Role of triggering receptor expressed on myeloid cells 2 in the pathogenesis of non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a progressive disease. Without effective interventions, NAFLD can gradually develop to non-alcoholic steatohepatitis, fatty liver fibrosis, liver cirrhosis and even hepatocellular carcinoma. It is still to investigate the precise molecular mechanism behind the pathophysiology of NAFLD. Triggering receptor expressed on myeloid cells 2 (TREM2) can sense tissue injury and mediate immune remodeling, thereby inducing phagocytosis, lipid metabolism, and metabolic transfer, promoting cell survival and combating inflammatory activation. NAFLD might develop as a result of TREM2's regulatory role. We here briefly summarize the biological characteristics of TREM2 and its functions in the disease progression of NAFLD. Moreover, we propose to broaden the therapeutic strategy for NAFLD by targeting TREM2.

Key Words: Triggering receptor expressed on myeloid cells 2; Non-alcoholic fatty liver disease; Macrophage; Lipid metabolism; Inflammation

Core Tip: Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunoglobulin superfamily receptor located on the cell membrane. In the liver, it is mainly expressed on macrophages. TREM2 plays an important regulatory role in lipid metabolism, inflammation, fibrosis, and hepatocellular carcinoma, and is considered a potential therapeutic target for non-alcoholic fatty liver disease (NAFLD) (for example, macrophages with high expression of TREM2 can effectively clear apoptotic liver cells in a timely manner), thereby inhibiting chronic liver inflammation and non-alcoholic steatohepatitis lesions caused by obesity. This article highlights the most recent research developments on TREM2 in NAFLD, in an attempt to identify new pathways for NAFLD treatment.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) usually refers to hepatic steatosis that occurs without additional triggers (for example, medication, drinking alcohol, or certain genetic factors), symbolized by the formation of big fat droplets in liver cells due to an excess of triglyceride (TG)[1]. NAFLD patients are often accompanied by obesity, hyperlipidemia, diabetes mellitus type 2, as well as other metabolic disorders, which makes its prevention and treatment more complex[2]. According to current statistics, 30% of people worldwide have NAFLD, and the number is still rising[3]. In the upcoming decades, NAFLD may even emerge as the primary cause of end-stage liver disease, making it one of the most prevalent liver illnesses[4]. The range of NAFLD manifestations encompasses NAFLD, non-alcoholic steatohepatitis (NASH), as well as the cirrhosis and hepatocellular carcinoma (HCC) that can result from them[5]. NAFLD is histologically characterized by the presence of hepatic steatosis accounting for ≥ 5% of the liver, in the absence of any signs of hepatocyte injury. NASH is a more severe form of NAFLD, characterized by the presence of at least 5% hepatic steatosis along with inflammation accompanied by hepatocyte injury, including inflammatory reaction and ballooning degeneration, whether or not fibrosis is present. Over time, NASH has the potential to progress to cirrhosis and even cancer of the liver. In addition, NASH raises the risk of diabetes and cardiovascular disease[6]. The precise molecular mechanisms underlying the pathophysiology of NAFLD have not yet been thoroughly investigated, despite the fact that several fundamental and clinical investigations have been carried out. Resmetirom, the first medication in the world to treat adult NASH patients, is currently authorized for sale by the Food and Drug Administration[7]. Although encouraging, the results of the latest phase 3 clinical trial (MAESTRO-NASH) in patients with F1-F3 fibrosis and NASH have raised important questions about the comprehensive risks and advantages of Resmetirom[8,9]. Considering the need for long-term treatment, it is necessary to balance the effectiveness, safety, tolerability, and cost of drugs. For the diagnosis of NAFLD, as of right now, no particular biomarker exists for evaluating the progression and outcome of NAFLD, except for invasive liver biopsy. Therefore, the current understanding of NAFLD remains insufficient.

The immunoglobulin superfamily triggering receptor expressed on myeloid cells (TREM) 2 is found on the cell membrane, which undergoes a single transmembrane transition. The TREM2 gene was initially discovered by Swiss scientists Axel and Jess during cDNA cloning of dendritic cells (DCs) derived from mouse macrophages and monocytes[10]. TREM2 is expressed in osteoclasts and microglia under physiological settings and is crucial for tissue growth and functional preservation[11]. Zhou et al[12] reported the TREM2 gene is also expressed in certain macrophages of human adipose tissue, adrenal gland, and placental tissue, according to single-cell RNA transcriptome sequencing, but its role still needs further confirmation. In a diseased state, TREM2, as a membrane receptor, can perceive tissue damage and mediate immune remodeling, inducing phagocytosis, lipid metabolism, and metabolic transfer, promoting cell survival, and suppressing inflammatory activation[13]. Currently, TREM2 has been linked to both the onset and progression of Alzheimer's disease (AD) and other potential neurodegenerative disorders, obesity related to metabolic syndrome, NAFLD, atherosclerosis, tumors and other diseases. In the past few years, the biological function of TREM2 in liver associated illnesses has gradually been demonstrated. Non-parenchymal cells (NPCs) in the liver, including hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, immature monocyte derived DCs, and Kupffer cells (KCs), express TREM2[14-17]. Numerous studies have demonstrated that TREM2 is a possible therapeutic target for NAFLD and regulates lipid metabolism, inflammation, fibrosis, and HCC. This article reviews the biological characteristics of TREM2 and the role it plays in NAFLD development. It is suggested that treatment strategies should be expanded for NAFLD by targeting TREM2.

