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Eosinophilic esophagitis (EoE) is a type 2 allergic disease characterized by esophageal inflammation and epithelial cell dysfunction. Acquired loss of the anti-serine protease of kazal type 7 (SPINK7) in the squamous epithelium of the esophagus has a causal role in EoE pathogenesis. Yet there is a limited understanding of the factors that regulate its expression and responsiveness to inflammatory stimuli. Herein, we identified the transcription factor, ovo like transcriptional repressor 1 (OVOL1) as an esophageal selective gene product that regulates SPINK7 promoter activity. Overexpression ofOVOL1increasedSPINK7expression, whereas, its depletion decreasedSPINK7expression, impaired epithelial barrier and increased production of the pro-atopy cytokine thymic stromal lymphopoietin (TSLP). Mechanistically, ligands of AHR induced nuclear translocation of OVOL1 which in turn promoted epithelial cell differentiation, barrier function andSPINK7expression. Interleukin (IL)-4 and IL-13 abolished AHR ligand-induced OVOL1 nuclear translocation. Stimulation with IL-13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein. This effect of IL-13 was dependent on the esophageal specific cysteine protease calpain-14. Translational studies demonstrated loss of OVOL1 protein expression in patients with EoE. In summary, AHR mediates its action via OVOL1-induced SPINK7 transcription, and IL-4 and IL-13 repress this pathway in EoE. As such, activation of the AHR pathway is a potential intervention strategy for reversing EoE.Graphical abstractThe influence of the exposome on regulatory networks in EoE pathogenesis.AHR is activated and influenced by diet nutrients, environmental toxicants, microbiome composition, tryptophan metabolites, and drugs. When AHR is activated, it promotes translocation of OVOL1 to the nucleus, which in turn promotes expression of epithelial genes includingSPINK7. SPINK7 expression promotes epithelial differentiation, barrier function, decreased proteolytic activity, and decreased TSLP production. IL-4 and IL-13 inhibit OVOL1 nuclear translocation and therefore, repressSPINK7expression. IL-13–stimulatedCAPN14expression decreases OVOL1 protein expression andSPINK7transcription.

Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. The main cellular location where the SARS-CoV-2 S protein priming occurs remains debatable, therefore hampering the development of targeted treatments. Herein, we identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease-inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Our data support the key role of extracellular serine proteases in SARS-CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells., Summary Delivery of extracellular serine protease inhibitors (serpins) such as A1AT has the capacity to reduce SARS-CoV-2 dissemination by binding and inhibiting extracellular proteases on the host cells, thus, inhibiting the first step in SARS-CoV-2 cell cycle (i.e. cell entry).

Introduction: Diagnostic and therapeutic strategies in eosinophilic esophagitis (EoE) are constantly evolving. Recently, the improved understanding of EoE pathogenesis has led to identification of a variety of other potential targets that have never been considered before.Areas covered: In September 2019, we performed structured literature searches in Medline and PubMed, Cochrane meta-analyses, and abstracts of international congresses to review new potential therapeutic approaches for EoE.Expert opinion: The advent of omics disciplines has been helping in finding new molecular targets in EoE pathogenesis and may provide future guidance for deep phenotyping of the disease and therefore facilitate the possibility of personalized medicine. Interestingly, these new treatments should be focused on the restoration of epithelial barrier dysfunction, downregulation of specific molecular pathways of eosinophilic inflammation, and finally, prevention of esophageal remodeling. In this review, we highlight the most recent insights in EoE pathogenesis, which open new pathways for developing new therapeutic targets for clinical practice.

Allergic asthma is a chronic inflammatory lung disease associated with increased cytokine secretion. Aspects of airway inflammation are also linked to a common genetic variant that corresponds to the small GTPase, Rab27, a protein involved in vesicular trafficking in immune cells. However, the mechanisms by which Rab27 contributes to airway inflammation and cytokine release remain ambiguous. In this issue of the JCI, Okunishi et al. explored the role that the Rab27 effector, exophilin-5, has in allergic inflammation. Exophilin-5-deficient mice and asthma mouse models revealed that exophilin-5 regulates IL-33 production and the Th2 response. Notably, exophilin-5 deletion enhanced IL-33 release and pathogenic Th2 responsiveness through the mTOR pathway and altered intracellular IL-33 trafficking. This work provides insights into the molecular mechanisms that underlie inflammatory lung disease.

