The Role of Neutrophils in Non-Alcoholic Fatty Liver Disease: Mechanisms and Clinical Significance

Abstract

This review methodically elucidates the pivotal role of neutrophils in the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). A body of research indicates that neutrophils are preactivated in a background of systemic metabolic dysregulation. Furthermore, neutrophils are specifically recruited to the liver via the CXCR2/CXCL1 axis. In the liver microenvironment, neutrophils directly mediate hepatocyte injury and potently amplify inflammation through synergistic mechanisms. These mechanisms include degranulation, the release of toxic proteases, the production of reactive oxygen species, and the formation of neutrophil extracellular traps. More crucially, neutrophils have been shown to promote disease progression from simple fatty liver to non-alcoholic steatohepatitis, liver fibrosis, and hepatocellular carcinoma by forming complex interaction networks with macrophages, hepatic stellate cells, and hepatocytes. These findings not only deepen our understanding of the immunopathological mechanisms of NAFLD but also highlight the immense clinical translational potential of neutrophils as novel biomarkers and therapeutic targets.

Introduction

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease on a global scale, affecting over a quarter of the world’s population.^1–3 Its epidemic rise is closely linked to the surge in obesity, type 2 diabetes, and metabolic syndrome.^1–4 NAFLD comprises a spectrum of progressive liver injury, ranging from the relatively benign form of simple hepatic steatosis (NAFL) to the more severe form of non-alcoholic steatohepatitis (NASH).^3,5 NASH is characterized by hepatocyte injury, ballooning degeneration, and inflammation. NASH signifies a pivotal juncture in the progression of liver disease, with a range of 20–30% of NASH patients progressing to liver fibrosis, cirrhosis, and, in some cases, liver failure or hepatocellular carcinoma (HCC).^6–8 NAFLD-associated HCC is emerging as a growing oncological burden, frequently occurring in NASH patients who have not yet progressed to cirrhosis. This underscores the need to deepen our understanding of its pathogenesis.^9,10

Historically, the pathophysiology of NAFLD has been dominated by the “two-hit” and subsequent “multiple-hit” theories. This theory posits that hepatic lipid accumulation, or the initial insult, renders hepatocytes more susceptible to subsequent injurious factors, such as oxidative stress and lipotoxicity.^11–13 However, recent research has begun to elucidate that the onset and progression of NAFLD is primarily driven by chronic, low-grade inflammation originating from the innate immune system.^14–16 In the context of metabolic dysfunction, persistent injury signals emerge from hepatocyte lipid overload, endoplasmic reticulum stress, and pathogen-related molecular patterns, such as lipopolysaccharide (LPS), derived from dysbiotic gut microbiota.^5,17–19 These signals activate liver-resident immune cells (eg, Kupffer cells) and recruit peripheral immune cells, leading to massive release of pro-inflammatory cytokines and chemokines. This process gives rise to a self-amplifying inflammatory microenvironment that significantly contributes to the progression from simple fatty liver to NASH.^15,20

Among liver-infiltrating immune cells, the role of macrophages/Kupffer cells has been the subject of extensive research.^21,22 However, the role of neutrophils—the most abundant effector cells of the innate immune system—in NAFLD has long been overlooked or oversimplified to a “first responders” role in acute infections. Traditionally regarded as terminally differentiated cells with a limited lifespan, neutrophils primarily execute defensive functions by phagocytosing pathogens, releasing antimicrobial granules, and generating reactive oxygen species (ROS) at infection sites.^23,24 However, mounting evidence suggests that in chronic metabolic diseases, neutrophils not only exhibit prolonged survival but also demonstrate complex functional plasticity.^25,26 Neutrophils have been shown to exert a profound influence on hepatocytes, stellate cells, and other immune cells by secreting a range of inflammatory mediators and forming distinctive neutrophil extracellular traps (NETs).^27–30 Consequently, the role of neutrophils has evolved from a marginal participant in acute inflammation to a pivotal actor in the progression of chronic inflammation and the tissue damage associated with NAFLD.

Although preliminary research has suggested the potential significance of neutrophils in NAFLD, the current understanding of this field remains fragmented. This is particularly evident in the area of neutrophil activation mechanisms, effector pathways, and specific roles at different disease stages, which have yet to be systematically elucidated. The objective of this review is twofold: first, to comprehensively integrate current research findings, and second, to delve into the multifaceted functions of neutrophils in the pathogenesis and progression of NAFLD. The article will methodically explore the mechanisms underlying neutrophil recruitment and activation. It will then analyze hepatocyte injury, as well as the role of degranulation, ROS release, and NETs formation in this process. Furthermore, the article will delve into the intricate interactions of these cells with other hepatic cells and will provide a comprehensive, step-by-step examination of the specific roles of neutrophils in the progression of NASH, liver fibrosis, and HCC. Finally, the study will evaluate the translational potential and challenges of targeting neutrophils as a therapeutic strategy, providing theoretical guidance for future research.

Neutrophil Recruitment and Activation in NAFLD

“Preactivated” State: Priming Effects of Systemic Metabolic Dysregulation

The process of neutrophil recruitment in NAFLD is initiated by functional preactivation, which is induced by alterations in the systemic metabolic environment. In the context of pathological conditions characterized by obesity, insulin resistance (IR), and hyperlipidemia, elevated circulating free fatty acids—particularly saturated fatty acids such as palmitic acid—have been observed to enhance baseline neutrophil activity significantly. This enhancement is attributed to the activation of Toll-like receptor 4 (TLR4) on the neutrophil surface, which induces endoplasmic reticulum stress.³¹ Concurrently, metabolic endotoxemia resulting from dysbiosis and compromised intestinal barrier function allows sustained entry of low-dose LPS into the circulation, providing a stable initial signal for persistent neutrophil activation.³² This metabolic preactivation state does not immediately trigger intense inflammatory responses; rather, it significantly lowers neutrophils’ response threshold to subsequent chemotactic signals. Furthermore, this process has been shown to enhance their migration, phagocytosis, and the potential to form NETs. This, in turn, establishes the functional foundation for their rapid accumulation and effector activities in the liver.