STRUCTURE AND FUNCTION OF TREM2

The human TREM2 gene has five exons and is found on chromosome 6's broken arm 21.1 (Figure 1)[18]. The five axons on the TREM2 gene encode 693 base pairs[19]. Structurally, TREM2 consists of a signal peptide, a V-type immunoglobulin domain, an extracellular domain with a short stalk sequence, a transmembrane helix, and a cytoplasmic tail[20]. Among them, V-type immunoglobulin domain is used to identify the ligand of TREM2, and extracellular domain with a short stalk sequence is used to connect V-type immunoglobulin domain and transmembrane helix[21,22]. The cytoplasmic tail receives external signals from the transmembrane helix, which is anchored on the cell membrane[23,24].

Figure 1 Structure and function of triggering receptor expressed on myeloid cells 2. A: Triggering receptor expressed on myeloid cells (TREM) 2 consists of a signal peptide, an extracellular domain containing a V-type immunoglobulin domain, a short stalk sequence, a transmembrane helix, and a cytoplasmic tail; B: After binding to TREM2, ligands such as high-density lipoprotein, low-density lipoproteins, lipopolysaccharide, apolipoprotein, and other ligands bind to adaptor proteins DNAX-activating proteins (DAP) 12 and DAP10 to form a ligand-receptor complex, which triggers the phosphorylation of the immune receptor tyrosine-based activation motif in the cytoplasmic region of DAP12, recruiting and mediating spleen tyrosine kinase (Syk) phosphorylation, thereby activating downstream signaling pathways such as phosphatidylinositol 3-kinase, mitogen-activated protein kinase, extracellular regulating kinase, and c-Jun N-terminal kinase signaling pathways; C: During the early formation of the TREM2-DAP12 complex, the recruitment of Syk to DAP12 and the activation of downstream signaling can be blocked by Src homology 2-containing inositol-5'-phosphatase 1; D: TREM2 can also be cleaved by a disintegrin and metalloproteinase (ADAM) 10, ADAM17, and γ-secretase to release soluble TREM2. ADAM: A disintegrin and metalloproteinase; APOE: Apolipoprotein E; DAP: DNAX-activating proteins; ERK: Extracellular regulating kinase; HDL: High-density lipoprotein; ITAM: Immune receptor tyrosine-based activation motif; JNK: C-Jun N-terminal kinase; LDL: Low-density lipoproteins; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase; STREM2: Soluble triggering receptor expressed on myeloid cells 2; Syk: Spleen tyrosine kinase; TREM2: Triggering receptor expressed on myeloid cells 2.

Transmembrane receptor TREM2 belongs to the immunoglobulin superfamily that requires binding to extracellular ligands to exert its physiological functions. The ligands of TREM2 include various free and bound anionic molecules on the plasma membrane and several apolipoproteins[25]. Through the positively charged residues in the transmembrane domain, TREM2 binds to ligands to form a receptor signaling complex that attaches to adaptor proteins: Signal transmission is mediated by DNAX-activating proteins (DAP) 12 and DAP10[26]. DAP12, often referred to as TYRO protein tyrosine kinase binding protein, is a signal transduction adaptor protein produced in cells involved in innate immunity responses. It possesses an immune receptor tyrosine-based activation motif (ITAM) structure in the cytosolic region[27]. Upon binding to ligands to form a ligand-receptor complex, TREM2 triggers phosphorylation of the ITAM in the cytosolic region of DAP12, recruiting and mediating phosphorylation of spleen tyrosine kinase (Syk), thereby downstream signaling pathways such phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), extracellular regulating kinase (ERK), and c-Jun N-terminal kinase (JNK) signaling pathways are activated[28]. In this process, Src homology 2-containing inositol-5'-phosphatase 1 (SHIP1) plays a facilitating role in the recruitment of PI3K to the TREM2-DAP12 complex[29]. Syk expression has been discovered to be positively connected with the severity of the disease, causing hepatocyte damage and exacerbating liver inflammation and fibrosis. It has been seen in both parenchymal (hepatocytes) and non-parenchymal (HSCs and KCs) cells in the liver[30]. In the early stage of TREM2-DAP12 complex formation, recruitment of Syk to DAP12 and activation of downstream signaling can be blocked by SHIP1, which contains inositol-5'-phosphatase 1[16]. By directly attaching its Src homology domain to the membrane proximal SPYQEL sequence inside the ITAM structure of DAP12, SHIP1 transforms the activating signaling complex into an inhibitory signaling complex, preventing Syk from signaling downstream and having anti-inflammatory and anti-fibrotic properties[31]. Kim et al[32] have revealed that a disintegrin and metalloproteinase (ADAM) 10, ADAM17, as well as gamma secretase can also cleave TREM2, releasing soluble TREM2 (sTREM2). The sTREM2 has been found in biological samples from people with inflammatory neurological disorders, including AD and amyotrophic lateral sclerosis, in several kinds of studies and the degree of sTREM2 is strongly connected with the severity of the condition[33,34]. Previous studies have shown that ADAM17 and ADAM10 can cleave human TREM2 at the H157-S158 peptide bond, releasing sTREM[35-38]. Experimental studies have shown that the level of sTREM2 dynamically reflects the infiltration of TREM2 + macrophages in the liver and can be used as a circulating biomarker to track the progression of NAFLD[39]. Clinical studies have shown that plasma sTREM2 is a better biomarker than conventional laboratory indicators for identifying distinct phases of NAFLD[40]. It is recommended that plasma sTREM2 be used as a non-invasive diagnostic marker for NAFLD.

