Determinants of oral anaphylaxis to food

口腔食物过敏的决定因素

• 批准号: 10586739
• 负责人: Stephanie Caroline Eisenbarth
• 金额: $ 80.48万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-07 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586739
• 关键词: Affect; Alleles; Allergens; Allergic; Allergy to eggs; Allergy to peanuts; Anaphylaxis; Antibodies; Antigens; Backcrossings; Basophils; Blood; Bone Marrow; C3H/HeJ Mouse; C57BL/6 Mouse; CRISPR/Cas technology; Candidate Disease Gene; Cell Count; Cell Differentiation process; Cell physiology; Cellular biology; Child; Chimera organism; Chromosome 8; Chromosome Mapping; Complement; DNA Sequence Alteration; Data; Enzymes; Epithelial Cells; Exhibits; FDA approved; Food; Food Hypersensitivity; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Goblet Cells; Human; IgE; In Vitro; Individual; Ingestion; Inherited; Intestinal permeability; Lead; Life; Link; Maps; Mediating; Molecular; Mouse Strains; Mucous Membrane; Mus; Oral; Pathway interactions; Patients; Permeability; Persons; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Predisposition; Process; Quantitative Trait Loci; Reaction; Recording of previous events; Regulation; Resistance; Resolution; Risk; SNP genotyping; Secretory Cell; Surface; System; Testing; Work; autosome; cell type; diagnostic biomarker; diagnostic strategy; experimental study; food allergen; forward genetics; gastrointestinal epithelium; genetic resistance; genomic locus; in vivo; intestinal epithelium; mast cell; microbiota; mouse model; novel; novel therapeutics; oral immunotherapy; pharmacologic; prevent; programs; resistance gene; single-cell RNA sequencing; targeted treatment; uptake; validation studies

PROJECT SUMMARY The goal of this proposal is to identify new ways of preventing anaphylaxis in those with food allergy by defining cellular and molecular mechanisms that transport intact food allergens across the gut epithelium. In its most severe form, food allergy can trigger life-threatening anaphylaxis. One factor that can determine risk of anaphylaxis is the integrity of the gut barrier, but there are no treatments to reduce intestinal permeability to food allergens. A major mode of allergen transport across the gut epithelium occurs transcellularly through secretory and goblet cell-associated antigen passages (GAPs). Using mouse models of peanut and egg allergy, we have made the exciting discovery that susceptibility to oral anaphylaxis was genetically determined and associated with an increased number and function of GAPs. Using a forward genetic screen we identified a single chromosomal region that tracks with this phenotype. Moreover, drug treatments targeting goblet cell biology reduced anaphylaxis in vivo. We hypothesize that the genetic regulation of gut goblet cell quantity and transport capability determines susceptibility to anaphylaxis by controlling intestinal permeability to intact food allergens. In Aim 1, using complementary mouse strains described above, we will perform quantitative trait locus (QTL) mapping with SNP genotyping to identify genetic resistance loci for oral anaphylaxis associated with reduced gut permeability. These data will be integrated with scRNA-Seq analyses of intestinal epithelium from anaphylaxis susceptible vs. resistant littermates to identify cell type-specific regulators of allergen transport. Using CRISPR/Cas9 gene editing in mice, we will test the contribution to gut permeability and oral anaphylaxis in vivo of known and novel candidate genes. In Aim 2 we will determine goblet cell-intrinsic vs. goblet cell extrinsic pathways that inhibit allergen transport. Our preliminary data in mice suggests that the number and function of GAPs prominently contribute to susceptibility to oral anaphylaxis. Using bone marrow chimeras and in vitro human or mouse enteroid cultures we will directly test whether transcellular transport of allergens is increased in anaphylaxis susceptible humans or mice and will determine whether these phenotypes are epithelial cell-intrinsic or -extrinsic. In Aim 3 we will perform a targeted screen of FDA approved drugs that could inhibit goblet cell differentiation and/or function for their ability to block fluorescent allergen uptake in vitro and oral anaphylaxis in vivo. Effective compounds will be validated using human gut enteroids from donors with or without food allergy. We have already identified multiple drugs that reduce allergen transport in oral anaphylaxis susceptible mice, highlighting that pharmaceutical blockade of these pathways could potentially prevent anaphylaxis in patients with food allergy. Impact: Identifying genes and cellular pathways regulating food allergen transport by gut epithelia could lead to new ways of preventing anaphylaxis and to diagnostic approaches to more accurately stratify anaphylaxis risk for food-allergic individuals.

项目概要 该提案的目标是通过定义跨肠道上皮运输完整食物过敏原的细胞和分子机制，确定预防食物过敏者过敏反应的新方法。在最严重的情况下，食物过敏会引发危及生命的过敏反应。决定过敏反应风险的因素之一是肠道屏障的完整性，但没有治疗方法可以降低肠道对食物过敏原的通透性。过敏原跨肠上皮转运的主要模式是通过分泌性和杯状细胞相关抗原通道 (GAP) 进行跨细胞运输。利用花生和鸡蛋过敏的小鼠模型，我们取得了令人兴奋的发现，即对口腔过敏反应的易感性是由基因决定的，并且与 GAP 数量和功能的增加有关。使用正向遗传筛选，我们鉴定了追踪这种表型的单个染色体区域。此外，针对杯状细胞生物学的药物治疗减少了体内过敏反应。我们假设肠道杯状细胞数量和运输能力的遗传调控通过控制肠道对完整食物过敏原的通透性来决定过敏反应的易感性。在目标 1 中，使用上述互补小鼠品系，我们将通过 SNP 基因分型进行数量性状位点 (QTL) 作图，以确定与肠道通透性降低相关的口腔过敏反应的遗传抗性位点。这些数据将与过敏反应易感性与耐药性同窝小鼠肠上皮的 scRNA-Seq 分析相结合，以确定过敏原转运的细胞类型特异性调节因子。在小鼠中使用 CRISPR/Cas9 基因编辑，我们将测试已知和新型候选基因对体内肠道通透性和口腔过敏反应的贡献。在目标 2 中，我们将确定抑制过敏原转运的杯状细胞内在途径和杯状细胞外在途径。我们在小鼠身上的初步数据表明，GAP 的数量和功能显着影响口腔过敏反应的易感性。使用骨髓嵌合体和体外人或小鼠肠样培养物，我们将直接测试过敏反应易感人或小鼠中过敏原的跨细胞转运是否增加，并将确定这些表型是上皮细胞内在的还是外在的。在目标 3 中，我们将对 FDA 批准的药物进行有针对性的筛选，这些药物可以抑制杯状细胞分化和/或功能，因为它们能够阻止体外荧光过敏原吸收和体内口腔过敏反应。有效的化合物将使用来自有或没有食物过敏的捐赠者的人肠道肠素进行验证。我们已经发现了多种药物可以减少口腔过敏反应易感小鼠的过敏原转运，这强调了药物阻断这些途径可能会预防食物过敏患者的过敏反应。影响：识别通过肠道上皮细胞调节食物过敏原转运的基因和细胞途径可能会带来预防过敏反应的新方法，以及更准确地对食物过敏个体的过敏反应风险进行分层的诊断方法。