduced. Importantly, FceR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1-/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting. Conclusions Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FceR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice.

Background Lung nociceptor neurons amplify immune cell activity and mucus metaplasia in response to an inhaled allergen challenge in sensitized mice. Objective We sought to identify the cellular mechanisms by which these sensory neurons are activated subsequent to allergen exposure. Methods We used calcium microscopy and electrophysiologic recording to assess whether vagal neurons directly respond to the model allergen ovalbumin (OVA). Next, we generated the first nociceptor-specific FceR1γ knockdown (TRPV1Cre::FceR1γfl/fl) mice to assess whether this targeted invalidation would affect the severity of allergic inflammation in response to allergen challenges. Results Lung-innervating jugular nodose complex ganglion neurons express the high-affinity IgE receptor FceR1, the levels of which increase in OVA-sensitized mice. FceR1γ-expressing vagal nociceptor neurons respond directly to OVA complexed with IgE with depolarization, action potential firing, calcium influx, and neuropeptide release. Activation of vagal neurons by IgE-allergen immune complexes, through the release of substance P from their peripheral terminals, directly amplifies TH2 cell influx and polarization in the airways. Allergic airway inflammation is decreased in TRPV1Cre::FceR1γfl/fl mice and in FceR1α–/– mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FceR1 to immune complexes in both mouse jugular nodose complex ganglion neurons and human induced pluripotent stem cell–derived nociceptors. Conclusions Allergen sensitization triggers a feedforward inflammatory loop between IgE-producing plasma cells, FceR1-expressing vagal sensory neurons, and TH2 cells, which helps to both initiate and amplify allergic airway inflammation. These data highlight a novel target for reducing allergy, namely, FceR1γ expressed by nociceptors.

Rationale: Cardiac fibrosis is an important feature of cardiac remodeling and is a hallmark of heart failure. Recent studies indicate that elevated IgE plays a causal role in pathological cardiac remodeling. However, the underlying mechanism of how IgE promotes cardiac fibrosis has not been fully elucidated. Methods and Results: To explore the function of IgE in cardiac fibrosis, we stimulated mouse primary cardiac fibroblasts (CFs) with IgE and found that both IgE receptor (FceR1) and fibrosis related proteins were increased after IgE stimulation. Specific deletion of FceR1 in CFs alleviated angiotensin II (Ang II)-induced cardiac fibrosis in mice. To investigate the mechanisms underlying the IgE-mediated cardiac fibrosis, deep miRNA-seq was performed. Bioinformatics and signaling pathway analysis revealed that IgE upregulated Col1a1 and Col3a1 expression in CFs by repressing miR-486a-5p, with Smad1 participating downstream of miR-486a-5p in this process. Lentivirus-mediated overexpression of miR-486a-5p was found to alleviate Ang II-induced myocardial interstitial fibrosis in mice. Moreover, miR-486-5p serum levels were lower in patients with heart failure than in healthy controls, and were negatively correlated with NT-proBNP levels. Conclusions: Our study demonstrates that elevated IgE promotes pathological cardiac fibrosis by modulating miR-486a-5p and downstream factors, such as Smad1. These findings suggest new targets for pathological cardiac fibrosis intervention.

Objective: By binding to its high-affinity receptor FcεR1, IgE activates mast cells, macrophages, and other inflammatory and vascular cells. Recent studies support an essential role of IgE in cardiometabolic diseases. Plasma IgE level is an independent predictor of human coronary heart disease. Yet, a direct role of IgE and its mechanisms in cardiometabolic diseases remain incompletely understood. Approach and Results: Using atherosclerosis prone Apoe −/− mice and IgE-deficient Ige −/− mice, we demonstrated that IgE deficiency reduced atherosclerosis lesion burden, lesion lipid deposition, smooth muscle cell and endothelial cell contents, chemokine MCP (monocyte chemoattractant protein)-1 expression and macrophage accumulation. IgE deficiency also reduced bodyweight gain and increased glucose and insulin sensitivities with significantly reduced plasma cholesterol, triglyceride, insulin, and inflammatory cytokines and chemokines, including IL (interleukin)-6, IFN (interferon)-γ, and MCP-1. From atherosclerotic lesions and peritoneal macrophages from Apoe −/− Ige −/− mice that consumed an atherogenic diet, we detected reduced expression of M1 macrophage markers (CD68, MCP-1, TNF [tumor necrosis factor]-α, IL-6, and iNOS [inducible nitric oxide synthase]) but increased expression of M2 macrophage markers (Arg [arginase]-1 and IL-10) and macrophage-sterol-responsive-network molecules (complement C3, lipoprotein lipase, LDLR [low-density lipoprotein receptor]-related protein 1, and TFR [transferrin]) that suppress macrophage foam cell formation. These IgE activities can be reproduced in bone marrow-derived macrophages from wild-type mice, but muted in cells from FcεR1-deficient mice, or blocked by anti-IgE antibody or complement C3 deficiency. Conclusions: IgE deficiency protects mice from diet-induced atherosclerosis, obesity, glucose tolerance, and insulin resistance by regulating macrophage polarization, macrophage-sterol-responsive-network gene expression, and foam cell formation.

Background Low vitamin D levels have been associated with allergic diseases. Vitamin D has potent immunomodulatory properties, but the mechanisms remain unclear. We have investigated the effect of oral vitamin D supplementation on circulating immune cell phenotypes in infants. Method A double‐blinded randomised controlled trial was conducted to investigate the effect of oral vitamin D supplementation (400 IU/d) on eczema and immune development. A subset of 78 infants was included in this analysis. Phenotypic analysis of immune cell subsets was performed using flow cytometry. Results Vitamin D supplementation resulted in median 25(OH)D levels of 80.5 vs 59.5 nmol/L in the placebo group at 3 months of age (P = .002) and 87.5 vs 77 nmol/L at 6 months of age (P = .08). We observed significant changes in immune cell composition from birth (cord blood) to 6 months of age. Vitamin D supplementation did not impact these changes, nor did immune cell composition correlate with plasma 25(OH)D levels. Through exploratory analysis, we identified possible associations with eczema development and increased abundance of naive CD4‐ T cells at birth, as well as associations with basophils, iNKT and central memory CD4+ T cells, and altered expression patterns of IgE receptor (FceR1) on monocytes and dendritic cells with eczema at 6 months. Conclusions Vitamin D supplementation in infants who were vitamin D sufficient at birth did not affect developmental changes in immune cells during the first 6 months of life. However, immune cell profiles at birth and at 6 months of age were associated with early life eczema.