TREM2 AND LIVER LIPID METABOLISM

Liver is the primary organ of the lipid metabolism in the human body, and plays a significant part in the metabolic processes of lipids including digestion, absorption, decomposition, synthesis, and transportation. Abnormal accumulation of lipids in liver cells is a pathological characteristic of NAFLD, and TG is the primary type of lipid accumulation in the liver of NAFLD patients[41]. TG deposition in the liver can be attributed to increased production of new fat in the liver, enhanced esterification of free fatty acids (FFA) (glycerol and FFA form TG through esterification), and reduced output of TG from very low density lipoprotein[42]. It has been demonstrated that insulin resistance (IR) is linked to the process of fat accumulation in the liver[43]. IR not only leads to dysfunction in the synthesis and transport of TG in liver cells, but also promotes the mobilization of peripheral fat and increases the concentration of FFA in plasma[44]. Excessive FFA can increase the production of lipotoxic substances in liver cells, which in turn promotes the progression of NAFLD to NASH[45,46]. There is mounting evidence that TREM2 regulates lipid metabolism (Figure 2)[47,48].

Figure 2 Regulatory role of triggering receptor expressed on myeloid cells 2 in liver lipid metabolism. On the one hand, macrophages lacking triggering receptor expressed on myeloid cells (TREM) 2 lead to the accumulation of hypertrophic adipocytes, causing systemic hypercholesterolemia. On the other hand, macrophages lacking TREM2 release exosomes, leading to mitochondrial fragmentation in hepatocytes and fatty acid oxidation dysfunction, accelerating the progression of non-alcoholic fatty liver disease (NAFLD). The deficiency of TREM2 may aggravate insulin resistance (IR) by increasing serum ceramide, leading to hepatic steatosis. The amount of soluble TREM2 in serum and IR are positively correlated. The increase of TREM1 and the decrease of TREM2 are involved in the pathological process of NAFLD. FAO: fatty acid oxidation; IR: insulin resistance; STREM2: Soluble triggering receptor expressed on myeloid cells 2; TREM: Triggering receptor expressed on myeloid cells 2.

Under the influence of metabolic disorders, obesity, and IR, abnormal formation of TG in liver cells results in hepatic steatosis, which is the first hit in the development of NAFLD[49]. One study used a high-fat diet (HFD) to induce a rat NAFLD model. HFD-induced NAFLD model rats exhibited severe metabolic dysfunction and hepatic steatosis, as evidenced by elevated plasma TG and total cholesterol (TC) levels and decreased TREM2 protein expression, in contrast to rats fed normal diet[50]. Knocking out the TREM2 gene in mice can inhibit the recruitment of macrophages to hypertrophic adipocytes and their clearance from hypertrophic adipocytes, leading to the accumulation of hypertrophic adipocytes, causing systemic hypercholesterolemia and inflammation[51]. TREM1 is a transmembrane receptor belonging to the immunoglobulin superfamily, just to TREM2[52,53]. NAFLD is closely associated with both an increase in TREM1 and a decrease in TREM2, and the pathological process of NAFLD involves an imbalance between TREM1 and TREM2[54]. Quercetin is one of the important flavonoids in nature, with high biological activity in vivo, and has the effects of improving lipid metabolism, anti-inflammatory, and anti-tumor[55-58]. According to studies, quercetin can lower plasma TC and TG levels, regulate the protein expression of TREM1 and TREM2, and alleviate HFD-induced NAFLD model rat liver lipid droplet accumulation[50]. However, whether TREM2 has an inhibitory effect on TG synthesis has not been further investigated.

The final product of lipid hydrolysis in the body is fatty acids, which are mainly oxidized and decomposed in mitochondria. TREM2 can protect the structure of mitochondria and ensure the oxidative decomposition of fatty acids[13]. Hepatocyte mitochondria and TREM2-expressing liver macrophages were discovered to have a metabolic coordination using in vivo and in vitro models of liver lipid excess[59]. TREM2-deficient macrophages can release exosomes rich in miR-106b-5p, which blocks downstream mitochondrial fusion protein 2 target molecules, leading to mitochondrial fragmentation in hepatocytes, fatty acid metabolism dysfunction (fatty acid oxidation dysfunction), and accelerated initial progression of NAFLD[60]. On the other hand, overexpression of TREM2 in liver macrophages improves the structure and energy supply of hepatocyte mitochondria, increases lipid catabolism, reduces de novo fatty acid synthesis, and alleviates NAFLD[61,62]. This suggests that TREM2 may protect the structure and energy supply of mitochondria by inhibiting the secretion of macrophage exosomes, ensuring the smooth oxidation of fatty acids, prevent hepatocytes from accumulating too much fatty acids, participating in regulating lipid metabolism, and alleviating NAFLD.

