CEBPB-mediated transcriptional regulation in Kupffer cells in metabolic dysfunction-associated steatohepatitis [RNA-seq]

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. KC-specific Cebpb monoallelic knockout (Clec4f-iCre;Cebpbfl/+) mice, and utilized littermate mice (Clec4f-iCre;Cebpb+/+) were fed with high-fat high-cholesterol diet (HFHCD) for 12 weeks. Total mRNA of murine liver tissue was extracted and subjected to RNA-seq.

背景与目的：Kupffer细胞（Kupffer cell, KC）参与代谢功能障碍相关脂肪性肝炎（metabolic dysfunction-associated steatohepatitis, MASH）的肝脏炎症与纤维化进程，但其潜在分子机制尚未完全阐明。转录因子CCAAT/增强子结合蛋白β（CCAAT/enhancer binding protein β, C/EBPβ）已被证实与代谢及免疫紊乱均相关。本研究旨在探究C/EBPβ在MASH发病过程中Kupffer细胞内的作用。
材料与方法及结果：本研究采用12周高脂高胆固醇饮食（high-fat and high-cholesterol diet, HFHCD）模型，对野生型或Kupffer细胞特异性Cebpb杂合敲除小鼠（Cebpbfl/+;Clec4f-iCre）进行造模，随后通过组织病理学、流式细胞术分析及RNA测序对肝脏进行评估。研究发现，野生型小鼠经HFHCD造模后可诱导显著的炎症性免疫细胞浸润，包括单核细胞源性巨噬细胞（monocyte-derived macrophages, MoMFs）比例升高，同时Kupffer细胞内C/EBPβ蛋白水平同步上升。Kupffer细胞特异性Cebpb杂合敲除可显著减轻HFHCD诱导的肝脏免疫细胞（包括MoMFs、中性粒细胞及CD8+ T细胞）浸润及炎症相关基因表达。本研究通过流式分选HFHCD处理小鼠的Kupffer细胞进行C/EBPβ CUT&Tag测序，发现MASH模型小鼠Kupffer细胞内C/EBPβ活性升高，进而选择性促进部分MASH诱导基因的表达。结合RNA-seq与CUT&Tag-seq联合分析，辅以双荧光素酶报告基因实验，证实Vcam1（编码血管细胞黏附分子1, vascular cell adhesion molecule 1, VCAM1）是小鼠MASH肝脏Kupffer细胞内C/EBPβ的关键下游靶基因。在小鼠肝脏及MASH患者样本中，VCAM1均主要表达于Kupffer细胞；其在肝脏内的表达在MASH发病后显著升高，并参与促进MASH中的免疫细胞浸润。
结论：在MASH发病过程中，Kupffer细胞内升高的C/EBPβ作为受MASH相关刺激调控的转录因子，可直接转录激活Vcam1启动子，导致Kupffer细胞内VCAM1表达异常升高，进而引发免疫细胞浸润。本研究结果提示，Kupffer细胞内的C/EBPβ可作为非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）炎症的潜在治疗靶点。本研究使用Kupffer细胞特异性Cebpb单等位基因敲除（Clec4f-iCre;Cebpbfl/+）小鼠，并以同窝野生型小鼠（Clec4f-iCre;Cebpb+/+）作为对照，给予12周高脂高胆固醇饮食（HFHCD）造模，提取小鼠肝脏组织总mRNA并进行RNA测序。