Roles of hepatic immunity in metabolic dysfunction-associated steatotic liver disease: Cellular and molecular mechanisms and clinical trials

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common type of chronic liver disease, encompassing a broad spectrum of pathology ranging from hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH). Characterized by hepatic inflammation, cell death, and different severities of fibrosis, MASLD can lead to liver cirrhosis and hepatocellular carcinoma (HCC). Although over 100 million people are affected in the United States, effective treatment remains limited, including the only United States Food and Drug Administration-approved resmetirom targeting thyroid hormone receptor-β, which failed to prevent MASLD progression to HCC based on clinic studies. It is necessary and urgent to develop new therapies by advancing the understanding of molecular mechanisms underlying MASLD. Hepatic innate immune cells play an essential role in maintaining liver physiologic homeostasis, as well as actively contributing to MASLD pathogenesis and progression by interacting with liver parenchymal cells and adaptive immune cells in the progression of MASLD and MASH. In this review, we summarize current knowledge about the function of various residential and infiltration innate immune cells in the pathogenesis of MASLD and discuss the molecular mechanisms by which they contribute to liver inflammation, metabolic dysregulation, and fibrogenesis. Additionally, we recapitulate current clinical trials focusing on targeted innate immune cell manipulation and metabolic modulation as therapeutic strategies for MASLD.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Metabolic dysfunction-associated steatohepatitis; Innate immune cells; Molecular mechanisms; Clinical trials; Drugs

Core Tip: Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a broad spectrum of pathology ranging from hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH). Effective treatments for MASLD remain limited. Hepatic innate immune cells play an essential role in maintaining liver physiological homeostasis and contributing to MASLD pathogenesis and progression by interacting with liver parenchymal cells and adaptive immune cells in the progression of MASLD and MASH. Treatments targeting innate immune cell manipulation and metabolic modulation, such as fibroblast growth factor 21 analogues, farnesoid X receptor agonists, and chemokine receptor antagonists, can provide therapeutic strategies for MASLD.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common type of chronic liver disease and is closely associated with obesity and type 2 diabetes (T2D)[1,2]. Its progressive form is metabolic dysfunction-associated steatohepatitis (MASH), characterized by advanced liver inflammation and cell death, as well as varying degrees of fibrosis[3,4]. Both MASLD and MASH can lead to liver cirrhosis and hepatocellular carcinoma (HCC). However, treatment options for MASLD or MASH remain very limited. Resmetirom (brand name rezdiffra), an oral thyroid hormone receptor-β (THR-β) agonist, is currently the only drug approved by the United States Food and Drug Administration for the treatment of adults with noncirrhotic MASH[5]. Currently, there is no sufficient research evidence that supports the beneficial effects of resmetirom on the prevention of advanced cirrhosis and HCC[6]. However, MASLD impacts the efficacy of immunotherapy in patients. Cohort studies have shown that patients with MASH-associated HCC who received treatments of anti-programmed death-1 (PD-1) or its ligand had decreased overall survival compared to patients with HCC caused by other etiologies[7]. Preclinical studies suggest that the failure of immunotherapy in MASH-associated HCC may be due to the expansion of PD-1⁺ cluster of differentiation (CD) 8⁺ T cells within tumors[7]. Therefore, effective therapeutic drugs or strategies are imperative to prevent the onset and/or progression of MASLD and MASH.

In the liver, innate immune cells primarily consist of dendritic cells (DCs), monocyte-derived macrophages (MDMs), liver-resident macrophages [Kupffer cells (KCs)], neutrophils, natural killer (NK) cells, NK T (NKT) cells, mucosal-associated invariant T (MAIT) cells, and innate lymphoid cells (ILCs). Hepatic innate immune cells comprise a large proportion of hepatic non-parenchymal cells and reciprocally communicate with injured hepatocytes to mediate the progression of liver inflammation and fibrosis during liver injury[8]. Both injured hepatocytes and activated innate immune cells (e.g., KCs) can induce the activation and proliferation of hepatic stellate cells (HSCs) and promote the recruitment of adaptive immune cells, thereby advancing liver fibrosis and cirrhosis.

Studies have used single-cell RNA sequencing (scRNA-seq) to unveil novel innate immune cell functions and effects on hepatocytes in the environment of MASH liver. For example, MASH-associated macrophages exhibit higher expression levels of triggering receptors expressed on myeloid cells 2 (Trem2) compared to KCs[9]. Another study demonstrates that lipid droplets released from damaged hepatocytes in MASH livers can substantially induce Trem2-expressing macrophages to exacerbate MASH[10]. As the most metabolically active organ in the human body, the functions of hepatic innate immune cells contribute to and are also influenced by both intrahepatic and extrahepatic factors. The activation and differentiation of innate immune cells happen in the development and progression of MASLD. In summary, these studies emphasized the crucial role of innate immune cells as a communication bridge among hepatocytes, adaptive immune cells, and other hepatic non-parenchymal cells. Nonetheless, we aim to systematically review the roles of innate immunity in MASLD, discuss key molecular signaling pathways involved in innate immune cell activation, and summarize the current therapeutic options in clinical trials that target innate immunity for the treatment of MASLD.

INNATE IMMUNE CELLS ARE CRITICAL IN THE PATHOGENESIS OF MASLD AND MASH

Among the complex, intertwined processes involved in the progression of MASLD, hepatic lobular inflammation represents a key driver of fibrosis and the transition from modest steatosis to steatohepatitis[11]. Continuous chronic inflammation exacerbates MASLD development and the associated metabolic-related stress, resulting in activation of the innate immune system. In this section, we discuss the roles of innate cells and their interactions with parenchymal cells during the development and progression of MASLD.

