Deletion of sphingosine 1-phosphate receptor 1 in myeloid cells reduces hepatic inflammatory macrophages and attenuates MASH

Background: Immune cell-driven inflammation is a key mediator of metabolic dysfunction-associated steatohepatitis (MASH) progression. We have previously demonstrated that pharmacological sphingosine 1-phosphate (S1P) receptor modulation ameliorates MASH and is associated with attenuated accumulation of intrahepatic macrophage and T cell subsets. Although S1P receptors are expressed on several immune cell types, given the prominent role of monocyte-derived recruited macrophages in the sterile inflammation of MASH, we hypothesized that deletion of S1P receptor 1 (S1P1) on myeloid cells may ameliorate MASH by reducing the accumulation of proinflammatory monocyte-derived macrophages into the liver. Methods: The LyzMCre approach was used to generate myeloid cell-specific knockout mice, termed S1pr1MKO. Littermate S1pr1loxp/loxp mice were used as wild-type (WT) controls. MASH was established by feeding mice a high-fat, fructose, and -cholesterol (FFC) diet for 24 weeks which leads to the development of steatohepatitis and MASH-defining cardiometabolic risk factors. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by mass cytometry and immunohistochemistry. Results: Histological examination demonstrated a reduction in liver inflammatory infiltrates and fibrosis in FFC-fed S1pr1MKO compared to WT. There was a corresponding reduction in alanine aminotransferase, a sensitive marker for liver injury. As determined by mass cytometry, a significant decrease in recruited macrophages was noted in the livers of FFC-fed S1pr1MKO mice compared to WT. Gene ontology pathway analysis revealed a significant suppression of the peroxisome proliferator-activated receptor gamma (PPAR) and mitogen activated protein kinase (MAPK) pathways in S1pr1MKO consistent with attenuated MASH in mice. Conclusion: Deletion of S1P1 in myeloid cells is sufficient to attenuate intrahepatic accumulation of monocyte-derived macrophages and ameliorate murine MASH. S1pr1 floxed mice, S1pr1loxp/loxp, (The Jackson Laboratory, stock #019141) were crossed with Lyz2-Cre mice (The Jackson Laboratory, stock #004781) to generate myeloid cell specific knockout mice S1pr1loxp/loxp;Lyz2-Cre/0, designated as S1pr1MKO henceforth. Littermate controls, S1pr1loxp/loxp referred to as wildtype (WT), were employed for all experiments. Starting at 12 weeks of age, mice were placed on a high-fat, -fructose, and -cholesterol (FFC) diet (AIN-76A Western Diet, originally manufactured as D12079B, TestDiet, St. Louis, MO) or standard rodent chow diet (CD) for 24 weeks. Cryopreserved mouse liver tissues were recovered and homogenized in TRIzol reagent (Ambion). After tissue debris was centrifuged down, the supernatant was transferred into separate tubes. Total RNA was extracted using the Quick-RNA MiniPrep Kit (Zymo Research, R1055). RNA quality and yield was assessed by NanoDrop ND1000 (Thermo Scientific, Waltham, MA), then reverse transcribed into cDNA with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, 1708891). Bulk RNA sequencing was performed at the Genome Analysis Core of Medical Genome Facility at Mayo Clinic, Rochester. mRNA sequencing libraries were prepared and sequenced on an Ilumina HiSeq 2000 instrument at the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility. RNA-seq data were analyzed using