As the initiating factor of the "first hit" in the pathogenesis of NAFLD, IR and the onset of NAFLD are tightly associated. IR is the term used to describe the increased secretion of excessive insulin to induce hyperinsulinemia in order to maintain blood glucose stability when insulin's effectiveness in promoting glucose uptake and utilization is reduced for a variety of reasons[63]. In the state of IR, high concentrations of insulin promote the massive synthesis and storage of fat, inhibit the decomposition and oxidation of fat, and deposit a significant liver fat, which may contribute to the development of NAFLD[64]. Therefore, IR can cause fat to build up in liver cells, which can result in NAFLD liver steatosis[65]. TREM2 expression was considerably lower in obese patients with IR than in obese patients without IR and non-obese patients with IR, according to a clinical investigation involving 27 patients[66]. Serum sTREM2 levels and IR were positively correlated, and waist circumference and fat mass index rose when serum sTREM2 levels rose, according to a survey of 2742 Japanese community members over 40[67]. Under 13 weeks of HFD conditions, TREM2 knockout mice showed more severe IR and liver steatosis compared with wild-type (WT) mice[68]. Further research found that the mechanism by which TREM2 deficiency aggravates IR may be related to the increase in serum ceramide[69]. Ceramide can cause impaired insulin sensitivity, and targeted inhibition of ceramide synthesis can restore insulin tolerance and reverse liver steatosis in TREM2 knockout mice[70,71]. These results imply that TREM2 might be crucial to the IR-induced metabolism of liver lipids.

TREM2 AND CHRONIC INFLAMMATION

Reactive oxygen species (ROS), free radicals, endoplasmic reticulum stress, and other tissue damage can all contribute to liver injury and impaired cell function when there is an excessive buildup of lipids in the liver. Liver tissue injury can also induce immune response, leading to significant accumulation of monocyte-derived macrophages and resident macrophages, increased numbers of neutrophils and natural killer cells, infiltration of DCs, activation of liver inflammatory pathways, hence the transition from benign fatty degeneration to NASH. Therefore, chronic inflammation is an important histological marker of NAFLD.

In WT and TREM2 mice, researchers created models of acute and chronic liver injury. For in vitro tests, they separated primary liver cells from both mouse genotypes[72]. When exposed to acute and recurrent carbon tetrachloride and acetaminophen poisoning, mice deficient in TREM2 show increased levels of liver damage and inflammation, with TREM2 deficiency significantly associated with deteriorating survival. Consistent with its role in inflammation-related injury, when inflammation levels are high, TREM2 can block the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and ROS, while stimulating the production of interferon-β and IL-10, and alleviating excessive immune injury in the liver[72]. According to in vitro research, TREM2 + macrophages can help reduce inflammation following liver injury by blocking the generation of pro-inflammatory reactions brought on by lipoproteins containing lipopolysaccharide[73]. Liver macrophages expressing TREM2 exhibit inhibition of toll like receptor (TLR) 4-driven ERK and MAPK phosphorylation activation, leading to pro-inflammatory responses, as well as lipid peroxidation and cell death caused by ROS[61]. In addition, TREM2 can also inhibit the production of TLR4 dependent monocyte chemoattractant protein-1 in HSCs[16]. In summary, TREM2 has anti-inflammatory properties in chronic liver inflammation. In a multicenter proteomics study, the researchers characterized 4730 circulating proteins in NAFLD 306 patients with histological characteristics, and performed transcriptome analysis of paired liver tissues[74]. In order to identify high-risk steatohepatitis, they ultimately developed a non-invasive diagnostic logistic regression analysis model that included four proteins (TREM2, ADAMTSL2, AKR1B10, CFHR4), body mass index, and type 2 diabetes status. As a result, TREM2 might be crucial in regulating the inflammatory response in NAFLD (Figure 3).

Figure 3 Regulatory role of triggering receptor expressed on myeloid cells 2 in non-alcoholic fatty liver disease-related inflammatory injury. The liver macrophages expressing Triggering receptor expressed on myeloid cells 2 (TREM2) exhibit inhibition of the pro-inflammatory response caused by toll like receptor (TLR) 4-driven extracellular regulating kinase and mitogen-activated protein kinase phosphorylation activation, as well as reactive oxygen species-mediated lipid peroxidation and hepatocyte death. High expression of TREM2 ensures that macrophages can effectively clear apoptotic hepatocytes through efferocytosis in a timely manner, thereby inhibiting the transformation of non-alcoholic fatty liver disease into non-alcoholic steatohepatitis (NASH). TREM2 can induce macrophage differentiation towards M2 macrophages and inhibit inflammatory response. Tumor necrosis factor and interleukin-1β can induce a disintegrin and metalloproteinase 17 to cleave TREM2, leading to an increase in soluble TREM2 levels and aggravation of inflammation in NASH. TREM2 can also inhibit the production of TLR4-dependent monocyte chemoattractant protein-1 in hepatic stellate cells. The TREM2 receptor expressed in dendritic cells inhibits nuclear factor-kappa B through binding with DNAX-activating proteins 12, thereby alleviating liver inflammation. ADAM: A disintegrin and metalloproteinase; DAP: DNAX-activating proteins; STREM2: Soluble triggering receptor expressed on myeloid cells 2; TREM: Triggering receptor expressed on myeloid cells 2.