Bee venom is a blend of biochemicals ranging from small peptides and enzymes to biogenic amines. It is capable of triggering severe immunologic reactions owing to its allergenic fraction. Venom components are presented to the T cells by antigen-presenting cells within the skin. These Th2 type T cells then release IL-4 and IL-13 which subsequently direct B cells to class switch to production of IgE. Generating venom-specific IgE and crosslinking FcεR1(s) on the surface of mast cells complete the sensitizing stage in allergic individuals who are most likely to experience severe and even fatal allergic reactions after being stung. Specific IgE for bee venom is a double-edged sword as it is a powerful mediator in triggering allergic events but is also applied successfully in diagnosis of the venom allergic patient. The healing capacity of bee venom has been rediscovered under laboratory-controlled conditions using animal models and cell cultures. The potential role of enzymatic fraction of bee venom including phospholipase A2 in the initiation and development of immune responses also has been studied in numerous research settings. Undoubtedly, having insights into immunologic interactions between bee venom components and innate/specific immune cells both locally and systematically will contribute to the development of immunologic strategies in specific and epitope-based immunotherapy especially in individuals with Hymenoptera venom allergy.

Introduction: Human plasmacytoid dendritic cells (pDCs) play key roles in anti-viral responses and induce regulatory T cells (Treg), which are essential for healthy immune responses to allergens and prevent airway remodelling. IgE-FceR1 (IgE) cross-linking on pDCs impairs anti-viral responses but the effects in Treg generation remain elusive. Methods: Blood pDCs were purified and incubated with Omalizumab or untreated. pDCs were then activated with CpG ODN 2006 (TLR9-L) in the presence of IgE crosslinker. Flow cytometry, ELISA, quantitative PCR and co-cultures were performed to assess the effects of IgE cross-linking in the capacity of TLR9-L-activated pDCs to generate Treg. Results: IgE cross-linking on TLR9-L-activated pDCs significantly reduced the expression of tolerogenic markers PD‐L1, IDO and ICOSL, inhibited IFN-α production whereas increased proinflammatory TNF-α. The capacity of TLR9-L-activated pDCs to induce functional CD4+CD25highFOXP3+Treg was significantly impaired upon IgE cross-linking, which was reverted by the pretreatment of pDCs with Omalizumab. Corticosteroids abolished the capacity of TLR9-L-activated pDCs to generate Treg. Mechanistically, IgE cross-linking on TLR9-L-activated pDCs promotes a misbalance in tolerogenic molecules and cytokine signature that impairs their capacity to generate Treg. Conclusion: IgE cross-linking breaks the capacity of pDCs to generate Treg in vitro, which can be restored by Omalizumab. We shed light into potential novel immunomodulatory mechanisms by which Omalizumab might exert beneficial clinical effects compared to the general suppression promoted by corticosteroids.

The tranquillizing effects of quercetin on allergic asthma are promising, but its poor water solubility and bioavailability is still a bottleneck. In this study, an ovalbumin (OVA) sensitized BALB/c mice asthma model was used to investigate the potential of quercetin nanocrystals (nQ) on relieving asthma aggravation. The water soluble nQ was prepared by the homogenization using the high energy sonication method. X-ray diffraction data showed the formation of nQ (10-30 nm) which was in agreement with transmission electron microscopy. The nQ was found to be more stable and soluble in PBS, and sera of BALB/c mice compared to bulk quercetin. Dose dependent experiments with nQ on OVA sensitized asthma mice exhibited significant anti-asthmatic potential of nQ at much lower dose (1 mg/kg body weight) compared to bulk quercetin. The treatment of nQ remarkably resulted in reduced OVA specific immunoglobulin E (sIgE) production, anaphylaxis signs and type 1 skin test. The nQ also significantly modulated the expression of Th2 cytokines like IL-4 and IL-5, which are responsible for IgE class switching and suppressed the degranulation/secretion of different chemical mediators (PGD2, mMCPT-1 Cys-L and TSLP) from activated mast cells. The levels of FcεR1, Syk, c-Yes, PI-3, p-PI-3, PLC-γ2, and p-PLC-γ2 were found to be reduced in the OVA sensitized BALB/c mice treated with nQ compared to those treated with OVA only. The results indicate that nQ alleviate pulmonary inflammation and airway hyporesponsiveness in allergic asthma at much lower dose compared to bulk quercetin and may be considered as a potential drug for the treatment of asthmatic patients.

Metformin, an anti-diabetic drug, possesses anti-inflammatory property beyond its glucose-lowering activity, but its regulatory effect on mast cells and allergic responses remains unknown, wherein the aryl hydrocarbon receptor (AhR)-ligand axis is critical in controlling mast cell activation. Herein, we provide evidence supporting the role of metformin in modulating mast cell activation by FceR1-, AhR-mediated signaling or their combination. Metformin at relatively low doses was shown to suppress FceR1-mediated degranulation, IL-13, TNF-α and sphingosine-1-phosphate (S1P) secretion in murine bone marrow-derived mast cells (BMMCs). In contrast, metformin at the same doses potently inhibited all parameters in mast cells stimulated with an AhR ligand, 5,11-dihydroindolo[3,2-b]carbazole-6-carbaldehyde (FICZ). Further, metformin was shown to inhibit FceR1- and AhR-mediated passive cutaneous anaphylaxis (PCA) in vivo, reversible by a S1P receptor 2 antagonist, JTE-013. Using AhR reporter cells, Huh7-DRE-Luc cells, a human mast cell line, HMC-1, and BMMCs, metformin's inhibitory effect was mediated through the suppression of FICZ-induced AhR activity, calcium mobilization and ROS generation. Notably, FICZ-mediated oxidation of S1P lyase (S1PL) and its reduced activity were reversed by metformin, resulting in decreased levels of S1P. Collectively, these results suggested the potential utility of metformin in treating allergic diseases, particularly in cases with comorbid type II diabetes mellitus.