Gastrointestinal (GI) allergic disease is an umbrella term used to describe a variety of adverse, food antigen-driven, immune-mediated diseases. Although these diseases vary mechanistically, common elements include a breakdown of immunologic tolerance, a biased type 2 immune response, and an impaired mucosal barrier. These pathways are influenced by diverse factors such as diet, infections, exposure to antibiotics and chemicals, GI microbiome composition, and genetic and epigenetic elements. Early childhood has emerged as a critical period when these factors have a dramatic impact on shaping the immune system and therefore triggering or protecting against the onset of GI allergic diseases. In this Review, we will discuss the latest findings on the molecular and cellular mechanisms that govern GI allergic diseases and how these findings have set the stage for emerging preventative and treatment strategies.

Significance MM3122 represents an advanced lead candidate for clinical development as a novel antiviral drug for COVID-19. In addition to being novel drugs, these selective TMRSS2 inhibitors can be used as valuable chemical probes to help elucidate mechanisms of viral pathogenesis. Since TMPRSS2 plays a key role as a viral protein processing protease in the pathogenesis of other coronaviruses (SARS-CoV, MERS-CoV) as well as influenza viruses, MM3122 and this class of TMPRSS2 inhibitors hold much promise as new drugs to not only treat SARS-CoV-2 infections but also potentially represent broad-spectrum antivirals., The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

The entry of the coronavirus SARS-CoV-2 into human lung cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations. In vitro assays confirm that both drugs inhibit the human protein TMPRSS2, a SARS-Cov-2 spike protein activator. As no experimental structure is available, we provide a model of the TMPRSS2 equilibrium structure and its fluctuations by relaxing an initial homology structure with extensive 330 microseconds of all-atom molecular dynamics (MD) and Markov modeling. Through Markov modeling, we describe the binding process of both drugs and a metabolic product of camostat (GBPA) to TMPRSS2, reaching a Michaelis complex (MC) state, which precedes the formation of a long-lived covalent inhibitory state. We find that nafamostat has a higher MC population than camostat and GBPA, suggesting that nafamostat is more readily available to form the stable covalent enzyme-substrate intermediate, effectively explaining its high potency. This model is backed by our in vitro experiments and consistent with previous virus cell entry assays. Our TMPRSS2-drug structures are made public to guide the design of more potent and specific inhibitors.

The hallmark of mast cell activation is secretion of immune mediators by regulated exocytosis. Measurements of mediator secretion from mast cells that are genetically manipulated by transient transfections provide a powerful tool for deciphering the underlying mechanisms of mast cell exocytosis. However, common methods to study regulated exocytosis in bulk culture of mast cells suffer from the drawback of high signal-to-noise ratio because of their failure to distinguish between the different mast cell populations, that is, genetically modified mast cells versus their non-transfected counterparts. In particular, the low transfection efficiency of mast cells poses a significant limitation on the use of conventional methodologies. To overcome this hurdle, we developed a method, which discriminates and allows detection of regulated exocytosis of transfected cells based on the secretion of a fluorescent secretory reporter. We used a plasmid encoding for Neuropeptide Y (NPY) fused to a monomeric red fluorescent protein (NPY-mRFP), yielding a fluorescent secretory granule-targeted reporter that is co-transfected with a plasmid encoding a gene of interest. Upon cell trigger, NPY-mRFP is released from the cells by regulated exocytosis, alongside the endogenous mediators. Therefore, using NPY-mRFP as a reporter for mast cell exocytosis allows either quantitative, via a fluorimeter assay, or qualitative analysis, via confocal microscopy, of the genetically manipulated mast cells. Moreover, this method may be easily modified to accommodate studies of regulated exocytosis in any other type of cell.

