Spatial proteomics and cell-cell cross-talk analysis reveal PD-L1 and IL-6 interaction in human primary sclerosing cholangitis

Background & aims Primary sclerosing cholangitis (PSC) is a chronic, immune-mediated cholangiopathy characterized by persistent inflammation and fibrotic remodelling of the biliary tree. We combined spatial proteomics of PSC-affected large bile ducts with a co-culture system of biliary epithelial cells (BECs) and activated peripheral blood mononuclear cells (aPBMCs) to dissect the molecular cross-talk at the epithelial-immune interface. Methods Spatial proteomic analysis was performed on N=6 PSC-affected bile ducts and N=5 healthy controls by GeoMx® DSP. Single Cell RNA-seq analysis from publicly available datasets of human control (N=22) and PSC-affected (N=8) livers was performed. We developed an in vitro inflammatory model of PSC by transwell co-culture of BECs (i.e. H69 and primary human biliary tree stem cells) and primary human PBMCs (N=6). Co-cultures were analysed by transcriptomics on nCounter®, by immunoassays and by culture with/without blocking agents. Results Peribiliary glands showed higher PD-L1 expression in PSC (score=1.2±0.4) compared to controls (0.3±0.5; p<0.05) associated with apoptosis in neighbouring immune cells at spatial proteomics. In vitro, aPBMCs induced the upregulation of 68 immunoregulatory genes in BECs; PD-L1 and IL-6 up-regulation was confirmed at protein level. In turn, BECs determined an increased apoptosis in T (CD4+/CD8+) and NK cells. In co-culture, the exposure of BECs to IL-6/sIL-6r complex induced significant PD-L1 up-regulation (12.7±1.3; p<0.001) compared to controls (2.7±1.0), blocked by IL-6 Receptor (2.6±1.2; p<0.001) or JAK1/2 inhibitors. Conclusions These findings define a critical immunosuppressive axis in PSC driven by BEC-derived IL-6 and PD-L1. Mechanistically, we identified IL-6 as an upstream regulator of PD-L1 via JAK/STAT3 and NF-κB pathways, predominantly through IL-6 trans-signalling. Our study provides a rationale for targeting epithelial-immune cell interactions as a therapeutic strategy in PSC. IMPACT AND IMPLICATIONS Pathogenetic basis of primary sclerosing cholangitis (PSC) is poorly understood, hampering the development of specific therapeutic approaches. We individuated IL-6 as a key upstream regulator of PD-L1 via the JAK/STAT3 and NF-κB pathways in biliary epithelial cells, representing a major player in regulating epithelial-immune cross-talk during biliary injury. The individuation of PD-L1 pathway in immune-epithelial cell interactions in PSC may be of help in the understanding of this disease, as well as in defining treatment strategies.