The high affinity immunoglobulin E (IgE) receptor-FcεR1 is mainly expressed on the surface of effector cells. Cross-linking of IgE Abs bound to FcεR1 by multi-valent antigens can induce the activation of these cells and the secretion of inflammatory mediators. Since FcεR1 plays a central role in the induction and maintenance of allergic responses, this study aimed to investigate the association of FcεR1 with the allergic phenotype of Cε expression and cytokine and histamine release from peripheral leukocytes. Peripheral leukocytes from 67 allergic and 50 non-allergic subjects were used for genotyping analysis. Peripheral mononuclear cells (PBMCs) were used for Cε expression and ELISpot analysis, while polymorphonuclear cells (PMNs) were used for histamine release. The association between genotype polymorphism of the FcεR1α promoter region (rs2427827 and rs2251746) and allergic features of Cε expression and histamine were analyzed, and their effects on leukocytes function were compared with wild type. The genotype polymorphisms of FcεR1α promoter region with CT and TT in rs2427827 and TC in rs2251746 were significantly higher in allergic patients than in non-allergic controls. Patients with single nucleotide polymorphism (SNP) of FcεR1α promoter region had high levels of total IgE, mite-specific Der p 2 (Group 2 allergen of Dermatophagoides pteronyssinus)-specific IgE and IgE secretion B cells. The mRNA expression of FcεR1α was significantly increased after Der p2 stimulation in PBMCs with SNPs of the FcεR1α promoter region. Despite the increased Cε mRNA expression in PBMCs and histamine release from PMNs and the up-regulated mRNA expression of interleukin (IL)-6 and IL-8 secretions after Der p2 stimulation, there was no statistically significant difference between SNPs of the FcεR1α promoter region and the wild type. SNPs of FcεR1α promoter region were associated with IgE expression, IgE producing B cells, and increased Der p2-induced FcεR1α mRNA expression. These SNPs may be used as a disease marker for IgE-mediated allergic inflammation caused by Dermatophagoides pteronyssinus.

Background African Americans (AA) are disproportionately impacted by atopic dermatitis (AD), with increased prevalence and therapeutic challenges unique to this population. Molecular profiling data informing development of targeted therapeutics for AD are derived primarily from European American (EA) patients. These studies are absent in AA, hindering development of effective treatments for this population. Objective We sought to characterize the global molecular profile of AD in the skin of AA patients as compared with that of EA AD and healthy controls. Methods We performed RNA-Seq with reverse transcription polymerase chain reaction validation and immunohistochemistry studies in lesional and nonlesional skin of AA and EA AD patients vs healthy controls. Results African American AD lesions were characterized by greater infiltration of dendritic cells (DCs) marked by the high-affinity immunoglobulin E (IgE) receptor (FceR1+) compared with EA AD (P Conclusion The molecular phenotype of AA AD skin is characterized by attenuated Th1 and Th17 but similar Th2/Th22-skewing to EA AD. Our data encourages a personalized medicine approach accounting for phenotype-specific characteristics in future development of targeted therapeutics and clinical trial design for AD.

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

IgE has a key role in the pathogenesis of allergic responses through its ability to activate mast cells via the receptor FcεR1. In addition to mast cells, many cell types implicated in atherogenesis express FcεR1, but whether IgE has a role in this disease has not been determined. Here, we demonstrate that serum IgE levels are elevated in patients with myocardial infarction or unstable angina pectoris. We found that IgE and the FcεR1 subunit FcεR1α were present in human atherosclerotic lesions and that they localized particularly to macrophage-rich areas. In mice, absence of FcεR1α reduced inflammation and apoptosis in atherosclerotic plaques and reduced the burden of disease. In cultured macrophages, the presence of TLR4 was required for FcεR1 activity. IgE stimulated the interaction between FcεR1 and TLR4, thereby inducing macrophage signal transduction, inflammatory molecule expression, and apoptosis. These IgE activities were reduced in the absence of FcεR1 or TLR4. Furthermore, IgE activated macrophages by enhancing Na+/H+ exchanger 1 (NHE1) activity. Inactivation of NHE1 blocked IgE-induced macrophage production of inflammatory molecules and apoptosis. Cultured human aortic SMCs (HuSMCs) and ECs also exhibited IgE-induced signal transduction, cytokine expression, and apoptosis. In human atherosclerotic lesions, SMCs and ECs colocalized with IgE and TUNEL staining. This study reveals what we believe to be several previously unrecognized IgE activities that affect arterial cell biology and likely other IgE-associated pathologies in human diseases.

To cite this article: Martino DJ, Bosco A, McKenna KL, Hollams E, Mok D, Holt PG, Prescott SL. T-cell activation genes differentially expressed at birth in CD4+ T-cells from children who develop IgE food allergy. Allergy 2012; 67: 191–200. Abstract Background: Presymptomatic immaturity in neonatal T-cell function is a consistent antecedent of allergic disease, including reduced responsiveness to polyclonal activation. Methods: To elucidate the underlying mechanisms, we examined for differences in T-cell gene expression in longitudinal samples collected at birth and at 1 year of age in children with (n = 30) and without IgE-mediated food allergy (n = 30). We employed a low-level soluble anti-CD3 stimulus to activate the T-cell receptor (TCR) and surveyed gene expression by DNA microarray in purified CD4+ T-cells. Allergen-specific responses were assessed in parallel functional studies. Results: At birth, the allergic group showed a reduced number of genes up regulated in response to anti-CD3 treatment on the microarray and a reduced lympho proliferative capacity, suggesting clear differences in T-cell signalling pathways. Polymerase chain reaction (PCR) validation of candidate genes confirmed significantly lower expression of a number of genes in the allergic group including RELB, NFKB2, LIF and FAS. By 12 months of age, there were marked changes in the anti-CD3 response in all infants, culminating in upregulation of cytokine genes (IL-5, IL-13, IL-17 and IL-22). Neonatal differences were no longer apparent. Instead, the allergic group, all symptomatic by this age, showed differential expression of T-cell lineage pathways including GATA-3, MAL and FcER1 in unstimulated T-cells. Allergen stimulation induced significantly higher cytokines production (IL-5, IL-13 and IFNγ) in the allergic group. Conclusion: Although transient, suboptimal neonatal T-cell activation pathways that signal through the NF-κB complex may affect the developmental transition of T-cell phenotypes in the periphery shortly after birth and may increase the risk of food allergy.