A direct correlation has been demonstrated between the preactivated state and IR, which exerts a profound modulatory effect on neutrophil function.³³ Within the domain of IR, where insulin signaling is compromised, neutrophils may manifest modified functional capacities.³⁴ For instance, impaired insulin signaling can lead to dysregulated chemotaxis, potentially due to disrupted actin cytoskeleton dynamics and polarization.^34,35 A body of research indicates a correlation between IR and a diminished phagocytic capacity of neutrophils. This diminished capacity could result in impaired clearance of debris and potentially protracted inflammatory stimuli.^34,35 Furthermore, the state of IR, often characterized by low-grade inflammation and increased oxidative stress, primes neutrophils for an exaggerated response to subsequent stimuli, including an increased propensity for NETosis upon encountering factors like free fatty acids or LPS.³⁴ Consequently, IR functions not solely as a background metabolic condition but also as an active modulator that directly reprograms neutrophil functional responses, rendering them more susceptible to a pro-inflammatory and tissue-damaging phenotype in the liver.

Furthermore, the dysregulated secretion of adipokines in obesity plays a critical role in neutrophil preactivation. Leptin, which is elevated in obesity, functions as a pro-inflammatory adipokine.^36,37 Leptin receptors have been identified on neutrophils, and leptin signaling has been demonstrated to directly enhance neutrophil chemotaxis, delay apoptosis, and potentiate the respiratory burst, thereby promoting a primed state.^37–40 It can also synergize with other stimuli to induce the release of pro-inflammatory cytokines from neutrophils.^38,41 Conversely, adiponectin, which is typically reduced in obese individuals, generally exerts anti-inflammatory effects.⁴² Adiponectin has been reported to suppress neutrophil phagocytic activity and the production of ROS.^43,44 Consequently, the shift in the leptin/adiponectin balance towards a pro-inflammatory profile in obesity (high leptin, low adiponectin) further contributes to neutrophil preactivation and heightened responsiveness, which in turn contributes to their pathogenic potential in NAFLD.

Beyond LPS, the dysbiotic gut microbiome produces a vast array of metabolites that enter the circulation and directly modulate neutrophil phenotype and function.⁴⁵ For instance, short-chain fatty acids (SCFAs), such as butyrate and propionate, which are often reduced in dysbiosis, have been demonstrated to exhibit anti-inflammatory effects on neutrophils.^46,47 These substances have been observed to impede various biological processes, including neutrophil chemotaxis, adhesion, and the production of pro-inflammatory mediators. This potential inhibitory effect may be attributed to the ability of these substances to bind to G-protein-coupled receptors (eg, GPR41/43) or to inhibit histone deacetylases (HDACs).^45,47 Conversely, other microbial metabolites may promote a pro-inflammatory state. Taurine-conjugated metabolites, including tauro-β-muricholic acid, have been implicated in shaping the immune landscape.⁴⁸ However, the direct effects of these metabolites on neutrophils are still under investigation. It has been demonstrated that certain gut microbes are capable of producing metabolites that function as damage-associated molecular patterns (DAMPs). These metabolites have been observed to either act as DAMPs or to directly prime the nod-like receptor protein 3 (NLRP3) inflammasome.^49,50 This gut microbiota-neutrophil axis indicates that alterations in the microbial composition can directly program neutrophils towards a more or less inflammatory state before they even reach the liver. These pre-programmed neutrophils, upon subsequent recruitment to the steatotic liver, may then exhibit an exaggerated pro-inflammatory and tissue-damaging response, thereby exacerbating the progression from simple steatosis to NASH.

Key Chemokines and Chemokine Receptors

The migration of preactivated neutrophils to the liver is a complex process that is primarily regulated by a network of chemokines produced locally in the liver. Among these, the signaling axis formed by C-X-C motif chemokine ligand 1 (CXCL1) and its receptor CXCR2 plays a central role in this process.^51,52 In NAFLD, the livers of affected individuals exhibit a heightened response to lipotoxic stress, characterized by the accumulation of fat within hepatocytes. This response is accompanied by the activation of early-activated hepatic stellate cells and Kupffer cells, which are stimulated by DAMPs. Consequently, these cells undergo significant synthesis and secretion of the chemokine CXCL1, leading to its accumulation in the affected livers.⁵³ This chemokine binds to the CXCR2 receptor, which is highly expressed on neutrophil surfaces. The result of this binding is the triggering of intracellular signaling that guides neutrophils to migrate directionally toward liver tissue along a concentration gradient.^52,54 In NASH animal models, blocking this pathway with CXCR2 antagonists or by knockout has been shown to reduce hepatic neutrophil infiltration and subsequent inflammatory damage significantly.⁵¹ This finding provides functional confirmation of the critical role of this axis. Furthermore, other chemokines, such as C-C motif chemokine ligand 3 (CCL3), and lipid mediators, including leukotriene B4 (LTB4), have been shown to play a synergistic role in this process.^39,55

