Single-Cell Profiling of Paracrine Network and Immune Landscape in Non-Alcoholic Steatohepatitis

Abstract

The liver is a heterogeneous organ comprising a diverse array of cells including parenchymal hepatocytes, and non-parenchymal cells (NPCs) including endothelial cells, hepatic stellate cells, cholangiocytes, and various immune cells. These different cell types function in concert to regulate hepatic metabolism and maintain tissue homeostasis. Non-alcoholic fatty liver disease (NAFLD) is a major hepatic comorbidity of metabolic syndrome that is characterized by pathogenic fat accumulation. Chronic NAFLD may progress to non-alcoholic steatohepatitis (NASH), an inflammatory condition that is associated with liver injury, immune cell infiltration, and liver fibrosis, and increases the risk for end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Despite the prominent roles of NPCs in NASH pathogenesis, the molecular nature of intercellular crosstalk among different liver cell types and their reprogramming in disease remains poorly understood.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for unraveling cellular heterogeneity in complex tissue through profiling transcriptomes of individual cells. In my thesis research, I performed scRNA-seq on NPCs isolated from healthy and diet-induced NASH mouse livers to dissect the paracrine signaling network and to uncover the immune cell landscape in NASH. We found that each cell type exhibited enriched expression of a unique subset of secreted ligands and membrane receptors, in a restricted pattern conserved from mouse to human. We confirmed expression for this paracrine network through quantitative proteomics and found macrophages and stellate cells to be major hubs of ligands and receptors which were upregulated in NASH. The hepatic stellate cells (HSCs) provide a source of stellakines which are predicted to act on endothelial and immune cells. Functionally, we showed that HSCs express a class of G-protein coupled receptors that regulate cellular contractility in response to vasoactive ligands.

ScRNA-seq revealed a highly disease-specific population of NASH-associated macrophages (NAMs) marked by abundant expression of marker genes including Trem2 and Gpnmb. NAMs were found at higher levels in both human and mouse NASH and decreased upon dietary and therapeutic interventions for NASH. Their transcriptional profile indicates a propensity for phagocytosis, antigen presentation, and extracellular matrix remodeling, illustrating a potentially important role for NAMs in NASH progression. Tracing hematopoietic cells through bone marrow transplantation, we demonstrated that NAMs originate from the bone marrow and not tissue-resident progenitors. We developed a Trem2 Cre knockin mouse strain to track the emergence of NAMs during NASH progression. Our mouse liver injury and fibrosis assays were unaffected by Trem2 knockout in NASH, indicating Trem2 itself may be unimportant in NASH, but the role of the NAMs they mark are not yet precluded from pathophysiology. Finally, we discovered that NASH is linked to liver CD8+ T cell exhaustion, which is characterized by high levels of PD1 and LAG3 expression and diminished IFNγ, IL2, and TNFα secretion upon T cell stimulation. NASH-associated T cell exhaustion is attenuated by the adipose hormone Neuregulin 4 (NRG4), which protects mice from diet-induced NASH and hepatocellular carcinoma. Taken together, my thesis work has revealed the transcriptomic nature of liver cell heterogeneity, the global landscape of cell-cell signaling in the liver, and NASH-associated NPC reprogramming at a single-cell resolution.