Neutrophils in MASLD and MASH

Abstract

Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) and its progressive form, Metabolic Dysfunction Associated Steatohepatitis (MASH), represent significant health concerns associated with the metabolic syndrome. These conditions are characterized by excessive hepatic fat accumulation, inflammation, and potential progression to cirrhosis and hepatocellular carcinoma. Neutrophils are innate immune cells that play a pivotal role in the development of MASLD and MASH. They can infiltrate the hepatic microenvironment in response to inflammatory cytokines and damage associated molecular patterns (DAMPs) derived from the liver and exacerbate tissue damage by releasing of reactive oxygen species (ROS), cytokines, and neutrophil extracellular traps (NETs). Moreover, neutrophils can disrupt the metabolism of hepatocytes through key factors such as neutrophil elastase (NE) and human neutrophil peptides-1 (HNP-1), leading to inflammation and fibrosis, while myeloperoxidase (MPO) and lipocalin (LCN2) are involved in inflammatory and fibrotic processes. In contrast, neutrophils contribute to liver protection and repair through mechanisms involving microRNA-223 and matrix metalloproteinase 9 (MMP9). This dual role of neutrophils highlights their significance in the pathogenesis of MASLD and MASH. This review summarizes current understanding from recent studies on the involvement of neutrophils in MASLD and MASH. Understanding complex roles of neutrophils within the liver’s unique microenvironment offers insights into novel therapeutic strategies, emphasizing the need for further research to explore neutrophil-targeted interventions for managing MASLD and MASH.  

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an epidemic chronic liver disease that affects almost one-quarter of the world’s population, with its incidence increasing due to lifestyle changes (1, 2). Metabolic dysfunction associated steatohepatitis (MASH), a more active form of MASLD, is characterized by active hepatic necroinflammation and rapid fibrosis progression. It is becoming the leading cause of hepatocellular carcinoma and liver transplantation (1, 3). At present, estimated global prevalence of MAFLD and metabolic dysfunction-associated steatohepatitis (MASH) in adults is 25-30%. For MASH, its prevalence is 3-5%. In children, MAFLD affects approximately 7% of the population (4-7). The incidence of MAFLD seems to increase in parallel with epidemics of obesity and diabetes. If current trends continue without changes, it is projected that over half of the adult population will be affected by MAFLD, with the incidence of MASH increasing by up to 56% (6-9). Furthermore, MAFLD and MASH have been identified as the fastest-growing causes of hepatocellular carcinoma (HCC), which has an annual incidence rate of approximately 0.5-2.6% (7).

The impact of MAFLD on healthcare expenses and resource utilization remains significant in the current era (9, 10). Individuals with MAFLD are at an increased risk of liver-related morbidity and mortality as well as metabolic comorbidities, which could further strain healthcare systems (4, 6, 7). The cumulative healthcare expenses linked to MAFLD are approximately 80-100% higher compared to similar controls. This discrepancy mainly arises from increased rates of hospitalizations and outpatient visits of MAFLD patients (4, 6, 7).

Neutrophils are the most abundant leukocytes. They serve as the first line of defense against infections, participating in almost all kind of acute inflammation and chronic inflammatory process (11-13). They play a crucial role in shaping both innate and adaptive immune responses and function as coordinators of overall immune and inflammatory responses. In sterile inflammation, neutrophils are activated and recruited to the site of injury, participating in inflammatory responses to restore the physiological function of the tissue. Due to their versatile functions, neutrophils have been highlighted as critical mediators of diseases in multiple organs, including the liver (14, 15). In MASLD, excessive neutrophil infiltration into the liver is a prominent feature (16, 17). This infiltration into the liver is associated with inflammation and subsequent inflammation-induced pathological injury related to the severity of MASH (16, 18). Depletion of neutrophils or inhibition of neutrophil-derived inflammatory mediators has been shown to delay the progression of liver diseases (19, 20). This review summarizes the pathological role of neutrophils during inflammation and fibrosis associated with progression and severity of MASLD.

