Epigenetic mechanisms in the development of peanut/tree nut allergy

表观遗传变化是基因表达的关键调控机制。在子项目B2中，我们的目标是解开DNA甲基化对花生/树坚果过敏的影响。在一项横断面研究中，我们比较了在临床项目A2中招募的过敏患者与非过敏对照的甲基化组，以找到花生/树坚果过敏的决定因素。我们的纵向方法比较之前和之后的饮食干预研究A2的甲基化模式将确定潜在的耐受性发展和治疗responsibility.In第一个资金周期，我们进行了外周血单核细胞（PBMC）的原理分析的证据甲基化组的变化。我们已经建立了从PBMC分离和DNA/RNA提取到甲基化分析的完整实验工作流程，包括数据处理，关联分析和差异甲基化位置和区域（DMPs和DMR）的功能评估。尽管由于COVID 19大流行，本研究是在缩减的数据集中进行的，但我们在全基因组显著性上鉴定了1个SNP，以及许多可能与免疫相关功能相关的候选DMP和DMR。有趣的是，两个候选DMR得到了先前关于IgE致敏的甲基化研究的支持。除了工作计划外，我们还建立了一个体外细胞刺激方案，使用花生和榛子来研究DNA甲基化和基因转录的过敏原特异性差异。事实上，横截面分析的结果揭示了花生/树坚果过敏个体在未刺激和刺激的PBMC之间的重叠和独特的甲基化差异，表明过敏原特异性刺激对DNA甲基化的影响。通过使用亚群，过敏性和非过敏性个体之间的甲基化差异应该更明显，并且将检测细胞类型特异性免疫机制。我们将研究CD 4+和CD 8 + T细胞，这两种细胞都被证明参与过敏反应和耐受性的发展。此外，我们将完成纵向分析，通过分析用变应原特异性提取物刺激PBMC后产生的甲基化差异来扩展分析。这两种方法，PBMC亚群及其过敏原特异性刺激的分析，将加强识别参与花生/树坚果过敏和耐受性诱导的免疫机制。最后，将与其他KFO项目联合分析所确定的DMR，以了解它们作为环境因素（如微生物组）的介体的作用，它们参与形成免疫表型，以及它们与miRNA表达的潜在相互作用。

Epigenetic changes are key regulatory mechanisms of gene expression. In subproject B2, we aim to unravel the impact of the DNA methylome on peanut/tree nut allergy. In a cross sectional study, we compare the methylome of allergic patients recruited in the clinical project A2 with non-allergic controls in order to find determinants of peanut/tree-nut allergy. Our longitudinal approach comparing the methylation patterns before and after the dietary intervention study of A2 will identify changes in the methylome underlying tolerance development and potential predictors of treatment response.In the first funding period, we performed a proof of principle analysis on peripheral blood mononuclear cells (PBMCs). We have established the complete experimental workflow from PBMC isolation and DNA/RNA extraction to methylation profiling including data processing, association analyses and functional assessment of differentially methylated positions and regions (DMPs and DMRs). Although due to COVID 19 pandemic this study was performed in a reduced data set, we identified 1 DMP at genome-wide significance and a number of candidate DMPs and DMRs which could be linked to immune-related functions. Interestingly, two of the candidate DMRs are supported by a previous methylation study on IgE sensitization. In addition to the work program, we have established an in vitro cell stimulation protocol using peanut and hazelnut to investigate allergen-specific differences in DNA methylation and gene transcription. Indeed, the results of the cross-sectional analysis revealed both overlapping and distinct methylation-differences between peanut/tree nut allergic individuals in unstimulated and stimulated PBMCs, indicating an effect of allergen-specific stimulation on DNA methylation.The main focus of the second funding period is on the analysis of the methylome in specific subpopulations of PBMCs. By using subpopulations, methylation differences between allergic and non-allergic individuals should be more pronounced and cell-type specific immune mechanisms will be detected. We will investigate CD4+ and CD8+ T-cells, both of which have been shown to be involved in the allergic response and tolerance development. In addition, we will complete the longitudinal analysis which will be extended by the analysis of methylation differences arising upon PBMC stimulation with allergen-specific extract. Both approaches, the analysis of PBMC subpopulations and their allergen-specific stimulation, will strengthen the identification of immune mechanisms involved in peanut/tree nut allergy and tolerance induction. Finally, the identified DMRs will be analyzed jointly with other KFO projects regarding their role as mediators of environmental factors such as the microbiome, their involvement in shaping immunological phenotypes, and their potential interaction with miRNA expression.