Mounting evidence suggests that helminth infections protect against autoimmune diseases. As helminths cause chronic IgE-mediated activation of basophils and mast cells we hypothesized that continuous activation of these cells could prevent diabetes onset in nonobese diabetic (NOD) mice in the absence of infection. Anti-FceR1 activated basophils and mast cells and resulted in the release of IL-4 and histamine into the bloodstream. Anti-FceR1-treated NOD mice showed a type 2 shift in insulin-specific antibody production and exhibited significant delays in diabetes onset. IL-4 responses played a partial role as the protective effect of anti-FceR1 therapy was diminished in IL-4-deficient NOD mice. In contrast, histamine signaling was not required as anti-FceR1-mediated protection was not reduced in mice treated with histamine receptor blockers. These results demonstrate that anti-FceR1 therapy delays diabetes onset in NOD mice and suggest that chronic basophil and mast cell activation may represent a new avenue of therapy for Th1-associated autoimmune diseases.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam 3 CSK 4 and MALP-2, on Ag (DNP–HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam 3 CSK 4 , but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam 3 CSK 4 , most probably by electrostatic forces, reduced the level of active DNP–HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE + Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam 3 CSK 4 indicate that one must be cautious when interpretating results obtained with “model” substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcɛR1) and TLR2 are examined.

Aim: QGE031 is a high-affinity humanized anti-IgE antibody in development for treatment of uncontrolled severe asthma. We predicted steady-state dose responses by fitting a mathematical model to PKPD data. Methods: Subjects with mild, atopic asthma (N=37) were randomised to 3 s.c. doses (240mg,N=8; 72mg,N=8; 24mg,N=8) of QGE031 every 2 weeks (wks) for 12wks, Omalizumab (OMA) (N=6; US dosing table) or placebo (N=7). Allergen challenges were conducted 6, 12 and 18 wks after the first dose. Mathematical functions were fitted to full time course drug, total IgE and basophil data. After curve fitting, 0-6 month responses of ∼1000 patients, each receiving an array of 4 wkly administrations of QGE031 (0-1200mg) and OMA (US dosing table), were simulated. Results: The model fitted the response data, down regulation of basophil FceR1 and surface IgE, and inhibition of bronchial and skin allergen reactivity from subjects treated with QGE031 and OMA. QGE031 36mg, dosed every 4 wks, was predicted to give similar skin wheal and allergen PC15 responses to OMA. Higher doses of QGE031 would be required for patients with higher baseline IgE. Conclusion: QGE031 is more effective than OMA in inhibiting bronchial and skin allergen response in a clinical setting. This mathematical model could allow better planning and prediction of future trial outcomes.

Basophils are, together with mast cells, typical innate effector cells of allergen-induced IgE-dependent allergic diseases. Both cell types express the high-affinity receptor for IgE (FceR1), release histamine, inflammatory mediators, and cytokines following FceR1 cross-linking. Basophils are rare granulocytes in blood, lymphoid, and non-lymphoid tissues, and the difficulties to detect and isolate these cells has hampered the study of their biology and the understanding of their possible role in pathology. Furthermore, the existence of other FceR1-expressing cells, including professional Ag-presenting dendritic cells, generated some controversy regarding the ability of basophils to express MHC Class II molecules, present Ag and drive naive T cell differentiation into Th2 cells. The focus of this review is to present the recent advances on the interactions between basophils and peripheral blood and tissue memory Th1, Th2, and Th17 cells, as well as their potential role in IgE-independent non-allergic chronic inflammatory disorders, including human inflammatory bowel diseases. Basophils interactions with the innate players of IgE-dependent allergic inflammation, particularly innate lymphoid cells, will also be considered. The previously unrecognized function for basophils in skewing adaptive immune responses opens novel perspectives for the understanding of their contribution to the pathogenesis of inflammatory diseases.

Here we report the optimization of small molecule inhibitors of human mast cell degranulation via anti-IgE-mediated tryptase release following cross-linking and activation of IgE-loaded FceR1 receptors. The compounds are selective upstream inhibitors of FceR1-dependent human mast cell degranulation and proved to be devoid of activity in downstream ionomycin mediated degranulation. Structure–activity relationship (SAR) leading to compound 26 is outlined.

Summary Background and objective The MS4A2 gene, the b chain of the high-affinity receptor for immunoglobulin (Ig)E, has previously been linked to atopy and asthma. The b-chain of FceR1 enhances receptor maturation and signal transduction capacity, leading to the release of proinflammatory mediators and cytokines that can exacerbate the symptom of asthma. This study was performed to evaluate whether two genetic polymorphisms of the FceR1b gene (FceR1b � 109T4C and FceR1b E237G) are associated with aspirin-intolerant asthma (AIA). Methods The MS4A2 gene polymorphisms (FceR1b � 109T4C and FceR1b E237G) were determined by SNP-IT TM assays in patients with AIA (N=164), aspirin-tolerant asthma (ATA, N=144) and normal controls (NC, N=264) recruited from a Korean population. Results The genotype frequencies of FceR1b � 109T4C and E237G polymorphisms were not significantly associated with the pathogenesis of AIA. However, FceR1b � 109T4C polymorphism was significantly associated with the presence of specific IgE to Staphylococcal enterotoxin B (SEB); the number of subjects carrying both homozygous TT genotype of FceR1b � 109T4C and specific IgE to SEB was significantly higher in the AIA group when compared with the other control groups (P=0.01, odds ratio (OR)=7.723, 95% confidence interval (CI)=1.327‐39.860 for AIAvs. ATA; P=0.02, OR=6.364, 95% CI=1.149� 35.229 for AIA vs. NC). In addition, luciferase reporter assays also showed that the FceR1b � 109T allele was associated with higher promoter activity of MS4A2 in both RBL-2H3 and A549 cell lines. Conclusion FceR1b � 109T4C polymorphism may increase expression of MS4A2 by mast cells, leading to enhanced release of proinflammatory mediators in the asthmatic airway, contributing to increased susceptibility to AIA.

Omalizumab (Xolair®, Novartis) is the first biologic agent introduced to specifically manage allergic disease. It is a recombinant DNA-derived humanized monoclonal immunogobulin (Ig)G1 antibody with unique antihuman IgE binding specificities. Dosed every 2 to 4 weeks subcutaneously, it has the ability to neutralize free IgE in the blood and interstitial space in less than a day. It almost completely downregulates the high-affinity IgE receptors on basophils and mast cells over a period of several weeks. In multiple Phase II and III clinical studies, omalizumab has been shown to be safe and efficacious for treating allergic asthma and has been approved in Australia and the USA for this indication. It has proven effective in allergic rhinitis, latex sensitivity and peanut sensitivity and may be used in combination with standard allergy immunotherapy, providing a safer and more efficacious approach. Numerous clinical studies are in progress evaluating its use beyond allergic asthma.