METABOLIC ASPECTS ON PATHOGENESIS OF MASLD AND MASH

MASLD is an umbrella term that encompasses a disease spectrum of liver diseases ranging from simple hepatic steatosis (fatty liver) to MASH, which involves hepatocyte damage with inflammation and/or fibrosis, potentially leading to cirrhosis (2, 21). MASLD is increasingly recognized as a critical component of metabolic syndrome, accompanying obesity, diabetes, and dyslipidemia. It is prevalent among patients with metabolic syndrome. MASLD typically arises when the synthesis of triglycerides in the liver exceeds the rate of triglyceride breakdown (22). This condition is triggered by an increase in the absorption of free fatty acids in the liver or an increase in the biosynthesis of triglycerides from carbohydrate metabolism. Free fatty acids serving as substrates for triglyceride synthesis in the liver originate from the diet, endogenous biosynthesis, and release from adipose tissues (23).

Carbohydrates can enhance the synthesis of free fatty acids from acetyl-coenzyme A by raising blood insulin levels (24). Conditions such as obesity and insulin resistance characterized by hyperinsulinemia can activate AKT2, liver receptor X, and mTOR, increasing the activity of sterol regulatory binding protein-1c (SREBP-1c), a key transcription factor in lipogenesis (25). Glucose can also enhance the activity of the carbohydrate responsive element-binding protein (ChREBP) and liver-type pyruvate kinase, strengthening pathways involved in fatty acid synthesis and providing necessary substrates (26, 27).

Approximately 60% of free fatty acids entering the liver come from adipocytes. Activities of adipocyte triglyceride hydrolase (ATGL) and hormone-sensitive lipase (HSL) involved in the breakdown of triglycerides in fat cells are normally inhibited by insulin (28). However, insulin resistance can enhance lipolysis in adipose tissue, increasing the influx of fatty acids into the liver and leading to MASLD. This further exacerbates insulin resistance in the liver (29). Additionally, adipocytes can secrete adipokines including adiponectin, which can regulate metabolism. Insulin resistance can alter the endocrine function of adipocytes and change the polarization of surrounding macrophages, leading to increased secretion of pro-inflammatory cytokines and worsening MASLD (30).

An increase in circulating free fatty acids can also induce insulin resistance in skeletal muscles. Normally, insulin binds to insulin receptors on muscle cells, leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and through the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, resulting in the translocation of glucose transporter type 4 (GLUT4) to the membrane and increased glucose uptake (31). However, lipid derivatives within muscle cells, such as diacylglycerol, ceramide, and acylcarnitine from incomplete oxidation, can inhibit normal tyrosine phosphorylation and disrupt glucose uptake by increasing serine phosphorylation of IRS-1 through Protein Kinase C (PKC) and inhibitor of nuclear factor kappa B (Iκβ)/NF-κβ pathways. Excess supply of amino acids, free fatty acids, and glucose to muscles can activate the mechanistic target of rapamycin (mTOR)-S6 kinase signaling, further impairing insulin-mediated glucose uptake by increasing IRS-1 serine phosphorylation (32). Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 can inhibit IRS-1 and increase inducible nitric oxide synthase (iNOS) through the IκB kinase (IKK)/c-Jun N-terminal kinase (JNK) pathway, impairing insulin-mediated glucose uptake in muscle cells (33). Chronic inflammation in adipocytes due to long-term nutritional excess can lead to increased lipolysis, elevated levels of circulating fatty acids and triglycerides, and macrophage infiltration, secreting pro-inflammatory cytokines (34). This inflammatory condition can increase fatty acid influx into skeletal muscle, leading to fat accumulation and insulin resistance.

In summary, the development of insulin resistance can enhance lipolysis and free fatty acid release in adipose tissues, increasing fatty acid influx into liver cells and impairing glucose utilization in muscle cells. This will lead to hyperinsulinemia and increase glucose uptake and de novo lipogenesis, further exacerbating hepatic fat accumulation. These conditions can lead to detrimental hepatic inflammation through lipotoxicity and oxidative stress, followed by activation of Kupffer cells (KCs), hepatic stellate cells (HSC), and infiltrating immune cells, further accelerating disease progression (Fig. 1) (35, 36).

Fig. 1.

Pathophysiology of fatty liver disease. A schematic illustration of the transition from a healthy liver through stages of Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) and Metabolic Dysfunction Associated Steatohepatitis (MASH) to cirrhosis and potential hepatocellular carcinoma (HCC). Fatty liver disease is driven by increased accumulation of triglycerides due to lipolysis-derived non-esterified fatty acids (NEFAs) from adipocytes, de novo lipogenesis in the liver, and dietary derived energy sources. This accumulation leads to lipotoxicity, which in turn triggers recruitment and activation of immune cells such as neutrophils, monocytes, T lymphocytes, dendritic cells, and macrophages including Kupffer cells, resulting in hepatic inflammation and fibrosis.

