Metabolic crosstalk between skeletal muscle cells and liver through IRF4-FSTL1 in nonalcoholic steatohepatitis

Inter-organ crosstalk has gained more and more attention recently. However, the mechanisms under this remain incompletely understood. Here, we revealed an endocrine pathway regulated by skeletal muscle IRF4 that manipulates liver pathology. We studied that skeletal muscle specifically deleted IRF4 (F4MKO) mice showed ameliorated liver steatosis, inflammation and fibrosis, without changes in body weight on NASH diet. Proteomics analysis of serum suggested that follistatin-like protein 1 (FSTL1), as a myokine, might linked the communication between skeletal muscle and liver. Dual luciferase assays showed that IRF4 can transcriptionally regulated FSTL1 and reconstitution of FSTL1 expression in skeletal muscle of F4MKO mice, using adeno-associated virus, was sufficient to restore the liver pathology. Furthermore, we performed co-culture experiments to verify different receptors contribute to FSTL1’s function in different cell types of liver. Finally, we found serum FSTL1 level was positively correlated with NASH progression in human, whereas the mRNA level of Fstl1 and its receptors were downregulated in liver biopsy from NASH patients. These data reveal a signaling pathway from skeletal muscle to liver via IRF4-FSTL1-receptors in the pathogenesis of NASH and implicate useful targets for the management of NASH. Three liver tissue samples of each group for RNA sequencing were collected. Total RNA was isolated using the Trizol method according to manufacturer’s instructions. RNA quality was measured using the Agilent 2100 Bioanalyzer (RNA 6000 Nano Kit; Agilent Technologies, Santa Clara, CA, USA). cDNA libraries for each sample were constructed as reported previously Libraries were sequenced on BGIseq500 platform (BGI-Shenzhen, China), using 150 bp paired-end reads aimed at 30 million reads per sample.The raw sequencing data was filtered with trim-galore (v0.6.7) by removing reads containing sequencing adapter and reads with low-quality base. The clean reads were mapped to the reference genome (mm10) using Hisat2 (2.2.1). Quantification of gene expression was calculated using FeatureCounts (v2.0.1), converted to transcripts per million (TPM) using the R software. We conducted a differential gene expression analysis comparing F4MKO versus control and overexpression of FSTL1 in F4MKO (F4MKO+FSTL1) versus F4MKO. Subsequently, differential expression analysis between groups was conducted using the DESeq2(1.3.40) with adjust P value < 0.05 and |FC|>1.5. We used the obtained log2FC to perform a gene-set enrichment analysis to detect pathways and biological processes enriched with profiling genes.

器官间串扰（inter-organ crosstalk）近年来受到越来越多的关注，但其背后的调控机制仍未完全阐明。本研究揭示了一条由骨骼肌干扰素调节因子4（IRF4）调控的内分泌通路，该通路可影响肝脏病理进程。我们发现，特异性敲除骨骼肌IRF4（以下简称F4MKO）的小鼠在非酒精性脂肪性肝炎（NASH）饮食喂养条件下，肝脏脂肪变性、炎症及纤维化程度均得到改善，且体重无明显变化。血清蛋白质组学分析显示，卵泡抑素样蛋白1（FSTL1）作为肌因子，可能介导了骨骼肌与肝脏之间的通讯。双荧光素酶报告基因实验结果表明，IRF4可对FSTL1进行转录调控；通过腺相关病毒（AAV）在F4MKO小鼠的骨骼肌中重新恢复FSTL1的表达，足以逆转其肝脏病理损伤。此外，我们通过共培养实验验证了不同受体在肝脏不同细胞类型中介导FSTL1的功能。最后，我们发现人体血清FSTL1水平与NASH进展呈正相关，而NASH患者肝活检组织中Fstl1及其受体的mRNA表达水平均下调。本研究揭示了一条在NASH发病机制中由骨骼肌经IRF4-FSTL1-受体轴向肝脏传递的信号通路，并为NASH的临床干预提供了潜在靶点。每组收集3份肝脏组织样本用于RNA测序。按照试剂盒说明书采用Trizol法提取总RNA，采用安捷伦2100生物分析仪搭配RNA 6000纳米试剂盒（Agilent Technologies, Santa Clara, CA, USA）检测RNA质量。参照既往报道的方法构建各样本的cDNA文库；随后在BGIseq500测序平台（深圳华大基因，中国）上进行测序，采用150 bp双端读长模式，每个样本预计产出3000万条读段。使用trim-galore（v0.6.7）对原始测序数据进行质控过滤，去除包含测序接头的读段及低质量碱基读段。使用Hisat2（2.2.1）将clean reads比对至参考基因组mm10。使用FeatureCounts（v2.0.1）计算基因表达量，并通过R软件转换为每百万转录本（TPM）值。我们开展了两组差异基因表达分析：分别为F4MKO组与对照组的比较，以及F4MKO+FSTL1组（F4MKO小鼠骨骼肌过表达FSTL1）与F4MKO组的比较。随后采用DESeq2（1.3.40）进行组间差异表达分析，筛选阈值为校正后P值<0.05且|倍数变化（FC）|>1.5。我们以得到的log2FC值进行基因集富集分析，以筛选出表达谱基因富集的通路及生物学过程。