Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

IgE receptor of mast cells signals mediator release and inflammation via adaptor protein 14-3-3ζ.

BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.

Targeting interleukin-33 and thymic stromal lymphopoietin pathways for novel pulmonary therapeutics in asthma and COPD.

Interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) are alarmins that are released upon airway epithelial injury from insults such as viruses and cigarette smoke, and play critical roles in the activation of immune cell populations such as mast cells, eosinophils and group 2 innate lymphoid cells. Both cytokines were previously understood to primarily drive type 2 (T2) inflammation, but there is emerging evidence for a role for these alarmins to additionally mediate non-T2 inflammation, with recent clinical trial data in asthma and COPD cohorts with non-T2 inflammation providing support. Currently available treatments for both COPD and asthma provide symptomatic relief with disease control, improving lung function and reducing exacerbation rates; however, there still remains an unmet need for further improving lung function and reducing exacerbations, particularly for those not responsive to currently available treatments. The epithelial cytokines/alarmins are involved in exacerbations; biologics targeting TSLP and IL-33 have been shown to reduce exacerbations in moderate-to-severe asthma, either in a broad population or in specific subgroups, respectively. For COPD, while there is clinical evidence for IL-33 blockade impacting exacerbations in COPD, clinical data from anti-TSLP therapies is awaited. Clinical data to date support an acceptable safety profile for patients with airway diseases for both anti-IL-33 and anti-TSLP antibodies in development. We examine the roles of IL-33 and TSLP, their potential use as drug targets, and the evidence for target patient populations for COPD and asthma, together with ongoing and future trials focused on these targets.

Phase 2 randomized clinical trial of astegolimab in patients with moderate to severe atopic dermatitis.

BACKGROUND: The binding of IL-33 to its receptor ST2 (alias of IL1RL1) leads to the release of inflammatory mediators and may play a role in the pathogenesis of atopic dermatitis. Astegolimab is a fully human, IgG2 mAb that binds to ST2 and inhibits IL-33 signaling. OBJECTIVES: This study sought to assess the efficacy, safety, and pharmacokinetics of astegolimab in patients with atopic dermatitis. METHODS: This was a randomized, placebo-controlled, phase 2 study in which adults with chronic atopic dermatitis were randomized 1:1 to receive astegolimab 490 mg every 4 weeks or placebo, for 16 weeks. The primary outcome was the percentage of change from baseline to week 16 of the Eczema Area and Severity Index score. RESULTS: A total of 65 patients were enrolled in the study (placebo, n = 32; astegolimab, n = 33). The adjusted mean percentage of change from baseline to week 16 in the Eczema Area and Severity Index score was -51.47% for astegolimab compared with -58.24% for placebo, with a nonsignificant treatment difference of 6.77% (95% CI: -16.57-30.11; P = .5624). No differences were observed between treatment groups for secondary efficacy outcomes and in exploratory biomarkers (blood eosinophils, serum IL-5, serum CCL13). With the use of loading dose, pharmacokinetic exposure was sufficient from week 1. Astegolimab was well-tolerated, with a safety profile consistent with that observed in previous clinical trials. CONCLUSIONS: In patients with atopic dermatitis, astegolimab did not show a significant difference compared to placebo for the primary or secondary outcomes.

Effects of inhaled JAK inhibitor GDC-4379 on exhaled nitric oxide and peripheral biomarkers of inflammation.