DCs

Although elevated frequencies of conventional DCs (cDCs) and plasmacytoid DCs in the liver are the hallmarks of MASH, the role of DCs in the pathogenesis and pathophysiology of MASH is dichotomous[12,13]. Over a decade ago, Henning et al[14] reported that intrahepatic DCs expand, mature, and assume an activated phenotype in MASH[14]. Depletion of DCs using diphtheria toxin injection in CD11c-DTR mice exacerbated intrahepatic inflammation, apoptosis, and fibrosis rather than assuaging the pathological phenotype of MASH, suggesting that DCs play a protective role in MASH[14]. In another study, the CD103⁺ cDC1 subset was identified as a protective DC subtype in a murine model, and adoptive transfer of CD103⁺ cDC1s to Batf3-deficient animals attenuated liver damage and inflammation[15]. On the contrary, single-cell transcriptomics analysis revealed that X-C motif chemokine receptor 1⁺ cDC1-T cell pairs in liver-draining lymph nodes showed that cDCs influence inflammatory T cell reprogramming and exacerbate MASH development. However, depletion of cDC1 in XCR1^DTA mice abolished liver pathology[16]. These findings suggest that distinct DC populations within this heterogeneous population may play discrete roles in MASH development.

Macrophages

In MASLD or MASH livers, infiltrated and liver-resident macrophages, including MDMs, KCs, and other macrophages, are a focal point in this field due to the supposed specific communication and functionality between cells. Moreover, macrophages are a heterogeneous population, with distinct transcriptional profiles and functions. For example, hepatic macrophages play different roles in HSC activation and collagen degradation[17]. Modern technologies, including scRNA-seq and spatial proteomics, have provided deep interrogation of hepatic macrophages in both diseased and healthy livers and have shifted the paradigm of macrophage function, which was previously implicated as a driver in the progression of steatosis toward end-stage disease, to being heterogeneous with a multitude of phenotypes, activation states, and functions. This new principle that resident and recruited cells display immense heterogeneity in MASLD and MASH could lead to viable therapy strategies as these complex populations are unraveled.

Clustering analysis from scRNA-seq data shows expansion of KCs and MDMs in diet-induced MASH liver, both of which exhibit a shift towards a proinflammatory phenotype, contributing to a proinflammatory environment in MASH liver[9]. Transcriptomic profiling of liver macrophages shows that inflammatory markers are significantly upregulated in MDMs compared to KCs in mice fed with a high-fat diet (HFD)[18]. This suggests that the lineage of macrophages in the liver may define their role in the development of MASH. For instance, Tran et al[19] observed that while MDMs maintained a high inflammatory status, whereas the self-renewing ability of KCs was impaired. Using clodronate liposomes or gadolinium chloride, depletion of liver macrophages inhibits hepatic steatosis and MASH progression[20,21]. Thus, the ability of macrophages to promote hepatic steatosis and ultimately MASH development highly depends on the recruitment of highly inflammatory MDMs at different stages of MASH development.

While subsets of macrophages and their high inflammatory status in the liver may advance the development of MASH, their activities are under tight regulation by a variety of proteins. MER proto-oncogene tyrosine kinase (MerTK) is a Tyro-Axl-MerTK family protein that is highly upregulated in macrophages. MERTK signaling in macrophages has been shown to induce the expression of transforming growth factor beta 1 (TGF-β1) via extracellular signal-regulated kinase (ERK) 1/2 activation and the subsequent activation of HSCs, leading to the progression of fibrosis[22]. Ablation of MERTK signaling by disintegrin and metalloproteinase domain-containing protein 17 decreased MASH progression[23]. Further, the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multimeric protein complex critical to caspase-1-dependent interleukin (IL)-1β secretion[24]. NLRP3 is richly expressed in MDMs, KCs, and other immune cells. Studies have shown that hepatic expression of NLRP3 increases in MASLD mice[25,26]. All components of the NLRP3 inflammasome are reportedly upregulated in macrophages isolated from MASH liver. In NLRP3-deficient mice, protection against HFD-induced fatty liver disease was observed[27], and treatment with NLRP3 inhibitors, such as obeticholic acid, improved MASH pathology[26,28]. Other molecules, such as pyruvate kinase M2[29], complement factor C5a receptor 1[30], yes-associated protein[31], and receptor for advanced glycation end products[32], have been shown to promote macrophage inflammatory status and ultimately advance MASH development. Ablation of these signaling molecules showed protective outcomes. In summary, targeting components of pervasive inflammasomes in liver macrophages may represent important therapeutic strategies in the treatment of MASH disease.

Recent studies have characterized MDMs to account for their heterogeneous roles in innate and adaptive immunity in both infiltrating and resident cells. Specifically, classically activated macrophages or M1 macrophages, which result from stimulation by type 1 stimuli, exhibit inflammatory phenotypes; whilst their counterparts, known as alternatively activated macrophages (AAMs) or M2 macrophages, which were induced by type 2 cytokines such as IL-4 and IL-13, are crucial for phagocytosis and anti-inflammatory function in the liver. Studies have demonstrated that AAMs can promote tissue repair through TGF-β and other factors, fibrosis restoration, and general protection against liver injury[33,34]. Interplays and counterbalance between M1 and M2 macrophages through cell-cell interaction or mediators such as IL-10 directly influence the overall liver homeostasis and the pathogenesis of MASLD. In an acute liver injury mouse model, Starkey et al[35] showed that AAMs exert positive effects in reversing the inflammatory environment of the injury via means of resolution and repair. Furthermore, this study observed a 60% reduction in necrotic liver area and an 8.4-fold increase in bromodeoxyuridine levels, indicative of liver regeneration following injections of AAMs[35]. This phenotype of macrophages is multi-faceted in its ability to reduce inflammatory cytokines [e.g., interferon-γ (IFN-γ) and IL-6], reduce necrotic liver tissue, and stimulate hepatocyte and endothelial cell proliferation[33,34]. During MASH, hepatocyte death is the primary trigger that activates the innate immune system, releasing damage-associated molecular patterns and proinflammatory cytokines and accelerating fibrosis, thus worsening disease severity. AAMs are formidable in the potential of reversing MASLD before MASH or end-stage disease; their highly phagocytic phenotype has been observed to effectively clear necrotic material, reduce pro-inflammatory cytokines, and aid in the proliferation of the major cell populations of the liver[34].

