TREM2 macrophages mediate the beneficial effects of bariatric surgery against MASH

Abstract

Background and aims: For patients with obesity and metabolic syndrome, bariatric procedures such as vertical sleeve gastrectomy (VSG) have a clear benefit in ameliorating metabolic dysfunction-associated steatohepatitis (MASH). While the effects of bariatric surgeries have been mainly attributed to nutrient restriction and malabsorption, whether immuno-modulatory mechanisms are involved remains unclear.

Approach and result: Using murine models, we report that VSG ameliorates MASH progression in a weight loss-independent manner. Single-cell RNA sequencing revealed that hepatic lipid-associated macrophages (LAMs) expressing the triggering receptor expressed on myeloid cells 2 (TREM2) repress inflammation and increase their lysosomal activity in response to VSG. Remarkably, TREM2 deficiency in mice ablates the reparative effects of VSG, suggesting that TREM2 is required for MASH resolution. Mechanistically, TREM2 prevents the inflammatory activation of macrophages and is required for their efferocytic function.

Conclusions: Overall, our findings indicate that bariatric surgery improves MASH through a reparative process driven by TREM2+ macrophages, providing insights into the mechanisms of disease reversal that may result in new therapies and improved surgical interventions.

Keywords: NASH; inflammation; lipid-associated macrophages; scRNA-seq; vertical sleeve gastrectomy.

Graphical abstract

FIGURE 1

VSG ameliorates MASH progression independent of weight loss. (A) Experimental design, (B) weekly body weights after surgery (^# p < 0.05 for VSG vs. Sham AL, ^$ p < 0.05 for VSG vs. Sham AL and VSG vs. Sham PF comparisons), (C) liver weight, (D) serum AST (left) and ALT (right), (E) representative H&E-stained liver sections (left) and hepatic triglyceride content (right) in C57BL6/J (WT) mice fed an HFHC diet for 14 weeks before assignment to Sham AL (n = 6–17), Sham PF (n = 6–17), or VSG (n = 6–25) surgeries. Mice were maintained on the HFHC diet for 5 weeks after surgery. A cohort of mice without any intervention were fed an NCD (n = 8–25) throughout the study. (F) Representative Picro Sirius Red–stained liver sections (left) and collagen area (right) 10 weeks after surgery; NCD (n = 8), Sham AL (n = 8), Sham PF (n = 7), and VSG (n = 9). Arrows identify regions with collagen deposition. Data are biological experimental units presented as mean ± SEM and were analyzed by one-way ANOVA with a Holm-Šídák multiple comparison test. Abbreviations: H&E, hematoxylin and eosin; HFHC, high-fat high-carbohydrate; MASH, metabolic dysfunction–associated steatohepatitis; NCD, normal chow diet; Sham AL, Sham ad libitum; Sham PF, Sham pair-fed; VSG, vertical sleeve gastrectomy; WT, wild type.

FIGURE 2

scRNA-seq shows a substantial heterogeneity of hepatic macrophages during MASH and after VSG. (A) Experimental design, (B) number of sequenced cells per sample, (C) integrated UMAP analysis of monocytes and macrophages and expression of marker genes, (D) heat and dot plot of the expression and coverage of marker genes, (E) pathway analysis of upregulated DEGs in LAMs, compared with all other subsets (gene ontology terms, adj. p < 0.1), and (F) slingshot trajectory analysis (top left) and gene expression over pseudotime for trajectories 1 (top right), 2 (bottom left), and 3 (bottom right) from scRNA-seq of hepatic macrophages and monocytes from Sham AL (n=8), Sham PF (n=8), and VSG (n=8) mice 5–10 weeks after surgeries. Differential expression testing was performed by a Wilcoxon rank-sum test. Abbreviations: DEG, differentially expressed gene; LAM, lipid-associated macrophage; MASH, metabolic dysfunction–associated steatohepatitis; scRNA-seq, single-cell RNA sequencing; Sham AL, Sham ad libitum; Sham PF, Sham pair-fed; UMAP, uniform manifold approximation and projection; VSG, vertical sleeve gastrectomy.

FIGURE 3

VSG enhances lipid metabolism and lysosomal gene programs in hepatic LAMs. (A) UMAPs with cluster IDs for all groups (top) and relative monocyte and macrophage cluster proportions (bottom), (B) UMAPs showing Trem2 expression in monocytes and macrophages (left) and expression of Trem2 in cluster 1 (LAMs) from the scRNA-seq analysis of monocytes and macrophages from Sham AL, Sham PF, or VSG groups 5 weeks after surgery (n = 4). (C) sTREM2 in the serum of NCD (n = 8), Sham AL (n = 7), Sham PF (n = 9), and VSG (n = 8) mice 5 weeks after surgeries. (D) Volcano plots showing DEGs between Sham AL and VSG (left), and Sham PF and VSG (right) LAMs, (E) pathway analysis of LAMs from VSG versus Sham AL (top) and VSG versus Sham PF (bottom) comparisons, (F) GSEA plots of the VSG versus Sham PF LAMs comparison (top), and heatmaps of respective pathways (bottom). sTREM2 levels were analyzed by one-way ANOVA with Holm-Šídák multiple comparison test. Differential expression testing was performed by a Wilcoxon rank-sum test. Pathway analysis was performed by GSEA (adj. p < 0.1). Data are biological experimental units presented as mean ± SEM. Abbreviations: DEG, differentially expressed gene; FC, fold change; GSEA, gene seat enrichment analysis; LAM, lipid-associated macrophage; scRNA-seq, single-cell RNA sequencing; Sham AL, Sham ad libitum; Sham PF, Sham pair-fed; sTREM2, soluble TREM2; TREM2, triggering receptor expressed on myeloid cells 2; UMAP, uniform manifold approximation and projection; VSG, vertical sleeve gastrectomy.

