FASN-dependent lipogenesis controls macrophage efferocytosis in alcohol-associated liver disease

Abstract

Background & Aims

Defective clearance of apoptotic hepatocytes contributes to inflammation of the liver and progression of alcohol-associated liver disease (ALD), but mechanisms regulating macrophage efferocytosis during alcohol exposure remain unclear. We investigated whether fatty acid synthase (FASN)-dependent lipid metabolism controls hepatic macrophage efferocytosis in ALD.

Methods

Human liver tissues from patients with alcohol-related cirrhosis (AC) and controls (n = 18 per group), together with experimental ALD mouse models (six per group), were analyzed for hepatocyte apoptosis and hepatic macrophage alterations. Transcriptomic profiling (three per group), pharmacological inhibition, and myeloid- or Kupffer cell-specific Fasn knockout mice (six per group) were used to define the role and mechanism of FASN-mediated lipogenesis in macrophage efferocytosis.

Results

In patients with AC, hepatocyte apoptosis and accumulation of CD68-positive inflammatory macrophages were significantly increased compared with controls (p <0.0001). In experimental ALD mice, hepatocyte apoptosis and monocyte-derived macrophage infiltration were also significantly increased (p <0.0001). Mechanistically, ethanol impaired macrophage efferocytosis by >80% (p <0.01). This was associated with inhibition of the PI3K/AKT/SREBP1 pathway, reduced FASN expression, and suppressed de novo lipogenesis. Reduced FASN expression decreased NRF2 activity and impaired TREM2 transcription, resulting in defective clearance of apoptotic cells. TREM2-positive hepatic macrophages were significantly decreased in patients with AC and experimental ALD mice (p <0.0001). Consistently, Kupffer cell-specific Fasn deletion significantly aggravated hepatocyte apoptosis and liver injury in vivo (p <0.01).

Conclusions

Alcohol impairs macrophage efferocytosis by suppressing the PI3K/AKT/SREBP1–FASN-NRF2-TREM2 axis. Disruption of this lipogenic program promotes hepatocyte apoptosis and liver inflammation in ALD.

Impact and implications

Alcohol-associated liver disease is characterized by hepatocyte death and ongoing inflammation, but mechanisms that connect these processes to macrophage efferocytosis remain poorly understood. In our research, we found that ethanol suppresses FASN-dependent de novo lipogenesis and downstream NRF2-TREM2 signaling in hepatic macrophages. Impairment of lipogenesis compromises efferocytosis, leading to accumulation of apoptotic hepatocytes and increased monocyte infiltration. These findings underscore the potential of targeting macrophage lipid metabolism as a therapeutic strategy in ALD. However, further translational validation is needed before clinical application.

Graphical abstract

Highlights

• •Alcohol disrupts hepatic macrophage efferocytosis in ALD.
• •A PI3K/AKT/SREBP1/FASN axis controls lipid metabolism during efferocytosis.
• •FASN-NRF2-TREM2 signaling links macrophage lipid metabolism to apoptotic cell clearance.
• •Kupffer cell-specific Fasn deletion aggravates liver injury in experimental ALD.

Introduction

Alcohol-associated liver disease (ALD) is a major cause of liver-related morbidity and mortality worldwide.¹ Hepatocyte death is a central feature of ALD; it occurs early during disease development and persists throughout disease progression.^2,3 Ethanol and its metabolite acetaldehyde exert direct hepatotoxic effects, and chronic alcohol consumption impairs intestinal barrier integrity, allowing gut-derived microbial products to reach the liver. These signals activate Toll-like receptors on hepatic immune cells, amplifying innate immune responses and promoting liver injury.⁴

Efferocytosis, the macrophage-mediated clearance of dying cells, is essential for maintaining tissue homeostasis and limiting inflammation.⁵ Failure to clear these apoptotic cells results in the release of damage-associated molecular patterns (DAMPs), leading to persistent immune activation and exacerbation of tissue injury. Impaired efferocytosis has been implicated in metabolic dysfunction-associated steatotic liver disease (MASLD), atherosclerosis, cancer, and infections.⁶ In the liver, Kupffer cells (KCs) are responsible for clearing dead cells and debris; however, whether alcohol exposure disrupts macrophage efferocytosis during ALD progression has not been explored.

Immune cells exhibit dynamic reprogramming of their metabolic pathways in response to tissue injury and pathogen stimuli. Lipids and their metabolites play crucial roles in signaling pathways that modulate macrophage responses to pathogens, damage, and inflammation.⁷ Alcohol differentially affects lipid metabolism in hepatocytes and hepatic macrophages. In hepatocytes, ethanol enhances fatty acid synthase (FASN)-dependent de novo lipogenesis (DNL) and suppresses fatty acid oxidation, whereas in hepatic macrophages, ethanol inhibits fatty acid synthesis and impairs bacterial clearance.^8,9 Whether ethanol regulates macrophage efferocytosis in ALD through metabolic reprogramming remains poorly understood.