Neutrophils

Neutrophil infiltration is a typical histological feature of MASH in animals and humans[36]. Chemokines such as C-X-C motif chemokine ligand 1 (CXCL1) play a crucial role in neutrophil infiltration. In patients with MASH, CXCL1 was highly elevated but not in obese individuals or HFD-fed mice. Hwang and colleagues demonstrated that ectopic expression of Cxcl1 alone in the liver could sufficiently drive the progression from simple steatosis to MASH in mice with a HFD via induction of neutrophil infiltration, neutrophil-derived reactive oxygen species (ROS) production, and stress kinase activation[37]. Knockout of neutrophil cytosolic factor 1 gene encoding a component of reduced nicotinamide adenine dinucleotide phosphate oxidase 2 involved in neutrophil oxidative burst significantly diminished CXCL1-induced MASH development and stress kinase activation in HFD-fed mice[37]. This study highlights that MASH liver has high neutrophil infiltration, and neutrophils contribute to MASH development by inciting redox imbalance in the liver. Mice with the deficiency of breast regression protein 39, a homolog of human YKL-40 (also known as chitinase 3-like 1), suppressed NLRP3 activation-induced liver inflammation and fibrosis by inhibiting the infiltration of Ly6C⁺OPN⁺ (osteopontin) lipid-associated macrophages (LAMs), as well as Ly6G⁺H3Cit⁺ (citrullinated histone H3 positive) neutrophils[38].

Neutrophil extracellular trap (NET) accumulation has been observed in MASH conditions, and these NET reservoirs may indeed be promoted via MASH-specific mechanisms[39]. In MASH liver, transcriptomic profiling revealed that NETs impacted gene expression profile in naïve CD4⁺ T cells, which metabolically reprogrammed these T cells to differentiate to regulatory T cells, all occurring in the presence of Toll-like receptor (TLR) 4[40]. This study robustly supports the ability of neutrophils to shape adaptive immune response during MASH progression. Notably, neutrophil count is crucial for the computation of systemic immune-inflammation index (SII). Specifically, (platelet count × neutrophil count/Lymphocyte count) has been used to compute SII. In a cross-sectional investigation involving 10505 participants (5937 of whom were diagnosed with hepatic steatosis), multivariable logistic regression showed that high SII level was an independent risk factor for hepatic steatohepatitis[41]. Therefore, SII may represent an affordable way to identify hepatic steatosis, although this needs to be validated by additional studies. On the other hand, neutrophil-specific microRNA production enriched in lipotoxic hepatocytes has been shown to hamper MASH progression via low-density lipoprotein receptor (LDLR)-dependent clearance of miR-223-enriched apolipoprotein E (APOE)-expressing extracellular vesicles (EVs), absence of LDLR and APOE-dependent uptake of miR-223-rich EVs amplify MASH progression[42]. This study suggests that neutrophils may play a beneficial role in MASH; however, this advantageous type of functional role is understudied. Overall, evidence from several studies has shown that neutrophil infiltration can contribute to the progression of MASH.

NK cells

The role of NK cells in MASH is complex and environment-dependent. In mice fed with a methionine- and choline-deficient (MCD) diet, MASH development was associated with high expression of NK cell activation markers, NK group 2D and CD107a (lysosomal-associated membrane protein 1 or LAMP-1), while NK cell-deficient Nfil3^-/- mice reversed diet-induced MASH development[43]. Further, NK cells isolated from MASH livers have been reported to secrete high levels of pro-inflammatory cytokines, including IFN-γ, IL-1β, IL-12, C-C motif chemokine ligand 4 (CCL4), CCL5, and granulocyte-macrophage colony-stimulating factor (GM-CSF). These cytokines could activate the hepatic Janus kinase (JAK)/signal transducers and activators of transcription (STAT)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) shunt and induce hepatic damage[43]. This study illustrated the damaging effects of NK cells during MASH progression via the secretion of pro-inflammatory cytokines. Contrary to these findings, other reports have shown that NK cells may play a protective role in MCD diet-induced MASH in mice. Specifically, MCD-induced MASH liver exhibited upregulated expression of CXCL10, resulting in the recruitment of C-X-C motif chemokine receptor 3 (CXCR3)⁺ NK cells. NK cells recruited via the CXCL10-CXCR3 interaction inhibited hepatic inflammation but not steatohepatitis compared to NK cell-deficient Nfil3^-/- mice[44]. Thus, tissue-specific characteristics may impact NK functions and their protective or damaging contribution to MASH. In the adipocytes of mice fed with a HFD, NK cell receptor 1 (NKp46)-activating ligands were upregulated on the adipocytes, leading to increased release of IFN-γ in NK cells. IFN-γ release influenced the infiltration and activation of macrophages. This NK cell-macrophage crosstalk is important to maintain an inflammatory status in MASH[45]. Overall, the role of NK cells in MASH development is paradoxical as it has both protective antifibrotic and deleterious proinflammatory effects during MASH progression.

