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Mast cells (MCs) are derived from CD34+ hematopoietic progenitors, consist of different subtypes, and are involved in several inflammatory conditions. However, our understanding of human MC developmental trajectories and subtypes has been limited by a scarcity of suitable cellular model systems. Herein, we developed an in vitro model of human MC differentiation from induced pluripotent stem cells (iPSC) to study human MC differentiation trajectories. Flow cytometry characterization of hemopoietic cells derived from the myeloid cells-forming complex (MCFC) revealed an initial increase in Lin- CD34+ hematopoietic progenitors within Weeks 1-3, followed by an increase in CD34- CD45RA- SSClow and SSChigh hematopoietic cells. The Lin- CD34+ hematopoietic progenitors consisted of SSClow CD45RA- CD123± c-Kit+ FcεRI+ populations that were ß7-integrinhigh CD203c+ and ß7-integrinhigh CD203c- cells consistent with CMPFcεRI+ cells. Flow cytometry and cytologic analyses of the CD34- Lin- (SSClow) population revealed hypogranular cell populations, predominantly characterized by CD45RA- CD123± c-Kit+ FcεRI- ß7-integrinlow and CD45RA- CD123± c-Kit- FcεRI+ ß7-integrinMid cells. Analyses of hypergranular SSChigh cells identified Lin- CD34- CD45RA- c-Kit+ FcεRI- and Lin- CD34- CD45RA- c-Kit+ FcεRI+ cells. scRNA-seq analysis of the cells harvested at week 4 of the MCFC culture revealed the presence of monocyte and granulocyte progenitors (n = 547 cells, 26.7 %), Erythrocyte / unknown (n = 85, 4.1 %), neutrophils / myelocytes (n = 211 cells, 10.2 %), mast cell progenitor 1 (n = 599, 29.1 %), mast cell progenitor 2 (n = 152, 7.4 %), committed mast cell precursor (n = 113, 5.5 %), and MCs (n = 353, 17.1 %). In silico analyses of the MC precursor and mature MC populations revealed transcriptionally distinct MC precursor subtype and mature MC states (CMA1+ and CMA1- subtypes). Culturing MC precursor populations in MC maturation media (mast cell media II) led to homogenous mature MC populations as evidenced by high expression of high-affinity IgE receptor, metachromatic granules, presence of MC granule proteins (Tryptase and Chymase) and activation following substance P stimulation and FcεRI crosslinking. This human iPSC-based approach generates MC precursors and phenotypically mature and functional MC populations. This system will be a useful model to generate human MC populations and broaden our understanding of MC biology and transcriptional regulation of MC differentiation trajectories.
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The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.
Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from different countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.
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Alternativas aos Testes com Animais , Trato Gastrointestinal , Humanos , Alternativas aos Testes com Animais/métodos , Animais , Modelos Biológicos , Medição de Risco/métodos , Testes de Toxicidade/métodosRESUMO
BACKGROUND: Mast cell-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We previously revealed a synergistic interaction between the cytokine IL-4 and the mast cell-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis is unknown. OBJECTIVE: We sought to identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions. METHODS: RNA sequencing, Western blot, Ca2+ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock. RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4-stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia. CONCLUSIONS: These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.
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BACKGROUND: IgA is the most abundant immunoglobulin at the mucosal surface and although its role in regulating mucosal immunity is not fully understood, its presence is associated with protection from developing allergic disease. OBJECTIVE: We sought to determine the role of IgA immune complexes for therapeutic application to mucosal allergic responses. METHODS: Trinitrophenol (TNP)-specific IgA immune complexes were applied, using TNP-coupled ovalbumin (OVA), to airway and gut mucosal surfaces in systemically sensitized allergic animals to regulate allergen challenge responses. Animals were assessed for both pathologic and immune-mediated responses in the lung and gut, respectively, using established mouse models. RESULTS: The mucosal application of IgA immune complexes in the lung and gut with TNP-OVA regulated TH2-driven allergic response in the lung and gut, reducing TH2 cytokines and mucus (lung) as well as diarrhea and temperature loss (gut), but increasing IL-10 and the number of regulatory T cells. The IgA-OVA immune complex did not alter peanut-induced anaphylaxis, indicating antigen specificity. Using OVA-specific DO.11-green fluorescent protein IL-4 reporter mouse-derived TH2-skewed cells in a transfer model demonstrated that mucosal IgA immune complex treatment reduced TH2-cell expansion and increased the number of regulatory T cells. To address a potential mechanism of action, TGF-ß and IL-10 were induced in bone marrow-derived dendritic cells when they were exposed to IgA immune complex, suggesting a regulatory phenotype induced in dendritic cells that also led to an altered primary T-cell-mediated response in in vitro OVA-specific assays. CONCLUSIONS: These studies highlight one possible mechanism of how allergen-specific IgA may provide a regulatory signal to reduce the development of allergic responses in the lung and gut.
