Expression profiling by high throughput sequencing
Summary
Crohn’s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identified a distinct epithelial cell type in both terminal ileum and ascending colon (termed “LND”) with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, were rare in non-IBD controls but significantly expanded in active CD. These cells actively interacted with immune cells and specifically expressed IBD/CD susceptibility genes, as demonstrated by multimodal data, suggesting a possible role in CD immunopathogenesis. Furthermore, we discovered early and late LND subpopulations with different origins and developmental potential. A higher ratio of late to early LND cells correlated with better response to anti-TNF treatment. These findings suggest a potential pathogenic role for LND cells in both Crohn’s ileitis and colitis.
Overall design
65 CD and 18 non-IBD control patients in both TI and AC were collected. Tissues were incubated in a chelating buffer (0.5M EDTA, 0.1M DTT in DPBS) for 1.25hrs, and then transferred to cold active protease (5 mg/ml Protease from Bacillus licheniformis, 2.5 mg/mL DNase in PBS) for 25 minutes at 4˚c. Tissues were then pipetted 10-20 times to yield single cells. Cell suspensions were filtered, washed, and inspected for count and quality before loading onto inDrops for microfluidic capture. inDrops scRNA-seq was performed on the Novaseq6000. Brief descriptions for the various CD types: Control - non-IBD control, patient without IBD with normal colon histology Moderate - CD with chronic epithelial changes and moderately active disease by histology Normal_CD - CD with no chronic epithelial changes or active disease by histology Mild - CD with chronic epithelial changes and mildly active disease by histology Quiescent - CD with chronic epithelial changes but no active disease by histology Severe - CD with chronic epithelial changes and severely active disease by histology
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