Human basophils are potent producers of IL-4 following cross-linking of the high affinity receptor for IgE (Fc epsilon R1). Elevated levels of both total- and malaria-specific IgE have been demonstrated in sera from people living in malaria-endemic regions. Whether or not these IgE antibodies are pathogenic is unclear. Serum containing high IgE levels obtained from malaria individuals was used to establish whether IgE-immune complexes could induce IL-4 production in human basophils. The basophils, obtained from healthy donors, were primed with 10 ng/ml of IL-3 before being transferred to wells containing goat anti-human IgE or human antimalarial IgE-immune complexes. IL-4 was induced upon stimulation of human basophils by plate bound IgE-containing immune complexes. Basophils treated similarly but with goat anti-IgG/human antimalarial- IgG-immune complexes did not secrete IL-4. Similarly mononuclear cells depleted of basophils in parallel culture did not secrete IL-4. Thus, human basophils may contribute to the polarization of T-helper type 2 in the (Th2) responses in malaria hosts via IgE-induced IL-4 production.

Atopic disorders are on the increase in the Western world and are due, at least in part, to an overactive mast cell response. A better understanding of the intracellular signalling pathways that regulate both mast cell degranulation and the secretion of arachidonic acid metabolites and inflammatory cytokines could help in the treatment of these disorders. The src homology 2-containing inositol-polyphosphate 5'-phosphatase, SHIP, has been shown to be a key 'gatekeeper' of mast cell degranulation. SHIP prevents degranulation from occuring when IgE alone binds to the high-affinity receptor for IgE (FcvarepsilonR1), SHIP restrains it when IgE-bound FcvarepsilonR1 are engaged by multivalent allergens, and SHIP inhibits it when an IgG against the same allergen co-clusters the inhibitory low-affinity receptor for IgG (FcgammaRIIB) with the IgE receptor. SHIP acts as a negative regulator of degranulation by hydrolyzing phosphatidylinositol-3,4,5-trisphosphate, a second messenger generated in activated cells by phosphatidylinositol 3-kinase. Our finding that binding of only IgE to the FcvarepsilonR1 of SHIP-deficient mast cells results in massive degranulation, led us to investigate the signalling pathways that are triggered in normal murine bone marrow-derived mast cells by monomeric IgE. We report here that monomeric IgE activates signalling pathways resulting in mast cell survival, without stimulating degranulation or proliferation. These studies demonstrate that mast cell sensitization by IgE is an active rather than a passive process.

Omalizumab (Xolair(®)) is a humanized, recombinant, IgG, anti-IgE monoclonal antibody that binds to the Fc region of free IgE and prevents it from binding to its high-affinity receptor (FcεR1) on mast cells and basophils. This reduction in free IgE leads to a reduction in mast cell/basophil degranulation and the release of histamine, and to the down-regulation of FcεR1 receptors on these cells. Omalizumab does not bind to cell-bound IgE or to IgG. In well-controlled clinical trials in patients with chronic spontaneous urticaria and persistent symptoms despite background treatment with antihistamines, add-on therapy with subcutaneous omalizumab 300 mg every 4 weeks for 12 or 24 weeks significantly reduced the severity of itching, and the number and size of hives, and increased patients' health-related quality of life and the proportion of days free from angioedema compared with placebo. Subcutaneous omalizumab was generally well tolerated; the incidence and severity of adverse events in omalizumab recipients were similar to those in placebo recipients, and most adverse events were of mild or moderate severity. The only adverse events occurring more frequently with omalizumab than with placebo during treatment in a safety study were headache and upper respiratory tract infection. Thus, omalizumab is an effective and well-tolerated add-on therapy in patients with chronic spontaneous urticaria who are symptomatic despite background therapy with H1 antihistamines.

Immunoglobulin E (IgE) activates mast cells (MCs). It remains unknown whether IgE also activates other inflammatory cells, and contributes to the pathogenesis of abdominal aortic aneurysms (AAAs). This study demonstrates that CD4+ T cells express IgE receptor FcεR1, at much higher levels than do CD8+ T cells. IgE induces CD4+ T-cell production of IL6 and IFN-γ, but reduces their production of IL10. FcεR1 deficiency (Fcer1a-/-) protects apolipoprotein E-deficient (Apoe-/-) mice from angiotensin-II infusion-induced AAAs and reduces plasma IL6 levels. Adoptive transfer of CD4+ T cells (but not CD8+ T cells), MCs, and macrophages from Apoe-/- mice, but not those from Apoe-/- Fcer1a-/- mice, increases AAA size and plasma IL6 in Apoe-/- Fcer1a-/- recipient mice. Biweekly intravenous administration of an anti-IgE monoclonal antibody ablated plasma IgE and reduced AAAs in Apoe-/- mice. Patients with AAAs had significantly higher plasma IgE levels than those without AAAs. This study establishes an important role of IgE in AAA pathogenesis by activating CD4+ T cells, MCs, and macrophages and supports consideration of neutralizing plasma IgE in the therapeutics of human AAAs.

Background: In 2003, this laboratory published an account of the human mast cell line LAD2 (Laboratory of Allergic Diseases 2) that expressed FcεRI, responded to recombinant human stem cell factor (rhSCF) and resembled CD34+-derived human mast cells. LAD2 cells have now been distributed worldwide. To study the impact of this transfer, we analyzed the number of investigators receiving LAD2 cells and resulting publications. Methods: Records maintained in our laboratory, the Technology Transfer and Intellectual Property Office and Office of Technology Transfer, were reviewed for material transfer agreements (MTAs) and licensing agreements (LAs). Journals and impact factors were obtained from PubMed.gov by cross-referencing LAD2 and human mast cells from 2003 through November 2013. Results: Over 300 MTAs and 40 LAs were approved. LAD2 cells were shipped to over 30 countries. More than 80 papers have been published in journals with impact factors from 1.31 to 13.21. Intended uses include the study of receptors, degranulation, and cell signaling. LAD2 cells continue to express described markers and have consistent FcεR1-mediated degranulation. Conclusions: Success of the LAD2 line reflects the demand for a human mast cell line in research, the uniqueness of this cell line, and that it continues to exhibit minimal variation from its original description. We hope that the awareness of the impact of this cell line on mast cell research will encourage others to develop and distribute other similar cell lines with additional characteristics so as to address the limitations of depending on the study of cultured human mast cells from tissues.