NKT cells

Like NK cells, NKT cells function as innate immune cells, as they can be rapidly activated to secrete a variety of cytokines such as IL-2, IL-10, and IFN-γ to regulate both innate and adaptive immune cells[46]. Moreover, NKT cells share important features of other T cells, including expression of a semi-invariant T cell receptor (TCR) in type I NKT cells (type II or variant NKT cells lack expression of the invariant TCR-alpha chain) that interacts with CD1d-bound glycolipid antigens, and expression of T cell markers such as CD25, CD44, and CD69[47]. NKT cells showed a protective effect in male mice with MASH induced by a choline-deficient HFD, such that male CD1d^-/- Balb/c mice displayed severe liver inflammation and fibrosis compared to wild-type Balb/c mice. However, this protective effect was absent in female mice counterparts, perhaps caused by the low number of hepatic type I NKT cells in female mice compared to male mice[48]. Another study showed that Akkermansia muciniphila (AM06) isolated from breast milk can ameliorate the severity of MASH and suppress MASH-associated HCC progression by increasing the infiltration of liver CXCR6-expressing NKT cells and reducing macrophage recruitment[49]. The role of NKT cells in MASH is also contradictory. For example, it has been demonstrated that NKT cells in MCD-induced MASH liver increased the secretion of osteopontin to promote liver fibrosis[50], whereas in another study, type I or invariant NKT (iNKT)-deficient Jα18^-/- gave rise to less severe hepatic steatosis and fibrosis following a choline-deficient L-amino acid-defined diet[51]. In addition, the number IL-17A-expressing iNKT cells was increased in peripheral blood mononuclear cells from patients with MASH compared to that in patients with MASLD or healthy controls[51]. These studies suggest that the subtypes of liver NKT cells play different roles in MASLD.

MAIT cells

MAIT cells are innate-like T cells in the liver and are known for their dual roles in tissue repair, inhibition of infection, and resolution of fibrosis under inflammatory conditions. The frequency of MAIT cells (TCRVa7.2⁺ CD161^high CD3⁺ T cells) in circulating blood in patients with MASH decreased compared to that in the group with steatosis or healthy controls[52]. Activation (CD69) and exhaustion (PD-1) markers were highly expressed on MAIT cells in MASLD patients compared to control groups. In addition, these cells highly expressed CXCR6 with a high tendency to migrate into MASLD liver upon injury. Functional study showed that activated MAIT cells could induce macrophage polarization to an M2-like phenotype by producing cytokines such as IL-4[53]. In contrast, mice deficient in MAIT cells under a MCD displayed severe hepatic inflammation and steatosis with an increased M1 macrophage polarization and a decreased M2 macrophage polarization in the livers[53]. Vα19 mice with high numbers of MAIT cells displayed low levels of serum triglyceride and non-esterified fatty acids compared to wild-type C57BL/6 mice after feeding with a HFD, which may be associated with a reduction of lipid accumulation in the liver or hepatic steatosis[54]. A recent study showed that MASLD-associated polyunsaturated fatty acids can selectively suppress the function of MAIT cells but not CD8⁺ T and NK cells to reduce MAIT cell expression of anti-tumor cytokines, such as tumor necrosis factor (TNF)-α, IFN-γ, and granzyme B[55]. The molecular mechanism of MAIT cells in fatty liver remains elusive, yet their dual roles in MASH pathogenesis underpin their potential for regulation within the tissue and their communication abilities to infiltrated innate immune cells.

ILCs

ILCs are derived from common lymphoid progenitors and can be classified into different groups based on their expression of transcription factors and cytokines[56]. This section excludes NK cells that belong to a group of ILCs. Increased production of IL-22 in intestinal type 3 ILCs with depletion of vasoactive intestinal peptide receptor 2 significantly reduced hepatic steatosis in mice with a HFD[57]. Gut microbiota transplantation from healthy donors suppressed HFD-induced hepatic steatosis and type 1 ILC (ILC1) cell population reduction in MASLD in male C57BL/6 mice. Similarly, oral administration of microbial metabolite indole-3-carbinol also improved hepatic steatosis and increased ILC1 cell population by activating aryl hydrocarbon receptor in the livers of MASLD mice[58].

Overall, hepatic innate immune cells are critical in liver inflammation, fibrosis, and lipid metabolism (Table 1), through their interactions with hepatocytes and adaptive immune cells and serving as cellular targets for MASLD and MASH therapy. Their plasticity creates a dynamic landscape throughout MASLD and MASH pathogenesis, which is particularly appealing for functional interrogation and/or drug targeting. Some typical markers can be utilized to differentiate these cell types in mouse and human tissues (Figure 1), which can further be applied to investigate their roles in MASLD.

Figure 1 The major innate immune cells in the metabolic dysfunction-associated steatotic liver disease liver. Some typical markers for mouse and human innate immune cells are listed. CD: Cluster of differentiation; MHC-II: Major histocompatibility complex II; HLA: Human leukocyte antigen; DC: Dendritic cell; ILC: Innate lymphoid cell; TCR: T cell receptor; MAIT: Mucosal-associated invariant T; NK: Natural killer cell; NKT: Natural killer T cell; MASLD: Metabolic dysfunction-associated steatotic liver disease.

Table 1 Roles of different innate immune cells in the progression of metabolic dysfunction-associated steatotic liver disease.