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Anafilaxia , Interleucina-10 , Animais , Camundongos , Interleucina-10/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Pulmão , Citocinas/metabolismo , Alérgenos , Anafilaxia/patologia , Ovalbumina , Células Th2 , Camundongos Endogâmicos BALB C , Modelos Animais de DoençasRESUMO
BACKGROUND: Basal zone hyperplasia (BZH) and dilated intercellular spaces (DISs) are thought to contribute to the clinical manifestations of eosinophilic esophagitis (EoE); however, the molecular pathways that drive BZH remain largely unexplored. OBJECTIVE: We sought to define the role of IL-13-induced transcriptional programs in esophageal epithelial proliferation in EoE. METHODS: We performed RNA sequencing, bioinformatics, Western blot, reverse transcriptase quantitative PCR, and histologic analyses on esophageal biopsies from healthy control and patients with EoE, primary esophageal cells derived from patients with EoE, and IL-13-stimulated esophageal epithelial keratinocytes grown at the air-liquid interface (EPC2-ALI). Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches and in vivo model of IL-13-induced esophageal epithelial remodeling (Krt5-rtTA x tetO-IL-13Tg) were used to define the role of signal transducer and activator of transcription 3 (STAT3) and STAT6 and secreted frizzled-related protein 1 (SFRP1) in esophageal epithelial proliferation. RESULTS: RNA-sequencing analysis of esophageal biopsies (healthy control vs EoE) and EPC2-ALI revealed 82 common differentially expressed genes that were enriched for putative STAT3 target genes. In vitro and in vivo analyses revealed a link between IL-13-induced STAT3 and STAT6 phosphorylation, SFRP1 mRNA expression, and esophageal epithelial proliferation. In vitro studies showed that IL-13-induced esophageal epithelial proliferation was STAT3-dependent and regulated by the STAT3 target SFRP1. SFRP1 mRNA is increased in esophageal biopsies from patients with active EoE compared with healthy controls or patients in remission and identifies an esophageal suprabasal epithelial cell subpopulation that uniquely expressed the core EoE proinflammatory transcriptome genes (CCL26, ALOX15, CAPN14, ANO1, and TNFAIP6). CONCLUSIONS: These studies identify SFRP1 as a key regulator of IL-13-induced and STAT3-dependent esophageal proliferation and BZH in EoE and link SFRP1+ esophageal epithelial cells with the proinflammatory and epithelial remodeling response in EoE.
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Esofagite Eosinofílica , Humanos , Esofagite Eosinofílica/patologia , Interleucina-13/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Epiteliais/metabolismo , RNA Mensageiro/metabolismo , Proliferação de CélulasRESUMO
BACKGROUNDFood allergy (FA) is a growing health problem requiring physiologic confirmation via the oral food challenge (OFC). Many OFCs result in clinical anaphylaxis, causing discomfort and risk while limiting OFC utility. Transepidermal water loss (TEWL) measurement provides a potential solution to detect food anaphylaxis in real time prior to clinical symptoms. We evaluated whether TEWL changes during an OFC could predict anaphylaxis onset.METHODSPhysicians and nurses blinded to the TEWL results conducted and adjudicated the results of all 209 OFCs in this study. A study coordinator measured TEWL throughout the OFC and had no input on the OFC conduct. TEWL was measured 2 ways in 2 separate groups. First, TEWL was measured using static, discrete measurements. Second, TEWL was measured using continuous monitoring. Participants who consented provided blood samples before and after the OFCs for biomarker analyses.RESULTSTEWL rose significantly (2.93 g/m2/h) during reactions and did not rise during nonreacting OFCs (-1.00 g/m2/h). Systemic increases in tryptase and IL-3 were also detected during reactions, providing supporting biochemical evidence of anaphylaxis. The TEWL rise occurred 48 minutes earlier than clinically evident anaphylaxis. Continuous monitoring detected a significant rise in TEWL that presaged positive OFCs, but no rise was seen in the OFCs that resulted in no reaction, providing high predictive specificity (96%) for anaphylaxis against nonreactions 38 minutes prior to anaphylaxis onset.CONCLUSIONSDuring OFCs, a TEWL rise anticipated a positive clinical challenge. TEWL presents a monitoring modality that may predict food anaphylaxis and facilitate improvements in OFC safety and tolerability.
