Il-17 Signaling in Primary Sclerosing Cholangitis Patient-Derived Organoids
The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as extrahepatic cholangiocyte organoids (ECO). The ECO were treated with vehicle or IL-17A and assessed by NanoString analysis, single cell RNA sequencing (scRNA-seq), and whole genome sequencing (WGS). The secretome was assessed by Olink analysis. Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between non-PSC and PSC ECO. However, PSC ECO cells demonstrated a much more robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) after the treatment with IL-17A, compared to non-PSC ECO.  After rigorous filtering, WGS identified the presence of somatic mutations that were shared between PSC ECO. However, no somatic mutations dysregulating genes in the IL-17 pathway were identified. The secretome of PSC ECO contained more abundant chemokines and cytokines under basal conditions and following IL-17A stimulation when compared to non-PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation regardless of mutational status, suggesting a change in epigenetic regulation and the implication of this pathway in the pathogenesis of PSC. Overall design: Extrahepatic cholangiocyte organoids (ECO) from PSC (n=4) and non-PSC patients (n=4), all at passage 3, were treated with vehicle or human recombinant IL-17A (100 ng/mL) for 24 hours. The organoids underwent trypsin digestion followed by mechanical dissociation to obtain single cell suspensions in PBS + 0.1% BSA and were submitted to the Gene Analysis Core of the Medical Genome Facility (Mayo Clinic, Rochester, MN) for scRNA-seq analysis.