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，我们的目标是揭示DNA甲基组对花生/坚果过敏的影响。在一项横断面研究中，我们将临床项目A2中招募的过敏性患者的甲基组与非过敏性对照进行比较，以找到花生/坚果过敏的决定因素。我们的纵向方法比较了A2饮食干预研究前后的甲基化模式，将确定潜在耐受性发展的甲基组的变化和治疗反应的潜在预测因素。在第一个资助期，我们对外周血单个核细胞(PBMC)进行了原理证明分析。我们已经建立了从PBMC分离、DNA/RNA提取到甲基化分析的完整实验流程，包括数据处理、关联分析和差异甲基化位置和区域(DMPS和DMRS)的功能评估。尽管由于COVID19大流行，这项研究是在一个减少的数据集中进行的，但我们发现了1个具有全基因组意义的DMP和一些可能与免疫相关功能相关的候选DMP和DMR。有趣的是，其中两个候选DMRS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，我们还建立了一个使用花生和榛子的体外细胞刺激方案，以研究DNA甲基化和基因转录中过敏原特异性的差异。事实上，横断面分析的结果显示，花生/坚果过敏个体在未刺激和刺激的PBMC中存在重叠和明显的甲基化差异，表明过敏原特异性刺激对DNA甲基化的影响。第二个资助期的主要重点是分析特定PBMC亚群中的甲基化。通过使用亚群，过敏和非过敏个体之间的甲基化差异应该更加明显，并将检测到细胞类型特定的免疫机制。我们将研究CD4+和CD8+T细胞，这两种细胞都已被证明与过敏反应和耐受性的形成有关。此外，我们还将完成纵向分析，通过分析过敏原特异性提取物刺激PBMC所产生的甲基化差异来扩展纵向分析。这两种方法，分析PBMC亚群及其过敏原特异性刺激，将加强对花生/坚果过敏和耐受性诱导所涉及的免疫机制的识别。最后，将与其他KFO项目一起分析已确定的DMRS，以了解它们作为微生物组等环境因素的介体的作用，它们参与形成免疫表型的过程，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.