BACKGROUND: Janus Kinases (JAKs) mediate activity of many asthma-relevant cytokines. GDC-0214, an inhaled small molecule JAK1 inhibitor, has previously been shown to reduce fractional exhaled nitric oxide (FeNO) in patients with mild asthma, but required an excessive number of inhalations. AIM: To assess whether GDC-4379, a new inhaled JAK inhibitor, reduces FeNO and peripheral biomarkers of inflammation. METHODS: This study assessed the activity of GDC-4379 in a double-blind, randomized, placebo-controlled, Phase 1 study in patients with mild asthma. Participants included adults (18-65y) with a diagnosis of asthma for ≥6 months, forced expiratory volume in 1 s (FEV1)> 70% predicted, FeNO >40 ppb, using as-needed short-acting beta-agonist medication only. Four sequential, 14-day, ascending-dose cohorts (10 mg QD, 30 mg QD, 40 mg BID, and 80 mg QD) of 12 participants each were randomized 2:1 to GDC-4379 or placebo. The primary activity outcome was percent change from baseline (CFB) in FeNO to Day 14 compared to the pooled placebo group. Safety, tolerability, pharmacokinetics, and pharmacodynamic biomarkers, including blood eosinophils, serum CCL17, and serum CCL18, were also assessed. RESULTS: Of 48 enrolled participants, the mean age was 25 years and 54% were female. Median (range) FeNO at baseline was 79 (41-222) ppb. GDC-4379 treatment led to dose-dependent reductions in FeNO. Compared to placebo, mean (95% CI) percent CFB in FeNO to Day 14 was: -6 (-43, 32) at 10 mg QD, -26 (-53, 2) at 30 mg QD, -55 (-78, -32) at 40 mg BID and -52 (-72, -32) at 80 mg QD. Dose-dependent reductions in blood eosinophils and serum CCL17 were also observed. Higher plasma drug concentrations corresponded with greater FeNO reductions. No serious AEs occurred. The majority of AEs were mild to moderate. The most common AEs were headache and oropharyngeal pain. Minor changes in neutrophils were noted at 80 mg QD, but were not considered clinically meaningful. CONCLUSIONS: In patients with mild asthma, 14-day treatment with GDC-4379 reduced FeNO levels and peripheral biomarkers of inflammation. Treatment was well tolerated without any major safety concerns. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12619000227190.

Astegolimab, an anti-ST2, in chronic obstructive pulmonary disease (COPD-ST2OP): a phase 2a, placebo-controlled trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous inflammatory airway disease. The epithelial-derived IL-33 and its receptor ST2 have been implicated in airway inflammation and infection. We aimed to determine whether astegolimab, a selective ST2 IgG2 monoclonal antibody, reduces exacerbations in COPD. METHODS: COPD-ST2OP was a single-centre, randomised, double-blinded, placebo-controlled phase 2a trial in moderate-to-very severe COPD. Participants were randomly assigned (1:1) with a web-based system to received 490 mg subcutaneous astegolimab or subcutaneous placebo, every 4 weeks for 44 weeks. The primary endpoint was exacerbation rate assessed for 48 weeks assessed with a negative binomial count model in the intention-to-treat population, with prespecified subgroup analysis by baseline blood eosinophil count. The model was the number of exacerbations over the 48-week treatment period, with treatment group as a covariate. Safety was assessed in the whole study population until week 60. Secondary endpoints included Saint George's Respiratory Questionnaire for COPD (SGRQ-C), FEV1, and blood and sputum cell counts. The trial was registered with ClinicalTrials.gov, NCT03615040. FINDINGS: The exacerbation rate at 48 weeks in the intention-to-treat analysis was not significantly different between the astegolimab group (2·18 [95% CI 1·59 to 2·78]) and the placebo group (2·81 [2·05 to 3·58]; rate ratio 0·78 [95% CI 0·53 to 1·14]; p=0·19]). In the prespecified analysis stratifying patients by blood eosinophil count, patients with 170 or fewer cells per µL had 0·69 exacerbations (0·39 to 1·21), whereas those with more than 170 cells per µL had 0·83 exacerbations (0·49 to 1·40). For the secondary outcomes, the mean difference between the SGRQ-C in the astegolimab group versus placebo group was -3·3 (95% CI -6·4 to -0·2; p=0·039), and mean difference in FEV1 between the two groups was 40 mL (-10 to 90; p=0·094). The difference in geometric mean ratios between the two groups for blood eosinophil counts was 0·59 (95% CI 0·51 to 0·69; p<0·001) and 0·25 (0·19 to 0·33; p<0·001) for sputum eosinophil counts. Incidence of treatment-emergent adverse events was similar between groups. INTERPRETATION: In patients with moderate-to-very severe COPD, astegolimab did not significantly reduce exacerbation rate, but did improve health status compared with placebo. FUNDING: Funded by Genentech and National Institute for Health Research Biomedical Research Centres.

