METTL14 downregulation drives S100A4+ monocyte-derived macrophages via MyD88/NF-κB pathway to promote MAFLD progression

Abstract

Without intervention, a considerable proportion of patients with metabolism-associated fatty liver disease (MAFLD) will progress from simple steatosis to metabolism-associated steatohepatitis (MASH), liver fibrosis, and even hepatocellular carcinoma. However, the molecular mechanisms that control progressive MAFLD have yet to be fully determined. Here, we unraveled that the expression of the N6-methyladenosine (m6A) methyltransferase METTL14 is remarkably downregulated in the livers of both patients and several murine models of MAFLD, whereas hepatocyte-specific depletion of this methyltransferase aggravated lipid accumulation, liver injury, and fibrosis. Conversely, hepatic Mettl14 overexpression alleviated the above pathophysiological changes in mice fed on a high-fat diet (HFD). Notably, in vivo and in vitro mechanistic studies indicated that METTL14 downregulation decreased the level of GLS2 by affecting the translation efficiency mediated by YTHDF1 in an m6A-depedent manner, which might help to form an oxidative stress microenvironment and accordingly recruit Cx3cr1⁺Ccr2⁺ monocyte-derived macrophages (Mo-macs). In detail, Cx3cr1⁺Ccr2⁺ Mo-macs can be categorized into M1-like macrophages and S100A4-positive macrophages and then further activate hepatic stellate cells (HSCs) to promote liver fibrosis. Further experiments revealed that CX3CR1 can activate the transcription of S100A4 via CX3CR1/MyD88/NF-κB signaling pathway in Cx3cr1⁺Ccr2⁺ Mo-macs. Restoration of METTL14 or GLS2, or interfering with this signal transduction pathway such as inhibiting MyD88 could ameliorate liver injuries and fibrosis. Taken together, these findings indicate potential therapies for the treatment of MAFLD progression.

Fig. 1

METTL14 is downregulated in the livers of mice with HFD and in human MAFLD. a Sections of human healthy liver (n = 5) and metabolic fat liver disease (MAFLD) (n = 5) were analyzed by immunohistochemical analysis for METTL14. Representative images are shown. b Relative quantitative analysis of METTL14 in normal human liver and metabolic fat liver tissues (n = 5). c Schematic diagram of the dietary feeding scheme. Six-week-old C57BL/6 wild-type mice were fed either a control diet (CON) or a 60% high-fat diet (HFD) for 24 weeks. d Mice were sacrificed, and liver sections were analyzed by immunohistochemical analysis for METTL14 expression (n = 3). e Relative quantitative analysis of METTL14 in liver tissues of mice fed the HFD and control diet (n = 3). f Comparison of the liver-to-body weight ratio of 8-week-old wild-type (WT, n = 10), heterozygous knockout mice (KO^+/−, n = 7) and homozygous knockout mice (KO, n = 9). g, h Serum ALT (g) and AST (h) in wild-type (WT, n = 5), heterozygous knockout mice (KO^+/−, n = 5) and homozygous knockout mice (KO, n = 3). i Schematic diagram of 6-week-old WT, KO^+/− and KO mice fed a HFD for 16 weeks. KO mice fed a HFD for 4 weeks were randomly injected with AAV-8 overexpressing METTL14 (AAV-OV) or control vector (AAV-NC) through the caudal vein, while WT mice and KO^+/− mice fed a HFD for 4 weeks were injected with AAV-NC. j–o Comparison of liver weight (j) and liver to body ratio (k), serum ALT (l) and AST (m), serum triglycerides (TG, n) and cholesterol (CHO, o) of WT (n = 4), KO^+/− (n = 4), KO injected with empty AAV-8 vector (KO-NC, n = 5) and KO injected with AAV-8 vector overexpressing METTL14 (KO-OV, n = 5). p H&E (left) and Oil Red O (right) staining of liver sections from HFD-treated WT (n = 4), KO^+/− (n = 4), KO-NC (n = 5) and KO-OV (n = 5) mice are shown. Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The mouse image was created with BioRender.com