The hallmark of mast cell activation is secretion of immune mediators by regulated exocytosis. Measurements of mediator secretion from mast cells that are genetically manipulated by transient transfections provide a powerful tool for deciphering the underlying mechanisms of mast cell exocytosis. However, common methods to study regulated exocytosis in bulk culture of mast cells suffer from the drawback of high signal-to-noise ratio because of their failure to distinguish between the different mast cell populations, that is, genetically modified mast cells versus their non-transfected counterparts. In particular, the low transfection efficiency of mast cells poses a significant limitation on the use of conventional methodologies. To overcome this hurdle, we developed a method, which discriminates and allows detection of regulated exocytosis of transfected cells based on the secretion of a fluorescent secretory reporter. We used a plasmid encoding for Neuropeptide Y (NPY) fused to a monomeric red fluorescent protein (NPY-mRFP), yielding a fluorescent secretory granule-targeted reporter that is co-transfected with a plasmid encoding a gene of interest. Upon cell trigger, NPY-mRFP is released from the cells by regulated exocytosis, alongside the endogenous mediators. Therefore, using NPY-mRFP as a reporter for mast cell exocytosis allows either quantitative, via a fluorimeter assay, or qualitative analysis, via confocal microscopy, of the genetically manipulated mast cells. Moreover, this method may be easily modified to accommodate studies of regulated exocytosis in any other type of cell.

The entry of the coronavirus SARS-CoV-2 into human cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations.In vitroassays confirm the hypothesis that both drugs act by inhibiting the human protein TMPRSS2. As no experimental structure is available, we provide a model of the TMPRSS2 equilibrium structure and its fluctuations by relaxing an initial homology structure with extensive 280 microseconds of all-atom molecular dynamics (MD) and Markov modeling. We describe the binding mode of both drugs with TMPRSS2 in a Michaelis complex (MC) state preceding the formation of a long-lived covalent inhibitory state. We find that nafamostat to has a higher MC population, which in turn leads to the more frequent formation of the covalent complex and thus higher inhibition efficacy, as confirmedin vitroand consistent with previous virus cell entry assays. Our TMPRSS2-drug structures are made public to guide the design of more potent and specific inhibitors.

Eosinophilic esophagitis (EoE) is a chronic, food antigen–driven, inflammatory disease of the esophagus and is associated with impaired barrier function. Evidence is emerging that loss of esophageal expression of the serine peptidase inhibitor, kazal type 7 (SPINK7), is an upstream event in EoE pathogenesis. Here, we provide evidence that loss of SPINK7 mediates its pro-EoE effects via kallikrein 5 (KLK5) and its substrate, protease-activated receptor 2 (PAR2). Overexpression of KLK5 in differentiated esophageal epithelial cells recapitulated the effect of SPINK7 gene silencing, including barrier impairment and loss of desmoglein-1 expression. Conversely, KLK5 deficiency attenuated allergen-induced esophageal protease activity, modified commensal microbiome composition, and attenuated eosinophilia in a murine model of EoE. Inhibition of PAR2 blunted the cytokine production associated with loss of SPINK7 in epithelial cells and attenuated the allergen-induced esophageal eosinophilia in vivo. Clinical samples substantiated dysregulated PAR2 expression in the esophagus of patients with EoE, and delivery of the clinically approved drug α1 antitrypsin (A1AT, a protease inhibitor) inhibited experimental EoE. These findings demonstrate a role for the balance between KLK5 and protease inhibitors in the esophagus and highlight EoE as a protease-mediated disease. We suggest that antagonizing KLK5 and/or PAR2 has potential to be therapeutic for EoE.