Although it is unknown if macrophages are friendly or hostile, they do contribute to liver inflammation as NAFLD progresses. The 90% of all macrophages in the human body are liver macrophages, which are extremely diverse and mostly comprise resident and infiltrating macrophages[75]. KCs, or liver-resident macrophages, are frequently seen in the liver's sinusoids. They are already established in the liver tissue throughout embryonic development and are derived from certain progenitor cells that were derived from the yolk sac[76]. In healthy liver tissue, KCs, as the sentinel cells of the liver, dominate the liver macrophages[77]. KCs can timely clean up damaged/dead cells to reduce tissue inflammation microenvironment and maintain liver homeostasis[78]. The three primary types of infiltrating macrophages are splenic, peritoneal, and monocyte-derived macrophages. Of these, the majority of infiltrating macrophages in the liver are monocyte-derived macrophages, which contribute to pathological diseases[79]. When the liver is affected by external factors and develops lesions, activated KCs can recruit a large number of monocytes in the blood to the liver and differentiate into CD11b+F4/80+ macrophages, known as classically activated M1 macrophages, which secrete pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, as well as ROS, accelerating liver tissue injury[80]. These macrophages have the capacity to release a multitude of chemokines simultaneously, such as C-C chemokine ligand (CCL) 2, CCL3, and CCL5, which recruit monocyte macrophages and T lymphocytes in the blood circulation into the liver to exert their innate immunity and initiate adaptive immune regulation[81]. On the other hand, another classic type of liver macrophages, the alternative activated M2 macrophages, are crucial for tissue healing and the inhibition of inflammatory reactions[82]. When stimulated by cytokines such as IL-4 and IL-13, macrophages can polarize towards M2 type, which secretes a large amount of inhibitory inflammatory factors such as transforming growth factor (TGF)-β, IL-10, IL-4, and IL-13, while promoting the activation of arginase to induce the apoptosis of pro-inflammatory M1 type macrophages, accelerating tissue repair and remodeling[83]. Therefore, macrophages may have a dual role in the onset and progression of NAFLD. On the one hand, they may accelerate the progress of NAFLD by releasing inflammatory factors, on the other hand, they may slow down the progression of NAFLD by engulfing dead cells through their phagocytic function. Previous studies have demonstrated that monocyte-derived macrophages infiltration is markedly elevated in NAFLD patients' livers and is implicated in the progression and reversal of NAFLD disease[84]. TREM2 is mostly expressed in liver macrophages generated from monocytes, although it is hardly expressed in liver resident macrophages[85]. NPCs taken from the livers of both healthy and NASH mice were subjected to single-cell RNA sequencing by researchers. A characteristic of both animal and human NASH that is linked to disease severity is the elevated expression of TREM2, which they discovered to be present in NASH-associated macrophages (NAMs)[86]. Research shows that TREM2 can induce macrophage differentiation into M2 macrophages, increase the expression of M2 cytokines, and inhibit the expression of M1 cytokine[87]. This may explain the mechanism of the anti-inflammatory effect of TREM2 + macrophages in NAFLD. A recent study found that TREM2 expressed by macrophages is a key factor in maintaining immune homeostasis in the liver to stop NASH from developing[88]. The study found that high expression of TREM2 ensures that macrophages can effectively clear apoptotic hepatocytes (also known as "efferocytosis") in a timely manner, thereby inhibiting obesity-related NASH lesions and chronic liver inflammation. Defects in TREM2-dependent phagocytosis can lead to chronic liver inflammation and ultimately promote the transformation of NAFLD to NASH[88]. Fredrickson et al[89] used spatial transcriptomics to confirm that vertical sleeve gastrectomy (VSG) improved the metabolic status in the hepatic macrophage microenvironment in mouse and human MASH, and they further revealed by animal tests that TREM2 + macrophages directly mediated the benefits of VSG on MASH by attenuating hepatic inflammation and boosting efferocytosis of lipid-laden apoptotic hepatocytes, which directly mediated the mitigating effect of VSG on MASH. Cao et al[90] used bioinformatics and machine learning algorithms to screen five potential biomarkers for the diagnosis of NASH, including TREM2, which was subsequently validated by single-cell transcriptome and animal experiments to find that TREM2 was up-regulated in NASH, highlighting the great potential of TREM2 in the diagnosis of NASH. Further research found that in NASH mice, TNF and IL-1β caused the activity and expression of ADAM17, which played a role in splicing TREM2, leading in a notable surge in the level of TREM2 hydrolytic product sTREM2[91]. The increase in sTREM2 level means that TREM2 is hydrolyzed extensively, TREM2 cannot effectively exert anti-inflammatory effects, and NASH-related inflammatory reactions are exacerbated, revealing an important mechanism for the reduction of TREM2 in the progression of NAFLD. The hydrolytic product sTREM2 can be used as a non-invasive diagnostic biomarker to accurately exclude or diagnose NASH, reducing liver biopsy. A cross-sectional clinical study confirmed that plasma sTREM2 had good accuracy (area under the receiver operating characteristic curve = 0.92; 95%CI: 0.84-0.99, P < 0.01) for the diagnosis of NASH, with a sensitivity of 100% (95%CI: 0.76-1.0) and specificity of 86% (95%CI: 0.71-0.94)[39]. To further investigate the exclusion and diagnostic threshold of plasma sTREM2 for NASH, researchers collected 218 patients from three hospitals, including 48 patients in experimental group (9 patients with NASH, 18 patients with NAFLD, 11 obese patients without NAFLD, and 10 healthy controls), and 170 patients in validation group (all patients were confirmed to have increased liver stiffness by liver biopsy). The study ultimately determined that plasma sTREM2 levels ≤ 38 ng/mL were the optimal threshold for excluding NASH (sensitivity 90% and specificity 52%), and plasma sTREM2 levels ≥ 65 ng/mL were the optimal threshold for diagnosing NASH (specificity 89% and sensitivity 54%). As a non-invasive diagnostic biomarker, plasma sTREM2 can accurately exclude or diagnose NASH, reducing liver biopsy. Blocking macrophage TREM2 cleavage to restore its ability to clear apoptotic liver cells may be an effective method for preventing and treating NAFLD.