Multiple forms of regulated cell death, including apoptosis, necroptosis, pyroptosis, and ferroptosis, contribute to ALD pathogenesis.¹⁰ As efferocytosis is essential for clearing dead cells and maintaining liver homeostasis, its disruption can remarkably affect ALD progression. Here, we demonstrate accumulation of apoptotic hepatocytes and increased monocyte infiltration in the livers of patients with alcohol-related cirrhosis (AC) and in murine ALD models. Mechanistically, ethanol inhibited the PI3K/AKT/SREBP1 pathway, thereby reducing FASN expression in hepatic macrophages. Loss of FASN suppressed NRF2 activity and impaired NRF2-dependent transcription of the efferocytosis receptor TREM2, resulting in defective macrophage efferocytosis and aggravated liver injury. In this study, we establish a connection between macrophage lipid metabolism and impaired efferocytosis in ALD, and provide a rationale for targeting macrophage lipogenesis in future therapeutic studies.

Materials and methods

Animal experiments

Female C57BL/6J wild-type mice (age: 8–10 weeks) were purchased from Vital River (Beijing, China). Clec4f-IRES-Cre-2A-tdTomato mice were obtained from Shanghai Model Organisms Center (Shanghai, China). Fasn^flox/flox mice were provided by Prof Zhinan Yin (Jinan University, Guangzhou, China). KC-specific Fasn knockout mice (Fasn^ΔKC) were generated by crossing Clec4f-Cre mice with Fasn^flox/flox mice. Myeloid-specific Fasn knockout mice (Fasn^ΔMye) were generated by crossing Fasn^flox/flox mice with Lyz2-Cre mice.

Mice were maintained under specific pathogen-free conditions. ALD was induced using the Lieber-DeCarli ethanol diet for 10 days followed by a single ethanol gavage, as described previously.¹¹ Samples were collected 9 h after the final gavage. All procedures were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of University of Science and Technology of China (Hefei, Anhui, China).

Human samples

Liver biopsy specimens were collected from The First Affiliated Hospital of University of Science and Technology of China and The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China). The study involved 18 patients with AC and 18 patients with benign liver conditions, including hepatic hemangioma and hepatolithiasis, as controls. Written informed consent was obtained from all participants.

Histology

Liver tissues were fixed in 4% paraformaldehyde (Biosharp, Beijing, China) and embedded in paraffin for H&E staining. Frozen sections were prepared for Oil Red O staining. Images were acquired using a Zeiss light microscope (Jena, Germany).

Serum biochemistry

Plasma AST and ALT levels were measured using commercial kits (Nanjing Jiancheng, Nanjing, China).

Cell culture

RAW264.7, Jurkat, and AML12 cells were cultured in DMEM with 10% FBS (Sigma, St. Louis, USA). Bone marrow-derived macrophages (BMDMs) were generated using macrophage colony-stimulating factor (20 ng/ml) (PeproTech, Inc., Cranbury, USA). Cells were treated with ethanol (100 mM, 24 h) unless indicated. Inhibitors included C75, LY294002, fatostatin, and ML385 (MedChemExpress, New Jersey, USA).

Efferocytosis assays

To induce apoptosis, we treated Jurkat cells with 10 μM staurosporine (MedChemExpress, New Jersey, USA) for 6 h and AML12 cells with 100 μM staurosporine for 16 h. BMDMs were seeded at 1 × 10⁵ cells per well and co-incubated with apoptotic cells at a 1:5 ratio. For efferocytosis assays, DiD-labeled (Beyotime, Shanghai, China) BMDMs and carboxyfluorescein succinimidyl ester (CFSE)-labeled (Invitrogen, Waltham, USA) apoptotic cells were co-incubated for 1 h, and then washed with PBS. Macrophage efferocytosis was assessed by immunofluorescence imaging (Zeiss, LSM800, Jena, Germany).

Neutral lipid staining

Neutral lipid droplets in efferocytic BMDMs were visualized using 4,4-difluoro- 1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene. After fixation, cells were incubated with 0.1 μg/ml4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a- diaza-s-indacene in the dark at 37 °C for 30 min, washed with PBS, and then counterstained with DAPI (Biosharp, Beijing, China). Fluorescence images were acquired using a Zeiss LSM800 confocal microscope.

RNA sequencing

Total RNA was extracted using acid guanidinium thiocyanate–phenol–chloroform extraction (TRIzol) (Invitrogen, Waltham, USA). Library preparation and sequencing were performed by Shanghai OE Biotech Co., Ltd. (Shanghai, China).