The high affinity receptor for immunoglobulin E (designated Fc epsilon RI) is the member of the antigen (Ag) receptor superfamily responsible for linking pathogen-or allergen-specific IgEs with cellular immunologic effector functions. This review provides background information on Fc epsilon RI function combined with more detailed summaries of recent progress in understanding specific aspects of Fc epsilon RI biology and biochemistry. Topics covered include the coordination and function of the large multiprotein signaling complexes that are assembled when Fc epsilon RI and other Ag receptors are engaged, new information on human receptor structures and tissue distribution, and the role of the FcR beta chain in signaling and its potential contribution to atopic phenotypes.

We investigated the binding of IgE and different types of allergen–IgE complexes to CD23-expressing human B cells. We performed the experiments using chimeric Bip 1 (CB1), a chimeric humanized IgE specific for the major birch allergen, Bet v 1, together with monomeric and oligomeric forms of recombinant Bet v 1 (rBet v 1), and Bet v 1-specific IgG antibodies. In this model IgE binding to CD23 was independent of variations in antibody affinities towards monomeric and oligomeric Bet v 1 as demonstrated by plasmon surface resonance. CB1 alone or in the form of small immune complexes consisting of one molecule of CB1 plus allergen, showed comparable binding to CD23 on B cells. Using anti-IgE antibody probes discriminating CD23-bound from CD23-unbound IgE, it is demonstrated that in large immune complexes obtained with oligomeric Bet v 1 or by super-crosslinking of small immune complexes with Bet v 1-specific IgG, anti-IgE staining of B cells increased. This increase of staining was due to the presence of IgE antibodies in the immune complexes that were not directly engaged in CD23 binding, and thus available for IgE detection. Our study thus reveals that CD23 can bind in a comparable manner to free IgE and IgE–allergen complexes of different size and composition, which may also include allergen-specific IgG. The interplay of free IgE with IgE–allergen immune complexes of different sizes and composition with CD23 binding represents a mechanism for the modulation of CD23-mediated immune responses such as IgE-facilitated allergen presentation in allergic diseases.

Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CDw17+ cells. Basophil purity was 99.5 ± 0.4% (range 98.7–99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with solCD117 c -kit mAb. Mast cell purity was 99.4 ± 0.7% (range: 97.5–99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (FceR1) α chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.

The purpose of this review is to collate current knowledge and recent advances in molecular mechanism behind the immediate type hypersensitivity of foods. Food allergy is a growing concern of human health in developed as well as developing countries now days. Food allergic reactions are mostly IgE mediated and also known as immediate type hypersensitivity or type I reaction. This review encompasses a wide range of molecular events during IgE mediated reactions like primary exposure of allergens, processing of allergens by antigen presenting cells, role of transcription factors like GATA-3, STAT-6, NF-AT, c-maf, c-kit and NF-κB, Treg cells, toll like receptors, cytokines and chemokines, class switch to IgE, FceR1 receptor, priming of IgE on mast cells or basophils, signaling events followed by secondary exposure of allergens, degranulation and release of mediators like leukotrienes, histamines, prostaglandins, β-hexosaminidase and ultimately anaphylaxis. This review may be helpful to beginners as well as experts working in the field of allergy and immunology because of the stepwise explanations of molecular mechanisms involved in IgE mediated reactions.

IgE antibodies are involved not only in the immune defense against parasites, but also in the pathogenesis of allergic diseases and anaphylaxis. Allergen-induced crosslinking of IgE bound to FceR1 on mast cells triggers degranulation and may initiate an allergic reaction.1 Although IgE is a major player in allergic responses, IgE regulation and the molecular pathway underlying IgE+ B-cell maturation remains elusive. In an exciting report published in the April issue of Nature Immunology, Talay and colleagues demonstrate a new model for IgE switching and memory in vivo.2 In contrast to the current paradigm, IgE+ memory B cells and plasma cells develop through a germinal-center (GC) IgE+ intermediate, without the need of a transitional IgG1 phase. Furthermore, IgE+ memory B cells, and not IgG1+ memory B cells, are the source of cellular IgE memory. The typical immune response to T cell-dependent antigens starts with B-cell differentiation into short-lived plasma cells that produce low-affinity antibodies, followed by the formation of GCs that are the origin of both memory B cells and long-lived plasma cells that produce high-affinity antibodies.3 Previous studies reported a pathway of IgE B-cell maturation that is distinct from the classical model of B-cell differentiation.4,5 IgE+ B cells were not found in GCs nevertheless differentiated into high-affinity plasma cells. It was proposed that GC IgG1+ intermediates switched to IgE+ plasma cells outside the GCs.4 This model was strengthened by the observation that IgG1-deficient mice were impaired in producing high-affinity IgE.5 Moreover, cellular IgE memory was reported to reside in IgG1+ memory B cells which undergo a sequential switch to IgE after re-exposure to antigen.4 IgE differentiation is difficult to study since IgE-expressing B cells are very rare under physiological conditions and the detection of IgE+ cells by antibody staining is complex as cells expressing Fce receptors can bind IgE.1 The previously described detection method, which consists of acid treatment to remove serum IgE bound to CD23, followed by staining with anti-IgE, may lack the sensitivity needed to detect the low surface expression of IgE on IgE+ GC B cells. In the current study by Talay and colleagues, this problem is circumvented by the use of sensitive IgE reporter mice. Here, a bicistronic reporter gene encoding-enhanced green fluorescent protein (GFP) is inserted into the murine locus encoding IgE downstream of the cytoplasmic domain of membrane IgE. This elegant method allows for the specific and direct detection of both IgE-switched B cells and plasma cells. After validation of the reporter, they investigate IgE switching and memory using two models: infection of mice with the helminth Nocardia brasiliensis and a classical immunization with TNP-ovalbumin, which both result in a strong T helper 2 response associated with specific IgG1 and IgE production. IgE-switched cells, identified as GFPhi B cells, are found in lymph nodes (mediastinal and mesenteric) and spleen 7–10 days after infection. Talay et al. demonstrate that almost 100% of the GFPhi cells display a GC B-cell phenotype (B220+IgD−GL7+CD95+) at day 10, which decreases to 60%–80% of GFPhi cells at day 35 after infection. Additionally, a small population of IgE+ memory B cells (B220+IgD−GL7−CD38hi) is detected. These results are in contrast to previous studies in which IgE+ cells were not found in GCs and had plasma cell characteristics. Talay and colleagues reveal that 15%–20% of all plasma cells (B220−CD138+) in the lymph node and 3% of plasma cells in the spleen are IgE+ 15 days after infection. Remarkably, in the bone marrow only 1% of all plasma cells are IgE+ at day 35, which suggests that IgE+ plasma cells are rather short-lived. Their findings are strengthened by confirming the results in the TNP-ovalbumin model. In line with the flow cytometry data, the authors find GFP+ cells in IgD−GL7+ GC structures in lymph nodes of infected mice by immunofluorescence microscopy. IgE+CD138+ plasma cells are located distant from GCs and IgE+ memory B cells are found in clusters next to GL7+ GCs. To validate that cellular IgE memory resides within IgE+ memory cells and to rule out a role for IgG1+ memory cells, adoptive transfer experiments were conducted. GFPhiCD38hi memory B cells were sorted from the reporter mice and transferred into B cell-deficient mice. They demonstrate a potent IgE serum response in the recipient mice starting 5 days after infection, with similar kinetics as a secondary infection in intact IgE reporter mice. Importantly, in mice that received sorted B220+IgD−GL7−CD38hiGFPint-neg non-IgE memory cells, no IgE serum response is generated after infection with N. brasiliensis. Therefore, a putative role for IgG1+ memory cells as the source of IgE memory can be excluded. A recent study by Yang et al., using different IgE reporter mice, confirms that IgE+ B cells differentiate into GC B cells.6 However, the presence of IgE+ B cells in the GC is transient, and IgE+ plasma cells show reduced affinity maturation compared to IgG1+ plasma cells. They conclude that IgE+ B cells show a bias toward differentiation into short-lived plasma cells. This fits with the study by Talay et al. considering the kinetics of serum IgE and the very low number of IgE+ plasma cells found in the bone marrow. The skewed differentiation into short-lived plasma cells may provide a mechanism to prevent aberrant IgE antibody production and systemic anaphylaxis. The new model of IgE production by Talay and colleagues provides us with a deeper understanding of IgE biology (Figure 1), and may reveal new opportunities for the prevention of allergic responses. Figure 1 A new model for IgE plasma-cell and memory B-cell differentiation. After T cell-dependent activation, naive IgM+ B cells switch to IgE and enter the germinal-center reaction. Germinal-center IgE+ B cells (B220+IgD−GL7+ ...

KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. Disclosures No relevant conflicts of interest to declare.

Background Previous studies have shown that constitutive and IgE-mediated histamine production by human lung mast cells is inhibited by a transferable factor produced by the airway epithelium. We have tested the hypothesis that a similar interaction exists between epithelial cell and mast cell lines. We have also investigated the effect of co-culture of epithelial cell lines and TH2 cells on interleukin (IL)-13 production. Methods A549 or BEAS-2B cells were grown to confluence overnight. Media was removed and LAD2, HMC1.2 or human-derived ex-vivo T-cells added for 16 h. For transwell experiments epithelial cells were added to a 24-well plate, replaced with fresh media after 16 h and mast cells media added to the insert, maintaining the mast cell/epithelium/volume ratio. Wells, and transwell insert media, were then centrifuged, supernatants harvested and mediator release quantified by histamine or IL-13 ELISA. Results Neither mast cell line consistently produced histamine in response to IgE and anti-IgE. Flow cytometry suggested that this was due to absence of the high-affinity IgE receptor FceR1. Constitutive histamine production by HMC1.2 was reduced from 191 ± 13 ng/106 cells by 60.9% (95% CI 54.1, 67.8; p < 0.0001) when co-cultured with A549 and 21% (95% CI 14.2, 28.1; p < 0.0001) with BEAS-2B cells. Similar findings were seen with the LAD2 mast cell line. Constitutive IL-13 production by TH2 cells was reduced from 18000 ± 1800 pg/106 cells by 68.6% (95% CI 62.0, 75.1; P < 0.0001) by A549 and 59.9% (95% CI 53.3, 66.5; p < 0.0001) by BEAS-2B. Epithelial inhibition was similar when cells were separated by a transwell suggesting involvement of a transferable factor. ![Abstract P103 Figure 1][1] Abstract P103 Figure 1 -hour constitutive histamine (left) and IL-13 (right) release from mast or T-cells in the presence or absence of epithelial cell lines Conclusion Epithelial cell lines inhibit a range of asthma-related immunological responses, probably by producing an inhibitory substance. [1]: pending:yes

In mast cells, crosslinking of IgE bound to the FceR1 receptor with multivalent antigen initiates cell activation and consequent inflammatory responses. Clustered IgE-FceR1 interacts with early signaling partners such as Lyn, Syk, and LAT, initiating a signaling cascade. Our previous scanning electron microscopy (SEM) studies quantified the nanoscale co-redistribution of FcRI with Lyn, Syk, and LAT within the plasma membrane upon antigen stimulation. In this study, we use stochastic optical reconstruction microscopy (STORM) to localize proteins on the cell surface with sub-diffraction (∼20nm half width) resolution using a simple fluorescence microscope set up with TIRF illumination. We present, in snapshots, the time-dependence of FcRI reorganization after antigen addition in chemically fixed cells and quantify the extent of clustering using the pair autocorrelation function. The results of STORM measurements agree well with conclusions from SEM experiments. With multi-color STORM, we image the distributions of multiple protein species on the same cell and calculate the degree of protein co-clustering, before and after antigen stimulation, using the pair cross-correlation function. Ongoing experiments are aimed at quantifying the co-redistribution of FcRI with early signaling partners. We have also extended this technique to imaging living cells at room temperature to measure FceRI redistribution in real time and study the organization and dynamics of proteins undergoing stimulated responses. Our results confirm that STORM is an effective tool for quantitative imaging of cellular components at the nanoscale, and our ongoing studies are providing new information to clarify the physical basis for spatial assembly of specific proteins in the plasma membrane during early signaling events.

