Immune Signaling Mediates Stromal Changes to Support Epithelial Reprogramming in Celiac Duodenum

Coeliac Disease (CeD) is a chronic autoimmune disorder affecting 0.5-1% of the general population with a wide geographical distribution. Despite recent efforts to deeply phenotype gluten-specific immune activation at the single cell level, recent clinical studies targeting gluten degradation and other immune tolerance mechanisms have been unsuccessful. To this end, a deeper understanding of immune and non-immune cellular dynamics and interactions are required to characterize tissue-specific mechanisms responsible for CeD pathogenesis, repair, and resolution. Here, we assembled the most comprehensive scRNAseq dataset in Coeliac Disease to date, including 203,555 cells across 21 active CeD and 11 control duodenal samples. Compared to control duodenum, CeD was characterized by single cell differential changes in abundance, gene expression and cell-cell interactions across cellular compartments. In the immune compartments, CeD samples showed expected increases in plasma cell abundance and shifts toward type 1 effector biology (e.g., increase in cycling CD8pos, ?d T cells and IFNG transcriptional shifts) and Tfh-related biology (e.g., increases in IL21 signaling to effector T cells). In addition, activated myeloid subsets, including DC2 and monocytes, were increased in disease and were characterized by increased pro-inflammatory pathway expression, including IL-1ß. Non-immune compartments showed increased stem/crypt and secretory enterocytes in CeD samples with a decrease in absorptive enterocytes, reflecting the villus atrophy and crypt hyperplasia hallmarks of CeD epithelial dysfunction. Accompanying the epithelial changes, distinct changes in stromal populations were identified, particularly with increases in abundance and transcriptional activity of NRG1 and SMOC2 fibroblasts. Cell-cell interaction analysis across multiple cellular compartments proposed a distinct increased role of fibroblasts to support the epithelial reprogramming of the increased stem/crypt epithelial fraction in CeD, mediated by myeloid derived IL-1ß signal and lymphoid-derived IFN-?. This dataset reveals a previously unknown role for T-myeloid-stromal-epithelial cell communication in CeD, highlighting key mechanisms of the tissue-level cellular dynamics in response to gluten ingestion. Overall design: Single cells were isolated from cryopreserved duodenal biopsies. Briefly, biopsies were dissociated using liberase/dnase solutions combined with removal of dead cells using EasySep™ Dead Cell Removal (Annexin V) Kit.

乳糜泻（Coeliac Disease, CeD）是一种慢性自身免疫性疾病，全球普通人群患病率为0.5%~1%，地理分布广泛。尽管近年来学界已尝试在单细胞水平深入解析谷蛋白特异性免疫激活特征，但针对谷蛋白降解及其他免疫耐受机制的临床研究均未取得成功。为此，亟需更深入地解析免疫与非免疫细胞的动态变化及相互作用，以明确驱动乳糜泻发病、组织修复及病情缓解的组织特异性机制。

本研究整合了目前最全面的乳糜泻单细胞RNA测序（single-cell RNA sequencing, scRNAseq）数据集，涵盖21例活动性乳糜泻患者及11例对照的十二指肠活检样本，共计203555个细胞。与对照十二指肠组织相比，乳糜泻患者的细胞组分在细胞丰度、基因表达及细胞间相互作用层面均出现显著的单细胞差异变化。

在免疫细胞组分中，乳糜泻样本呈现出预期的浆细胞丰度升高，且向1型效应细胞生物学特征偏移（例如增殖性CD8阳性细胞、γδ T细胞增多，以及干扰素γ（Interferon gamma, IFNG）转录特征改变），同时向滤泡辅助T细胞（Follicular helper T cells, Tfh）相关生物学特征偏移（例如向效应T细胞传递白细胞介素21（Interleukin 21, IL21）信号的能力增强）。此外，疾病组中活化的髓系细胞亚群（包括DC2亚群及单核细胞）丰度升高，且表现出促炎通路表达上调的特征，包括白细胞介素1β（Interleukin 1β, IL-1β）。

非免疫细胞组分中，乳糜泻样本的干细胞/隐窝肠上皮细胞与分泌型肠上皮细胞占比升高，而吸收型肠上皮细胞占比降低，这一特征与乳糜泻上皮功能障碍的典型表现——绒毛萎缩及隐窝增生相吻合。伴随上皮细胞的改变，基质细胞群也出现显著变化，尤其是神经调节蛋白1（Neuregulin 1, NRG1）及富含半胱氨酸的酸性分泌蛋白样2（Secreted protein acidic and cysteine rich 2, SMOC2）阳性成纤维细胞的丰度与转录活性均有所升高。

跨多细胞组分的细胞间相互作用分析表明，在髓系来源的IL-1β信号与淋巴系来源的IFN-γ介导下，成纤维细胞在支持乳糜泻中增多的干细胞/隐窝上皮组分的上皮重编程过程中发挥了此前未被发现的重要作用。本数据集揭示了T细胞-髓系细胞-基质细胞-上皮细胞通信网络在乳糜泻中的全新功能，阐明了机体针对谷蛋白摄入产生的组织水平细胞动态变化的关键机制。

实验设计概述：从冷冻保存的十二指肠活检组织中分离单细胞。简要流程为：使用Liberase/脱氧核糖核酸酶解离活检组织，并通过EasySep™ Dead Cell Removal (Annexin V) Kit去除死细胞。