CEBPB-mediated transcriptional regulation in Kupffer cells in metabolic dysfunction-associated steatohepatitis [CUT&Tag]

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 ß (C/EBPß) has been implicated in both metabolic and immune dysregulations. In this study, we aimed to investigate the role of C/EBPß 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/EBPß 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/EBPß CUT&Tag-seq. We found that C/EBPß 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/EBPß 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/EBPß 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/EBPß in KCs can be a potential therapeutic target for NASH inflammation. Overall design: Wildtype mice were fed with HFHCD or normal diet (ND) for 12 weeks. FACS-sorted KCs were used for library preparation of C/EBPb CUT&Tag