ROLES OF IMMUNE CELLS IN MASLD AND MASH PATHOGENESIS

Immune response plays a critical role in the development and progression of MASLD and MASH. Liver resident immune cells comprise roughly 20% of non-hepatocytes. They include macrophages (KCs), dendritic cells (DCs), and a diverse array of lymphocytes such as Natural Killer (NK) cells, CD4⁺ T cells, CD8⁺ T cells, unconventional T cells such as NKT cells, mucosal-associated invariant T (MAIT) cells, γδT cells, double-negative CD4⁻CD8⁻ T cells, and homo-dimeric CD8⁺ T cells that express only the α chain of the T-cell receptor, along with B cells (37, 38). This unique composition creates a liver microenvironment enriched in cytokines and growth factors, predominantly regulatory, derived from these liver resident immune cells. Indeed, this environment provides immunological tolerance and facilitates KCs and DCs, which encourage the activation of regulatory T cells (Tregs) and suppress effector T cells through incomplete activation, exhaustion, and premature apoptosis of T cells (39-42).

However, inflammatory responses in MASLD and MASH can disrupt this balance (Fig. 2). The chronic inflammatory characteristic of MASH can dysregulate innate and adaptive immune cells. Activated by hepatic inflammation, KCs and HSCs can release chemokines and pro-inflammatory cytokines, which subsequently recruit and activate circulating innate immune cells such as neutrophils and monocytes (43-45). These monocytes can differentiate into pro-inflammatory macrophages, while activated KCs can further intensify inflammation through TNF-α, C-C motif chemokine ligand (CCL)2, and IL-1β production (46). Neutrophils can contribute to MASH by releasing toxic granules such as elastase and myeloperoxidase (MPO), production of pro-inflammatory mediators, and generation of reactive oxygen species (ROS), leading to death of hepatocytes (47). DCs are transformed into mature pro-inflammatory subsets in hepatic inflammation. They can facilitate monocyte recruitment and regulate NK and CD8⁺ T cell activities. They are involved in tissue remodeling. They can also produce pro-inflammatory cytokines (such as TNF-α, interferon [IFN]-γ, and IL-6) and induce fat deposition in the liver (48).

Fig. 2.

Interactions between immune cells and hepatocytes during fatty liver disease. This schematic figure depicts cellular interactions and processes involved in progression involved in hepatic inflammation during MASLD and MASH. Dendritic cells (DCs) and Kupffer cells (KCs) are activated in response to fat deposition and inflammation in hepatocytes, contributing to the recruitment of monocytes and neutrophils. Hepatic stellate cells (HSCs) are shown to undergo TRAIL-mediated apoptosis by natural killer (NK) cells. Monocytes differentiate into proinflammatory macrophages upon enteric hepatic environment, while neutrophils directly contribute to hepatocyte damage. The overall dynamic depicted illustrates the multifaceted nature of hepatic inflammation, comprising both innate and adaptive immune components.

In initial stages of MASLD, NK cells can promote TRAIL-mediated HSC apoptosis and activate liver infiltrating immune cells through IFN-γ, promoting MASH progression (49). B cells through the generation of antibodies against oxidatively damaged molecules and expression of B cell activating factors are known to contribute to hepatocyte ballooning and fibrosis and produce pro-inflammatory cytokines affecting T cell functions (50). T cells, particularly in the early phase of liver inflammation, play a pivotal role in the pathogenesis of MAFLD and MASH. T helper type 1 (Th1) cells can induce hepatocyte apoptosis through production of IFN-γ, and activate KCs to differentiate into pro-inflammatory phenotype. Th2 cells, despite their anti-inflammatory properties, contribute to liver fibrosis under IL-13 influence. Notably, the proportion of Th17 cells, which are pro-inflammatory, is higher in MASH than in MASLD. They can promote macrophage recruitment through upregulation of CXCL10 via IL-17 and lipid synthesis in hepatocyte through IL-17 receptor A (IL-17RA). Similarly, an increase of CD8⁺ T cells can exacerbate inflammation through IFN-γ, IL-17A, and IL-17F production (51). In contrast, percentages of Tregs are decreased in MASH patients, resulting in augmentation of inflammatory responses. Overall, the intricate interaction between resident and circulating immune cells, coupled with HSC activation, drives liver inflammation and fibrosis in MASLD and MASH.