Immunofluorescence

Frozen liver sections were fixed in 4% paraformaldehyde, permeabilized, blocked, and incubated with primary antibodies against CLEC4 (R&D systems, Minneapolis, USA), IBA1 (Abcam, Cambridge, UK), CD68 (Proteintech, Wuhan, China), TREM2 (Abcam, Cambridge, UK), or HNF4α (Abcam, Cambridge, UK), followed by the appropriate fluorescent secondary antibodies and DAPI staining. For detection of apoptotic hepatocytes, liver sections were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Abbkine, Wuhan, China) staining and co-stained with anti-HNF4α antibody. TUNEL⁺HNF4α⁺ cells were quantified as apoptotic hepatocytes. For cellular immunofluorescence, cells were fixed, blocked, and incubated with anti-TREM2 antibody, followed by fluorescent secondary antibody and DAPI staining. Images were acquired using a Zeiss LSM800 confocal microscope and quantified using ImageJ.

Quantitative PCR

BMDMs were co-incubated with apoptotic cells (1:5, 12 h), washed to remove unengulfed cells, and collected for RNA extraction using TRIzol. cDNA was synthesized using HiScript III RT SuperMix (Vazyme, Nanjing, China), and quantitative PCR (qPCR) was performed using the QuantiNova kit (Qiagen, Hilden, Germany). Relative expression was calculated using the 2^−ΔΔCt method with 18S as a control.

Western blot

BMDMs were co-incubated with apoptotic cells (1:5, 12 h), and proteins were extracted using RIPA buffer (Proteintech, Wuhan, China). Nuclear and cytoplasmic fractions were prepared using a commercial kit (Beyotime, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with antibodies against FASN (Cell Signaling Technology, Danvers, USA), SREBP1 (Immunoway, San Jose, USA), PI3K (Bioss, Beijing, China), AKT, p-PI3K, p-AKT, NRF2, LaminB1, and β-actin (Proteintech, Wuhan, China), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech, Wuhan, China) and chemiluminescence detection.

Statistical analysis

Data are shown as mean ± SEM or median (Q1, Q3). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, USA). Differences were evaluated using the unpaired Student’s t test or the Mann-Whitney U test. A p value <0.05 was considered significant.

Results

Hepatocyte apoptosis is increased in human and experimental ALD

We first examined hepatocyte apoptosis in liver samples from patients with AC (Fig. 1A). TUNEL staining combined with HNF4α labeling demonstrated marked hepatocyte apoptosis in AC livers compared with controls (Fig. 1B and C). We next evaluated apoptosis in a murine ALD model. Mice that were fed the Lieber-DeCarli ethanol diet showed significantly increased serum ALT and AST levels compared with controls (Fig. S1A). Histological analysis revealed pronounced steatosis and lipid accumulation, as shown by H&E and Oil Red O staining (Fig. 1D and Fig. S1B), confirming successful model establishment. Consistent with the human data, there was increased hepatocyte apoptosis in ALD mice (Fig. 1E and F). To determine the cellular source of apoptosis, liver sections were co-stained with TUNEL and markers for T cells (CD3), endothelial cells (CD31), and macrophages (IBA1). Only minimal apoptosis was detected in these cell populations, indicating that hepatocytes represent the predominant apoptotic cell type in ALD (Fig. S1C–E). During ALD progression, hepatic macrophage populations are expanded by infiltrating monocytes, which contribute to inflammation and liver injury.^12,13 Immunofluorescence analysis demonstrated a significant increase in IBA1⁺ CLEC4F⁻ monocyte-derived macrophages (MoMFs) in the livers of ALD mice (Fig. 1G and H). In parallel, increased numbers of CD68⁺ macrophages were observed in liver tissues from patients with AC (Fig. 1I and J). In these patients, the systemic inflammatory response index was more pronounced (Table S1). Together, these data demonstrate increased accumulation of apoptotic hepatocytes during ALD and enhanced inflammation.

Fig. 1.

Hepatocyte apoptosis is markedly increased in human and experimental ALD.

