Spatial proteomics and cell-cell crosstalk analysis reveal PD-L1 and IL-6 interaction in human primary sclerosing
Giulia Orlandi1, Diletta Overi2, Rosanna Di Tinco1
1Department of Surgery, Medicine Dentistry and Morphological Sciences with Interest in Transplant, University of Modena and Reggio Emilia, Modena, Italy.
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 crosstalk at the epithelial-immune interface.
Methods:
Spatial proteomic analysis was performed on six PSC-affected bile ducts and five healthy controls by GeoMx® DSP. Single-cell RNA-sequencing 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), which was associated with apoptosis in neighbouring immune cells on spatial proteomics. In vitro, aPBMCs induced the upregulation of 68 immunoregulatory genes in BECs; PD-L1 and IL-6 upregulation was confirmed at the protein level. BECs induced increased apoptosis in T (CD4+/CD8+) and NK cells. In co-culture, the exposure of BECs to the IL-6/sIL-6R complex induced significant PD-L1 upregulation (12.7 ± 1.3; p <0.001), which was inhibited by IL-6 receptor blockade (2.6 ± 1.2; p <0.001) or JAK1/2 inhibition.
Conclusions:
These findings define a critical immunosuppressive axis in PSC driven by BEC-derived IL-6 and PD-L1. Mechanistically, IL-6 acts as an upstream regulator of PD-L1 via JAK/STAT3 and NF-κB signalling, predominantly through IL-6 trans-signalling. This study provides a rationale for targeting epithelial-immune cell interactions as a therapeutic strategy in PSC.
Impact And Implications:
The pathogenic basis of primary sclerosing cholangitis (PSC) is poorly understood, hampering the development of specific therapeutic approaches. We identified IL-6 as a key upstream regulator of PD-L1 via the JAK/STAT3 and NF-κB pathways in biliary epithelial cells, highlighting its central role in regulating epithelial-immune crosstalk during biliary injury. The identification of the PD-L1 pathway in epithelial-immune cell interactions in PSC may improve understanding of the disease, as well as inform the development of therapeutic strategies.