RATIONALE: Benralizumab is a humanized, afucosylated, anti-interleukin-5 receptor alpha (IL-5Ra) monoclonal antibody with enhanced antibody-dependent cell cytotoxicity that has been shown to reduce blood and airway eosinophils in early-phase clinical investigations. Basophils express IL-5Ra, are associated with asthma, and could be a potential target for benralizumab. The aim of this analysis was to determine whether benralizumab reduced the number of blood basophils in asthma. METHODS: MI-CP220 (NCT01238861) was a 52 week, double-blind, placebo controlled phase 2 study to evaluate the efficacy and safety of benralizumab (2, 20 and 100mg administered subcutaneously on weeks 1, 4, 8, 16, 24, 32, and 40) in 606 adults with uncontrolled asthma. In an exploratory analysis, blood basophils were assessed by flow cytometry using FCeR1 expression paired with CRTH2, CD123, and CCR3 at week -1 (baseline) and week 52. RESULTS: The mean baseline absolute basophil counts (N = 549 subjects) were 45.2 (placebo), 51.2 (2mg), 54.7 (20mg) and 48.7 (100mg) cells/mm 3 . Mean basophil counts (N = 451 subjects) decreased following 20 or 100mg benralizumab at week 52 relative to baseline- percent change from baseline was: placebo (+16.3%), 2mg (-4.17%, p = 0.152), 20mg (-46.97%, p = 0.039), and 100mg (-60.3%, p 1 . CONCLUSIONS: In adults with uncontrolled asthma, benralizumab reduced blood basophil counts. Benralizumab holds promise as a novel therapy for asthma by removing two key cell types, eosinophils and basophils, associated with asthma pathobiology and warrants continued investigation.

The manifestation of atopic dermatitis (AD) results from a complex interaction of environmental factors, skin barrier defects and immunological phenomena. The initial events in AD remain unclear while much progress has been made in identifying the mechanisms leading to the manifestation and maintenance of allergic inflammation. AD pathogenesis can be approached from many different points of view. In this review, we describe findings in mouse models and in humans separately. We focus on recent findings that underscore the importance of Langerhans cells in initial mechanisms of skin inflammation in AD.

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.

Tissue-resident human mast cells may modulate tissue remodeling by the secretion of angiogenin, a potent angiogenic factor. ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated in tumor growth, heart failure, wound healing, asthma, and the composition of the adult gut microflora. Human mast cells (HuMC) are similarly associated with modulation of vascular permeability, angiogenic processes, wound healing, and asthma. We hypothesized that HuMC express and secrete ANG in response to divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or neuropeptides and following FcεRI aggregation. Expression and production of ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC constitutively expressed ANG mRNA and that it was up-regulated by exposure to E. coli. Activation of HuMC by FcεRI aggregation resulted in release of small amounts of ANG (160 pg/mL ANG. These observations demonstrate that HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived ANG is available in the subsequent inflammatory response.

Asthma is an inflammatory and remodeling disorder of the conducting airways, which contract too much and too easily spontaneously and in response to a wide range of exogenous and endogenous stimuli. This airway hyperresponsiveness is accompanied by enhanced sensory irritability and increased mucus secretion. The different clinical expressions of asthma involve varying environmental factors that interact with the airways to excite both acute and chronic inflammation as well as the varying pathophysiological contributions that smooth muscle contraction, edema and remodeling contribute to disease expression (Fig. 1). Asthma is a heterogeneous disorder with multiple clinical sub-phenotypes that have different interactions with the environment, responses to treatment and outcomes. Overall, asthma is a good example of gene–environment interactions, although no single gene or environmental factor accounts for the disease. The great majority of asthma is associated with atopy but while over 40% of the population in the developed world is atopic, only 7–11% expresses this as asthma. In order to explain this paradox, in addition to allergy, there must be important factors that translate the atopic phenotype into the lower airways to manifest this as Th-2-type airway inflammation. Asthma is a common inflammatory disorder of the airway that classically involve allergen driven Th-2 lymphocyte polarisation with coordinate production of IL-3, IL-4, IL-5, IL-9, GM-CSF, and IL-13 which are all encoded in a gene cluster on chromosome 5 q 31–34 [1]. These cytokines drive the allergic inflammatory response through the recruitment and activation of T cells, eosinophils, basophils, and monocyte/macrophages. Interleukins-4, -9, and -13 in the presence of MHC-Class II-restricted allergen presentation by T cells and CD40 or OX40 costimulation causes enhanced IgE production with subsequent sensitization of mast cells and basophils through the binding of allergen-specific IgE to high-affinity receptors (FceR1). In the case of nonallergic or intrinsic asthma, Th-2 pathways are dominant but IgE production is either localized to the airways [2, 3] or is no longer prominent. Many aspects of the inhaled environment influence the maturational direction of the subsequent T cell

The receptor with high affinity for immunoglobulin E (FcERI) is a key molecule in triggering the allergic reaction. It isα tetrameric complex of oneα subunit, oneβ subunit, and two disulfide-linkedγ subunits. This receptor is present exclusively on mast cells and basophils. Molecules identical to theγ subunit of FceRI also formα cell surface complex with other Fc receptors such as mouse FcγRIIa in macrophages and most probably with human FcγRIII (CD16) in natural killer (NK) cells. Here we show by in situ hybridization that the human genes for theα (FCER1A) and γ subunits (FCER1 G) of FcERI and the gene for FcγRIII (FCGR3, CD16) are located on human chromosome band 1823.

Protein kinase (PK) C-epsilon is strongly expressed in mast cells (MCs) and activated in response to antigen-mediated high-affinity receptor for IgE (Fc epsilonR1) engagement. A critical role of PKC-epsilon in antigen-triggered activation of various signaling pathways was observed in basophilic leukemia cells. To study the function of PKC-epsilon in MCs differentiated in vitro from murine bone marrow, we used our established PKC-epsilon null mice. Unexpectedly, we did not reveal any difference in antigen-induced activation of many central signaling molecules (PKB, mitogen-activated protein kinase, p38, Jun-N-terminal kinase, phospholipase C-gamma1, Bruton's tyrosine kinase, PKD, Fos and PKC-delta) in time-course as well as dose-response studies between PKC-epsilon-deficient and wild-type MCs. In correlation, antigen-triggered degranulation, release of arachidonic acid and secretion of IL-6 were unaltered by the loss of PKC-epsilon. Furthermore, stimulation of MCs via different receptor systems [Steel factor receptor (c-kit) and toll-like receptor 4] did not lead to differences in the measured responses between both cell types. These results strongly suggest that PKC-epsilon plays a redundant role in MCs stimulated by antigen as well as other well-known MC stimuli.