Adhesion and Transendothelial Migration

Upon reaching the hepatic microvascular system guided by chemokines, neutrophil infiltration into the hepatic parenchyma relies on a multi-step cascade mediated by adhesion molecules.^56,57 The process commences with a process known as selectin-mediated transient adhesion. In the presence of inflammatory stimuli, E-selectin and P-selectin, which are expressed on hepatic sinusoidal endothelial cells, bind to the neutrophil surface ligand P-selectin glycoprotein-1 (PSGL-1). This binding mediates the deceleration of neutrophils in the bloodstream and their subsequent rolling along the vascular wall.^58,59 Subsequently, local chemokine gradients activate rolling neutrophils, inducing a conformational change in their integrin Mac-1 (CD11b/CD18) to a high-affinity state.⁶⁰ The activated Mac-1 forms a stable bond with intercellular adhesion molecule-1 (ICAM-1), which is highly expressed on endothelial cells, firmly anchoring neutrophils to the sinusoidal wall.⁶¹ Finally, adherent neutrophils traverse the hepatic sinusoidal endothelium through endothelial junctions or by traversing the cell bodies. This process results in the cells entering the interstitial space, thereby completing transendothelial migration and reaching the damaged liver tissue (Figure 1A).^62,63

Figure 1.

Recruitment and activation of neutrophils in NAFLD and the core mechanism of liver injury. (A) Systemic Preactivation and Liver-Specific Recruitment. (B) Local Activation and Effect Mechanism.

Abbreviations: FFA, Free Fatty Acids; LPS, Lipopolysaccharide; TLR4, Toll-Like Receptor 4; CXCL1, C-X-C Motif Chemokine Ligand 1; CXCR2, C-X-C Chemokine Receptor Type 2; DAMPs, Damage-Associated Molecular Patterns; ICAM-1, Intercellular Adhesion Molecule 1; PSGL-1, P-Selectin Glycoprotein Ligand-1; Mac-1, Macrophage-1 Antigen; HSC, Hepatic Stellate Cell; NE, Neutrophil Elastase; MPO, Myeloperoxidase; NOX2, NADPH Oxidase 2; ROS, Reactive Oxygen Species; NETs, Neutrophil Extracellular Traps; CXCL8, C-X-C Motif Chemokine Ligand 8; TNF-α, Tumor Necrosis Factor-alpha; IL-1β, Interleukin-1beta; IL-6, Interleukin-6; IL-12, Interleukin-12; IL-23, Interleukin-23.

Sustained Activation in the Local Liver Microenvironment

Neutrophils that successfully migrate into liver tissue are ultimately fully activated by highly concentrated local injury signals, releasing their terminal effector functions.⁶⁴ The high concentrations of free fatty acids, LPS, complement activation fragments, and DAMPs released from necrotic or apoptotic hepatocytes in the local hepatic microenvironment provide potent activation signals for neutrophils.^65–67 These signals are generated by binding to their corresponding pattern recognition receptors. This local activation directly triggers their robust antimicrobial defense mechanisms, including the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and the explosive production of ROS, the degranulation of cytoplasmic granule contents (such as neutrophil elastase, myeloperoxidase, and cathepsins), and initiation of NETosis under specific stimuli like cholesterol crystals or activated platelets, leading to NET formation.^68–70 Consequently, neutrophils undergo a comprehensive transformation from a preactivated state in circulation to a local effector cell in the liver, profoundly contributing to the inflammatory and deleterious processes of NAFLD (Figure 1B).

Mechanisms by Which Neutrophils Drive Liver Injury and Inflammation

Direct Cytotoxic Effects of Degranulation and Proteases

Upon activation, neutrophils release pre-synthesized proteases and antimicrobial peptides into the extracellular space through degranulation, a core mechanism that mediates direct hepatocyte damage.^68,70,71 Neutrophil elastase (NE) and cathepsin G have been identified as pivotal factors in this process.⁷² NE has been shown to specifically degrade key junctional proteins, such as E-cadherin, on the hepatocyte membrane, thereby disrupting the integrity of intercellular junctions. Furthermore, it has been demonstrated to directly cleave intracellular signaling proteins, thereby activating the caspase cascade and inducing hepatocyte apoptosis.^73,74 Concurrently, cathepsin G has been shown to exhibit intrinsic cellular toxicity while synergistically enhancing NE’s proteolytic activity.⁷¹ Myeloperoxidase (MPO), another significant granule component, has been shown to catalyze the generation of highly oxidative hypochlorous acid. This process occurs through direct binding to hepatocyte surfaces, which in turn triggers intracellular oxidative stress and death signaling pathways.^75–77 The release of these proteases leads to the formation of high-concentration zones within the local hepatic microenvironment. The hydrolysis of extracellular matrix components, combined with direct targeting of hepatocytes, results in the degradation of liver tissue architecture and the subsequent loss of hepatocyte function.

Burst Production of Reactive Oxygen Species and Oxidative Stress

Upon activation, neutrophils rapidly assemble the NADPH oxidase complex (NOX2), transferring electrons to oxygen molecules to explosively generate large amounts of superoxide anion (O₂•⁻)—a process termed “respiratory burst”.^78,79 The superoxide anion can further convert into multiple ROS species, including hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), and hydroxyl radicals (OH•).^78,80 It has been established that these highly reactive ROS exert multifaceted damaging effects on hepatocytes. These effects include peroxidation of membrane lipids, altered membrane fluidity, and increased permeability. In addition, there is an attack on thiol groups and amino acid residues of proteins, causing enzyme inactivation and functional abnormalities. Furthermore, ROS inducers cause mitochondrial DNA damage, disrupt energy metabolism, and ultimately drive hepatocytes toward apoptosis or necrosis.^81–83 In NAFLD, exogenous ROS produced by neutrophils synergize with endogenous ROS generated by lipid-overloaded hepatocytes, thereby exacerbating hepatic oxidative stress. This molecular event is a critical component in the progression from simple fatty liver disease to NASH.^82,83