Targeting the Human ßc Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.

Lysophosphatidic acid species are associated with exacerbation in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation. PATIENTS AND METHODS: LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups. RESULTS: The levels of LPA species were intercorrelated (rho 0.29-0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8-1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8-2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2-0.9); LPA20:4-low = 1.4 (0.9-2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8-1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2-0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1-6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2-6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2-6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2-6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1-8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1-8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3-10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2-9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup. CONCLUSIONS: The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development.

Comparison of genome-wide gene expression profiling by RNA Sequencing versus microarray in bronchial biopsies of COPD patients before and after inhaled corticosteroid treatment: does it provide new insights?

More DEGs are detected by RNA-Seq than microarrays in COPD lung biopsies and are associated with immunological pathways. Performing bulk tissue cell-type deconvolution in microarray lung samples, using the SVR method, reflects RNA-Seq results. https://bit.ly/2N8sY3s.

Genome-wide Analyses of Chromatin State in Human Mast Cells Reveal Molecular Drivers and Mediators of Allergic and Inflammatory Diseases.

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.

IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization.

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.

Meningeal Mast Cells as Key Effectors of Stroke Pathology.

Stroke is the leading cause of adult disability in the United States. Because post-stroke inflammation is a critical determinant of damage and recovery after stroke, understanding the interplay between the immune system and the brain after stroke holds much promise for therapeutic intervention. An understudied, but important aspect of this interplay is the role of meninges that surround the brain. All blood vessels travel through the meningeal space before entering the brain parenchyma, making the meninges ideally located to act as an immune gatekeeper for the underlying parenchyma. Emerging evidence suggests that the actions of immune cells resident in the meninges are essential for executing this gatekeeper function. Mast cells (MCs), best known as proinflammatory effector cells, are one of the long-term resident immune cells in the meninges. Here, we discuss recent findings in the literature regarding the role of MCs located in the meningeal space and stroke pathology. We review the latest advances in mouse models to investigate the roles of MCs and MC-derived products in vivo, and the importance of using these mouse models. We examine the concept of the meninges playing a critical role in brain and immune interactions, reevaluate the perspectives on the key effectors of stroke pathology, and discuss the opportunities and challenges for therapeutic development.

microRNA-mRNA regulatory networks underlying chronic mucus hypersecretion in COPD.

Chronic mucus hypersecretion (CMH) is a common feature in chronic obstructive pulmonary disease (COPD) and is associated with worse prognosis and quality of life. This study aimed to identify microRNA (miRNA)-mRNA regulatory networks underlying CMH.The expression profiles of miRNA and mRNA in bronchial biopsies from 63 COPD patients were associated with CMH using linear regression. Potential mRNA targets of each CMH-associated miRNA were identified using Pearson correlations. Gene set enrichment analysis (GSEA) and STRING (search tool for the retrieval of interacting genes/proteins) analysis were used to identify key genes and pathways.20 miRNAs and 539 mRNAs were differentially expressed with CMH in COPD. The expression of 10 miRNAs was significantly correlated with the expression of one or more mRNAs. Of these, miR-134-5p, miR-146a-5p and the let-7 family had the highest representation of CMH-associated mRNAs among their negatively correlated predicted targets. KRAS and EDN1 were identified as key regulators of CMH and were negatively correlated predicted targets of miR-134-5p and let-7a-5p, let-7d-5p, and let-7f-5p, respectively. GSEA suggested involvement of MUC5AC-related genes and several other relevant gene sets in CMH. The lower expression of miR-134-5p was confirmed in primary airway fibroblasts from COPD patients with CMH.We identified miR-134-5p, miR-146a-5p and let-7 family, along with their potential target genes including KRAS and EDN1, as potential key miRNA-mRNA networks regulating CMH in COPD.

Investigating the roles of regulatory T cells, mast cells and interleukin-9 in the control of skin inflammation by vitamin D.