Liver tissues from patients with alcohol-related cirrhosis (AC) and controls (Ctrl) were analyzed (n = 18 per group). (A) H&E staining of liver sections (scale bar = 20 μm). (B, C) TUNEL and HNF4α immunofluorescence staining showing apoptotic hepatocytes and quantification (scale bar = 50 μm). Female C57BL/6J mice (age: 8–10 weeks) were fed the Lieber-DeCarli ethanol diet followed by a single ethanol gavage (n = 6 per group). (D) H&E staining of liver sections (scale bar = 20 μm). (E, F) TUNEL and HNF4α staining for hepatocyte apoptosis, with quantification (scale bar = 50 μm). (G, H) Immunofluorescence staining of hepatic macrophages (Scale bar = 50 μm). IBA1⁺CLEC4F⁻, monocyte-derived macrophages; IBA1⁺CLEC4F⁺, Kupffer cells. (I, J) CD68 immunofluorescence staining of hepatic macrophages in human liver samples (scale bar = 50 μm). Bars represent mean ± SEM. Statistical significance was assessed using an unpaired two-tailed Student’s t test. ∗∗∗p <0.001, ∗∗∗∗ p <0.0001. ALD, alcohol-associated liver disease. ALD, alcohol-associated liver disease; CLEC4F, C-type lectin domain family 4 member F; HNF4α, hepatocyte nuclear factor 4 alpha; IBA1, ionized calcium-binding adapter molecule 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Essential role of DNL in macrophage efferocytosis

Efficient clearance of apoptotic cells is required for liver homeostasis. To determine whether metabolic reprogramming influences macrophage efferocytosis, we performed RNA sequencing in BMDMs co-cultured with apoptotic cells. Transcriptomic analysis revealed marked metabolic remodeling, with substantial enrichment of genes involved in fatty acid metabolism (Fig. 2A). Among these, Fasn, a key enzyme in DNL, was strongly upregulated (Fig. 2B). Western blot analysis confirmed increased FASN expression in efferocytic macrophages (Fig. 2C and Fig. S2A), together with elevated intracellular neutral lipid levels (Fig. 2D). qPCR analysis further revealed increased mRNA expression of anti-inflammatory cytokines (Il-10, Tgf-β) and decreased expression of pro-inflammatory cytokines (Il-1β) (Fig. 2E and Fig. S2B and C). Using PKH-26/CFSE-based efferocytosis assays, we found that pharmacological inhibition of FASN with C75 significantly impaired macrophage engulfment of apoptotic cells (Fig. 2F and G and Fig. S2D). Similarly, BMDMs derived from Fasn^ΔMye mice exhibited defective efferocytosis (Fig. 2H and I and Fig. S2E).

Fig. 2.

De novo lipogenesis is required for macrophage efferocytosis.

BMDMs were co-cultured with apoptotic Jurkat or AML12 cells (1:5) for 12 h. RNA sequencing was performed after efferocytosis of apoptotic Jurkat cells (n = 3), whereas AML12 cells were used in all other efferocytosis assays. (A) Gene Ontology (GO) analysis of metabolic pathways. (B) Heatmap of lipid metabolism-related genes. (C) FASN protein levels in efferocytic BMDMs by Western blot (n = 3). (D) Neutral lipid staining (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) in efferocytic BMDMs (scale bar = 50 μm). (E) mRNA levels of Il-10 in efferocytic BMDMs (n = 6). (F, G) Efferocytosis assay. BMDMs were pretreated with C75 (40 μM, 4 h), labeled with DiD (red), and co-incubated with CFSE-labeled apoptotic AML12 cells (green) for 1 h (scale bar = 100 μm, n = 6). (H, I) Efferocytosis in BMDMs from Fasn^ΔMye mice (scale bar = 100 μm, n = 6). Bars represent mean ± SEM. Two-tailed unpaired t test. ∗∗p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001. ACs, apoptotic cells; BMDMs, bone marrow-derived macrophages; Ctrl, controls; Fasn^fl/fl, Fasn^flox/flox mouse; Fasn^ΔMye, myeloid cell-specific Fasn knockout mouse. ACs, apoptic cells; BMDMs, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; Ctrl, controls; DMSO, dimethylsulfoxide; FASN, fatty acid synthase; Fasn^fl/fl, Fasn^flox/flox mouse; Fasn^Δmye, myeloid cell–specific Fasn knockout mouse.

Ethanol suppresses FASN-dependent lipogenesis to impair macrophage efferocytosis

RNA sequencing revealed that ethanol exposure markedly altered metabolic pathways and reduced expression of genes involved in lipogenesis, including Fasn (Fig. 3A and B) in BMDMs. BODIPY staining showed that ethanol-treated efferocytic macrophages exhibited reduced intracellular lipid accumulation (Fig. 3C). Ethanol reduced FASN mRNA and protein levels in efferocytic BMDMs (Fig. 3D and E and Fig. S3A and B). Ethanol-treated macrophages exhibited a considerably decreased ability to engulf apoptotic cells (Fig. 3F and G and Fig. S3C and D). Decreased mRNA levels of anti-inflammatory cytokines (Il-10) and increased expression of pro-inflammatory cytokines (Il-1β, Tnf-α) were observed in efferocytic BMDMs exposed to ethanol (Fig. 3H and Fig. S3E).