Currently acknowledged as the most efficient antigen presenting cells, DCs contribute significantly to the body's innate immune response, initiate adaptive immune responses, shape the type of immune response, and aid in the exertion of immune effects[92,93]. Signals in the liver microenvironment and the lipid content of DC cells can cause them to change from a tolerogenic to an immunogenic phenotype[94]. It is still unclear how DCs participate in the inflammatory process of NAFLD, as conflicting results have emerged from limited studies. Henning et al[95] have shown that DCs may minimize the inflammatory reaction of NASH by removing apoptotic bodies and necrotic material from the liver. According to certain research, liver DCs contribute to the development of illness in models of thioacetamide-induced liver fibrosis or major cellular (MCD)-induced NASH[96,97]. The immune regulatory ability of DCs is related to pattern recognition and immune regulation of DC surface receptors. DCs' expression of the TREM2 receptor contributes to reducing inflammatory reactions in the body by specifically pairing with DAP12 Ligands[98,99]. Research shows that in DCs with acute liver injury, TREM2 inhibits nuclear factor-kappa B (NF-κB) by binding with DAP12, thereby reducing liver inflammation[100].

TREM2 AND LIVER FIBROSIS

The primary cells in the liver that produce collagen are HSCs, which are quiescent, non-proliferative peri-sinusoidal cells that are situated between hepatocytes and sinusoidal endothelial cells in a healthy liver[87]. In the state of chronic liver injury, HSCs are in a sustained activated state, which disrupts the balance between the dissolution and deposition of extracellular matrix (ECM) in liver cells, resulting in progressive liver fibrosis. Liver fibrosis is one of the main factors contributing to the higher death rate from NAFLD[101-103]. If not intervened in time, liver fibrosis will eventually develop into liver cirrhosis and even HCC, and a series of serious issues like portal hypertension and liver failure, hepatic encephalopathy, etc. will occur, which seriously threatens the health of patients[104,105]. As an important immune cell in the liver, macrophages can regulate NAFLD-related liver fibrosis[106]. The study found that eliminating liver macrophages in mice might prevent liver fibrosis from developing in the early stages of fibrosis formation, while in the recovery stage of fibrosis, it increased the deposition of intrahepatic fibrosis and prevented the regression of liver fibrosis[107]. Why do these different effects occur? The reason is that in different stages of liver fibrosis, different subtypes of liver macrophages affect the appearance and regression of liver fibrosis pathology through different mechanisms. HSCs are the primary mediators of liver fibrosis, while the conversion of quiescent HSCs into myofibroblasts is the core of the pathogenesis of liver fibrosis. Macrophages regulate the activation of HSCs in a crucial way[108,109]. During the initial and progressive stages of NASH progression to liver fibrosis, injury-related pattern molecules and apoptotic bodies activate macrophages in the Disse space, causing their phenotype to shift toward M1 macrophages[110]. Activated macrophages can produce pro-fibrotic factors which include TGF-β and platelet-derived growth factor (PDGF), stimulate HSC, and promote their transformation into myofibroblasts, thus boosting the occurrence of liver fibrosis[111]. In addition, macrophages may result in inflammatory elements including TNFγ and IL-1β, which recruit extrahepatic inflammatory cells into the liver, further aggravating liver cell injury[112]. At the same time, inflammatory factors can activate HSCs via the pathway of NF-κB, maintain HSCs activity, and promote the development of liver fibrosis[113]. During the regression of liver fibrosis, the phenotype of liver macrophages is dominated by M2 macrophages, and they may have two ways of promoting anti-fibrosis: (1) Macrophages recruit and activate monocyte-derived macrophages and natural killer cells, leading to the apoptosis of activated HSCs, thereby exerting an anti-fibrotic effect[114]; and (2) Macrophages can phagocytize damaged hepatocytes, reduce inflammation, thus slowing the development of liver fibrosis[115].

The role of TREM2 in NAFLD-related liver fibrosis is unclear, as different studies have provided conflicting experimental results (Figure 4). According to research, male C57Bl6/J mice were given bone marrow from their littermates that were TREM2+/+ (TREM2+/+-tp, grey) and TREM2-/- (TREM2-/--tp, green) after being lethally irradiated. When mice were put on an MCD diet for four weeks following six weeks of recuperation, TREM2-/--bone marrow-chimeric animals displayed more pronounced fibrosis, indicating that TREM2 on macrophages has a protective function in NAFLD liver fibrosis. Further research showed that TREM2 could promote the reduction of mitochondrial membrane potential (MMP) 12 and increase the expression of tissue inhibitors of metalloproteinase 1, thereby exerting an anti-fibrotic effect[91]. Additionally, in vitro studies have demonstrated that TREM2 Loss can result in abnormalities in ECM remodeling and lipid processing, which can cause steatohepatitis and cell death, exacerbate fibrosis[91]. In NAFLD, plasma sTREM2 levels can be utilized as a biomarker to track the development of liver fibrosis. During the stages of liver fibrosis F0-F2, plasma sTREM2 levels increase proportionally with fibrosis stage, but no gradual increase was observed during the stages of fibrosis F3 and F4, indicating that plasma sTREM2 has a low ability to differentiate advanced liver fibrosis[40]. The increase in plasma sTREM2 means that TREM2 is cleaved. Does this imply that in the early phases of the development of liver fibrosis, TREM2 is cleaved extensively during the transition of macrophages to M1 macrophages, which in turn leads to the promotion of liver fibrosis by macrophages? In the late stages of liver fibrosis, the gradual increase in plasma sTREM2 does not mean that TREM2 cleavage is inhibited during the transition of macrophages to M2 macrophages, which in turn plays a role in inhibiting liver fibrosis?