Fig. 2

GLS2 was downregulated in both KO mice and HFD mice and consequently promoted the oxidative stress microenvironment. a Heat maps presenting differentially expressed proteins (DEPs) in HFD vs control diet-fed mice (n = 3) and KO vs WT mice (n_KO= 4, n_WT = 6), respectively. b Nine quadrant diagrams presenting the overlapping upregulated proteins and/or downregulated proteins among HFD and KO mice from proteomics sequencing (the dashed lines indicating the fold changes are at 1.5). c GO and KEGG analyses of differentially expressed proteins indicating lipid metabolic alterations among KO and WT mice. d The levels of specific markers related to lipid metabolism including fatty acid oxidation (CPT1A, ACOX1, ACAT2), de novo lipogenesis (FASN, SCD1, ACACA), TAG synthesis (GPAT3, DGAT1, MGAT1) and lipid uptake (CD36, FABP7, LDLR). e Overview of the pathway analysis based on metabolite alterations in KO mice from metabolomics analysis. f Western blot indicating the levels of GLS2 in 8-week-old WT and KO mice (top) and HFD-fed and control diet-fed mice (bottom). Representative images are shown (n = 4). g Relative quantitative analysis showing the levels of GLS2 in WT and KO mice (top) and HFD and control diet mice (bottom). h Immunohistochemical staining analysis of GLS2 in WT and KO mice, HFD-fed mice and control diet-fed mice (n = 4, scar bar = 50 μm). i Relative quantitative analysis of GLS2 in WT and KO mice (top) and HFD-fed and control diet-fed mice (bottom) (n = 4 respectively). j DCFH-DA was used to display the levels of intracellular ROS in primary cultured hepatocytes isolated from WT and KO mice. Representative images are shown(scar bar = 500 μm). k TAC levels showing total antioxidant capacity using ABTS methods in KO (n = 4) and WT (n = 5) mice. l ELISA showing the levels of 8-OHdG in KO and WT mice (n = 6 respectively). Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

METTL14 regulates GLS2 expression in an m6A-dependent manner via YTHDF1. a Western blot showing METTL14 and GLS2 expression in HUH7 (left) and Hep3B (right) cells infected with METTL14 overexpression (OE) or shRNA lentivirus vector (SH). b, c Relative m6A enrichment in Gls2 mRNA in liver tissues of WT and KO mice (b), HFD and control diet mice (c) by MeRIP-qPCR. n = 4. d The image showing the position and sequences of three potential m6A-binding sites with very high confidence of GLS2 mRNA using online SRAMP database (https://www.cuilab.cn/sramp). e Relative luciferase activity of three mutant plasmids and their wild-type plasmids of GLS2 in HUH7 (left) and Hep3B (right) cells with knockdown of METTL14. f Relative luciferase activity of three mutant plasmids and their wild-type plasmids of GLS2 in HUH7 (left) and Hep3B (right) cells with overexpression of METTL14. g Western blot (left) and relative quantitative analysis (right) showing METTL14 and GLS2 expression in HUH7 cells infected with YTHDF1 overexpression or shRNA lentivirus vector. h Western blot (left) and relative quantitative analysis (right) showing METTL14 and GLS2 expression in Hep3B cells infected with YTHDF1 overexpression or shRNA lentivirus vector. i Relative luciferase activity of three mutant plasmids and their wild-type plasmids of GLS2 in HUH7 (left) and Hep3B (right) cells with knockdown of YTHDF1. j Relative luciferase activity of three mutant plasmids and their wild-type plasmids of GLS2 in HUH7 (left) and Hep3B (right) cells with overexpression of YTHDF1. k Western blot showing YTHDF1 and GLS2 expression in HUH7 (left) and Hep3B (right) cells transfected with YTHDF1 overexpression (oe-YTHDF1), YTHDF1 mutation (K395A & Y397A, Mut) and vector plasmid (Vector) respectively. l Relative luciferase activity of HUH7 (top) and Hep3B (bottom) cells transfected with YTHDF1 overexpression, YTHDF1 mutant (K395A and Y397A) and vector plasmids, respectively. m Western blot showing METTL14, YTHDF1 and GLS2 expression in HUH7 (left) cells and Hep3B cells (right) infected with YTHDF1 overexpression (oe-YTHDF1) and/or METTL14 shRNA (sh-METTL14) lentivirus vector as well as control vectors. Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Increased abundance of Cx3cr1⁺Ccr2⁺ macrophages in the liver of the KO group. a UMAP plot displaying the distributions of 6 Kupffer cell clusters, 4 Mo-mac clusters, 3 dendritic cell clusters, 1 neutrophil cluster and 1 other cluster. b The percentage of cell counts, cell clusters and cell types in the KO and WT groups. c Violin plots showing marker genes and markers of lipid associated macrophages (LAMs) or scar associated macrophages (SAMs) across each cluster. d DEGs for Kupffer cells, monocyte-derived macrophages, dendritic cells and neutrophil clusters. e Bar plot showing different proportions of each cluster in the KO and WT groups