Cells	Markers	Functions	Ref.
cDCs	CD103	Adoptive transfer of CD103-expressing cDCs protected liver inflammation and damage in Batf3-deficient animals with a high sucrose diet or MCD diet during MASH progression	Heier et al[15]
cDCs	XCR1+	XCR1+ cDC1 cells influence inflammatory T cell reprogramming and exacerbate MASH development	Deczkowska et al[16]
Macrophages	MERTK	Ablation of MERTK signaling by disintegrin and metalloproteinase domain-containing protein 17 decreased MASH progression	Cai et al[23]
Macrophages	Ly6C+OPN+	Infiltration of Ly6C+OPN+ lipid-associated macrophages promoted MASH progression	Kui et al[38]
Neutrophils	Ly6G+H3Cit+	Infiltration of Ly6G+H3Cit+ neutrophils promoted MASH progression	Kui et al[38]
NK cells	CXCR3+	CXCR3+ NK cells decreased liver inflammation in MCD-induced MASH in mice	Fan et al[44]
NKT cells	IL-17A+	IL-17A-expressing invariant NKT cells in peripheral blood mononuclear cells were associated with MASH progression in patients	Maricic et al[51]
MAIT cells	TCRVa7.2+ CD161high CD3+	The frequency of MAIT cells in circulating blood in patients with MASH decreased compared to that in the group with steatosis or healthy controls	Waller et al[52]
ILCs	CD45+ TCRβ- B220-; NK1.1+ CD49a+ CD49b-	Increasing the frequency of ILC1 frequency by fecal microbiota transplantation and indole-3-carbinol inhibited MASLD	Hou et al[58]

cDCs: Conventional dendritic cells; ILC: Innate lymphoid cell; MAIT: Mucosal-associated invariant T; NK: Natural killer cell; NKT: Natural killer T cell; TCR: T cell receptor; CD: Cluster of differentiation; IL: Interleukin; MCD: Methionine- and choline-deficient; MASH: Metabolic dysfunction-associated steatohepatitis; XCR: X-C motif chemokine receptor; CXCR: C-X-C motif chemokine receptor.

MOLECULAR REGULATORS IN INNATE IMMUNE CELLS CONTRIBUTE TO ACTIVATION AND DYSFUNCTION IN MASLD

Innate immunity plays a pivotal role in the progression of MASLD by regulating liver inflammation and fibrosis[59-61]. Treatments, such as bariatric surgery and anti-inflammatory and antifibrotic medicines, can modulate the function and profiles of innate immune cells to suppress MASLD progression[62,63]. In this section, we review the many molecular regulators in innate immune cell activation and dysfunction in MASLD and MASH pathogenesis, some of which may serve as therapeutic targets for the treatment of liver disease.

Adenosine 5’-monophosphate-activated protein kinase

Adenosine 5’-monophosphate-activated protein kinase (AMPK) serves as a therapeutic target for MASLD and MASH. The AMPK signaling pathway is implicated in lipid and glucose metabolism by interacting with downstream and upstream genes, such as sterol regulatory element-binding protein 1 (SREBP1)[64] and carnitine palmitoyl transferase 1 A[65]. Cre-lox system knock-out AMPK catalytic subunit alpha 1 or 2 in bone marrow-derived and tissue-resident myeloid cells can accelerate HFD-induced liver fibrosis in both female and male mice[66]. In addition, AMPK activation upregulates peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α expression and decreases SREBP1 expression to suppress lipid accumulation in hepatocytes[67].

Glycolysis and mitochondrial respiration were dramatically increased in monocytes of patients with MASH compared to healthy controls, which was associated with proton leak and increased expression of proinflammatory cytokines (IL-1β and TNF-α) in serum. A reduction of AMPK phosphorylation was associated with the metabolic adaptation of monocytes in patients with MASH, which favored the activation of peroxisome-proliferator-activated receptor-gamma coactivator-1α, a transcription regulator of mitochondrial biogenesis and oxidative metabolism[68].

Farnesoid X receptor

Inhibiting hepatic farnesoid X receptor (FXR) expression can ameliorate HFD-induced lipid accumulation, oxidative stress, inflammation, and insulin resistance in mice[69,70]. In vitro and in vivo mechanistic investigations demonstrated that HFD treatment can inhibit hepatic expression of zinc finger and BTB domain containing 18a, which regulates hepatic lipid accumulation and inhibits macrophage activation by upregulating FXR-mediated clathrin heavy chain expression to suppress NLRP3 inflammasome activation[71]. Therefore, a semi-synthetic bile acid analog that binds to FXR has therapeutic potential in the treatment of MASH.

Additionally, obeticholic acid can directly suppress the activation of macrophage NLRP3 inflammasome to result in a reduction of hepatic lipid accumulation[28]. Treatment with a dual agonist (INT-767) of FXR and Takeda G protein-coupled receptor 5 increased the proportion of anti-inflammatory liver monocytes and the expression of M2-like macrophage marker genes such as CD206, Fizz1 (resistin-like molecule alpha 1), and IL-10[72].

JAK/STAT

Granulocyte colony-stimulating factor (GCSF)-deficient mice had decreased lipid accumulation in hepatic and adipose tissues and insulin resistance compared to wild-type mice[73]. Mechanistically, GCSF can regulate hepatic infiltration of neutrophils and macrophages during MASLD progression, and disruption of GCSF/GCSF receptor interaction can suppress liver lipid accumulation by increasing phosphorylation of JAK1/2 and STAT3 and their activation through inhibition of suppressor of cytokine signaling 3 expression[73]. Activated NK cells in mice with diet-induced MASH secreted pro-inflammatory cytokines and chemokines such as IL-β, IL-12, GM-CSF, IFN-γ, CCL4, and CCL5, which activated hepatic JAK-STAT1/3 and NF-κB signaling pathways to increase the production of ROS and induce apoptosis, thereby fostering liver damage[43].

Mitogen-activated protein kinases

Mitogen-activated protein kinases, including ERK, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase, play an essential role in modulating liver inflammation via regulation of proinflammatory cytokine products, immune cell recruitment, and the expression of key enzymes in inflammation (e.g., iNOS or inducible nitric oxide synthase)[74,75]. Blocking JNK/NF-κB signaling pathway in macrophages can inhibit liver inflammation and suppress macrophage-hepatocyte interaction to reduce liver injury[76].

