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Abstract：
BACKGROUND: Crohn\'s disease complicated by perianal fistulae is more prevalent and severe in patients of African ancestry.
METHODS: We profiled single cells from diverse patients with Crohn\'s disease with perianal fistula from colorectal mucosa and fistulous tracts. Immunofluorescence was performed to validate predicted cell-cell interactions. Unstimulated monocytes were chronically cultured in diverse cohorts. A subset was analyzed by single-nucleus RNA + ATAC sequencing.
RESULTS: Fistulous tract cells from complete proctectomies demonstrated enrichment of myeloid cells compared to paired rectal tissues. Ligand-receptor analysis highlights myeloid-stromal cross-talk and cellular senescence, with cellular co-localization validated by immunofluorescence. Chitinase-3 like-protein-1 (CHI3L1) is a top upregulated gene in stromal cells from fistulae expressing both destructive and fibrotic gene signatures. Monocyte cultures from patients of African ancestry and controls demonstrated differences in CHI3L1 and oncostatin M (OSM) expression upon differentiation compared to individuals of European ancestry. Activating protein-1 footprints are present in ATAC-seq peaks in stress response genes, including CHI3L1 and OSM; genome-wide chromatin accessibility including JUN footprints was observed, consistent with reported mechanisms of inflammatory memory. Regulon analyses confirm known cell-specific transcription factor regulation and implicate novel ones in fibroblast subsets. All pseudo-bulked clusters demonstrate enrichment of genetic loci, establishing multicellular contributions. In the most significant African American Crohn\'s genetic locus, upstream of prostaglandin E receptor 4, lymphoid-predominant ATAC-seq peaks were observed, with predicted RORC footprints.
CONCLUSIONS: Population differences in myeloid-stromal cross-talk implicate fibrotic and destructive fibroblasts, senescence, epigenetic memory, and cell-specific enhancers in perianal fistula pathogenesis. The transcriptomic and epigenetic data provided here may guide optimization of promising mesenchymal stem cell therapies for perianal fistula.
BACKGROUND: This work was supported by grants U01DK062422, U24DK062429, and R01DK123758.
METHODS: We profiled single cells from diverse patients with Crohn\'s disease with perianal fistula from colorectal mucosa and fistulous tracts. Immunofluorescence was performed to validate predicted cell-cell interactions. Unstimulated monocytes were chronically cultured in diverse cohorts. A subset was analyzed by single-nucleus RNA + ATAC sequencing.
RESULTS: Fistulous tract cells from complete proctectomies demonstrated enrichment of myeloid cells compared to paired rectal tissues. Ligand-receptor analysis highlights myeloid-stromal cross-talk and cellular senescence, with cellular co-localization validated by immunofluorescence. Chitinase-3 like-protein-1 (CHI3L1) is a top upregulated gene in stromal cells from fistulae expressing both destructive and fibrotic gene signatures. Monocyte cultures from patients of African ancestry and controls demonstrated differences in CHI3L1 and oncostatin M (OSM) expression upon differentiation compared to individuals of European ancestry. Activating protein-1 footprints are present in ATAC-seq peaks in stress response genes, including CHI3L1 and OSM; genome-wide chromatin accessibility including JUN footprints was observed, consistent with reported mechanisms of inflammatory memory. Regulon analyses confirm known cell-specific transcription factor regulation and implicate novel ones in fibroblast subsets. All pseudo-bulked clusters demonstrate enrichment of genetic loci, establishing multicellular contributions. In the most significant African American Crohn\'s genetic locus, upstream of prostaglandin E receptor 4, lymphoid-predominant ATAC-seq peaks were observed, with predicted RORC footprints.
CONCLUSIONS: Population differences in myeloid-stromal cross-talk implicate fibrotic and destructive fibroblasts, senescence, epigenetic memory, and cell-specific enhancers in perianal fistula pathogenesis. The transcriptomic and epigenetic data provided here may guide optimization of promising mesenchymal stem cell therapies for perianal fistula.