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Anafilaxia , Hipersensibilidade Alimentar , Humanos , Anafilaxia/diagnóstico , Anafilaxia/etiologia , Hipersensibilidade Alimentar/diagnóstico , Alimentos , AlérgenosRESUMO
INTRODUCTION: Food allergic reactions can be severe and potentially life-threatening and the underlying immunological processes that contribute to the severity of reactions are poorly understood. The aim of this study is to integrate bulk RNA-sequencing of human and mouse peripheral blood mononuclear cells during food allergic reactions and in vivo mouse models of food allergy to identify dysregulated immunological processes associated with severe food allergic reactions. METHODS: Bulk transcriptomics of whole blood from human and mouse following food allergic reactions combined with integrative differential expressed gene bivariate and module eigengene network analyses to identify the whole blood transcriptome associated with food allergy severity. In vivo validation immune cell and gene expression in mice following IgE-mediated reaction. RESULTS: Bulk transcriptomics of whole blood from mice with different severity of food allergy identified gene ontology (GO) biological processes associated with innate and inflammatory immune responses, dysregulation of MAPK and NFkB signalling and identified 429 genes that correlated with reaction severity. Utilizing two independent human cohorts, we identified 335 genes that correlated with severity of peanut-induced food allergic reactions. Mapping mouse food allergy severity transcriptome onto the human transcriptome revealed 11 genes significantly dysregulated and correlated with severity. Analyses of whole blood from mice undergoing an IgE-mediated reaction revealed a rapid change in blood leukocytes particularly inflammatory monocytes (Ly6Chi Ly6G- ) and neutrophils that was associated with changes in CLEC4E, CD218A and GPR27 surface expression. CONCLUSIONS: Collectively, IgE-mediated food allergy severity is associated with a rapid innate inflammatory response associated with acute cellular stress processes and dysregulation of peripheral blood inflammatory myeloid cell frequencies.
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Fenômenos Biológicos , Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Humanos , Animais , Camundongos , Leucócitos Mononucleares , Hipersensibilidade Alimentar/genética , Alérgenos , Imunoglobulina E , Receptores Acoplados a Proteínas GRESUMO
BACKGROUND: Pulmonary eosinophils comprise at least two distinct populations of resident eosinophils (rEOS) and inflammatory eosinophils (iEOS), the latter recruited in response to pulmonary inflammation. Here, we determined the impact of complement activation on rEOS and iEOS trafficking and function in two models of pulmonary inflammation. METHODS: BALB/c wild-type and C5ar1-/- mice were exposed to different allergens or IL-33. Eosinophil populations in the airways, lung, or mediastinal lymph nodes (mLN) were characterized by FACS or immunohistochemistry. rEOS and iEOS functions were determined in vivo and in vitro. RESULTS: HDM and IL-33 exposure induced a strong accumulation of iEOS but not rEOS in the airways, lungs, and mLNs. rEOS and iEOS expressed C3/C5 and C5aR1, which were significantly higher in iEOS. Initial pulmonary trafficking of iEOS was markedly reduced in C5ar1-/- mice and associated with less IL-5 production from ILC2 cells. Functionally, adoptively transferred pulmonary iEOS from WT but not from C5ar1-/- mice-induced airway hyperresponsiveness (AHR), which was associated with significantly reduced C5ar1-/- iEOS degranulation. Pulmonary iEOS but not rEOS were frequently associated with T cells in lung tissue. After HDM or IL-33 exposure, iEOS but not rEOS were found in mLNs, which were significantly reduced in C5ar1-/- mice. C5ar1-/- iEOS expressed less costimulatory molecules, associated with a decreased potency to drive antigen-specific T cell proliferation and differentiation into memory T cells. CONCLUSIONS: We uncovered novel roles for C5aR1 in iEOS trafficking and activation, which affects key aspects of allergic inflammation such as AHR, ILC2, and T cell activation.