Oxysterol binding protein like 8

Oxysterol binding protein like 8 is encoded by gene Osbpl8, which is highly expressed in EVs derived from bone-marrow-derived macrophages. Osbpl8-enriched EVs display anti-inflammatory and reduce lipotoxicity in MASH livers by regulating lipid metabolism and ameliorating endoplasmic reticulum (ER) stress through suppression of inositol-requiring kinase 1α/X-box binding protein-1 signaling pathway[77]. Hepatic expression of Osbpl3 was increased in patients with advanced MASLD compared to those with early MASLD, which was also observed in mice with MASLD. Further studies illustrated that the expression of Osbpl3 can be regulated by PPARγ[78].

PPARs

Overexpression of PPARγ can dampen palmitic acid-induced inflammatory cytokine gene expression in macrophages, whereas knockout of PPARg in macrophages worsens liver injury and accelerates MASLD progression in mice[79]. The beneficial function of PPARγ in MASLD is associated with the activation of Keap1/nuclear factor, erythroid 2-like transcription factor 2 signaling pathway[79]. Activation of PPAR signaling pathway was associated with macrophage-mediated inflammation in human MASH and murine MASH models[80]. Another study showed that activation of the PPARγ signaling pathway significantly inhibited M1 macrophage polarization and reduced lipid accumulation in vitro and in vivo[81]. Macrophage-specific knockout of PPARg can exacerbate liver inflammation and damage in mice with MASLD[79]. In addition, PPARγ can upregulate the expression of matrix metallopeptidase 10 to regulate IL-4-stimulated M2 macrophage polarization[82].

Protein tyrosine phosphatase receptor type O truncated isoform

During MASH progression in both mice and humans, protein tyrosine phosphatase receptor type o truncated isoform (PTPROt) expression has been observed to be increased in liver macrophages[83]. Overexpression of PTPROt in macrophages has been demonstrated to be associated with increased inflammation and mitochondrial damage via the activation of the NF-κB signaling pathway, along with increased expression of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and the production of ROS. In contrast, PTPROt deficiency in primary liver macrophages decreased the expression of NLRP1, NLRP3, NLRC4, and absent in melanoma 2, reducing ROS production[83].

T-cell membrane protein 4

T-cell membrane protein 4 (Tim-4), encoded by gene T-cell immunoglobulin and mucin domain containing 4, is highly expressed in liver macrophages. In macrophages of MASLD liver, Tim-4 interacts with liver kinase B1 and AMPKα to suppress NLRP3 inflammasome activation through phosphorylated AMPKα-regulated autophagy. Therefore, Tim-4 activation inhibits macrophage expression of IL-1β and IL-18 to ameliorate liver inflammation[84]. A recent study also demonstrates that impairing Tim-4 expression in KCs, either through neutralization antibody or genetic depletion, can decrease macrophage efferocytosis of apoptotic hepatocytes and increase the activation of HSCs, thereby promoting the progression of liver fibrosis[85].

TLRs

A brief two-week treatment with HFD in mice can significantly increase the frequency and cell number of ILC1 cells in MASLD livers[86], which is associated with an increase in the production of proinflammatory cytokines and chemokines, such as TNF-α and CXCR3. RNA-sequencing results demonstrate that TLR9 plays an important role in ILC1 differentiation by activating T-bet (T-box transcription factor) expression[86]. Another study shows that long-term treatment of TLR4 activator lipopolysaccharides (LPS) and HFD induces a more severe form of MASLD by activating NF-κB kinase subunit epsilon/NF-κB signaling, compared to single treatment of a HFD or LPS alone[87].

Trem2

Liver resident macrophages (KCs) are decreased during MASH progression, whereas the population of MDMs is increased. The expression of the scavenger receptor Trem2 in phagocytes is important for their phagocytic function as well as the clearance of apoptotic cells. Trem2^hi phagocytes have been implicated in hepatic injury[88,89]. In MASH liver, over 93% of KCs have been shown to harbor high Trem2 expression. This cell population, also termed MASH-associated macrophages, is responsible for the clearance of apoptotic cells and ECM during liver injury, which alludes to the functional reprogramming of macrophages during MASH pathogenesis[9]. Similarly, Chan and colleagues deemed a similar phenotype in macrophages, calling them LAMs that express Trem2, demonstrating that these LAMs aggregate into hepatic crownlike structures, believed to form around dying hepatocytes and localize them to areas rich in HSCs. These hepatic crownlike structures were observed in human MASH and seen to correlate with fibrosis severity. Based on this information, this phenotype of macrophages is seemingly involved in profibrotic pathways. Yet, the same researchers observed in C-C motif chemokine receptor 2 (CCR2) knockout mice with a HFD, meaning those with reduced LAMs and hepatic crownlike structures, liver fibrosis was increased[17]. This contradiction supports the notion that MDMs and even KCs, altogether, have dynamic phenotypes, altering their crosstalk, fibrotic effects, and overall impact on MASH liver tissue remodeling.

ATP6V0D2, a lysosomal gene in macrophages, regulates efferocytosis in Trem2-expressing macrophages, which supports maintenance of lipid metabolism and evades MASH progression. ATP6V0D2 deficiency in macrophages induces ER stress and promotes lipid accumulation and apoptosis of hepatocytes[90].

In summary, the several signaling pathways described above are uniquely involved with innate immune cell functionality, such as macrophages (Figure 2), thus, consideration of these cells as targets for therapeutic strategies to treat MASLD or MASH is imperative.

Figure 2 Molecular signaling pathways involved in macrophage function in metabolic dysfunction-associated steatotic liver disease. AMPKα: Adenosine monophosphate-activated protein kinase α; PTPRO: Protein tyrosine phosphatase receptor type O; ROS: Reactive oxygen species; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; IL: Interleukin; TNF: Tumor necrosis factor; NLRP3: NOD-like receptor family pyrin domain containing 3; Tim-4: T-cell membrane protein 4; LKB1: Liver kinase B1; Trem2: Triggering receptor expressed on myeloid cells 2; ER: Endoplasmic reticulum.