Figure 4 Bidirectional regulation of triggering receptor expressed on myeloid cells 2 in non-alcoholic fatty liver disease-related liver fibrosis. Triggering receptor expressed on myeloid cells 2 (TREM2) can promote the reduction of mitochondrial membrane potential (MMP) 12 and increase the expression of tissue inhibitors of metalloproteinase 1, thereby exerting an anti-fibrotic effect. The level of plasma soluble TREM2 can be used as a biomarker to monitor the progression of liver fibrosis in non-alcoholic fatty liver disease. Conversely, studies have shown that CD9+TREM2+ non-alcoholic steatohepatitis-associated macrophages or scar-associated macrophages accumulate in the liver and promote liver fibrosis by enhancing the expression of MMP12, MMP13, and MMP14. MMP: Mitochondrial membrane potential; SAM: S-adenosyl methionine; STREM2: Soluble triggering receptor expressed on myeloid cells 2; TIMP1: Tissue inhibitors of metalloproteinase 1.

Other studies have revealed the role of TREM2 in promoting the formation of liver fibrosis. The researchers identified a TREM2+CD9+ macrophage subset that shares characteristics of both monocytes and KCs. They called this cell type CD9+TREM2+ NAMs or scar-associated macrophages. This cell type is amplified in fibrotic livers in humans and has a fibrogenic role[116]. TREM2 + macrophages build up in the liver as diet-induced NASH and drug-induced cirrhosis advance, and TREM2 can exacerbate liver fibrosis by upregulating the production of MMP12, MMP13, and MMP14[117]. Triiodothyronine (T3) can inhibit pro-fibrotic TREM2+CD9+ macrophages and participate in the inhibition of liver fibrosis progression[118]. A traditional Chinese medicine formula consisting of five herbs including Scutellaria, Sophora flavescens, Saposhnikovia divaricata, Citrus reticulata, and Panax quinquefolium can effectively inhibit the expansion of TREM2+CD9+ macrophage subsets in damaged livers, and remold the fibrotic niche by regulating ligand-receptor interactions (including TGF-β/epidermal growth factor receptor, PDGF-β/PDGFRα, and TNF superfamily 12/TNF receptor superfamily 12A signaling) to inhibit the progression of liver fibrosis[119]. Interestingly, in the experimental model of HCC in a fibrogenic environment, TREM2 promoted the incidence of fibrosis in liver, but interfered with the occurrence of liver tumors[17].

TREM2 AND HCC

Since the first report of the correlation between NAFLD and HCC in 1990[120], the incidence of NAFLD-related HCC has shown a significant upward trend, and in patients with NASH, HCC can occur directly without going through the cirrhosis stage[121-123]. Among NASH patients with cirrhosis, the annual incidence of HCC is 0.5%-2.6%, while the annual incidence of HCC in patients with non-cirrhosis NAFLD or NASH is 0.01%-0.13%[124]. The incidence of HCC is higher in patients with higher NASH degree or higher fibrosis grade[125]. In the presence of NAFLD/NASH, due to the effects of metabolism, oxidative stress, inflammation, immune response, and fibrosis, sustained destruction and compensatory proliferation of hepatocytes create an environment conducive to carcinogenesis[126]. Therefore, it is critical to treat NAFLD/NASH by comprehending the process by which it progresses to liver cancer.

In the acute diethylnitrosamine (DEN) model, TREM2 knockout mice display hepatocyte injury, inflammation, elevated oxidative stress, and increased liver tumor number. Studies conducted in vitro have demonstrated that the formation of human HCC spheroids was inhibited by conditioned media derived from human HSCs overexpressing TREM2[17], suggesting that TREM2 can protect the liver from HCC. Similarly, the researchers observed that TREM2 could also inhibit the growth of liver tumors in fibrosis-related HCC. The researchers observed in HSCs that overexpression of TREM2 could regulate the secretion of Wnt ligands and reduced the tumorigenicity of HCC. Another study showed that overexpression of TREM2 inhibited the progression of HCC by regulating the PI3K/protein kinase B (AKT)/β-catenin pathway[127]. In addition, during the development of advanced HCC, TREM2 in KCs and tumor-associated macrophages (TAMs) may counteract the initiation of liver tumors in HSCs by preventing TLR1 receptor-dependent responses[128]. According to the aforementioned studies, TREM2 protects against liver cancer development via a variety of mechanisms (Figure 5).

Figure 5 Bidirectional regulatory role of triggering receptor expressed on myeloid cells 2 in Non-alcoholic fatty liver disease-related hepatocellular carcinoma. Overexpressed triggering receptor expressed on myeloid cells 2 (TREM2) in hepatic stellate cells can regulate the secretion of Wnt ligands and protect the liver from hepatocellular carcinoma (HCC). Overexpressed TREM2 in HCC cells can inhibit HCC progression by regulating the phosphatidylinositol 3-kinase/protein kinase B/β-catenin pathway. During the development of end-stage HCC, TREM2 in Kupffer cells and tumor-associated macrophages may inhibit the occurrence of liver tumors by preventing toll like receptor-1 receptor-dependent responses. Another study revealed that TREM2 is a carcinogenic gene in the development of HCC. AKT: Protein kinase B; GSK3β: Glycogen synthase kinase-3β; HCC: Hepatocellular carcinoma; PI3K: Phosphatidylinositol 3-kinase; TAMs: Tumor-associated macrophages; TLR: Toll like receptor; TREM2: Triggering receptor expressed on myeloid cells 2.