Fig. 5

Cx3cr1⁺Ccr2⁺ Mo-macs in the late stage of the developmental trajectory highly expressed S100 family protein genes. a Volcano plot comparing DEGs for Cx3cr1⁺Ccr2⁺ Mo-macs. b Feature plots showing the top 20 DE genes. c KEGG pathway enrichment analysis of upregulated DE genes in Cx3cr1⁺Ccr2⁺ Mo-macs. d Dot plot presenting the expression strength of key KEGG pathway genes among Cx3cr1⁺Ccr2⁺, Cx3cr1^-Ccr2^-and Ccr2⁺ Mo-macs and Kupffer cells. e Violin plots displaying the expression levels of polarization state marker genes and S100a family protein genes. f Volcano plot comparing DEGs between M06 and M09. g Heatmap displaying the expression of selected marker genes in Mo-macs arranged along the pseudotime trajectory. h Scatter plots and fitting curves presenting the expression trend of S100a4, S100a6, S100a10 and S100a11

Fig. 6

CX3CR1/MYD88/NF-κB regulated the expression of S100A4 in MAFLD progression. a Violin plots showing the expression of Toll-like receptors (TLRs) and adaptor MYD88 across each myeloid cell cluster. b, c Immunofluorescence analysis showing the distributions of CX3CR1⁺CCR2⁺MYD88⁺ cells in the KO and WT mice, HFD-fed and control diet-fed mice (n = 3, scar bar = 10 μm). d Immunohistochemical staining analysis of MYD88 in WT and KO mice (n = 3, scar bar = 50 μm). e Immunofluorescence analysis showing the colocalization of CX3CR1 and MYD88 in PMA-treated THP-1 cells by confocal fluorescence microscopy (scar bar = 20 μm). f Co-Immunoprecipitation analysis showing interaction between CX3CR1 and MYD88 in PMA-treated THP-1 cells. g Western blot showing the expression of NF-κB family proteins and S100A4 after treated with CX3CR1 inhibitor JMS-17-2. h Schematic diagram showing the procedures of RELA (P65) binding to the promoter of S100A4 and subsequent assays. i CUT-RUN analysis showing the binding capacity of RELA (p65) on three binding sites of S100A4 promoter regions in PMA-treated THP-1 cells. j Relative luciferase activity of mutant plasmid and wild-type plasmid of RELA binding sequences (site 3 in h) in 293T cells. k QRT-PCR analysis indicating mRNA expression of S100A4 after treated with CX3CR1 inhibitor JMS-17-2 in PMA-treated THP-1 cells. l Western blot showing the expression of NF-κB family proteins and S100A4 after treated with Diprovocim (NF-κB activator) or JSH-23 (NF-κB inhibitor). m QRT-PCR analysis showing S100A4 mRNA expression after treated with NF-κB activator/inhibitor and MyD88 inhibitor (ST2825) in PMA-treated THP-1 cells. n Oil red O staining and ROS analysis of HUH7 cells with HF treatment (12 μmol/l oleic acid and 6 μmol/l palmitic acid) and control solution for 48 h (scar bar = 100 μm). o QRT-PCR analysis showing the expression of S100A4 of PMA-treated THP-1 cells after cultured by HF conditional medium for 24 h. p Rescue assay showing the expression of S100A4 in PMA-treated THP-1 cells after cultured by HF conditional medium with/without CX3CR1 inhibitor, MyD88 inhibitor or NF-κB inhibitor. q QRT-PCR analysis showing the expression of HSC activation markers such as ACTA2, COL1A1, and TGFB1 of LX2 cells with/without high-fat supernatant stimulation as well as cocultivation with monocyte-derived macrophages (Mo-macs). Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