NETs Formation and Multifaceted Pathogenic Effects

The formation of NETs represents a distinctive immunodefensive and injurious mechanism of neutrophils.^84–86 In the context of NAFLD, a multitude of factors, including elevated levels of free fatty acids (specifically palmitic acid), LPS, and activated platelets, have been shown to trigger NETosis potently.^87–89 This process involves the depolymerization of chromatin, the mixing of granule proteins with chromatin, and the rupture of cell membranes, which ultimately results in the release of a fibrous reticular structure composed of DNA scaffolding, histones, NE, MPO, and other components.^29,84 NETs have been shown to mediate a variety of pathological effects through their physical structure and toxic components. In one potential pathway, the NET structure itself and the attached cytotoxic histones can directly damage hepatocyte membranes, leading to cell lysis. Of greater significance is the role of NETs as a persistent inflammatory platform. The components in question (eg, HMGB1 and DNA) can be recognized by toll-like receptors on macrophage surfaces, thereby activating the NLRP3 inflammasome. This process leads to the sustained release of potent proinflammatory factors, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), thereby transforming acute neutrophil infiltration into a chronic, self-amplifying inflammatory state.^29,30,90

However, it is imperative to acknowledge that the role of NETs in NAFLD may not be exclusively pathogenic, and a nuanced view considering disease stage and context is necessary. In the early stages of simple steatosis (NAFL), or even in a healthy liver, NETs might play a beneficial role in maintaining homeostasis. In the context of increased intestinal permeability, for instance, NETs could function as a physical and functional barrier to entrap and kill translocating bacteria or fungi, thereby preventing them from reaching the liver parenchyma and causing overt infection.^29,91 This could be a critical mechanism to contain low-level bacteremia associated with metabolic endotoxemia. Furthermore, NETs have been implicated in the resolution of inflammation by degrading cytokines and chemokines through proteases associated with their scaffold, potentially helping to limit the extent of tissue damage in a controlled response.⁹² This dual nature underscores the notion that the pathological effects of NETs observed in established NASH and fibrosis likely represent a failure of these homeostatic mechanisms. In this scenario, excessive and sustained NET formation overwhelms their protective functions, thereby tipping the balance from containment to chronic injury.

Release of Pro-Inflammatory Cytokines and Chemoattractants

Beyond the pre-formed mediators previously discussed, activated neutrophils also serve as a substantial source of de novo synthesized cytokines and chemokines, which significantly influence the hepatic immune microenvironment.^93,94 Upon stimulation by DAMPs, LPS, or other neutrophil-derived mediators, neutrophils have been observed to produce and release substantial amounts of IL-1β,^95,96 although the maturation of IL-1β is often dependent on inflammasome activation, which can occur within the neutrophil itself. This neutrophil-derived IL-1β exerts a paracrine function, amplifying the inflammatory cascade by activating Kupffer cells. This activation leads to the secretion of downstream effectors, such as tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6), and the enhancement of the recruitment of additional immune cells.⁹⁷ Furthermore, neutrophils have been identified as a pivotal source of chemokines, particularly CXC ligand 8 (CXCL8), in humans and its functional homologs in mice, thereby establishing a positive feedback loop that facilitates further neutrophil recruitment.⁹⁴ Furthermore, the secretion of TNF-α by these cells has been observed to directly induce hepatocyte stress and apoptosis, with a concomitant synergistic activation of hepatic stellate cells (HSCs) in response to other mediators.⁶⁴ More recently, the significance of neutrophils in influencing adaptive immunity has come to the fore. Neutrophils secrete cytokines such as Interleukin-12 (IL-12) and Interleukin-23 (IL-23), which are pivotal for the differentiation and maintenance of T helper 17 (Th17) cells.⁹⁴ The subsequent production of Interleukin-17 (IL-17) by these Th17 cells can then act back on neutrophils, as well as on Kupffer cells and HSCs, further fueling inflammation and fibrosis.⁹⁸ Consequently, neutrophil-derived cytokines function as pivotal nodes in the intricate cytokine network of NAFLD, thereby integrating innate and adaptive immune responses and amplifying the signals initiated by degranulation, ROS, and NETs.

Synergistic Amplification of Multiple Effect Mechanisms

Neutrophil-mediated liver injury is not the result of a single, isolated mechanism. Instead, it is the consequence of a series of interconnected, synergistically amplified effects originating from multiple pathways, including degranulation, ROS production, and NET formation. This synergy has been shown to amplify the overall pathological impact significantly. For instance, NE and MPO released during degranulation further promote ROS generation, while ROS itself serves as both a key inducer of NETosis and a crucial structural component of NETs.⁹⁹ Conversely, the DNA scaffold of NETs immobilizes and concentrates toxic proteases such as NE and MPO, shielding them from rapid clearance by protease inhibitors and thereby sustaining a locally high-concentration, damaging microenvironment.⁹¹ This positive feedback network enables activated neutrophils to rapidly establish and maintain a highly efficient hepatic microenvironment characterized by proteolytic activity, oxidative toxicity, and potent pro-inflammatory properties. This process has been shown to accelerate hepatocyte death significantly, the spread of inflammation, and the subsequent initiation of the fibrosis process (Figure 1B).¹⁰⁰

Interactions Between Neutrophils and Other Hepatic Cells and Their Stage-Specific Functions

Interaction with Macrophages/Kupffer Cells and Early Initiation Role in NASH Development