However, some studies have revealed that TREM2 is a carcinogenic gene during the development of HCC[129]. The researchers used in situ injection of Hepa1-6 cells and spontaneous HCC based on NASH to construct two HCC models. In both HCC models, tumor growth in TREM2-WT mice was progressive, while tumor growth in TREM2-KO mice was considerably diminished. Knocking down TREM2 reconstitutes TAMs into an immune stimulatory state and enhances programmed death-1 immune checkpoint blockade's therapeutic impact in HCC[130]. Echinacoside (ECH), a phenylacetyl glucoside extracted from the medicinal herb Cistanche deserticola, significantly reduces the TREM2 protein levels in HepG2 cells and DEN-induced hepatoma cells[131]. Moreover, overexpression of TREM2 significantly reduced ECH-mediated proliferation inhibition and inactivation of the AKT signaling pathway, indicating that ECH exerts its anti-tumor activity by reducing TREM2 expression and the PI3K/AKT signaling pathway.

INTERACTION OF TREM2 WITH OTHER MOLECULAR PATHWAYS

In NAFLD

Ganguly et al[132] showed by bioinformatics analysis that in NASH-related macrophages, pathways like phagocytosis, anti-fibrotic, and enhanced lipid/lipoprotein handling were enriched in genes positively associated with elevated TREM2 expression. Among the lipid and lipoprotein handling-related pathways are involved in lipid metabolism, plasma lipoprotein assembly, remodeling, and clearance, glycosphingolipid metabolism, sphingolipid metabolism, low-density lipoproteins clearance, and the metabolism of fat-soluble vitamins. Phagocytosis pathway is involved in lysosomal vesicle biogenesis and production of ROS and reactive nitrogen species in phagocytes. The anti-fibrotic pathway involves the degradation of collagen and ECM, down-regulation of TGF-β signaling, and down-regulation of SMAD 3/4 activity. In addition, low TREM2 expression in NAMs was concentrated in metapathways associated with cell death, increased cytokine signaling, and inflammation. Among them, the inflammatory pathway involves TLR4 signaling, TLR9 signaling, co-stimulation of CD28 molecules, NF-κB activation, C-type lectin receptor, ERK signaling, and nucleotide-binding oligomerization domain (NOD) 1 and NOD 2 signaling. Enhanced cytokine signaling involves IL-1 signaling, IL signaling, TNF signaling pathways, and cytokine signaling in the immune system. Cell death pathways involve pyroptosis, apoptosis, and regulated necrosis. Yu et al[133] demonstrated that TREM2 deficiency increased inflammation and hepatic steatosis in mouse models. Through additional research, they have confirmed that TREM2 controls macrophage pyroptosis via the PI3K/AKT signaling pathway and is necessary for the macrophages' anti-inflammatory phenotypic transition. Furthermore, they have found that TREM2 additionally regulates the inflammatory reaction by affecting the expression of TGF-β1. These results imply that TREM2 is able to prevent or delay NAFLD progression by acting in correlation with other molecular pathways (Figure 6).

Figure 6 Interaction of triggering receptor expressed on myeloid cells 2 with other molecular pathways in non-alcoholic fatty liver disease. Triggering receptor expressed on myeloid cells 2 (TREM2) high-expressing macrophages have phagocytosic and anti-fibrotic functions, and improve lipid/lipoprotein processing. TREM2 Low-expressing macrophages are enriched in metapathways associated with inflammation, enhanced cytokine signaling, and cell death. AKT: Protein kinase B; ERK: Extracellular regulating kinase; IL: Interleukin; NF-κB: Nuclear factor-kappa B; NOD: Nucleotide-binding oligomerization domain; PI3K: Phosphatidylinositol 3-kinase; ROS: Reactive oxygen species; TGF: Transforming growth factor; TLR: Toll like receptor; TNF: Tumor necrosis factor.

CONCLUSION

Although previous studies have focused on elucidating the role of TREM2 in neurodegenerative diseases such as AD, researchers have begun to pay attention to its possible contribution to NAFLD.

In summary, TREM2 is involved in every facet of the pathophysiology of NAFLD. Currently, the mainstream view is that TREM2 has a protective effect on liver lipid metabolism, inflammation, liver fibrosis, and HCC. Nevertheless, we should also note that there is still some controversy over whether TREM2 plays a protective or harmful role in different animal models of liver fibrosis and HCC. Therefore, further basic research on the TREM2 pathway in liver fibrosis and HCC models during the natural course of NAFLD is needed. Due to its important regulatory role in disease, research on targeted TREM2 treatment has become a hot topic. At present, monoclonal antibodies against TREM2 that target the microglial inflammatory response have been applied in clinical phase I trials in AD patients[134,135], but potential therapeutic strategies for NAFLD are still being explored. In addition, TREM2 may benefit NAFLD progression through interactions with other molecular pathways. However, these summaries were mainly from correlation analyses through bioinformatics and were rarely validated experimentally. The mechanism by which TREM2 interacts with other molecular pathways to participate in the remission of NAFLD progression need to be explored through more studies in the future. Although some research teams have validated the possibility of sTREM2 as a diagnostic biomarker for NAFLD/NASH in the clinic, there is a lack of validation with multicenter and large-sample clinical data. In the future, there is a need to develop easy-to-operate and lower-cost sTREM2 detection kits so that they can be widely applied in clinical practice.