Table_1_Effects of In Vivo Gluten Challenge on PBMC Gene Expression Profiles in Diet Treated Celiac Disease.xlsx

The pathological mechanisms that lead to the onset and reactivation of celiac disease (CD) remain largely unknown. While gluten free diet (GFD) improves the intestinal damage and associated clinical symptoms in majority of cases, it falls short of providing full recovery. Additionally, late or misdiagnosis is also common as CD presents with a wide range of symptoms. Clear understanding of CD pathogenesis is thus critical to address both diagnostic and treatment concerns. We aimed to study the molecular impact of short gluten exposure in GFD treated CD patients, as well as identify biological pathways that remain altered constitutively in CD regardless of treatment. Using RNAseq profiling of PBMC samples collected from treated CD patients and gluten challenged patient and healthy controls, we explored the peripheral transcriptome in CD patients following a short gluten exposure. Short gluten exposure of just three days was enough to alter the genome-wide PBMC transcriptome of patients. Pathway analysis revealed gluten-induced upregulation of mainly immune response related pathways, both innate and adaptive, in CD patients. We evaluated the perturbation of biological pathways in sample-specific manner. Compared to gluten exposed healthy controls, pathways related to tight junction, olfactory transduction, metabolism of unsaturated fatty acids (such as arachidonic acid), metabolism of amino acids (such as cysteine and glutamate), and microbial infection were constitutively altered in CD patients regardless of treatment, while GFD treatment appears to mostly normalize immune response pathways to “healthy” state. Upstream regulator prediction analysis using differentially expressed genes identified constitutively activated regulators relatively proximal to previously reported CD associated loci, particularly SMARCA4 on 19p13.2 and CSF2 on 5q31. We also found constitutively upregulated genes in CD that are in CD associated genetic loci such as MEF2BNB-MEF2B (BORCS8-MEF2B) on 19p13.11 and CSTB on 21q22.3. RNAseq revealed strong effects of short oral gluten challenge on whole PBMC fraction and constitutively altered pathways in CD PBMC suggesting important factors other than gluten in CD pathogenesis.

乳糜泻（celiac disease, CD）的发病与复发病理机制迄今仍未完全明确。尽管无麸质饮食（gluten free diet, GFD）可改善多数患者的肠道损伤及相关临床症状，但无法实现完全康复。此外，由于CD的临床表现谱广泛，延误或误诊的情况也较为常见。因此，明确CD的发病机制对解决诊断与治疗难题均至关重要。
本研究旨在探究接受无麸质饮食治疗的CD患者接受短期麸质暴露后的分子效应，并识别无论是否接受治疗，CD患者体内始终存在持续性异常改变的生物学通路。我们对接受无麸质饮食治疗的CD患者、麸质激发后的CD患者以及健康对照的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）进行RNA测序（RNAseq）分析，探究短期麸质暴露后CD患者的外周转录组特征。
仅3天的短期麸质暴露即可改变CD患者全基因组范围内的PBMC转录组。通路富集分析显示，在CD患者中，麸质暴露主要上调了天然免疫与适应性免疫相关的免疫应答通路。
我们以样本为单位评估了生物学通路的扰动情况。与麸质暴露后的健康对照相比，无论是否接受治疗，CD患者体内均存在持续性异常改变的通路，包括紧密连接通路、嗅觉转导通路、不饱和脂肪酸（如花生四烯酸）代谢通路、氨基酸（如半胱氨酸与谷氨酸）代谢通路以及微生物感染相关通路；而无麸质饮食治疗似乎主要将免疫应答通路恢复至"健康"状态。
利用差异表达基因进行上游调控因子预测分析，我们发现了与既往报道的CD相关基因座相对邻近的持续性激活调控因子，尤其是位于19p13.2的SMARCA4以及位于5q31的CSF2。
我们还发现CD患者体内存在持续性上调的基因，这些基因位于CD相关遗传基因座中，例如19p13.11区域的MEF2BNB-MEF2B（BORCS8-MEF2B）以及21q22.3区域的CSTB。
RNA测序结果显示，短期口服麸质激发对全PBMC组分具有显著影响，且CD患者的PBMC存在持续性异常的生物学通路，这表明CD的发病机制中除麸质外还存在其他重要致病因素。