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. Overall design: 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.