Neutrophils form a critical synergistic pro-inflammatory network with liver-resident Kupffer cells and recruited monocyte-derived macrophages.^67,101 At the crucial juncture of transition from simple fatty liver to NASH, neutrophils directly act on macrophages by releasing their granule contents (eg, NE and MPO) and NET components.^76,102 These substances, particularly histones and DNA within NETs, function as DAMPs, efficiently activating the NLRP3 inflammasome signaling pathway within macrophages. This process culminates in the maturation and secretion of potent proinflammatory cytokines, including IL-1β and IL-18.¹⁰³ Neutrophil-derived lipocalin-2 (LCN2) has been shown to promote crosstalk between neutrophils and hepatic macrophages by inducing CXCR2 expression.¹⁰⁴ Concurrently, the presence of proteases, such as NE, has been observed to encourage macrophage polarization toward the classically activated M1 phenotype.¹⁰⁵ In response, activated macrophages secrete substantial amounts of chemokines, such as CXCL1 and CXCL8, also known as interleukin-8 (IL-8), along with TNF-α. These chemokines further enhance neutrophil chemotaxis and potentiate their activity.^97,106,107 This positive feedback loop establishes and sustains a self-amplifying inflammatory microenvironment within the liver. This microenvironment serves as a core driver of progression from NAFL to NASH. Clinical studies have revealed the presence of significant neutrophil infiltration in liver biopsy specimens from patients diagnosed with NASH. Furthermore, the severity of this neutrophil infiltration is positively correlated with the severity of hepatitis.²³ Consequently, neutrophils are not only among the earliest participants in NASH inflammation but also key effector cells that propel simple fatty liver disease beyond the inflammatory threshold into NASH.

Interaction with Hepatic Stellate Cells and Sustained Propulsive Role in Liver Fibrosis

Neutrophils have been demonstrated to be potent inducers of the transdifferentiation of HSCs from a quiescent state to activated myofibroblasts. This process serves as a critical link between inflammation and fibrosis.¹⁰⁸ As the disease progresses to the fibrotic stage, the role of neutrophils evolves from a purely inflammatory effector to a pro-fibrotic regulatory function. Neutrophil-derived NE and MPO have been shown to directly activate HSCs, inducing proliferation, migration, and the synthesis of extracellular matrix components (eg, collagen I and III) through signaling pathways, including protease-activated receptors (PARs).^76,109 It is imperative to acknowledge the multifaceted effects of NETs throughout this process.^{30,103,110,111} Research has demonstrated that NETs enhance mitochondrial function and aerobic glycolysis, thereby providing additional bioenergetic and biosynthetic support to activate, proliferate, and migrate HSCs.¹¹⁰ The DNA scaffold of NETs provides a platform for the activation and stabilization of transforming growth factor-β1 (TGF-β1), while the proteases they carry, such as NE, can directly cleave and activate latent TGF-β1.¹¹² TGF-β1, the most potent known pro-fibrotic factor, exhibits sustained activation that markedly enhances HSC activation and fibrogenic capacity.^113,114 Results from animal model experiments suggest that inhibiting NET formation (eg, with PAD4 inhibitors) or degrading NETs (eg, with DNase I) significantly attenuates hepatic fibrosis.^110,115,116 This finding provides functional confirmation of the neutrophil-mediated fibrotic drive via the NET pathway.

Interactions with Hepatocytes and Other Immune Cells and Dual Role in HCC

A bidirectional pathophysiological interaction exists between neutrophils and hepatocytes. On one hand, the presence of neutrophils in the liver can exert direct cytotoxic effects on hepatocytes by releasing NE, MPO, ROS, and NETs, which can induce ballooning degeneration, apoptosis, and necrosis.^30,76,117 Conversely, damaged hepatocytes have been observed to actively contribute to inflammation by releasing DAMPs, including high mobility group box 1 (HMGB1), ATP, and mitochondrial DNA.¹¹⁸ These DAMPs can be recognized by Toll-like receptors (TLRs) and purinergic receptors on the neutrophil surface, further intensifying neutrophil activation, promoting increased production of inflammatory mediators, and facilitating NET formation. This sequence of events gives rise to a self-perpetuating cycle, which has been termed “hepatocyte injury →DAMPs release →neutrophil reactivation”.¹¹⁸

Beyond innate immune cells, neutrophils also engage in functional crosstalk with adaptive immune cells, particularly T lymphocytes.¹¹⁹ Neutrophils have been demonstrated to influence the differentiation of Th17 cells into the state above by secreting cytokines such as IL-12 and IL-23.^120,121 Furthermore, NETs directly activate T cells through their histones and specifically enhance Th17 cell differentiation.¹²² Th17 cells and their effector factor IL-17 have been demonstrated to play a pro-inflammatory role in the pathogenesis of NASH.^123,124 Furthermore, the expression of co-stimulatory molecules, such as CD86, on the neutrophil surface, in conjunction with neutrophil-secreted chemokines, can directly or indirectly modulate T cell activation and function.¹²⁵ In certain pathological conditions, neutrophils have also been observed to suppress T cell proliferation and effector functions by depleting L-arginine in the local microenvironment through arginase-1 expression.^126,127 These interactions imply that neutrophils may function as a link between innate and adaptive immunity, thereby playing a precisely tuned regulatory role in shaping the immune response specific to NAFLD/NASH (Table 1).

Table 1.