Fig. 3.

Ethanol disrupts macrophage efferocytosis by suppressing FASN-dependent lipogenesis.

BMDMs were treated with ethanol (100 mM, 24 h) unless indicated. RNA sequencing was performed (n = 3). (A) Gene Ontology (GO) analysis of the top 10 metabolic pathways. (B) Heatmap of lipid metabolism-related genes. BMDMs were exposed to ethanol and co-cultured with apoptotic AML12 cells (1:5) for 12 h. (C) Neutral lipid staining in BMDMs (Scale bar = 50 μm). (D) Fasn mRNA levels by qPCR (n = 6). (E) Western blot analysis of FASN protein levels (n = 3). (F, G) Efferocytosis assay. DiD-labeled BMDMs (red) were co-incubated with CFSE-labeled apoptotic AML12 cells (green) (scale bar = 100 μm, n = 6). (H) mRNA levels of Il-10, Il-1β, and Tnf-α in efferocytic BMDMs (n = 6). Bars represent mean ± SEM. Two-tailed unpaired t tests were used for the comparisons. ∗∗p <0.01, ∗∗∗∗p <0.0001. ACs, apoptotic cells; BMDMs, bone marrow-derived macrophages; Ctrl, controls; EtOH, ethanol; qPCR, quantitative PCR. ACs, apoptic cells; BMDMs, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; Ctrl, controls; EtOH, ethanol; FASN, fatty acid synthase.

Ethanol inhibits macrophage efferocytosis via the PI3K/AKT/SREBP1/FASN signaling axis

To explore the mechanism underlying FASN downregulation, we examined the PI3K/AKT/SREBP1 pathway, a key regulator of lipid synthesis. Ethanol exposure reduced both total and phosphorylated PI3K and AKT in macrophages (Fig. 4A). Pharmacologic inhibition of PI3K with LY294002 decreased SREBP1 and FASN expression in efferocytic macrophages (Fig. 4B) and significantly impaired efferocytosis (Fig. 4C). Il-10 mRNA expression was also reduced (Fig. 4D). Likewise, treatment with the SREBP1 inhibitor fatostatin decreased FASN levels (Fig. 4E), reduced efferocytosis (Fig. 4F), and suppressed Il-10 mRNA expression (Fig. 4G).

Fig. 4.

PI3K/AKT/SREBP1/FASN signaling mediates ethanol-induced impairment of macrophage efferocytosis.

(A) RAW264.7 cells treated with ethanol (100 mM, 24 h). Western blot analysis of PI3K, p-PI3K, AKT, and p-AKT protein levels (n = 3). BMDMs were treated with LY294002 (50 μM, 4 h) and co-cultured with apoptotic AML12 cells. (B) Western blot of FASN, cytoplasmic SREBP1(cSREBP1), and nuclear SREBP1(nSREBP1) protein levels (n = 3). (C) Efferocytosis assay. DiD-labeled BMDMs (red) and CFSE-labeled apoptotic cells (green) (Scale bar = 100 μm, n = 6). (D) Il-10 mRNA levels in efferocytic BMDMs by qPCR (n = 6). BMDMs were treated with fatostatin (50 μM) for 4 h before efferocytosis. (E) FASN protein levels by Western blot. (F) Efferocytosis assay (Scale bar = 100 μm, n = 6). (G) Il-10 mRNA levels by qPCR (n = 6). Bars represent mean ± SEM. Two-tailed unpaired t test. ∗∗∗p <0.001, ∗∗∗∗p <0.0001. ACs, apoptotic cells; BMDMs, bone marrow-derived macrophages; qPCR, quantitative PCR. ACs, apoptic cells; p-AKT, phosphorylated AKT; BMDMs, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; DMSO, dimethylsulfoxide; FASN, fatty acid synthase; PI3K, phosphoinositide 3-kinase; p-PI3K, phosphorylated PI3K; qPCR, quantitative PCR; SREBP1, sterol regulatory element-binding protein 1.

Ethanol suppresses TREM2 expression in hepatic macrophages

RNA sequencing identified Trem2 as an efferocytosis-related gene upregulated after engulfment of apoptotic cells but downregulated following ethanol treatment (Fig. 5A). qPCR and immunofluorescence analyses confirmed that TREM2 expression increased after efferocytosis and was markedly reduced by ethanol in efferocytic macrophages (Fig. 5B–D and Fig. S4A–D). Reduced TREM2 expression was also observed in BMDMs from Fasn^ΔMye mice after efferocytosis (Fig. 5F and G). Likewise, TREM2 expression was decreased in hepatic macrophages in the ALD model (Fig. 5H and I) and in liver tissues from patients with AC (Fig. 5J and K). These findings suggest that ethanol impairs macrophage efferocytosis, at least in part by downregulating TREM2, thereby promoting liver injury.

