BACKGROUND AND AIMS 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. METHODS 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. RESULTS 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. CONCLUSION 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.

中文翻译：

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在糖尿病性，非酒精性脂肪性肝炎诱导的肝细胞癌小鼠模型中，髓样特异性IRE1alpha缺失减少了肿瘤的发展。

背景和目的肥胖，糖尿病和相关的非酒精性脂肪性肝炎（NASH）是肝细胞癌（HCC）的上升危险因素。巨噬细胞是参与炎症和肿瘤发展的重要免疫细胞。已证明需要巨噬细胞肌醇的酶1α（IRE1α）参与了巨噬细胞的细胞因子生产，而髓样特异性IRE1α敲除（髓样IRE1α-KO）小鼠在高脂血症期间体重减轻了脂肪饮食喂养。但是，尚未研究髓样IRE1α对NASH和随后的HCC发育的影响。在这里，我们表征了糖尿病-NASH-HCC小鼠模型中肝巨噬细胞群体的转录特征，并研究了髓样特异性IRE1α缺失对肝巨噬细胞亚型表型和实验性NASH-HCC发育的影响。方法通过将Ire1a浮游小鼠与Lysm-Cre小鼠杂交，产生具有非功能性髓样IRE1α的小鼠。对两天大的IRE1α-KO小鼠和野生型（WT）小鼠皮下注射链脲佐菌素（STZ），并从18岁开始给雄性小鼠喂食高脂，蔗糖，胆固醇饮食（Western Diet，WD）。 4周到21周。对照髓样IRE1α-KO和野生型小鼠接受PBS注射，并饲喂匹配的对照饮食。评价这些小鼠的肥胖，糖尿病，NASH和HCC。通过流式细胞术和RNA测序对FACS分离的巨噬细胞亚群评估肝巨噬细胞群体。结果STZ注射和WD喂养导致葡萄糖耐量降低，患有纤维化的晚期NASH和HCC的发展。骨髓IRE1α-KOSTZ小鼠在WD喂养开始时表现出较低的空腹血糖水平，并且在WD喂养之后17周，尽管与野生型小鼠相比肝脏脂肪变性和炎症程度相似，但其葡萄糖耐量有所改善，并且HCC发育减弱。WT肝库普弗细胞，巨噬细胞和单核细胞的转录组学分析揭示了NASH-HCC发育过程中这些细胞亚群的表型变化。来自具有髓样IRE1α缺失的小鼠的分离的肝Kupffer细胞和巨噬细胞显示出与免疫系统激活和代谢途径有关的通路被下调（仅在Kupffer细胞中），而与细胞分裂和代谢有关的通路在单核细胞中被上调。这些转录差异在NASH-HCC发育过程中减弱。