CLINICAL TRIALS: DIAGNOSIS AND THERAPY

Biomarkers expressed in innate immune cells can be applied for the diagnosis and treatment of MASLD. For example, the frequency of sialic acid-binding immunoglobulin-like lectin 7⁺ CD56^dim NK cells was decreased in patients with MASLD[91]. These NK cells highly expressed CD57 and PD-1, which could be targeted for MASLD therapy. Inflammatory markers or obesity-associated biomarkers, such as leptin, adiponectin, leukocyte cell-derived chemotaxin-2, chemerin, and circulating full-length and caspase-cleaved cytokeratin 18, can serve as non-invasive biomarkers in MASLD screening and treatment evaluation[92]. The expression level of IL-1β in circulating mononuclear cells was positively associated with body mass index, hemoglobin A1C, visceral adipose tissue, and MASLD extent or degree in obese patients with prediabetes or T2D[93]. Furthermore, weight loss achieved by treatment of glucagon-like peptide receptor agonist liraglutide or lifestyle management reduced the expression level of IL-1β and MASLD degree[93].

THR-β agonist HSK31679 can ameliorate diet-induced MASH by modulating gut microbiota metabolism and decreasing peripheral DCs and macrophages[94]. Treatment of resmetirom at a dose of 80 mg or 100 mg, once daily for 52 weeks, can improve liver fibrosis and reduce low-density lipoprotein cholesterol (LDL-C) level from the baseline[95]. Compared to placebo, THR-β agonist TERN-501 significantly reduced liver fat content in a dose-dependent manner, which was measured using magnetic resonance imaging proton density fat fraction[96].

CCL24 (eotaxin-2) plays multiple roles in diseases, including the recruitment of immune cells (e.g., eosinophils and neutrophils), macrophage polarization, and tumorigenesis[97]. CCL24 contributes to liver inflammation and fibrosis in MASLD and MASH[98]. Patients with MASLD who received intravenous or subcutaneous injection of CM-101, an anti-human CCL24 monoclonal antibody, every 3 weeks for 12 weeks, had decreased serum levels of inflammatory and fibrotic biomarkers compared with those in the placebo group[99].

Tang and Borlak[100] reported that the expression levels of CD163 and CD206 were negatively correlated with the expression of fibroblast growth factor 21 (FGF21) in patients with MASLD, indicating that FGF21 inhibits macrophage M2 polarization[100]. A phase 2 clinical trial has also demonstrated the beneficial effects of an engineered long-acting FGF21 analogue efimosfermin alfa (BOS-580) in reducing hepatic lipid accumulation[101].

A meta-analysis study shows that FXR agonists can mitigate liver injury by reducing alanine aminotransferase and gamma-glutamyltransferase (GGT) levels, as well as liver fat content in total, and they also can ameliorate liver fibrosis in patients with MASLD compared to placebo[102]. Another meta-analysis also shows that FXR agonists can reduce hepatic steatosis, which is evaluated by non-invasive imaging technology, magnetic resonance imaging-derived proton density fat fraction[103]. Treatment with FXR agonist vonafexor reduced liver fat content and liver enzymes, resulting in a reduction in body weight[104].

As previously described, PPARs play important roles in liver metabolism, inflammation, and fibrosis[105-107]. Treatment with lanifibranor, a PPAR agonist, suppressed the infiltration of MDMs in MASLD livers, which also inhibited palmitic acid-induced activation of bone-marrow-derived macrophages from mice and monocytes from patients with MASLD[108]. Lanifibranor significantly ameliorated hepatic insulin resistance and reduced levels of fasting glucose, glycosylated hemoglobin, and HDL-C[109].

Pemafibrate, a PPARα agonist, can dramatically reduce levels of LDL-C, non-HDL-C, and apolipoprotein B from the baseline in patients with MASLD[110]. Studies have shown that pemafibrate can promote M2 polarization of both murine and human macrophages by increasing the expression of CD163[111]. In addition, it can also inhibit IFN-γ-induced human THP-1 cell inflammation by suppressing the activation of NLRP3/caspase-1 signaling pathway[111].

A randomized, placebo-controlled, phase 2 clinical trial (ClinicalTrials.gov, trial number: No. NCT04667377) showed that treatment with survodutide, a dual agonist of glucagon receptor and glucagon-like peptide-1 receptor, decreased body weight in a dose-dependent manner compared to placebo[112]. Another phage 2 trial showed that survodutide can significantly reduce liver fat content and improve liver fibrosis in patients with MASH compared to placebo treatment[113].

Treatment with icosabutate, a free fatty acid receptor 1 and 4 agonist, at a dose of 600 mg for 52 weeks, reduced liver injury biomarkers (aspartate aminotransferase, GGT, and alkaline phosphatase) compared to placebo, which attained a stage improvement of liver fibrosis by artificial intelligence-assisted digital pathology analysis[114]. Overall, innate immune cells are therapeutic targets for MASLD treatment (Table 2).

Table 2 Clinical trials of metabolic dysfunction-associated steatotic liver disease treatments.