BACKGROUND: This work was supported by grants U01DK062422, U24DK062429, and R01DK123758.
摘要：
背景：克罗恩病并发肛周瘘在非洲血统的患者中更为普遍和严重。
方法：我们从结肠直肠粘膜和瘘道分析了克罗恩病合并肛周瘘的不同患者的单细胞。进行免疫荧光以验证预测的细胞-细胞相互作用。将未刺激的单核细胞在不同的队列中慢性培养。通过单核RNA+ATAC测序分析子集。
结果：与成对的直肠组织相比，来自完整的直肠切除术的尿道细胞显示出骨髓细胞的富集。配体受体分析突出了骨髓基质串扰和细胞衰老，通过免疫荧光验证细胞共定位。几丁质酶-3样蛋白-1（CHI3L1）是表达破坏性和纤维化基因特征的瘘基质细胞中的最高上调基因。与欧洲血统的个体相比，来自非洲血统和对照组患者的单核细胞培养物显示出分化后CHI3L1和制瘤素M（OSM）表达的差异。激活蛋白-1足迹存在于应激反应基因的ATAC-seq峰中，包括CHI3L1和OSM；观察到包括JUN足迹在内的全基因组染色质可及性，与报道的炎症记忆机制一致。调节子分析证实了已知的细胞特异性转录因子调节，并在成纤维细胞亚群中暗示了新的转录因子。所有假性聚集簇都显示出遗传基因座的富集，建立多细胞贡献。在最重要的非裔美国人克罗恩的基因位点，在前列腺素E受体4的上游，观察到以淋巴为主的ATAC-seq峰，预测的RORC足迹。
结论：骨髓基质串扰的群体差异涉及纤维化和破坏性成纤维细胞，衰老，表观遗传记忆,和细胞特异性增强剂在肛瘘发病机制中的作用。本文提供的转录组和表观遗传学数据可以指导有前途的间充质干细胞治疗肛瘘的优化。
背景：这项工作得到了授权U01DK062422，U24DK062429和R01DK123758的支持。
方法：我们从结肠直肠粘膜和瘘道分析了克罗恩病合并肛周瘘的不同患者的单细胞。进行免疫荧光以验证预测的细胞-细胞相互作用。将未刺激的单核细胞在不同的队列中慢性培养。通过单核RNA+ATAC测序分析子集。
结果：与成对的直肠组织相比，来自完整的直肠切除术的尿道细胞显示出骨髓细胞的富集。配体受体分析突出了骨髓基质串扰和细胞衰老，通过免疫荧光验证细胞共定位。几丁质酶-3样蛋白-1（CHI3L1）是表达破坏性和纤维化基因特征的瘘基质细胞中的最高上调基因。与欧洲血统的个体相比，来自非洲血统和对照组患者的单核细胞培养物显示出分化后CHI3L1和制瘤素M（OSM）表达的差异。激活蛋白-1足迹存在于应激反应基因的ATAC-seq峰中，包括CHI3L1和OSM；观察到包括JUN足迹在内的全基因组染色质可及性，与报道的炎症记忆机制一致。调节子分析证实了已知的细胞特异性转录因子调节，并在成纤维细胞亚群中暗示了新的转录因子。所有假性聚集簇都显示出遗传基因座的富集，建立多细胞贡献。在最重要的非裔美国人克罗恩的基因位点，在前列腺素E受体4的上游，观察到以淋巴为主的ATAC-seq峰，预测的RORC足迹。
结论：骨髓基质串扰的群体差异涉及纤维化和破坏性成纤维细胞，衰老，表观遗传记忆,和细胞特异性增强剂在肛瘘发病机制中的作用。本文提供的转录组和表观遗传学数据可以指导有前途的间充质干细胞治疗肛瘘的优化。
背景：这项工作得到了授权U01DK062422，U24DK062429和R01DK123758的支持。