Interactions Between Neutrophils and Other Hepatic Cells

Interacting Cell Type	Key Interaction Modes/Mediators	Functional Consequences & Disease Stage	Research Evidence
Macrophages/Kupffer Cells	Neutrophil → Macrophage: NETs release (Histones/DNA) Release of NE, MPO Secretion of LCN2 Macrophage → Neutrophil: Release of CXCL1, TNF-α, IL-8	Disease Stage: NAFL to NASH transition Consequences: Establishes a positive feedback loop, leading to self-amplifying inflammation; core driver of NASH initiation and maintenance.	Neutrophil infiltration in patient liver biopsies correlates with inflammation severity; disrupting this interaction in animal models alleviates hepatitis.
Hepatic Stellate Cells	Neutrophil → HSC: NE and MPO directly activate HSCs NETs activate and stabilize TGF-β1 NETs enhance metabolic reprogramming of HSCs	Disease Stage: Hepatic Fibrosis Consequences: Drives HSC activation into myofibroblasts, promoting excessive ECM deposition; a key bridge linking inflammation to fibrosis.	Inhibiting or degrading NETs significantly reduces liver fibrosis in animal models.
Hepatocytes	Neutrophil → Hepatocyte: Release of NE, ROS, NETs induces damage Hepatocyte → Neutrophil: Release of DAMPs (HMGB1, mtDNA)	Disease Stage: Throughout (NASH, HCC initiation) Consequences: Creates a vicious cycle of “damage-DAMP release-reactivation”, directly causing hepatocyte death and perpetuating inflammation.	Direct toxicity of neutrophils to hepatocytes confirmed in vitro co-culture experiments.
T Lymphocytes	Neutrophil → T cell: NETs promote Th17 differentiation Secretion of Arginase-1 suppresses T cell function	Disease Stage: NASH & HCC immune microenvironment shaping Consequences: Acts as a bridge between innate and adaptive immunity, potentially shaping disease-specific immune responses via promoting Th17 response or suppressing T cell function.	Evidence is still emerging; represents an important future research direction.

Abbreviations: NETs, Neutrophil Extracellular Traps; NE, Neutrophil Elastase; MPO, Myeloperoxidase; LCN2, Lipocalin-2; CXCL1, C-X-C Motif Chemokine Ligand 1; TNF-α, Tumor Necrosis Factor-alpha; IL-8, Interleukin-8; TGF-β1, Transforming Growth Factor-beta 1; ROS, Reactive Oxygen Species; DAMPs, Damage-Associated Molecular Patterns; HMGB1, High Mobility Group Box 1; mtDNA, Mitochondrial DNA; Th17, T helper 17 cells; NAFL, Non-Alcoholic Fatty Liver; NASH, Non-Alcoholic Steatohepatitis; ECM, Extracellular Matrix; HCC, Hepatocellular Carcinoma.

In the development and progression of NAFLD-associated HCC, neutrophils play a complex, controversial dual role. On the one hand, they have been shown to indirectly promote tumor growth and metastasis by fostering a pro-inflammatory, pro-proliferative, and pro-angiogenic microenvironment.¹²⁸ The matrix metalloproteinases (MMPs) secreted by neutrophils, such as MMP-9, have been shown to degrade the extracellular matrix, thereby promoting tumor cell invasion.^129,130 The formation of NETs has also been demonstrated to capture circulating tumor cells, promote their extrahepatic metastatic colonization, and potentially activate pro-survival signals within cancer cells via receptors such as TLR9.^131–135 Conversely, under specific conditions, certain neutrophil subsets may exhibit anti-tumor properties, such as through antibody-dependent cellular cytotoxicity (ADCC) or direct killing of tumor cells.¹³⁶ This functional ambivalence may be associated with distinct neutrophil functional subtypes (eg, N1 antitumor vs N2 pro-tumor phenotypes) or dynamic signaling equilibria within the tumor microenvironment.¹³⁷ In the context of NAFLD-HCC, neutrophil functions driven by chronic inflammation—represented by NETs formation—are generally considered dominant in promoting tumorigenesis and metastasis (Figure 2).

Figure 2.

Neutrophil-mediated intercellular interactions drive NAFLD disease progression.

Abbreviations: NASH, Non-Alcoholic Steatohepatitis; NE, Neutrophil Elastase; NLRP3, Nod-like Receptor Protein 3; MPO, Myeloperoxidase; NETs, Neutrophil Extracellular Traps; IL-8, Interleukin-8; TNF-α, Tumor Necrosis Factor-alpha; CXCL1, C-X-C Motif Chemokine Ligand 1; HSC, Hepatic Stellate Cell; TGF-β1, Transforming Growth Factor-beta 1; HCC, Hepatocellular Carcinoma; MMP-9, Matrix Metalloproteinase-9; ROS, Reactive Oxygen Species; DAMPs, Damage-Associated Molecular Patterns.

Clinical Significance and Translational Medicine Prospects

As Biomarkers for Disease Diagnosis and Prognosis

Neutrophils and their associated active products have been shown to have significant potential as non-invasive biomarkers in the clinical management of NAFLD/NASH. The peripheral blood neutrophil-lymphocyte ratio (NLR), an easily accessible and cost-effective inflammatory marker, has been demonstrated in multiple clinical studies to correlate significantly with the degree of hepatic steatosis, inflammatory activity, and fibrosis staging in patients with NAFLD.^138–141 Furthermore, the presence of specific molecular markers of neutrophil activation, particularly circulating NET components, provides more precise information on disease activity. Levels of MPO, NE, and proteinase 3 (PR3) are significantly elevated in NASH patients and correlate closely with the histological severity of hepatic inflammation and fibrosis.^109,142 The integration of these biomarkers with conventional liver enzymes and imaging studies holds promise for developing more precise, non-invasive diagnostic models for screening high-risk patients, monitoring disease activity in real time, and evaluating treatment response.

