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Spatiotemporal single cell transcriptomic analysis of human gut macrophages reveals multiple functional and niche-specific subsets

ABSTRACT: Colonic resections were obtained from patients operated for sigmoid colon cancer at Akershus University Hospital (Ahus, 1478 LÃƒÂ¸renskog, Norway). The resected colon was immediately examined by an experienced pathologistand macroscopically normal colon, at least 10 cm from the tumor, were placed in vials with RPMI 1640 and put on ice for transport. The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants, and the study was approved by the Regional Committee for Medical Research Ethics (REK, 2018/703, Health Region South-East, Norway). For scRNA-seq, 4 colon specimens from sigmoid or ascending colon were obtained (age 62-78, 3 males). Immunofluorescence stainings were done on samples from 8 patients (age 62-78, 4 males) from ascending, transverse, descending and sigmoid colon. None of the patients had autoimmune, infectious or inflammatory diseases, nor received neoadjuvant chemotherapy or radiation therapy before the operation. All patients were operated following the national guidelines.

PROVIDER: EGAS00001005377 | EGA |

REPOSITORIES: EGA

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Project description:Background and aims: Mucosal abnormalities are potentially important in the primary pathogenesis of ulcerative colitis (UC). We investigated the mucosal transcriptomic expression profiles of biopsies from patients with UC and healthy controls (HC), taken from macroscopically non-inflamed tissue from the terminal ileum and three colonic locations with the objective of identifying abnormal molecules that might be involved in disease development. Methods: Whole-genome transcriptional analysis was performed on intestinal biopsies taken from 24 UC, 26 HC and 14 patients with CrohnM-bM-^@M-^Ys disease. Differential gene expression analysis was performed at each tissue location separately and results were then meta-analysed using FisherM-bM-^@M-^Ys method. Significantly differentially expressed genes were validated using qPCR. Gene location within the colon was determined using immunohistochemistry, subcellular fractionation, electron and confocal microscopy. DNA methylation was quantified by pyrosequencing. Results: Seven probes were abnormally expressed throughout the colon in UC patients with Family with sequence similarity member 5 C (FAM5C) being the most significantly underexpressed. Attenuated expression of FAM5C in UC was independent of inflammation, unrelated to phenotype or treatment, and remained low at rebiopsy approximately 23 months later. FAM5C is localised to the brush border of the colonic epithelium and expression is influenced by DNA methylation within its promoter. Conclusion: Genome-wide expression analysis of non-inflamed mucosal biopsies from UC patients identified FAM5C as significantly under-expressed throughout the colon in a major sub-set of patients with UC. Low levels of this gene could predispose to or contribute to the maintenance of the characteristic mucosal inflammation seen in this condition. Total RNA was extracted from the intestinal biopsies taken from macroscopically normal mucosa in the rectum, descending colon, ascending colon and terminal ileum in clinically quiescent Ulcerative colitis and Crohn's disease patients and compared to healthy controls. Normalized data for 26,261 probes out of 47,323 only. Criteria for inclusion not specified. The non-normalized matrix contains the complete non-normalized data for all probes.

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Spatiotemporal single cell transcriptomic analysis of human gut macrophages reveals multiple functional and niche-specific subsets

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