Restoration of METTL14 function in mice ameliorates liver inflammation, injury, and fibrosis. a Western blot showing METTL14 and GLS2 expression in mouse livers in the WT-NC, KO-NC, and KO-OV groups (n = 4). b, c Relative quantitative analysis of METTL14 (b) and GLS2 (c) expression in mouse livers in the WT-NC, KO-NC, and KO-OV groups. d DCFH-DA was used to display the levels of intracellular ROS in primary cultured hepatocytes isolated from WT-NC, KO-NC, and KO-OV mouse livers (n = 3, scar bar = 50 μm). e Relative quantitative analysis of ROS levels in primary cultured hepatocytes isolated from WT-NC, KO-NC, and KO-OV mouse livers. f TAC levels showing the total antioxidant capacity of mouse livers using ABTS methods in the WT-NC (n = 4), KO-NC (n = 5), and KO-OV (n = 5) groups. g Representative immunofluorescence images displaying the distributions of S100A4-positive, F4/80-positive, and S100A4 F4/80 double-positive macrophages among liver sections in the WT-NC, KO-NC, and KO-OV groups (scar bar = 50 μm or 5 μm). h Relative quantitative analysis of S100A4 F4/80 double-positive macrophages in the WT-NC (n = 4), KO-NC (n = 5), and KO-OV (n = 5) groups. FOV, field of view. i–l ELISA array detecting the levels of 8-OHdG (i), FGF2 (j), MMP2 (k), and S100A4 (l) in mouse liver tissues from the WT-NC (n = 4), KO-NC (n = 5), and KO-OV (n = 5) groups. m Immunofluorescence staining detecting α-SMA expression (left), Sirius Red staining (middle) and Masson staining (right) displaying the activation of HSCs and the severity of fibrosis of liver sections in the WT-NC (n = 4), KO-NC (n = 5), and KO-OV (n = 5) groups (scar bar = 20 μm or 25 μm). Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, *** p < 0.001

Fig. 8

Restoration of GLS2 ameliorates liver inflammation, injury, and fibrosis in Western diet/CCl₄-treated mice. a Schematic diagram of the dietary feeding scheme. Six-week-old C57/BL6 wild-type mice were fed a Western diet (WD) for 16 weeks and also treated with CCl₄ intraperitoneal injection. Mice fed a WD for 4 weeks were randomly injected with AAV-8 overexpressing Gls2 (OE-AAV) or control vector (CTL-AAV) through the caudal vein (n = 15). b, c Western blot (b) and quantitative analysis (c) showing the expression of GLS2 in the OE-AAV and CTL-AAV mice (n = 12). d Body weight changes of OE-AAV and CTL-AAV mice (n = 15). e Comparison of the liver-to-body weight ratio of OE-AAV and CTL-AAV mice (n = 12). f Representative image of resected livers from OE-AAV and CTL-AAV mice (n = 12). g Serum ALT, AST, TBIL, TG and CHO in OE-AAV and CTL-AAV mice (n = 12). h Tissue ROS analysis of liver sections from OE-AAV and CTL-AAV mice (n = 4, scar bar = 50 μm). i Total antioxidant capacity analysis of OE-AAV and CTL-AAV mice (n = 12). j ELISA analysis showing serum S100A4 levels of OE-AAV and CTL-AAV mice (n = 12). k HE, Oil Red O, Sirius Red, Masson staining and α-SMA immunofluorescence analysis of OE-AAV and CTL-AAV mice (n = 12, scar bar = 20 μm). l QRT-PCR analysis showing the expression of fibrosis markers such as Acta2, Col1a1 and Mmp2 of OE-AAV and CTL-AAV mice (n = 8). Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001. The mouse image was created with BioRender.com