As Potential Therapeutic Targets

Given the central role of neutrophils in the progression of NAFLD, therapeutic strategies targeting their recruitment, activation, and effector functions have emerged as desirable research directions. The following approaches are currently identified as primary potential targets: The development of small-molecule antagonists targeting the core chemotactic axis of CXCR2 is underway. In preclinical models of NASH, the inhibition of CXCR2 has been shown to reduce hepatic neutrophil infiltration and improve pathological damage, thereby providing a rationale for the treatment of human NASH.^143,144 Concurrently, inhibiting neutrophil activation and the release of toxic mediators is another critical strategy. NE inhibitors, such as sivelestat, and myeloperoxidase inhibitors, which have already been tested in clinical trials for other diseases, offer a viable pathway for repurposing in NASH treatment.^145,146 The objective of these agents is to neutralize the primary toxic substances produced by neutrophils, thereby reducing tissue damage while potentially preserving some host defense functions.^145,146 Moreover, the targeting of NET formation is identified as a pivotal intervention strategy. Peptidyl-L-arginine deiminase 4 (PAD4), a core enzyme in chromatin depolymerization and NETosis,¹⁴⁷ has shown substantial anti-inflammatory and anti-fibrotic effects when inhibited in animal models.¹⁴⁸ A therapeutic approach that involves directly neutralizing NET toxicity by injecting recombinant DNase I to degrade DNA is an exploratory strategy (Table 2).^149,150

Table 2.

Potential Therapeutic Strategies Targeting Neutrophils

Intervention Level	Target/Strategy	Representative Candidate Agents/Methods	Mechanism of Action	Preclinical/Clinical Evidence & Considerations
Recruitment & Infiltration	CXCL1/CXCL8-CXCR2 Axis	CXCR2 Small Molecule Antagonists	Blocks chemotactic recruitment of neutrophils to the liver.	Effective in reducing hepatic neutrophil infiltration and inflammation in NASH animal models.
Activation & Effector Function	NE	NE Inhibitors (eg, Sivelestat)	Neutralizes a key cytotoxic protease, reducing direct cellular injury.	Tested in clinical trials for other diseases (eg, ALI); drug repurposing for NASH is a feasible path.
	MPO	MPO Inhibitors	Inhibits the generation of toxic oxidative products like hypochlorous acid.	Under development; aims to mitigate oxidative stress damage.
NETs Formation	PAD4	PAD4 Inhibitors	Inhibits chromatin decondensation, blocking NET formation at its source.	Shows significant anti-inflammatory and anti-fibrotic effects in animal models.
	NETs DNA Backbone	Recombinant DNase I	Degrades the DNA scaffold of formed NETs, neutralizing their toxicity.	An exploratory therapeutic approach; long-term efficacy and specificity require further validation.
Combination Therapy	Multi-pathway Synergy	eg, CXCR2 Antagonist + GLP-1 Receptor Agonist	Combines anti-inflammatory (neutrophil-targeting) with metabolic improvement.	Represents a future direction for synergistic multi-target attacks on the disease; requires careful evaluation of efficacy and safety.

Abbreviations: CXCL1, C-X-C Motif Chemokine Ligand 1; CXCL8, C-X-C Motif Chemokine Ligand 8; CXCR2, C-X-C Chemokine Receptor Type 2; NE, Neutrophil Elastase; MPO, Myeloperoxidase; PAD4, Peptidyl Arginine Deiminase 4; NETs, Neutrophil Extracellular Traps; DNase I, Deoxyribonuclease I; GLP-1, Glucagon-Like Peptide-1; ALI, Acute Lung Injury; NASH, Non-Alcoholic Steatohepatitis.

Challenges and Future Directions

Limitations of Technical Methods and Innovation Needs

Current research on the roles of neutrophils in NAFLD faces substantial technical challenges. Conventional techniques, including static tissue section analysis and in vitro cell culture, are limited in their ability to accurately mirror the dynamic behavior of neutrophils within the intricate microenvironment of the living liver and their transient interactions with other cells. Consequently, the development and implementation of advanced in vivo imaging techniques, such as intravital multiphoton microscopy, are imperative for real-time monitoring of neutrophil migration pathways, residence times, and dynamic interactions with hepatocytes, hepatic stellate cells, and other components in both normal and fatty liver tissues.¹⁵¹ Moreover, existing knockout models generally target all neutrophils or their shared pathways, lacking cell-type specificity. Subsequent endeavors should prioritize the development of conditional, cell-specific knockout models (eg, employing MRP8-Cre or S100A8-Cre systems) to deplete target genes (eg, PAD4 or CXCR2) in neutrophils at designated time points.^152,153 This approach will enable precise in vivo analysis of the causal roles of specific molecular pathways in neutrophil pathogenic functions, avoiding developmental compensation or systemic effects associated with systemic knockout.

Exploring Neutrophil Heterogeneity

The prevailing perspective regarding neutrophils as a homogeneous population is undergoing a rapid transformation. A mounting body of evidence signifies considerable functional heterogeneity and plasticity among neutrophils across diverse pathological states.^154–156 In the context of NAFLD/NASH, a central scientific question is whether a distinct “NASH-associated neutrophil” subpopulation exists that drives disease progression. A critical initial step in identifying pathogenic subpopulations is the systematic mapping of the transcriptomic and surface marker profiles of hepatic and peripheral blood neutrophils across different stages of the NAFLD disease spectrum. This is best achieved by using high-throughput technologies, such as single-cell RNA sequencing (scRNA-seq) and mass cytometry. The subsequent identification of specific surface markers (eg, specific chemokine receptors or activation markers) will facilitate flow cytometric sorting and functional validation. This, in turn, has the potential to lay the foundation for the development of precision therapies targeting specific harmful subpopulations.