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Asma , Eosinófilos , Camundongos , Animais , Eosinófilos/patologia , Interleucina-33/genética , Imunidade Inata , Linfócitos/patologia , Asma/patologia , Pulmão/patologiaRESUMO
BACKGROUND: Food allergy diagnosis and management causes a number of social and emotional challenges for individuals with food allergies and their caregivers. This has led to increased interest in developing approaches to accurately predict food allergy diagnosis, severity of food allergic reactions, and treatment outcomes. However, the utility of these approaches is somewhat conflicting. OBJECTIVE: We sought to develop and utilize a murine model that mimics the disease course of food allergy diagnosis and treatment in humans and to identify biomarkers that predict reactivity during food challenge (FC) and responsiveness during oral immunotherapy (OIT) and how these outcomes are modified by genetics. METHODS: Skin-sensitized intestinal IL-9 transgenic (IL9Tg) and IL9Tg mice backcrossed onto the IL-4RαY709F background received a single intragastric exposure of egg antigen (ovalbumin), underwent oral FC and OIT; food allergy severity, mast cell activation, and ovalbumin-specific IgE levels were examined to determine the predictability of these outcomes in determining reactivity and treatment outcomes. RESULTS: Subcutaneous sensitization and a single intragastric allergen challenge of egg antigen to BALB/c IL9Tg mice and Il4raY709F IL9Tg induced a food allergic reaction. Enhanced IL-4Rα signaling altered the symptoms induced by the first oral exposure, decreased the cumulative antigen dose, increased the severity of reaction during oral FC, and altered the frequency of adverse events and OIT outcomes. Biomarkers after first oral exposure indicated that only the severity of the initial reaction significantly correlated with cumulative dose of oral FC. CONCLUSION: Collectively, these data indicate that single nucleotide polymorphisms in IL-4Rα can alter clinical symptoms of food allergic reactions, severity, and reactive dose during FC and OIT, and that severity of first reaction can predict the likelihood of reaction during FC in mice with IL-4Rα gain of function.
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Alérgenos , Hipersensibilidade Alimentar , Humanos , Camundongos , Animais , Ovalbumina , Imunoterapia , Camundongos Transgênicos , Biomarcadores , Administração Oral , Dessensibilização ImunológicaRESUMO
Background: Anaphylaxis is an acute life-threatening allergic reaction and a concern at a global level; therefore, further progress in understanding the underlying mechanisms and more effective strategies for diagnosis, prevention and management are needed. Objective: We sought to identify the global architecture of blood transcriptomic features of anaphylaxis by integrating expression data from human patients and mouse model of anaphylaxis. Methods: Bulk RNA-sequencings of peripheral whole blood were performed in: i) 14 emergency department (ED) patients with acute anaphylaxis, predominantly to Hymenoptera venom, ii) 11 patients with peanut allergy undergoing double-blind, placebo-controlled food challenge (DBPCFC) to peanut, iii) murine model of IgE-mediated anaphylaxis. Integrative characterisation of differential gene expression, immune cell-type-specific gene expression profiles, and functional and pathway analysis was undertaken. Results: 1023 genes were commonly and significantly dysregulated during anaphylaxis in ED and DBPCFC patients; of those genes, 29 were also dysregulated in the mouse model. Cell-type-specific gene expression profiles showed a rapid downregulation of blood basophil and upregulation of neutrophil signature in ED and DBPCFC patients and the mouse model, but no consistent and/or significant differences were found for other blood cells. Functional and pathway analysis demonstrated that human and mouse blood transcriptomic signatures of anaphylaxis follow trajectories of upregulation of cell movement, migration and neuroinflammatory signalling, and downregulation of lipid activating nuclear receptors signalling. Conclusion: Our study highlights the matched and extensive blood transcriptomic changes and suggests the involvement of discrete cellular components and upregulation of migration and neuroinflammatory pathways during anaphylaxis.
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Anafilaxia , Humanos , Camundongos , Animais , Anafilaxia/diagnóstico , Transcriptoma , Modelos Animais de Doenças , Basófilos , Alérgenos , Movimento CelularRESUMO
Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.