Background Proton pump inhibitors (PPIs) have been recognized as a primary treatment of eosinophilic esophagitis (EoE), an allergic inflammatory disease of the esophageal mucosa. The mechanisms underlying esophageal epithelial responses to PPIs remain poorly understood. Objective We hypothesized that PPIs can counteract IL-13–mediated esophageal epithelial responses that are germane for EoE pathogenesis. Methods Transcriptional responses of human esophageal cells to IL-13 and the PPIs omeprazole and esomeprazole were assessed by RT-PCR and RNA sequencing. Cytokine secretion was measured by multiplex analysis and ELISA. Results Human esophageal epithelial cells robustly responded to PPI stimulation by inducing a set of 479 core genes common between omeprazole and esomeprazole treatments. The transcriptional response to PPIs was partially mediated through the aryl hydrocarbon receptor signaling pathway, as the aryl hydrocarbon receptor antagonist GNF-351 modified approximately 200 genes, particularly those enriched in metabolic processes and regulation of cell death. PPI treatment reversed approximately 20% of the IL-13 transcriptome. Functional analysis of the PPI-responsive, upregulated genes revealed enrichment in metabolic and oxidation processes, and the unfolded protein response. In contrast, downregulated genes were overrepresented in functional terms related to cell division and cytoskeletal organization, which were also enriched for the genes in the EoE transcriptome reversed by PPIs. Furthermore, PPI treatment decreased the IL-13–induced proliferative response of esophageal epithelial cells. Conclusions These results demonstrate broad effects of PPIs on esophageal epithelium, including their ability to curtail transcriptomic processes involved in cellular proliferation and IL-13–induced responses, and they highlight the importance of AHR signaling in mediating these responses.

Compound exocytosis is considered the most massive mode of exocytosis, during which the membranes of secretory granules (SGs) fuse with each other to form a channel through which the entire contents of their granules is released. The underlying mechanisms of compound exocytosis remain largely unresolved. Here we show that the small GTPase Rab5, a known regulator of endocytosis, is pivotal for compound exocytosis in mast cells. Silencing of Rab5 shifts receptor-triggered secretion from a compound to a full exocytosis mode, in which SGs individually fuse with the plasma membrane. Moreover, we show that Rab5 is essential for FcεRI-triggered association of the SNARE protein SNAP23 with the SGs. Direct evidence is provided for SNAP23 involvement in homotypic SG fusion that occurs in the activated cells. Finally, we show that this fusion event is prevented by inhibition of the IKKβ2 kinase, however, neither a phosphorylation-deficient nor a phosphomimetic mutant of SNAP23 can mediate homotypic SG fusion in triggered cells. Taken together our findings identify Rab5 as a heretofore-unrecognized regulator of compound exocytosis that is essential for SNAP23-mediated granule-granule fusion. Our results also implicate phosphorylation cycles in controlling SNAP23 SNARE function in homotypic SG fusion.

IL-13 and IL-4 are potent mediators of type 2–associated inflammation such as those found in atopic dermatitis (AD). IL-4 shares overlapping biological functions with IL-13, a finding that is mainly explained by their ability to signal via the type 2 IL-4 receptor (R), which is composed of IL-4Rα in association with IL-13Rα1. Nonetheless, the role of the type 2 IL-4R in AD remains to be clearly defined. Induction of two distinct models of experimental AD in Il13ra1−/− mice, which lack the type 2 IL-4R, revealed that dermatitis, including ear and epidermal thickening, was dependent on type 2 IL-4R signaling. Expression of TNF-α was dependent on the type 2 IL-4R, whereas induction of IL-4, IgE, CCL24, and skin eosinophilia was dependent on the type 1 IL-4R. Neutralization of IL-4, IL-13, and TNF-α as well as studies in bone marrow–chimeric mice revealed that dermatitis, TNF-α, CXCL1, and CCL11 expression were exclusively mediated by IL-13 signaling via the type 2 IL-4R expressed by nonhematopoietic cells. Conversely, induction of IL-4, CCL24, and eosinophilia was dependent on IL-4 signaling via the type 1 IL-4R expressed by hematopoietic cells. Last, we pharmacologically targeted IL-13Rα1 and established a proof of concept for therapeutic targeting of this pathway in AD. Our data provide mechanistic insight into the differential roles of IL-4, IL-13, and their receptor components in allergic skin and highlight type 2 IL-4R as a potential therapeutic target in AD and other allergic diseases such as asthma and eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus featuring increased esophageal interleukin-13 (IL-13) levels and impaired barrier function. Herein, we investigated leucine-rich repeat-containing protein 31 (LRRC31) in human EoE esophageal tissue and IL-13-treated esophageal epithelial cells. LRRC31 had basal mRNA expression in colonic and airway mucosal epithelium. Esophageal LRRC31 mRNA and protein increased in active EoE and strongly correlated with esophageal eosinophilia and IL13 and CCL26 (chemokine (C-C motif) ligand 26) mRNA expression. IL-13 treatment increased LRRC31 mRNA and protein in air-liquid interface-differentiated esophageal epithelial cells (EPC2s). At baseline, differentiated LRRC31-overexpressing EPC2s had increased barrier function (1.9-fold increase in transepithelial electrical resistance (P