Topical application of biologically active vitamin D [1,25-dihydroxyvitamin D (1,25(OH)2D)], or low-calcemic analogues, curb skin inflammation through mechanisms that involve migratory dendritic cells (DCs) and regulatory T (TReg) cells. 1,25(OH)2D also promotes immunoregulation by mast cells, and inhibits the development of T helper type-9 (Th9) cells that secrete interleukin-9 (IL-9). Here, we investigated the ability of topical 1,25(OH)2D to suppress contact dermatitis through an IL-9-dependent process, examining mast cells and IL-9-secreting T cells. Contact dermatitis was modelled in adult BALB/c female mice by initiating a "biphasic ear swelling response" following a single application of 2,4-dinitrofluorobenzene (DNFB). Topical 1,25(OH)2D (125 ng) applied to ear pinnae prior to (but not after) DNFB sensitisation suppressed the efferent phase of the ear swelling response. This dose of 1,25(OH)2D did not cause hypercalcemia. At the peak of the efferent ear swelling response, proportions of TReg (CD3 + Foxp3+) cells and numbers of mast cells were increased in ear skin of 1,25(OH)2D-treated mice. Topical 1,25(OH)2D increased the proportion of Foxp3 + IL-9 + TReg cells and the capacity of TReg cells to secrete IL-9 ex vivo. However, the proportion of the IL-9 + cells of the total TReg cell population was small (< 1%), and the amount of IL-9 secreted by TReg cells from mice treated with IL-9 was low (< 50 pg/ml). Furthermore, injection of anti-IL-9 neutralising antibody (100 µg, intraperitoneally) prior to sensitisation did not significantly reverse the suppressive effects of 1,25(OH)2D. In conclusion, topically applied 1,25(OH)2D suppressed the efferent phase of a biphasic cutaneous ear swelling response through mechanism(s) that may be dependent on mast cells and TReg cells; however, the role of IL-9 in mediating these responses is uncertain. More studies are needed to further characterise the mechanisms by which topical 1,25(OH)2D modulates cell-mediated immune responses central to its suppressive effects upon contact dermatitis.

Role of the ß Common (ßc) Family of Cytokines in Health and Disease.

The ß common ([ßc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use ßc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the ßc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the ßc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease.

Imaging protective mast cells in living mice during severe contact hypersensitivity.

Contact hypersensitivity (CHS) is a common skin disease induced by epicutaneous sensitization to haptens. Conflicting results have been obtained regarding pathogenic versus protective roles of mast cells (MCs) in CHS, and this has been attributed in part to the limitations of certain models for studying MC functions in vivo. Here we describe a fluorescent imaging approach that enables in vivo selective labeling and tracking of MC secretory granules by real-time intravital 2-photon microscopy in living mice, and permits the identification of such MCs as a potential source of cytokines in different disease models. We show using this method that dermal MCs release their granules progressively into the surrounding microenvironment, but also represent an initial source of the antiinflammatory cytokine IL-10, during the early phase of severe CHS reactions. Finally, using 3 different types of MC-deficient mice, as well as mice in which IL-10 is ablated specifically in MCs, we show that IL-10 production by MCs can significantly limit the inflammation and tissue pathology observed in severe CHS reactions.

The Nedd4-2/Ndfip1 axis is a negative regulator of IgE-mediated mast cell activation.

Cross-linkage of the high-affinity immunoglobulin E (IgE) receptor (FcɛRI) on mast cells by antigen ligation has a critical role in the pathology of IgE-dependent allergic disorders, such as anaphylaxis and asthma. Restraint of intracellular signal transduction pathways that promote release of mast cell-derived pro-inflammatory mediators is necessary to dampen activation and restore homoeostasis. Here we show that the ligase Nedd4-2 and the adaptor Ndfip1 (Nedd4 family interacting protein 1) limit the intensity and duration of IgE-FcɛRI-induced positive signal transduction by ubiquitinating phosphorylated Syk, a tyrosine kinase that is indispensable for downstream FcɛRI signalosome activity. Importantly, loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo. Our findings reveal an important negative regulatory function for Nedd4-2 and Ndfip1 in IgE-dependent mast cell activity.