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Esôfago , Organoides , Adulto , Humanos , Células-Tronco , Células Epiteliais/metabolismo , Diferenciação CelularRESUMO
Acute and chronic intestinal inflammation is associated with epithelial damage, resulting in mucosal wounds in the forms of erosions and ulcers in the intestinal tract. Intestinal epithelial cells (IECs) and immune cells in the wound milieu secrete cytokines and lipid mediators to influence repair. Leukotriene B4 (LTB4), a lipid chemokine, binds to its receptor BLT1 and promotes migration of immune cells to sites of active inflammation; however, a role for intestinal epithelial BLT1 during mucosal wound repair is not known. Here we report that BLT1 was expressed in IECs both in vitro and in vivo, where it functioned as a receptor not only for LTB4 but also for another ligand, resolvin E1. Intestinal epithelial BLT1 expression was increased when epithelial cells were exposed to an inflammatory microenvironment. Using human and murine primary colonic epithelial cells, we reveal that the LTB4/BLT1 pathway promoted epithelial migration and proliferation leading to accelerated epithelial wound repair. Furthermore, in vivo intestinal wound repair experiments in BLT1-deficient mice and bone marrow chimeras demonstrated an important contribution of epithelial BLT1 during colonic mucosal wound repair. Taken together, our findings show a potentially novel prorepair in IEC mechanism mediated by BLT1 signaling.
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Lipídeos , Humanos , Animais , CamundongosRESUMO
Eosinophilic esophagitis (EoE) is an emerging chronic inflammatory disease of the oesophagus and is clinically characterized by upper gastrointestinal (GI) symptoms including dysphagia and esophageal food impaction. Histopathologic manifestations, which include intraepithelial eosinophilic inflammation and alterations of the esophageal squamous epithelium, such as basal zone hyperplasia (BZH) and dilated intercellular spaces (DIS), are thought to contribute to esophageal dysfunction and disease symptoms. Corroborative clinical and discovery science-based studies have established that EoE is characterized by an underlying allergic inflammatory response, in part, related to the IL-13/CCL26/eosinophil axis driving dysregulation of several key epithelial barrier and proliferative regulatory genes including kallikrein (KLK) serine proteases, calpain 14 (CAPN14) and anoctamin 1 (ANO1). The contribution of these inflammatory and proliferative processes to the clinical and histological manifestations of disease are not fully elucidated. Herein, we discuss the immune molecules and cells that are thought to underlie the clinical and pathologic manifestations of EoE and the emerging therapeutics targeting these processes for the treatment of EoE.
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Esofagite Eosinofílica , Anoctamina-1 , Calpaína , Esofagite Eosinofílica/tratamento farmacológico , Esofagite Eosinofílica/terapia , Eosinófilos , Humanos , Interleucina-13/metabolismo , Calicreínas , Serina ProteasesRESUMO
Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis - in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances.
Cells in the gut need to be protected against the many harmful microbes which inhabit this environment. Yet the immune system also needs to 'keep an eye' on intestinal contents to maintain tolerance to innocuous substances, such as those from the diet. The 'goblet cells' that are part of the gut lining do both: they create a mucus barrier that stops germs from invading the body, but they also can pass on molecules from the intestine to immune cells deep in the tissue to promote tolerance. This is achieved through a 'GAP' mechanism. A chemical messenger called acetylcholine can trigger both mucus release and the GAP process in goblet cells. Gustafsson et al. investigated how the cells could take on these two seemingly opposing roles in response to the same signal. A fluorescent molecule was introduced into the intestines of mice, and monitored as it pass through the goblet cells. This revealed how the GAP process took place: the cells were able to capture molecules from the intestines, wrap them in internal sack-like vesicles and then transport them across the entire cell. To explore the role of acetylcholine, Gustafsson et al. blocked the receptors that detect the messenger at the surface of goblet cells. Different receptors and therefore different cascades of molecular events were found to control mucus secretion and GAP formation; this explains how the two processes can be performed in parallel and independently from each other. Understanding how cells relay molecules to the immune system is relevant to other tissues in contact with the environment, such as the eyes, the airways, or the inside of the genital and urinary tracts. Understanding, and then ultimately harnessing this mechanism could help design of new ways to deliver drugs to the immune system and alter immune outcomes.
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Antígenos/metabolismo , Células Caliciformes/metabolismo , Transcitose , Vesículas Transportadoras/fisiologia , Animais , CamundongosRESUMO
BACKGROUND & AIMS: CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. METHODS: We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non-inflammatory bowel disease patients and sorted human memory CD4+ T cells. RESULTS: We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B- Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1-dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. CONCLUSIONS: Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.
