Hepatology Research Unit, Department of Internal Medicine and Pediatrics, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Entrance 36 - Floor 2, 9000 Ghent, Belgium; Gut-Liver Immunopharmacology Unit, Department of Basic and Applied Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Entrance 36 - Floor 3, 9000 Ghent, Belgium.
Lab of Pharmaceutical Biotechnology, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
Metabolism. 2020 Jun;107:154220. doi: 10.1016/j.metabol.2020.154220. Epub 2020 Mar 31.
Obesity, diabetes and associated non-alcoholic steatohepatitis (NASH) are rising risk factors for hepatocellular carcinoma (HCC). Macrophages are important immune cells involved in inflammation and tumour development. Macrophage inositol-requiring enzyme 1 alpha (IRE1α), an ER-stress protein, has been shown to be involved in macrophage cytokine production, and myeloid-specific IRE1α knock-out (myeloid IRE1α-KO) mice showed reduced weight gain during high-fat diet feeding. However, the effect of myeloid IRE1α on NASH and subsequent HCC development has not been examined. Here, we characterized the transcriptional profile of the hepatic macrophage population in a diabetes-NASH-HCC mouse model, and investigated the effect of myeloid-specific IRE1α deletion on the phenotype of hepatic macrophage subsets and experimental NASH-HCC development.
Mice with non-functional myeloid IRE1α were created by crossing Ire1a floxed mice with Lysm-Cre mice. Two-day old myeloid IRE1α-KO and wild type (WT) mice were subcutaneously injected with streptozotocin (STZ), and male mice were fed a high-fat, -sucrose, -cholesterol diet (Western diet, WD) from the age of 4 weeks until 21 weeks. Control myeloid IRE1α-KO and WT mice received a PBS injection and were fed a matched control diet. These mice were evaluated for obesity, diabetes, NASH and HCC. The hepatic macrophage population was evaluated by flow cytometry and RNA sequencing on FACS-isolated macrophage subsets.
STZ-injection and WD feeding resulted in an impaired glucose tolerance, advanced NASH with fibrosis, and HCC development. Myeloid IRE1α-KO STZ mice showed lower fasting glucose levels at the start of WD feeding, and an improved glucose tolerance and attenuated HCC development after 17 weeks of WD feeding despite a similar degree of liver steatosis and inflammation compared to WT mice. Transcriptomic analysis of WT liver Kupffer cells, macrophages and monocytes revealed phenotypical changes in those cell subsets during NASH-HCC development. Isolated liver Kupffer cells and macrophages from mice with a myeloid IRE1α deletion showed downregulated pathways involved in immune system activation and metabolic pathways (only in Kupffer cells), whereas pathways involved in cell division and metabolism were upregulated in monocytes. These transcriptional differences were attenuated during NASH-HCC development.
Our results show that myeloid-specific IRE1α deletion results in an altered transcriptional profile of hepatic macrophages and dampens diabetes-induced NASH-HCC development, possibly by attenuated diabetes induction.
肥胖、糖尿病和相关的非酒精性脂肪性肝炎（NASH）是肝细胞癌（HCC）的上升风险因素。巨噬细胞是参与炎症和肿瘤发展的重要免疫细胞。内质网应激蛋白肌醇需求酶 1α（IRE1α）已被证明参与巨噬细胞细胞因子的产生，髓系特异性 IRE1α 敲除（髓系 IRE1α-KO）小鼠在高脂肪饮食喂养期间体重增加减少。然而，髓系 IRE1α 对 NASH 及随后 HCC 发展的影响尚未被研究。在此，我们对糖尿病-NASH-HCC 小鼠模型中的肝巨噬细胞群的转录谱进行了表征，并研究了髓系特异性 IRE1α 缺失对肝巨噬细胞亚群表型和实验性 NASH-HCC 发展的影响。
通过将 Ire1a 基因敲除小鼠与 Lysm-Cre 小鼠杂交，创建了不具有功能的髓系 IRE1α 的小鼠。2 天大的髓系 IRE1α-KO 和野生型（WT）小鼠接受链脲佐菌素（STZ）皮下注射，雄性小鼠从 4 周龄开始至 21 周龄喂食高脂肪、高蔗糖、高胆固醇饮食（西方饮食，WD）。对照髓系 IRE1α-KO 和 WT 小鼠接受 PBS 注射并喂食匹配的对照饮食。评估这些小鼠的肥胖、糖尿病、NASH 和 HCC。通过流式细胞术和 FACS 分离的巨噬细胞亚群的 RNA 测序评估肝巨噬细胞群。
STZ 注射和 WD 喂养导致葡萄糖耐量受损、晚期 NASH 伴纤维化和 HCC 发展。IRE1α-KO STZ 小鼠在开始 WD 喂养时空腹血糖水平较低，尽管与 WT 小鼠相比，肝脏脂肪变性和炎症程度相似，但在 WD 喂养 17 周后，葡萄糖耐量得到改善，HCC 发展得到抑制。WT 肝库普弗细胞、巨噬细胞和单核细胞的转录组分析显示，在 NASH-HCC 发展过程中，这些细胞亚群的表型发生变化。具有髓系 IRE1α 缺失的分离肝库普弗细胞和巨噬细胞显示参与免疫系统激活和代谢途径的下调途径（仅在库普弗细胞中），而参与细胞分裂和代谢的途径在单核细胞中上调。这些转录差异在 NASH-HCC 发展过程中减弱。
我们的结果表明，髓系特异性 IRE1α 缺失导致肝巨噬细胞的转录谱发生改变，并抑制糖尿病诱导的 NASH-HCC 发展，可能是通过减弱糖尿病的诱导。