the MAP-RSeq pipeline (17). In brief, paired-end reads were aligned to the mouse genome reference mm10 using TopHat (v2.1.0) and gene counts were estimated using the feature Counts (v1.4.6) software based on the Ensembl gene definition files. Gene expression was quantified as reads per kilobase per million mapped reads (RPKM). Protein-coding genes with RPKM 1 in at least one sample were extracted and the 43295 genes with the largest between-sample variation were used in hierarchical clustering. A subset of protein-coding genes with RPKM 1 with intra-group statistical significance were selected for differential analysis (p-value 0.5). The differentially expressed genes were identified using the edgeR package (v3.18.1). Differentially expressed genes were selected using p-value 0.5 (1442 genes). Volcano plot was prepared with R-studio and the package ggplot2. Genes in the integrated analysis were analyzed with Ingenuity pathway analysis (IPA) to uncover common regulatory pathways. IPA mapped 1349 genes. The IPA filter for log Fold change used for further analysis was -0.5 for downregulated and +0.5 on upregulated genes. 505 genes were analyzed for top differentially activated pathways and results along with predicated upstream regulators were displayed.背景：免疫细胞介导的炎症是代谢功能障碍相关脂肪性肝炎（metabolic dysfunction-associated steatohepatitis, MASH）进展的关键介导因素。我们此前的研究证实，药理学调控1-磷酸鞘氨醇（sphingosine 1-phosphate, S1P）受体可改善MASH，且与肝内巨噬细胞和T细胞亚群的聚集减少相关。尽管S1P受体在多种免疫细胞类型中均有表达，但鉴于单核细胞源性招募巨噬细胞在MASH无菌性炎症中的关键作用，我们提出假说：髓系细胞上的S1P受体1（S1P receptor 1, S1P1）缺失可通过减少促炎单核细胞源性巨噬细胞在肝脏内的聚集，从而改善MASH。 方法：本研究采用LyzMCre技术构建髓系细胞特异性敲除小鼠，命名为S1pr1MKO。以同窝出生的S1pr1loxp/loxp小鼠作为野生型（wild-type, WT）对照。通过给小鼠喂食高脂、高果糖、高胆固醇（high-fat, fructose, and -cholesterol, FFC）饲料24周，构建MASH模型，该模型可诱导脂肪性肝炎及符合MASH定义的心脏代谢危险因素的发生。通过组织学和基因表达分析评估肝脏损伤与炎症情况。采用质谱流式细胞术和免疫组织化学分析肝内白细胞群体。 结果：组织学检查显示，与WT小鼠相比，喂食FFC饲料的S1pr1MKO小鼠肝脏炎症浸润和纤维化程度均有所降低。作为肝损伤敏感标志物的丙氨酸氨基转移酶水平也相应下降。质谱流式细胞术检测结果显示，喂食FFC饲料的S1pr1MKO小鼠肝脏内的招募巨噬细胞数量显著低于WT小鼠。基因本体（gene ontology, GO）通路分析显示，S1pr1MKO小鼠体内过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma, PPARγ）和丝裂原活化蛋白激酶（mitogen activated protein kinase, MAPK）通路显著受抑，这与小鼠MASH症状减轻的结果一致。 结论：髓系细胞中S1P1的缺失足以减少单核细胞源性巨噬细胞在肝内的聚集，并改善小鼠MASH。 S1pr1 floxed小鼠（即S1pr1loxp/loxp，购自The Jackson Laboratory，货号019141）与Lyz2-Cre小鼠（购自The Jackson Laboratory，货号004781）杂交，构建髓系细胞特异性敲除小鼠S1pr1loxp/loxp;Lyz2-Cre/0，此后将其命名为S1pr1MKO。所有实验均以同窝出生的S1pr1loxp/loxp小鼠作为野生型（WT）对照。小鼠自12周龄起，分别喂食高脂、高果糖、高胆固醇饲料（FFC饲料，即AIN-76A西式饲料，原产品为TestDiet生产的D12079B，购自密苏里州圣路易斯市TestDiet公司）或标准啮齿类动物饲料（chow diet, CD），持续24周。 将冻存的小鼠肝脏组织复苏后，在TRIzol试剂（Ambion公司）中进行匀浆。离心去除组织碎片后，将上清液转移至不同离心管中。采用Quick-RNA MiniPrep试剂盒（Zymo Research公司，货号R1055）提取总RNA。通过NanoDrop ND1000分光光度计（Thermo Scientific公司，马萨诸塞州沃尔瑟姆市）评估RNA质量与产量，随后使用iScript cDNA合成试剂盒（Bio-Rad Laboratories公司，货号1708891）将RNA反转录为cDNA。 批量RNA测序在罗切斯特市梅奥诊所医学基因组学中心基因组分析核心实验室完成。mRNA测序文库的制备及测序在梅奥诊所个体化医学中心医学基因组学实验室的Illumina HiSeq 2000测序仪上进行。RNA测序数据采用MAP-RSeq分析流程（文献17）进行分析。简言之，将双端测序reads与小鼠基因组参考序列mm10进行比对（使用TopHat v2.1.0软件），并基于Ensembl基因定义文件，采用featureCounts v1.4.6软件估算基因计数。基因表达量以每百万映射读数每千碱基片段数（reads per kilobase per million mapped reads, RPKM）进行量化。提取至少在1个样本中RPKM≥1的蛋白编码基因，并选取样本间差异最大的43295个基因进行层次聚类。选取组内具有统计学显著性且RPKM≥1的蛋白编码基因子集进行差异表达分析（p值≤0.5）。采用edgeR软件包（v3.18.1）鉴定差异表达基因，筛选标准为p值≤0.5，最终得到1442个差异表达基因。使用R-studio及ggplot2包绘制火山图。采用Ingenuity通路分析（Ingenuity pathway analysis, IPA）对整合分析中的基因进行分析，以揭示常见调控通路。IPA共映射到1349个基因。本次分析采用的log Fold change筛选阈值为：下调基因≤-0.5，上调基因≥+0.5。选取505个基因进行Top差异激活通路分析，并展示分析结果及预测的上游调控因子。