Translating From Animal Models to Human Evidence

While preclinical animal models provide indispensable evidence for elucidating mechanisms, their conclusions ultimately require validation in prospectively designed human cohort studies. At present, the corpus of direct human evidence demonstrating a causal relationship between neutrophils and human NAFLD/NASH remains relatively limited. Future research must urgently leverage large-scale, multicenter clinical cohorts to systematically analyze associations between neutrophil characteristics in peripheral blood and liver tissue samples (eg, NLR, NETs markers, transcriptomic features) and liver histopathology, non-invasive imaging, and long-term clinical outcomes (eg, fibrosis progression, HCC development). Furthermore, analyzing changes in neutrophil-related parameters before and after treatment (eg, with GLP-1 receptor agonists or FXR agonists) using samples from interventional clinical trials could provide indirect human evidence for the role of neutrophils in treatment response.

Considerations for Combination Therapy Strategies

Given the highly complex pathogenesis of NAFLD/NASH, monotherapy targeting neutrophils alone may be insufficient to reverse disease progression. Consequently, future therapeutic strategies will likely transition towards combination therapies. A promising direction for further research is combining neutrophil-targeting agents (eg, CXCR2 antagonists, PAD4 inhibitors) with existing therapies targeting distinct pathological pathways. For instance, a combination of anti-inflammatory agents (targeting neutrophils) with drugs that improve metabolism (eg, GLP-1 receptor agonists reducing insulin resistance and hepatic steatosis) or anti-fibrotic agents (eg, FXR agonists) could theoretically produce synergistic or additive effects, enabling a coordinated, multi-faceted attack on the disease’s multifactorial, multi-pathway nature. However, implementing this strategy requires meticulous preclinical research and systematic clinical trials to assess synergistic efficacy, the optimal timing of administration, and potential safety risks (Table 2).

Safety Considerations and the Risk-Benefit Balance of Targeting Neutrophils

While the targeting of neutrophils holds immense therapeutic promise, it is imperative to conduct a balanced discussion of the potential risks associated with such strategies. Neutrophils represent the primary frontline defense against invading pathogens, particularly bacteria and fungi. Consequently, long-term or systemic inhibition of neutrophil recruitment (eg, CXCR2 antagonists), key effector functions (eg, NE/MPO inhibitors), or NETs formation (eg, PAD4 inhibitors) could theoretically increase the host’s susceptibility to infections. This is a critical safety concern, especially in a chronic disease like NAFLD/NASH where patients may require prolonged treatment. A patient’s medical history, particularly the presence of recurrent infections, may contraindicate the use of certain therapeutic interventions.

A concomitant consideration is the potential impact on cancer immunosurveillance. As previously discussed, neutrophils exhibit a multifaceted, dualistic role in the context of HCC. While pro-tumorigenic N2 phenotypes and NETs can promote tumor growth and metastasis, certain neutrophil subsets and functions are also involved in anti-tumor immunity. In principle, compromising neutrophil function indiscriminately has the potential to impair the body’s capacity to eliminate nascent tumor cells, particularly in a population already exhibiting an elevated risk for HCC. This concern underscores the necessity for therapeutic strategies that are highly specific to the pathogenic neutrophil subsets or effector pathways driving NASH and fibrosis, while preserving the essential functions of neutrophils in host defense and anti-tumor surveillance. The development of such “precision” immunomodulatory approaches, potentially delivered locally to the liver, and careful patient selection will be paramount to maximizing the therapeutic window and minimizing off-target risks. In the context of future clinical trials, it is imperative that rigorous monitoring procedures be implemented to identify and address potential infectious complications and long-term cancer incidence.

Conclusion

This review methodically elucidates the pivotal role of neutrophils in the pathogenesis and progression of NAFLD. The extant evidence suggests that neutrophils are no longer regarded as mere acute inflammatory cells. Instead, they are considered to play an active, multifunctional “promoter” role in NAFLD, a chronic metabolic liver disease. At the initiation stage, neutrophils are “preactivated” against a backdrop of systemic metabolic disorders. These cells are then recruited explicitly to the liver via intricate chemotactic signals centered on the CXCL1/8-CXCR2 axis, ultimately undergoing full activation within the local microenvironment. At the effector mechanism level, neutrophils directly cause hepatocyte damage and significantly amplify hepatic inflammatory responses through multiple synergistic pathways, including degranulation, the release of toxic proteases, the explosive production of reactive oxygen species, and the formation of NETs.

More crucially, neutrophils establish intricate interaction networks with other hepatic cells, including macrophages, hepatic stellate cells, and hepatocytes. These networks translate acute inflammatory signals into chronic inflammation maintenance and fibrosis progression. This has been demonstrated to be a significant factor in the progression of the disease, from the initial stage of simple fatty liver to more advanced stages such as NASH, liver fibrosis, and ultimately, hepatocellular carcinoma. These findings not only deepen our understanding of the immunopathological mechanisms underlying NAFLD but also reveal the significant clinical translational value of neutrophils and their specific products (such as NETs) as potential non-invasive biomarkers and novel therapeutic targets.

Despite the persistent challenges posed by technical approaches, cellular heterogeneity, and human evidence, the development of enhanced research tools, in-depth analysis of neutrophil functional subpopulations, and integration with state-of-the-art multi-omics technologies, in conjunction with large-scale clinical cohort validation, holds considerable promise for elucidating the intricate regulatory mechanisms of neutrophils in NAFLD. The integration of neutrophil-specific strategies, including the inhibition of neutrophil recruitment, activation, or NETs formation, with existing metabolic interventions, has the potential to yield novel breakthroughs in the effective curbing of NAFLD progression.

Funding Statement

There is no funding to report.

Data Sharing Statement

Data sharing is not applicable to this article as no data were created or analysed in this study.

Author Contributions

Conceptualization and methodology, F.Z. and W.L.; visualization and supervision, W.L.; Writing - original draft, F.Z.; Writing - review and editing, W.L.; All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflicts of interest in this study.