Project description:Background & Aims: Kupffer cell (KC) participates in hepatic inflammation and fibrosis in metabolic dysfunction-associated steatohepatitis (MASH), but underlying molecular mechanisms are not fully resolved. Transcription factor CCAAT/enhancer binding protein β (C/EBPβ) has been implicated in both metabolic and immune dysregulations. In this study, we aimed to investigate the role of C/EBPβ in KCs under the context of MASH pathogenesis. Approach & Results: A 12-week high-fat and high-cholesterol diet (HFHCD) model was used in wild-type or KC-specific Cebpb heterozygous knockout mice (Cebpbfl/+;Clec4f-iCre), followed by evaluation of liver using histopathology, analytical flow cytometry and RNA-seq. We found that HFHCD in wild-type mice induced significant inflammatory immune cell infiltration, including increased proportion of monocyte-derived macrophages (MoMFs), accompanied with concomitant increase in C/EBPβ protein level in KCs. KC-specific Cebpb heterozygous knockout significantly reduced HFHCD-induced immune cell (including MoMFs, neutrophils and CD8+ T cells) infiltration and inflammation-related gene expression in liver. FACS-sorted KCs of HFHCD-treated mice were used for C/EBPβ CUT&Tag-seq. We found that C/EBPβ activity was increased in MASH KCs, leading to selective promotion on part of MASH-induced genes. Combined analysis of RNA-seq and CUT&Tag-seq, along with dual luciferase reporter assay identified Vcam1 (encoding vascular cell adhesion molecule 1, VCAM1) as a key downstream gene of C/EBPβ in KCs of murine MASH liver. In both murine liver and MASH patients, VCAM1 was predominantly expressed in KCs. Its expression in liver was significantly increased after MASH onset, and it was involved in promoting immune cell infiltration in MASH. Conclusions: In MASH pathogenesis, increased C/EBPβ in KCs act as a MASH-associated stimulus-regulated TF, and has a direct transcriptional activation effect on Vcam1 promoter, leading to abnormal elevation in VCAM1 expression in KCs and subsequent immune cell infiltration. Our findings suggest that C/EBPβ in KCs can be a potential therapeutic target for NASH inflammation.

Project description:There is an evident, unmet need to develop a commercially available in vitro system that can model inflammatory states of the liver and predict immune-mediated hepatotoxicity of drugs and xenobiotics taken under inflamed conditions. Hepatocyte-Kupffer cell co-cultures can model inflammation-mediated hepatotoxicity; however, Kupffer cell (KC) source remains an important bottleneck for the development of such models. Primary human Kupffer cells (PHKCs) are costly, limited in availability and exhibit donor variability. An alternative cell source for KCs has not been reported. Important paradigm shift from the classical dogma of adult blood-circulating monocyte-derived macrophages to intrahepatic precursor/fetal monocyte-derived macrophages has shed new light into the origin of KCs in vivo. Based on these recent findings, we report here, a novel method to generate human KCs in vitro from stem cells (hPSC-KCs) via fetal monocytes. hPSC-KCs expressed macrophage markers, CD11, CD14, CD68, CD163 and CD32 at gene and protein level and exhibited functional properties such as phagocytosis and Interleukin-6 and Tumor Necrosis Factor-4alpha production upon activation. Importantly, molecular signature, liver-macrophage specific CLEC-4F expression and cytokines production levels of hPSC-KCs were similar to PHKCs but different from non-liver macrophages. We used an inflammatory liver co-culture model to demonstrate that activated hPSC-KCs, but not non-liver macrophages, were able to recapitulate effects of PHKCs when stimulated with paradigm hepatotoxicants. hPSC-KCs developed in this study offer a renewable human cell source for liver-specific macrophages which can be used to develop in vitro systems for modelling the inflammatory state of the liver. Gene expression profiles of 9 samples were determined using Human Gene 2.0 ST Array. These 9 samples included three replicates each of PHKCs (primary human Kupffer cells), human pluripotent stem cell-dervied Kupffer cells (hPSC-KCs) and non-liver macrophages (NL-Mφ).

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Viral-directed augmentation of Kupffer cell cross-presentation provokes anti-tumor immunity against liver metastasis

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