Treatments	Drugs	Trials	Phases	Functions	Ref.
THR-β agonist	HSK31679	NCT05531097	1	Ameliorate diet-induced MASH by modulating gut microbiota and peripheral dendritic cells and macrophages	Zhang et al[94]
THR-β agonist	MGL-3196 (resmetirom)	NCT03900429	3	Treatment of resmetirom at a dose of 80 mg or 100 mg, once daily for 52 weeks, can improve liver fibrosis and reduce low-density lipoprotein cholesterol level from the baseline	Harrison et al[95]
THR-β agonist	TERN-501	NCT05415722	2	Reduce liver fat content compared to placebo in a dose-dependent manner	Noureddin et al[96]
Anti-human CCL24 monoclonal antibody	CM-101	NCT06044467	1	It can reduce serum levels of inflammatory and fibrotic biomarkers in patients with MASLD	Mor et al[99]
		NCT06037577	1
		NCT06025851	1
A long-acting fibroblast growth factor 21 analogue	Efimosfermin alfa	NCT04880031	2	Reduce hepatic glycogen storage, attenuate lipid accumulation, ameliorate fibrosis, and suppress immune response	Tang and Borlak[100]; Loomba et al[101]
Farnesoid X receptor agonist	Vonafexor (EYP001a)	NCT03812029	2	Reduce body weight, liver fat, and biomarker enzymes in patients with suspected fibrotic MASH	Ratziu et al[104]
PPAR agonist	Lanifibranor	NCT03459079	2	It significantly improved hepatic insulin sensitivity and reduced intrahepatic triglyceride content	Barb et al[109]
PPARα agonist	Pemafibrate	NCT03350165	2	Decrease levels of low-density lipoprotein cholesterol, non-HDL-C, and apolipoprotein B from the baselines in patients with MASLD	le Roux et al[112]
		NCT05923281	3
A dual agonist of glucagon receptor and glucagon-like peptide-1 receptor	Survodutide	NCT04771273	2	Reduce liver fat content and improve liver fibrosis in patients with MASH	Sanyal et al[113]
Free fatty acid receptor 1 and 4 agonist	Icosabutate (NST-4016)	NCT04052516	2	Reduce liver injury biomarkers and decrease liver inflammation and fibrosis in patients with MASH and mild to severe fibrosis	Harrison et al[114]

THR-β: Thyroid hormone receptor-β; CCL24: C-C motif chemokine ligand 24; PPAR: Peroxisome proliferator-activated receptor; MASH: Metabolic dysfunction-associated steatohepatitis; MASLD: Metabolic dysfunction-associated steatotic liver disease; HDL-C: High-density lipoprotein cholesterol.

In addition, clinical trials have also demonstrated the beneficial effects of treatments, such as low-dose aspirin (No. NCT04031729)[115], vitamin D[116] or E[117] supplementation, wheat amylase trypsin inhibitors or gluten-free diet (No. NCT04066400)[118], in reducing liver fat accumulation, serum levels of fibrogenic factors, and homeostatic model assessment of insulin resistance in patients with MASLD and MASH.

CONCLUSION

MASLD is a comorbidity associated with many other metabolic disorders and infections, such as T2D and cardiovascular disease[119]. It impacts the progression and treatment of these diseases (Figure 3), such as the gut-liver axis[120]. Accumulating evidence indicates that innate immune cells play essential roles in the development and progression of MASLD. Therefore, it is critically important to investigate it at the cellular and molecular levels to pinpoint the mechanistic shifts in pathogenic hallmarks, such as fibrotic deposits or lipid handling. The regulation of hepatic lipid accumulation and inflammation can alter the proportions of these immune cells, thereby ameliorating liver injury and MASLD progression. Several strategies in the clinic show beneficial effects in preventing MASLD or MASH progression. Physical exercise is beneficial for maintaining body weight and preventing obesity before the onset of MASLD[121]. It is also considered a non-pharmacological treatment option for MASLD or MASH. Interactions of diet-gut microbiota and their associated products, such as metabolites, bile acids, and microbial components, collectively modulate the profile and activation of intrahepatic immune cells. This occurs through the regulation of lipid content, insulin signaling pathway, and various other pathways discussed in this paper, ultimately influencing MASLD progression. Numerous studies have characterized changes in the gut microbiome of patients with MASLD or MASH[112]. For example, the relative abundance of Phocaeicola dorei (P. dorei) has been linked to changes in MASLD severity[122]. Supplementation with P. dorei was shown to reduce Western diet-induced MASLD severity in mice by reducing the expression of liver inflammatory cytokines and chemokines and increasing gene expression involved in lipid β-oxidation. Furthermore, the cell-free supernatant of P. dorei was found to inhibit LPS-induced macrophage inflammation by modulating p38 phosphorylation[123]. Current studies also validate that dietary intervention[123], calorie restriction[124], or weight loss programs[125] can improve liver fat composition, insulin sensitivity, and reduce the risk of hypertension, cardiovascular disease, and T2D[126,127]. However, it remains to be determined whether the combination therapies, including physical activity, diet modifications, and pharmacological treatments, can reverse liver injury in patients with MASH and MASH-associated advanced liver diseases. Several therapies, such as FGF21 analogues, FXR agonists, CCR2 and CCR5 dual antagonists, calorie restriction, bariatric surgery, and exercise, show promise for the prevention and treatment of MASLD in clinical trials. Models like organoids and human-like animal models are important for evaluating the effectiveness of pharmaceutical treatments. Serum biomarkers linked to hepatic immune cell activation are non-invasive methods for MASLD diagnosis and prognosis, as well as for evaluating the treatment efficacy in MASLD or MASH. Emerging biomarkers, such as microRNAs, require extensive evaluation. Additionally, many of the above-mentioned molecular targets identified in pre-clinical models await clinical evaluation.

Figure 3 Metabolic dysfunction-associated steatotic liver disease impacts the health of other organs in the body. Metabolic dysfunction-associated steatotic liver disease is a comorbidity associated with many other metabolic disorders, such as cardiovascular disease, kidney disease, neurological disorders, obesity, and intestinal disease. MASLD: Metabolic dysfunction-associated steatotic liver disease.