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Regulação da Expressão Gênica , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Receptores Imunológicos/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Colite/etiologia , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Imuno-Histoquímica , Memória Imunológica , Imunofenotipagem , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Receptores Imunológicos/metabolismo , Transdução de SinaisAssuntos
Hipersensibilidade Alimentar/etiologia , Imunidade Inata , Células Apresentadoras de Antígenos/imunologia , Basófilos/fisiologia , Hipersensibilidade Alimentar/imunologia , Microbioma Gastrointestinal , Humanos , Mastócitos/fisiologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologiaRESUMO
Food allergy is an emerging epidemic, and the underlying mechanisms are not well defined partly due to the lack of robust adjuvant free experimental models of dietary antigen sensitization. As housing mice at thermoneutrality (Tn) - the temperature of metabolic homeostasis (26-30°C) - has been shown to improve modeling various human diseases involved in inflammation, we tested the impact of Tn housing on an experimental model of food sensitization. Here we demonstrate that WT BALB/c mice housed under standard temperature (18-20°C, Ts) conditions translocated the luminal antigens in the small intestine (SI) across the epithelium via goblet cell antigen passages (GAPs). In contrast, food allergy sensitive Il4raF709 mice housed under standard temperature conditions translocated the luminal antigens in the SI across the epithelium via secretory antigen passages (SAPs). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg allergens at standard temperature predisposed Il4raF709 mice to develop an anaphylactic reaction. Housing Il4raF709 mice at Tn altered systemic type 2 cytokine, IL-4, and the landscape of SI antigen passage patterning (villus and crypt involvement). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg antigen under Tn conditions led to the robust induction of egg-specific IgE and development of food-induced mast cell activation and hypovolemic shock. Similarly, Tn housing of WT BALB/c mice altered the cellular patterning of SI antigen passage (GAPs to SAPs). Activation of SI antigen passages and the oral challenge of WT BALB/c mice with egg antigen led to systemic reactivity to egg and mast cell activation. Together these data demonstrate that Tn housing alters antigen passage cellular patterning and landscape, and concurrent oral exposure of egg antigens and SAP activation is sufficient to induce oral antigen sensitization.
Assuntos
Alérgenos/metabolismo , Anafilaxia/metabolismo , Hipersensibilidade a Ovo/metabolismo , Proteínas do Ovo/metabolismo , Abrigo para Animais , Intestino Delgado/metabolismo , Temperatura , Administração Oral , Alérgenos/administração & dosagem , Alérgenos/imunologia , Anafilaxia/imunologia , Anafilaxia/microbiologia , Animais , Modelos Animais de Doenças , Hipersensibilidade a Ovo/imunologia , Hipersensibilidade a Ovo/microbiologia , Proteínas do Ovo/administração & dosagem , Proteínas do Ovo/imunologia , Microbioma Gastrointestinal , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Células Caliciformes/microbiologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Permeabilidade , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Allergic asthma is a chronic inflammatory airway disease characterized by dysregulated type 2 immune responses, including degranulating airway eosinophils that induce tissue damage and airway hyperresponsiveness (AHR). The type 2 cytokines interleukin 5 (IL-5) and IL-13 and the eosinophil-specific chemokine CCL11/CCL24/CCL26 axis recruit, activate, and regulate eosinophils in the airways. In this issue of the JCI, Karcz et al. identified a mechanism involving the nucleotide sugar UDP-glucose (UDP-G) and the purinergic receptor P2Y14R in amplifying eosinophil accumulation in the lung. During type 2 inflammation, UDP-G activates P2Y14R on eosinophils, inducing the cells to move and migrate into the lung. Pharmacologically or genetically inhibiting P2Y14R on eosinophils attenuated eosinophil infiltration and AHR. Future experiments, including identifying additional type 2 factors regulating P2Y14R expression on lung eosinophils, are necessary to ascertain the impact of targeting P2Y14R as an alternative or adjunctive therapy to current type 2 biologics for the treatment of asthma.
Assuntos
Asma , Eosinófilos , Asma/tratamento farmacológico , Asma/genética , Glucose , Humanos , Interleucina-13 , Uridina Difosfato GlucoseRESUMO
BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.