ROLES OF NEUTROPHILS IN MASLD AND MASH PATHOGENESIS

Neutrophils are recruited to the liver in response to cytokines and chemokines such as CXCL1 and IL-8 (CXCL2) liberated from the liver microenvironment. In response to IL-1β derived from KCs, sinusoidal endothelial cells in the liver can upregulate intercellular adhesion molecule-1 (ICAM-1), allowing neutrophils to adhere via integrin α_Mβ₂ (Mac1) (52-54). Upon arriving at an injury site, neutrophils can migrate toward damage-associated molecular patterns (DAMPs) signals or activated complement system, regardless of the CXCL1 and CXCL2 gradients (52, 55). During migration, neutrophils become activated and play dual roles in either tissue damage and inflammation or tissue regeneration and immune suppression by releasing various cytokines and active molecules (Fig. 3) (54).

Fig. 3.

Modulation of hepatic immune responses by neutrophils. This schematic represents the intricate relationship among different immune cells within the liver and their impact on hepatocytes. Neutrophils activated by various signals within the hepatic microenvironment produce reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and granules, contributing to liver inflammation and hepatocyte damage. Neutrophil-derived granules such as neutrophil elastase (NE), human neutrophil peptides-1 (HNP-1), myeloperoxidase (MPO), and lipocalin2 (LCN2) activate Kupffer cells (KCs), macrophages, and hepatic stellate cells (HSCs) in the liver. NETs activate macrophages and induce exhaustion of CD8⁺ T cells and differentiation of regulatory T cells (T_regs). These interactions highlight the role of neutrophils in hepatic inflammation, with potential implications for the progression of liver diseases.

Neutrophils can contribute to hepatocyte damage and fibrosis by enhancing local inflammation through ROS generation, cytokine production, neutrophil extracellular trap (NET) formation, and granule-mediated proteolysis (47, 54, 55). Granule proteins mediate most of the neutrophil-mediated pathogenic processes in the liver (56). Neutrophil elastase (NE) can induce KC activation (47, 57) and disrupt lipid homeostasis through hepatic ceramide synthesis (58), resulting in insulin resistance. Furthermore, the absence of NE can reduce liver inflammation, hepatocellular ballooning, and steatosis in the early stage of MASH development (59). This suggests that NE participates in the progression of MAFLD and the onset of MASH. Human neutrophil peptides (HNP-1) can activate KCs, recruit and activate macrophages, and induce proliferation of activated HSCs, leading to liver inflammation and hepatic fibrosis, thereby contributing to progression of MASH (60). HNP-1 can also mediate fibrosis and promote collagen synthesis through the upregulation of heat shock protein 47 (HSP47) and collagen-1 in HSCs (61). Myeloperoxidase (MPO) can induce tissue damage through generation of aggressive oxidants and regulate inflammatory and fibrosis pathways. MPO deficiency can diminish chronic inflammation and reduce hepatic cholesterol accumulation and fibrosis (19). MPO can also induce death of hepatocytes (resulting in the progression of MAFLD), activate HSC (resulting in fibrosis), and promote liver injury (62). Lipocalin (LCN2) can induce the expression of CXCR2 in neutrophils and hepatic macrophages, leading to the production of proinflammatory chemokines and inducing inflammation, infiltration of macrophages, and liver injury (63). However, hepatocyte-derived LCN2 can protect against diet-induced MASLD by regulating lipid metabolism through induction of lipolysis, fatty acid oxidation, and lipid peroxidation (64). ROS generated from activated neutrophils can activate HCSs. A feed-forward signaling loop prolongs neutrophil survival, thus promoting liver inflammation and fibrosis (65).

NETs triggered by hepatic fat accumulation can promote MASLD progression through the augmentation of pro-inflammatory responses. Despite a non-causative role of NETs, NET formation can affect patterns of liver inflammation and trigger hepatic injury through macrophage activation and regulation of T cells rather than directly inducing hepatocyte injury (66-70). Moreover, S1PR2 can skew the cell death mechanism of neutrophils from apoptosis into NETosis in initial stages of liver diseases (71). NETs can shorten coagulation time and increase fibrin formation, enhancing the pro-inflammatory environment in the liver (72, 73). They are accompanied by increased neutrophil and macrophage infiltration and inflammatory cytokine production (68). NET formation does not affect the overall T cell population in the inflamed liver. However, NETs can enhance the differentiation of naïve T cells into Tregs via the induction of oxidative phosphorylation and induce exhaustion of CD8⁺ T cells via PD-L1 in MASH (66, 67). Thus, while NETs themself are not the culprit, their impact on immune responses can contribute to the progression of liver inflammation and fibrosis.

