Optimization of high throughput spectral flow cytometry for immune cell profiling in mouse liver

The liver plays an important role in both metabolism and immunity.Disruption of the hepatic immune microenvironment is closely associated with various liver diseases.To gain a better understanding of how different types of immune cells contribute to the progression of liver diseases,it is crucial to thoroughly characterize hepatic immune cells.Although direct digestion of liver tissue is a relatively simple method for isolating immune cells,it often induces excessive hepatocyte death,which causes a release of intracellular components that leads to the activation of stress responses and injury in the surrounding cells.This injury can lead to excessive death in the hepatic immune cells,making isolation and accurate characterization of the immune profile challenging,especially in diseased livers.The method described here addresses these challenges by utilizing Phosphate buffered saline(PBS)and digestion buffer perfusions to eliminate contaminating blood cells,ensure a pure hepatic immune population,and minimize hepatic immune cell death.Further ex vivo digestion of the liver enables the isolation of the immune cells from the hepatic tissues and the generation of a single-cell suspension that can be stained for spectral flow cytometry.To enhance intracellular cytokine detection and maintain signaling under different physiological and pathological conditions,this protocol uses an in vivo administration of Brefeldin A,a less toxic inhibitor of cytokine secretion.This in vivo administration of Brefeldin A allows for a more accurate representation of the immune cell function and cytokine expression compared to the traditionally used ex vivo Brefeldin A administration.A comprehensive spectral flow cytometry panel,comprising extracellular and intracellular staining,is used for deep immunophenotyping and immune cell effector function profiling.While this protocol is specifically designed for liver digestion of Mdr2 knockout mice(a model for primary sclerosing cholangitis)and flow cytometry staining,it can also be applied to other liver diseases and sensitive tissues.

Bile acids and sphingolipids in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is one of the fastest-growing diseases, and its global prevalence is estimated to increase >50% by 2030. NAFLD is comorbid with metabolic syndrome, obesity, type 2 diabetes, and insulin resistance. Despite extensive research efforts, there are no pharmacologic or biological therapeutics for the treatment of NAFLD. Bile acids and sphingolipids are well-characterized signaling molecules. Over the last few decades, researchers have uncovered potential mechanisms by which bile acids and sphingolipids regulate hepatic lipid metabolism. Dysregulation of bile acid and sphingolipid metabolism has been linked to steatosis, inflammation, and fibrosis in patients with NAFLD. This clinical observation has been recapitulated in animal models, which are well-accepted by experts in the hepatology field. Recent transcriptomic and lipidomic studies also show that sphingolipids are important players in the pathogenesis of NAFLD. Moreover, the identification of bile acids as activators of sphingolipid-mediated signaling pathways established a novel theory for bile acid and sphingolipid biology. In this review, we summarize the recent advances in the understanding of bile acid and sphingolipid-mediated signaling pathways as potential contributors to NAFLD. A better understanding of the pathologic effects mediated by bile acids and sphingolipids will facilitate the development of new diagnostic and therapeutic strategies for NAFLD.