Camat, D. | Search bioRxiv

bioRxiv2023

Single-cell and spatial transcriptomics reveals the human liver immunological landscape and myeloid dysfunction in PSC.

BackgroundPrimary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease characterized by bile retention, biliary tree destruction, and progressive fibrosis leading to end stage liver disease and transplantation. There is an unmet need to understand the cellular composition of the PSC liver and how it underlies disease pathogenesis. As such, we generated a comprehensive atlas of the PSC liver and a reference healthy liver dataset using multiple multi-omic modalities and functional validation. MethodsIn this work, we employed single-cell (12,000 cells), single-nuclei (23,000 nuclei), and spatial transcriptomics (1 sample by 10x Visium and 3 samples with multi-region profiling by Nanostring GeoMx DSP) to profile the cellular ecosystem in 5 patients with PSC. Transcriptomic profiles were compared to 100k single cell transcriptomes and spatial transcriptomics controls from 24 healthy neurologically deceased donor (NDD) livers. Flow cytometry and intracellular cytokine staining was performed to validate PSC-specific differences in immune phenotype and function. ResultsPSC explants with cirrhosis of the liver parenchyma and prominent periductal fibrosis were associated with a unique population of hepatocytes which transformed to a cholangiocyte-like phenotype. These hepatocytes were surrounded by diverse immune cell populations, including monocyte-like macrophages, liver-resident and circulating natural killer (NK) cells. Inflamed cholangiocytes, fibrosis-resident hepatic stellate cells, and endothelial cells released cytokines that recruited CD4+T-cells, dendritic cells, and neutrophils to the PSC liver. Tissue-resident macrophages, by contrast, were reduced in number and exhibited a dysfunctional inflammatory response to LPS and IFN-{gamma} stimulation. ConclusionsWe present the first comprehensive atlas of the PSC liver and demonstrate hyper-activation and exhaustion-like phenotypes of myeloid cells and markers of chronic cytokine expression in late-stage PSC lesions. Lay SummaryPrimary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic inflammation and irreparable damage to the bile ducts. Due to a limited understanding of the underlying pathogenesis of disease, there remains a paucity of treatment options. As such, we sequenced healthy and diseased livers to compare the activity, interactions, and localization of immune and non-immune cells. This revealed that outside PSC scar regions, hepatocytes are transitioning to bile duct cells, whereas within the scars, there is an accumulation of immune cells. Of these cells, macrophages that typically contribute to tissue repair were enriched in immunoregulatory genes and were less responsive to stimulation. These cells are likely involved in maintaining hepatic inflammation and could be targeted in novel therapeutic development.

cell biology↗

bioRxiv2022

Single cell profiling reveals strain-specific differences in myeloid inflammatory potential in the rat liver

Liver transplantation is currently the only treatment for end-stage liver disease and acute liver failure. Liver transplant rejection is among the most lethal complications of transplantation, and therapeutic development is limited by our lack of a comprehensive understanding of the cellular landscape of the liver. The laboratory rat (Rattus norvegicus), ideal in size as a model for surgical procedures, is a strong platform to study liver biology in the context of liver transplantation. Liver allograft rejection is known to be strain-specific in the rat model, although the transplantation is accepted without rejection in some strains, it leads to acute rejection in others. To shed light on the cellular landscape of the rat liver and build a foundation for strain comparison, we present a comprehensive single-cell transcriptomics map of the healthy rat liver of Lewis and Dark Agouti strains. Using a novel computational pipeline we developed to guide the detailed annotation of our rat liver atlas, we discovered that hepatic myeloid cells have strong Lewis and Dark Agouti strain-specific differences focused on inflammatory signaling pathways. We experimentally validated these strain-specific differences in myeloid inflammatory potential in vitro using intracellular cytokine staining. Our work provides the first examination of the multi-strain healthy rat liver by single cell transcriptomics and uncovers key insights into strain-specific differences in this valuable model animal. SummaryThe laboratory rat (Rattus norvegicus) is a standard model animal for orthotopic liver transplantation. Transplanting a liver from a Dark agouti (DA) to a Lewis (LEW) strain rat leads to transplant rejection and the reverse procedure leads to tolerance. Understanding this strain difference may help explain the cellular drivers of liver allograft rejection post-transplant. This study uses single-cell transcriptomics to better understand the complex cellular composition of the rat liver and unravels cellular and molecular sources of inter-strain hepatic variation. We generated single-cell transcriptomic maps of the livers of healthy DA and LEW rat strains and developed a novel, factor analysis-based bioinformatics pipeline to study data covariates, such as strain and batch. Using this approach, we discovered variations within hepatocyte and myeloid populations that explain how the states of these cells differ between strains in the healthy rat, which may explain why these strains respond differently to liver transplants.

genomics↗