Fig. 5.

Ethanol-mediated suppression of TREM2 expression in hepatic macrophages.

(A) RNA sequencing heatmap of efferocytosis-related genes in BMDMs exposed to ethanol 100 mM for 24 h, and co-cultured with apoptotic Jurkat cells (n = 3). (B) Trem2 mRNA levels in efferocytic BMDMs (n = 6). (C) Immunofluorescence staining of TREM2 in efferocytic BMDMs (scale bar = 50 μm, n = 6). BMDMs exposed to ethanol before efferocytosis. (D) Trem2 mRNA levels by qPCR (n = 6). (E) Immunofluorescence staining of TREM2 (Scale bar = 50 μm, n = 6). BMDMs from Fasn^fl/fl and Fasn^ΔMye mice were analyzed after efferocytosis. (F) Trem2 mRNA levels by qPCR (n = 6). (G) Immunofluorescence staining of TREM2 (scale bar = 50 μm, n = 6). (H, I) TREM2 expression in hepatic macrophages in ALD mice (IBA1 staining) (scale bar = 50 μm, n = 6). (J, K) TREM2 expression in hepatic macrophages in patients with AC and controls (CD68 staining) (scale bar = 50 μm, n = 18). Bars represent mean ± SEM. Statistical significance was assessed using a two-tailed unpaired t test. ∗∗∗p <0.001, ∗∗∗∗p <0.0001. AC, alcohol-related cirrhosis; ACs, apoptotic cells; ALD, alcohol-associated liver disease; Ctrl, controls; EtOH, ethanol; Fasn^fl/fl, Fasn^flox/flox mouse; Fasn^ΔMye, myeloid cell-specific Fasn knockout mouse. AC, alcohol-related cirrhosis; ALD, alcohol-associated liver disease; BMDMs, bone marrow-derived macrophages; Ctrl, controls; EtOH, ethanol; Fasn^fl/fl, Fasn^flox/flox mouse; Fasn^ΔMye, myeloid cell-specific Fasn knockout mouse; IBA1, ionized calcium-binding adapter molecule 1; qPCR, quantitative PCR; TREM2, triggering receptor expressed on myeloid cells 2.

FASN regulates TREM2 expression through NRF2 signaling in efferocytic macrophages

NRF2 reportedly regulates TREM2 transcription.¹⁴ Efferocytosis increased NRF2 levels in both cytoplasmic and nuclear fractions, whereas ethanol or C75 treatment reduced NRF2 expression in efferocytic macrophages (Fig. 6B and C). Inhibition of NRF2 using ML385 decreased TREM2 expression (Fig. 6D and E) and impaired efferocytosis (Fig. 6F and G). Restoration of NRF2 expression in Fasn^ΔMye macrophages rescued TREM2 levels (Fig. 6H and I) and partially restored efferocytosis (Fig. 6J and K). These findings indicate that FASN-dependent lipogenesis enhances efferocytosis, at least in part, through NRF2-mediated induction of TREM2 expression.

Fig. 6.

Regulation of TREM2 expression by FASN through NRF2 signaling.

(A) Cytoplasmic and nuclear NRF2 protein levels in efferocytic BMDMs (n = 3). (B, C) NRF2 levels in BMDMs treated with ethanol (100 mM, 24 h) or C75 (40 μM, 4 h) during efferocytosis (n = 3). (D) Trem2 mRNA levels after NRF2 inhibition (ML385) (n = 6). (E) Immunofluorescence staining of TREM2 (scale bar = 50 μm). (F, G) Efferocytosis assay after ML385 treatment. DiD-labeled BMDMs (red) and CFSE-labeled apoptotic cells (green) (scale bar = 100 μm, n = 6). Fasn^ΔMye BMDMs transfected with Nrf2 overexpression plasmid (Nrf2-OE). (H) Trem2 mRNA levels by qPCR (n = 6). (I) Immunofluorescence staining of TREM2 (Scale bar = 50 μm). (J, K) Efferocytosis assay in Fasn^ΔMye BMDMs with Nrf2 overexpression (scale bar = 100 μm, n = 6). Bars represent mean ± SEM or median (Q1, Q3). Two-tailed unpaired t test for D, G, and H, and the Mann-Whitney U test for part label K. ∗∗p <0.01, ∗∗∗∗p <0.0001. ACs, apoptotic cells; cNRF2, cytoplasmic NRF2 protein; Ctrl, controls; EtOH, ethanol; Fasn^fl/fl, Fasn^flox/flox mouse; Fasn^ΔMye, myeloid cell-specific Fasn knockout mouse; NC, negative control; nNRF2, nuclear NRF2; Nrf2-OE, Nrf2 overexpression; qPCR, quantitative PCR. ACs, apoptotic cells; BMDMs, bone marrow-derived macrophages; cNRF2, cytoplasmic NRF2 protein; CFSE, carboxyfluorescein succinimidyl ester; Ctrl, controls; DMSO, dimethylsulfoxide; EtOH, ethanol; Fasn^fl/fl, Fasn^flox/flox mouse; Fasn^ΔMye, myeloid cell-specific Fasn knockout mouse; NC, negative control; NRF2, nuclear factor erythroid 2-related factor 2; nNRF2, nuclear NRF2; Nrf2-OE, Nrf2 overexpression; qPCR, quantitative PCR; TREM2, triggering receptor expressed on myeloid cells 2.

