P038 A single-cell transcriptomics approach reveals high-resolution cellular signatures in Crohn’s disease

Abstract

Abstract Background Single-cell RNA sequencing (sc-RNAseq) provides high-resolution analysis of individual cells and cell clusters within complex tissues like the intestinal mucosa. No study to date has applied this novel technology to understand the complexity of intestinal inflammation in Crohn’s disease (CD) patients. Our aim is to identify the cellular subsets present in healthy and inflamed colon, and to describe how individual subsets are regulated in active CD. Methods Colon biopsies from a healthy control (HC) and two patients with endoscopically active colonic CD were collected and processed by enzymatic digestion and cell sorting to discard dead cells. Single-cell suspensions were processed by droplet-based protocol 10× chromium system followed by library preparation and sequencing. Sequencing data were aligned and quantified using Cell Ranger. Downstream analysis was performed using the R package Seurat. Results scRNA-seq analysis identified 14 transcriptionally different cell subsets in the healthy colon, including diverse types of T cells, plasma cells, mesenchymal cells, epithelial cells and myeloid populations, as well as a subset of MHCII+ CD20+ B cells. Overall, the number of transcriptionally differentiated cellular subsets was greater (16 and 17 subsets per sample) in the two CD colonic samples revealing higher cellular heterogeneity in active CD. In particular, in the HC we identified three T-cell subsets including a CD4+, CD8+ and a smaller subset of intraepithelial lymphocytes. In contrast, samples from CD patients presented four to six clearly differentiated T-cell clusters depending on the patient, with a marked enrichment of cytotoxicity genes (GZMA, GZMB, NKG7, CCL5 and AOAH) and T-regulatory genes (FOXP3, CTLA4, BATF and IL2RA). In addition, the HC sample presented three differentiated subsets of antibody-secreting plasma cells with predominant IgA and IgM production. In contrast, a clear expansion of IgG-producing plasma cells was observed in both CD patients. Moreover, the profiles of specific subsets significantly changed revealing transcriptional regulation within the individual subsets. Specifically, macrophages within the HC mucosa expressed the M2 markers CD163 and CD209 together with a whole signature of 163 additional genes, while in CD, they lost expression of these markers and upregulated CXCL8 and IL1B which were co-expressed with 268 additional genes common to macrophage populations in both CD patients. Conclusion We show that sc-RNAseq can be applied to study at a high cellular resolution human intestinal biopsies. Understanding the transcriptomic profile of the CD-related subsets will help dissect disease pathophysiology and provide knowledge for therapeutic targets.