Secretory granule (SG) transport is a critical step in regulated exocytosis including degranulation of activated mast cells. The latter process results in the release of multiple inflammatory mediators that play key roles in innate immunity, as well as in allergic responses. In this study, we identified the small GTPase Rab12 as a novel regulator of mast cell SG transport, and we provide mechanistic insights into its mode of action. We show that Rab12 is activated in a stimulus-dependent fashion and promotes microtubule-dependent retrograde transport of the SGs in the activated cells. We also show that this minus end transport of the SGs is mediated by the RILP–dynein complex and identify RILP as a novel effector of Rab12. Finally, we show that Rab12 negatively regulates mast cell degranulation. Taken together, our results identify Rab12 as a novel regulator of mast cell responses and disclose for the first time, to our knowledge, the mechanism of retrograde transport of the mast cell SGs.

Eosinophilic esophagitis (EoE) is a chronic, allergen-driven inflammatory disease of the esophagus characterized predominantly by eosinophilic inflammation, leading to esophageal dysfunction. Converging data have placed the esophageal epithelium at the center of disease pathogenesis. In particular, the main EoE disease susceptibility loci at 2p23 and 5p22 encode for gene products that are produced by the esophageal epithelium: the intracellular protease calpain 14 and thymic stromal lymphopoietin, respectively. Furthermore, genetic and functional data establish a primary role for impaired epithelial barrier function in disease susceptibility and pathoetiology. Additionally, the EoE transcriptome, a set of genes dysregulated in the esophagi of patients with EoE, is enriched in genes that encode for proteins involved in esophageal epithelial cell differentiation. This transcriptome has a high proportion of esophagus-specific epithelial genes that are notable for the unexpected enrichment in genes encoding for proteases and protease inhibitors, as well as in IL-1 family genes, demonstrating a previously unappreciated role for innate immunity responses in the esophagus under homeostatic conditions. Among these pathways, basal production of the serine protease inhibitor, Kazal-type 7 (SPINK7) has been demonstrated to be part of the normal differentiation program of esophageal epithelium. Profound lost expression of SPINK7 occurs in patients with EoE and is sufficient for unleashing increased proteolytic activity (including urokinase plasminogen activator), impaired barrier function, and production of large quantities of proinflammatory and proallergic cytokines, including thymic stromal lymphopoietin. Collectively, we put forth a model in which the esophagus is normally equipped as an anti-inflammatory sensing organ and that defects in this pathway, mediated by epithelial protease/protease inhibitor imbalances, unleash inflammatory responses resulting in disorders, such as EoE.