Kupffer cell-specific Fasn deletion exacerbates ALD progression through impaired efferocytosis

We next examined the impact of suppressed macrophage DNL in an experimental model of ALD. There were increased plasma ALT and AST levels in ALD mice with KC-specific Fasn deletion (Fasn^ΔKC; Fig. 7A) and aggravated steatosis (Fig. 7B). TREM2 expression in hepatic macrophages was reduced in Fasn^ΔKC mice (Fig. 7C and D), accompanied by increased hepatocyte apoptosis (Fig. 7E and F). Infiltration of MoMFs (IBA1⁺CLEC4F⁻) was also higher in the livers of Fasn^ΔKC mice (Fig. 7G and H).

Fig. 7.

Kupffer cell-specific Fasn deficiency aggravates ALD through impaired efferocytosis.

Kupffer cell-specific Fasn knockout mice (Fasn^ΔKC) were subjected to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) model of ALD (n = 6). (A) Plasma ALT and AST levels. (B) H&E staining of liver sections (scale bar = 20 μm). (C) TREM2 expression in hepatic macrophages (IBA1 staining) and quantification (scale bar = 50 μm). (D, E) TUNEL and HNF4α staining showing hepatocyte apoptosis and quantification (scale bar = 50 μm). (F, G) Immunofluorescence staining of hepatic macrophages (scale bar = 50 μm). IBA1⁺CLEC4F⁻, monocyte-derived macrophages; IBA1⁺CLEC4F⁺, Kupffer cells. Bars represent mean ± SEM or median (Q1, Q3). Two-tailed t test (B, F, H, and AST in C) or the Mann-Whitney U test (ALT in C). ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Fasn^fl/fl, Fasn^flox/flox mouse. ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CLEC4F, C-type lectin domain family 4 member F; Fasn^fl/fl, Fasn^flox/flox mouse; HNF4α, hepatocyte nuclear factor 4 alpha; IBA1, ionized calcium-binding adapter molecule 1; TREM2, triggering receptor expressed on myeloid cells 2; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Discussion

Hepatocyte death is a central feature of ALD; therefore, efficient and timely efferocytosis is required to limit secondary necrosis and promote resolution of inflammation.^5,10 Although defective efferocytosis has been implicated in several liver diseases,⁶ whether hepatic macrophage efferocytosis is altered during ALD has not been well defined. In this study, we observed increased hepatocyte apoptosis and accumulation of MoMFs in the livers of patients with AC and in experimental ALD. Our data further show that ethanol impairs macrophage efferocytosis by suppressing FASN-dependent DNL, leading to accumulation of apoptotic cells, enhanced inflammation, and aggravated liver injury.

Effective clearance of apoptotic cells is crucial to prevent secondary necrosis.¹⁰ Because macrophage-mediated efferocytosis is required to maintain hepatic immune homeostasis, disruption of this process would worsen liver inflammation.^15,16 Although ethanol reportedly impairs macrophage phagocytic activity in vitro,¹² direct evidence for defective efferocytosis in the liver tissues during ALD has been limited. Here, the livers of both AC and ALD mice showed a marked buildup of apoptotic hepatocytes and increased infiltration of MoMFs. This finding supports the notion that the removal of dying cells is inadequate in the injured liver.

A notable finding of this study is that macrophage efferocytosis is closely linked to lipid metabolism. Efferocytic macrophages exhibited enrichment of fatty acid metabolism-related genes, including Fasn, and increased FASN expression at the protein level. Inhibition of FASN, as well as myeloid-specific Fasn deletion, impaired engulfment of apoptotic cells. KC-specific Fasn deletion further aggravated hepatocyte apoptosis and accumulation of inflammatory macrophages in the ALD model. These data extend previous work linking lipogenesis to macrophage function^13,17 and suggest that FASN-mediated DNL is required for efficient efferocytosis during ALD.

SREBP1 is a key transcriptional regulator of lipid synthesis genes.[18], [19], [20] The PI3K/AKT pathway promotes SREBP1 activation and downstream lipogenesis genes expression.²¹ Upstream of FASN, ethanol suppressed PI3K/AKT/SREBP1 signaling in macrophages. This is of particular interest because ethanol enhances lipogenesis in hepatocytes by activating PI3K/AKT signaling.²² Our results, therefore, point to a cell type-specific effect of ethanol on lipid metabolism—lipogenesis is enhanced in hepatocytes but suppressed in macrophages. This divergence may help explain how alcohol simultaneously promotes steatosis in parenchymal cells while weakening reparative responses in hepatic macrophages.

Efferocytosis requires recognition of apoptotic cells through specific receptors, including TREM2, MerTK, and TIM4.^23,24 Among these, TREM2 has emerged as an important regulator of macrophage survival, phagocytosis, and inflammatory responses.^25,26 In our study, efferocytosis increased TREM2 expression, whereas ethanol decreased TREM2 in macrophages and in the livers of both ALD mice and patients with AC. Reduced TREM2 expression was associated with impaired efferocytosis and aggravated liver injury, indicating that TREM2 is required for efficient clearance of apoptotic hepatocytes during ALD.

Our data also place NRF2 between FASN and TREM2 in this pathway. NRF2 is upregulated by numerous metabolic and stress-responsive signals²⁷ and has been identified as a transcriptional regulator of Trem2.¹⁴ Inhibition of FASN reduced NRF2 expression and nuclear accumulation, whereas restoration of NRF2 in Fasn-deficient macrophages rescued TREM2 expression and partially restored efferocytosis. These results present a model in which FASN-dependent lipogenesis maintains macrophage efferocytosis by regulating TREM2 expression through NRF2.

TREM2 is predominantly expressed in hepatic non-parenchymal cells and is induced during liver injury. Previous studies have linked TREM2 deficiency to increased inflammation and fibrosis in different liver diseases.^28,29 In non-alcoholic fatty liver disease, loss of TREM2 accelerates disease progression and increases susceptibility to sepsis, whereas increased TREM2 expression in hepatic macrophages improves outcomes during sepsis.³⁰ We observed reduced TREM2 expression in hepatic macrophages in ALD, with increased hepatocyte apoptosis and inflammation. This indicates a role for TREM2 in maintaining hepatic macrophage homeostasis during alcohol-induced liver injury.

This study has several limitations. TREM2 can be cleaved to generate a soluble form with potentially distinct biological functions.³¹ However, whether altered lipogenesis affects TREM2 shedding in ALD remains unknown. Neutrophil infiltration is another hallmark of ALD, but the impact of ethanol on neutrophil lipid metabolism was not addressed here. Furthermore, the clinical cohort was relatively small, and validation in larger, independent cohorts will be important to strengthen the clinical relevance of these findings. Nevertheless, our data support a model in which ethanol suppresses macrophage lipogenesis and disrupts efferocytosis, leading to the accumulation of apoptotic hepatocytes and increased liver inflammation.

Conclusions

In this study, we define FASN-dependent lipogenesis as a critical regulator of macrophage efferocytosis in ALD. Ethanol suppresses the PI3K/AKT/SREBP1/FASN pathway, leading to reduced NRF2-TREM2 signaling, impaired clearance of apoptotic hepatocytes, and enhanced liver inflammation. Targeting macrophage lipid metabolism may represent a therapeutic strategy to restore efferocytosis and limit liver injury in ALD.

Abbreviations

AC, alcohol-related cirrhosis; AKT, protein kinase B; ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMDMs, bone marrow-derived macrophages; DNL, de novo lipogenesis; FASN, fatty acid synthase; HNF4α, hepatocyte nuclear factor 4 alpha; IBA1, ionized calcium-binding adapter molecule 1; KCs, Kupffer cells; MoMFs, monocyte-derived macrophages; NRF2, nuclear factor erythroid 2-related factor 2; PI3K, phosphatidylinositol 3-kinase; qPCR, quantitative polymerase chain reaction; SREBP1, sterol regulatory element-binding protein 1; TREM2, triggering receptor expressed on myeloid cells 2; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Authors’ contributions

CH, HW, and HX conceived and designed the study. CC and XJ performed major experiments. HL and PH contributed to animal experiments. CH, HW, HX, and CC drafted and revised the manuscript. All authors discussed the results and approved the final version of the manuscript.

Data availability

The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1438973. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82370594 and 82470626), and from Natural Science Foundation of Anhui Province (2508085Y044).

Conflicts of interest

The authors declare no conflicts of interest that pertain to this work.

Please refer to the accompanying ICMJE disclosure forms for further details.

Supplementary data

The following are the Supplementary data to this article.

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