Determinants of oral anaphylaxis to food

项目摘要 该提案的目标是通过定义将完整的食物过敏原运输穿过肠道上皮的细胞和分子机制来确定预防食物过敏反应的新方法。在最严重的情况下，食物过敏会引发危及生命的过敏反应。可以确定过敏反应风险的一个因素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渗透性。过敏原跨肠上皮转运的主要模式是通过分泌和杯状细胞相关抗原通道（GAP）进行跨细胞转运。使用花生和鸡蛋过敏的小鼠模型，我们已经取得了令人兴奋的发现，即对口服过敏反应的易感性是由遗传决定的，并与GAP的数量和功能增加有关。使用正向遗传筛选，我们确定了一个单一的染色体区域，跟踪这种表型。此外，靶向杯状细胞生物学的药物治疗减少了体内过敏反应。我们推测肠道杯状细胞数量和转运能力的遗传调节通过控制肠道对完整食物过敏原的渗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，我们将使用SNP基因分型进行数量性状基因座（QTL）作图，以鉴定与肠道通透性降低相关的口服过敏反应的遗传抗性基因座。这些数据将与过敏反应敏感与耐药同窝仔肠上皮的scRNA-Seq分析相结合，以鉴定过敏原转运的细胞类型特异性调节剂。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肠道通透性和体内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源性与杯状细胞外源性途径。我们在小鼠中的初步数据表明，GAP的数量和功能显著有助于对口服过敏反应的敏感性。使用骨髓嵌合体和体外人或小鼠肠道培养物，我们将直接测试过敏易感人或小鼠中过敏原的跨细胞转运是否增加，并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批准的药物进行靶向筛选，这些药物可以抑制杯状细胞分化和/或功能，因为它们能够阻断体外荧光过敏原摄取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供体的人肠类物质进行验证。我们已经确定了多种药物，减少过敏原运输在口服过敏敏感小鼠，强调这些途径的药物阻断可能会预防过敏反应的患者与食物过敏。影响力：确定基因和细胞途径调节食物过敏原运输的肠道上皮细胞可能会导致新的方法来预防过敏反应和诊断方法，以更准确地分层过敏反应的风险，食物过敏的个人。

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.