FIGURE 4

TREM2 is required for the reparative effects of VSG against MASH. (A) Body weight change after surgery, (B) liver weight, (C) serum ALT (left) and AST (right), (D) hepatic triglyceride content, (E) representative H&E-stained liver sections, (F) unsupervised PCA of bulk RNA-seq gene expression data from F4/80⁺-sorted macrophages, (G) Venn diagram with the number of DEGs between Sham and VSG in WT and TREM2 KO macrophages, and (H) pathway analysis (KEGG and gene ontology) for the TREM2 KO VSG versus WT VSG comparison. C57BL6/J (WT) and TREM2 KO (KO) mice were fed an HFHC diet for 14 weeks, assigned to either Sham AL or VSG, and analyzed 5 weeks after surgery (WT Sham, n = 4–8; WT VSG, n = 4–7; KO Sham, n = 4–8; and KO VSG, n = 4–8). (I) Gene expression of inflammatory cytokines in bone marrow–derived macrophages from WT (n = 3–7) or TREM2 KO (n = 4–6) mice left unstimulated (left) or stimulated with palmitate (right). (J) Representative flow plots showing apoptosis of AML12 hepatocytes that were either left unstimulated or stimulated with PA (left), and representative plots (middle) and quantification (right) of CellTracker Green (CMFDA)-positive macrophages following coculture of peritoneal macrophages from WT (n = 7) and TREM2 KO (n =5) mice with CMFDA-labeled apoptotic AML12 hepatocytes. Data from 4 experimental groups were analyzed by one-way ANOVA with Holm-Šídák multiple comparison test. Pathway analysis was performed by GSEA (adj. p < 0.1). Data from 2 experimental groups were analyzed by Mann-Whitney tests. Data are biological experimental units presented as mean ± SEM. Abbreviations: DEG, differentially expressed gene; GSEA, gene seat enrichment analysis; H&E, hematoxylin and eosin; HFHC, high-fat high-carbohydrate; KO, knockout; MASH, metabolic dysfunction–associated steatohepatitis; PA, palmitate; PCA, principal component analysis; Sham AL, Sham ad libitum; TREM2, triggering receptor expressed on myeloid cells 2; VSG, vertical sleeve gastrectomy; WT, wild type.

FIGURE 5

VSG increases the content of inflammatory lipid species in hepatic macrophages. (A) Composition of detectable metabolites in macrophages, (B) unsupervised PCA of metabolite data, (C) concentration of TGs, (D) chain length enrichment analysis of all lipid species, (E) concentration of major lipid species including PC, CEs, FAs, HexCer, Cer, SM, and DGs, (F) PC subspecies such as PCs (aa), PCs (ae), MUFA PCs, PUFA PCs, and SFA PCs, (G) SM subspecies including LCFA SMs and VLCFA SMs, (H) Cer subspecies including VLCFA-Cer and LCFA-Cer, (I) FA subspecies including MUFAs, PUFAs, and w-3 FAs, and (J) CE subspecies determined by mass spectrometry and liquid chromatography of hepatic macrophages isolated from the livers of HFHC-fed mice assigned to Sham AL (n = 4) or VSG (n = 4) 5 weeks after surgery. (K) Heatmap of the VSG/Sham AL ratio for lipid species in the liver and macrophages (mac) showing the p values for each comparison. Data were analyzed by the Welch 2-sided t tests. Data are biological experimental units presented as mean ± SEM. Abbreviations: CE, cholesterol ester; Cer, ceramides; DG, diacylglycerol; FA, fatty acid; HexCer, glycosylceramides; HFHC, high-fat high-carbohydrate; LCFA, long chain fatty acid; MUFA, monounsaturated fatty acid; PC, phosphatidylcholines; PCA, principal component analysis; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; Sham AL, Sham ad libitum; SM, sphingolipids; TG, triacylglycerols; VLCFA, very-long-chain fatty acid; VSG, vertical sleeve gastrectomy; w-3, omega-3.

FIGURE 6

Spatial transcriptomics of MASH livers following bariatric surgery reveals an improved metabolic status in the macrophage microenvironment. (A) Representative immunofluorescence images of ROIs (left) and magnified fields showing CD68⁻ (middle) and CD68⁺ (right) areas in liver specimens analyzed by spatial transcriptomics (Geomx). (B) Unsupervised PCA of gene expression data from CD68⁺ and CD68⁻ ROIs. (C) Volcano plots showing differentially expressed genes between NCD and Sham AL (left), Sham AL and VSG (middle), Sham PF and VSG (right) CD68⁻ ROIs. (D) Pathway analysis of VSG versus Sham AL (left) and VSG versus Sham PF (right). (E) Heatmap of zonation marker genes for PP and PCL ROIs. (F) Volcano plots showing DEGs between PP and PCL ROIs. (G) Pathway analysis of PP and PCL ROIs for the NCD and Sham AL (left), Sham AL and VSG (middle), and Sham PF and VSG comparisons (right). Liver specimens were collected from C57BL6/J (WT) fed either an NCD or an HFHC diet for 14 weeks, assigned to Sham AL, Sham PF, or VSG surgeries, and analyzed 5 weeks after surgery (n = 4). Data were analyzed using a Mann-Whitney test and corrected with a Benjamini-Hochberg procedure. Pathway analysis was performed by GSEA (adj. p < 0.1). Abbreviations: DEG, differentially expressed gene; FC, fold change; FDR, false discovery rate; HFHC, high-fat high-carbohydrate; NCD, normal chow diet; PCA, principal component analysis; PCL, pericentral; PP, periportal; ROI, regions of interest; Sham AL, Sham ad libitum; Sham PF, Sham pair-fed; VSG, vertical sleeve gastrectomy; WT, wild type.