In contrast, recent studies have indicated beneficial roles of neutrophils in fatty liver disease. MicroRNA-223 (miR-223) in neutrophils can inhibit MASLD progression through immune regulatory functions. Deletion of miR-223 promotes a full range of fatty liver diseases, from simple fatty liver to hepatocellular carcinoma, with increased inflammation (74, 75). Matrix metalloproteinase 9 (MMP9) derived from neutrophils is essential not only for collagenase activity, but also for collagen resorption during liver repair, promoting liver regeneration facilitated by hepatocyte-derived MMPs (76, 77).

TARGETING NEUTROPHILS FOR DEVELOPING THERAPEUTICS OF MAFLD AND MASH

The rising prevalence of MAFLD and MASH, along with their significant global health impact, has sparked extensive research interest in developing treatments for these conditions (9, 10, 78). Despite numerous pathophysiological mechanisms and genetic variants have been identified, no approved drugs currently exist for treating MAFLD or MASH. Lifestyle modifications including diet, physical activity, and exercise remain the cornerstone of treatment for MAFLD and MASH (9, 10, 78, 79). However, several clinical trials for therapeutics of MAFLD and MASH (9, 78) have shown promising improvements, encouraging researchers and pharmaceutical companies to continue developing safe and effective therapeutic strategies.

Immunotherapy through intervention of neutrophil activation and function may serve as one of the effective treatments for MAFLD and MASH. For example, inhibiting CXCR2, a chemokine receptor with high affinity for CXCL1, CXCL2, and CXCL8, can reduce neutrophil accumulation in the liver and affect liver metabolic activity independently of its effects on neutrophils, thereby reducing MAFLD and MASH (7, 63, 80). Additionally, combining CXCR2 inhibition with anti-programmed cell death protein 1 (anti-PD-1) therapy can reprogram tumor-associated neutrophils and resensitize MASH and HCC to immunotherapy (7, 81). Similarly, depletion of neutrophils during early stages of MAFLD and MASH can reduce liver inflammation (59). Genetic depletion of NE can decrease neutrophil accumulation in the liver, reducing liver damage and fibrosis (7, 58, 59, 82). Knockout of MPO or administration of MPO inhibitors can reduce liver injury and fibrosis in MASH mouse models (7, 19, 83). Furthermore, intervention of NET formation by DNase treatment or deletion of the gene encoding PAD4 can reduce hepatic macrophage infiltration, liver inflammation, injury, fibrosis, and MASH (7, 67, 68, 70). Therefore, Inhibitors targeting NETosis, granule proteins, and chemokines are promising approaches for treating MASH and liver inflammation. Similarly, infusing recombinant LCN2 has shown potential as a therapeutic strategy against MAFLD and MASH (7, 63). Overexpression of LCN2 in hepatocytes can protect against diet-induced MAFLD in mice (64). On the other hand, transfection or overexpression of miR-223 can attenuate fibrogenesis during liver injury, whereas knock-down of the miR-223 leads to a full spectrum of MASH (63, 74, 75). However, effects of these inhibitors have been investigated mainly using cell cultures and animal models. Furthermore, the abundance and complex signature of innate and adaptive immune cells in the liver may directly or indirectly affect immunotherapy outcomes (84). To provide a reliable theoretical basis for clinical treatment of liver diseases targeting neutrophils, more studies on inhibitors targeting activation or dysregulation of neutrophils during MAFLD and MASH are needed. Additionally, identifying subsets of neutrophils that may participate in MAFLD-MASH progression is crucial.

CONCLUSIONS

Recent advancements have significantly broadened our understanding of the role of neutrophils in MASLD and MASH. Additionally, it is becoming evident that the neutrophil population is more heterogeneous than previously recognized. New analytical techniques such as single-cell RNA sequencing have shed light on various subsets of neutrophils that may play distinct roles in the pathogenesis of MASLD and MASH. Exploring these subsets not only could deepen our understanding of neutrophil involvement in these conditions, but also could open avenues for novel therapeutic strategies.