Loss of barrier integrity has an important role in eliciting type 2 immune responses, yet the molecular events that initiate and connect this with allergic inflammation remain unclear. We reveal an endogenous, homeostatic mechanism that controls barrier function and inflammatory responses in esophageal allergic inflammation. We show that a serine protease inhibitor, SPINK7 (serine peptidase inhibitor, kazal type 7), is part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis. Experimental manipulation strategies reducing SPINK7 in an esophageal epithelial progenitor cell line and primary esophageal epithelial cells were sufficient to induce barrier dysfunction and transcriptional changes characterized by loss of cellular differentiation and altered gene expression known to stimulate allergic responses (for example, FLG and SPINK5 ). Epithelial silencing of SPINK7 promoted production of proinflammatory cytokines including thymic stromal lymphopoietin (TSLP). Loss of SPINK7 increased the activity of urokinase plasminogen-type activator (uPA), which in turn had the capacity to promote uPA receptor–dependent eosinophil activation. Treatment of epithelial cells with the broad-spectrum antiserine protease, α1 antitrypsin, reversed the pathologic features associated with SPINK7 silencing. The relevance of this pathway in vivo was supported by finding genetic epistasis between variants in TSLP and the uPA-encoding gene, PLAU . We propose that the endogenous balance between SPINK7 and its target proteases is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory responses and that protein replacement with antiproteases may be therapeutic for select allergic diseases.

Background A key question in the allergy field is to understand how tissue-specific disease is manifested. Eosinophilic esophagitis (EoE) is an emerging tissue-specific allergic disease with an unclear pathogenesis. Objective Herein we tested the hypothesis that a defect in tissue-specific esophageal genes is an integral part of EoE pathogenesis. Methods We interrogated the pattern of expression of esophagus-specific signature genes derived from the Human Protein Atlas in the EoE transcriptome and in EPC2 esophageal epithelial cells. Western blotting and immunofluorescence were used for evaluating expression of esophageal proteins in biopsy specimens from control subjects and patients with active EoE. Whole-exome sequencing was performed to identify mutations in esophagus-specific genes. Results We found that approximately 39% of the esophagus-specific transcripts were altered in patients with EoE, with approximately 90% being downregulated. The majority of transcriptional changes observed in esophagus-specific genes were reproduced in vitro in esophageal epithelial cells differentiated in the presence of IL-13. Functional enrichment analysis revealed keratinization and differentiation as the most affected biological processes and identified IL-1 cytokines and serine peptidase inhibitors as the most dysregulated esophagus-specific protein families in patients with EoE. Accordingly, biopsy specimens from patients with EoE evidenced a profound loss of tissue differentiation, decreased expression of keratin 4 (KRT4) and cornulin (CRNN) , and increased expression of KRT5 and KRT14 . Whole-exome sequencing of 33 unrelated patients with EoE revealed 39 rare mutations in 18 esophagus-specific differentially expressed genes. Conclusions A tissue-centered analysis has revealed a profound loss of esophageal tissue differentiation (identity) as an integral and specific part of the pathophysiology of EoE and implicated protease- and IL-1–related activities as putative central pathways in disease pathogenesis.

Exocytosis is a key event in mast cell functions. By this process, mast cells release inflammatory mediators, contained in secretory granules (SGs), which play important roles in immunity and wound healing but also provoke allergic and inflammatory responses. The mechanisms underlying mast cell exocytosis remained poorly understood. An essential step toward deciphering the mechanisms behind exocytosis is the identification of the cellular components that regulate this process. Because Rab GTPases regulate specific trafficking pathways, we screened 44 Rabs for their functional impacts on exocytosis triggered by the FcεRI or combination of Ca2+ ionophore and phorbol ester. Because exocytosis involves the continuous reorganization of the actin cytoskeleton, we also repeated our screen in the presence of cytochalasin D that inhibits actin polymerization. In this paper, we report on the identification of 30 Rabs as regulators of mast cell exocytosis, the involvement of 26 of which has heretofore not been recognized. Unexpectedly, these Rabs regulated exocytosis in a stimulus-dependent fashion, unless the actin skeleton was disrupted. Functional clustering of the identified Rabs suggested their classification as Rabs involved in SGs biogenesis or Rabs that control late steps of exocytosis. The latter could be further divided into Rabs that localize to the SGs and Rabs that regulate transport from the endocytic recycling compartment. Taken together, these findings unveil the Rab networks that control mast cell exocytosis and provide novel insights into their mechanisms of action.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs’ mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules (SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition.