Mechanistic insights into hepatic cell type-specific contributions to acetaminophen-induced acute liver injury

Abstract

Acetaminophen [N-acetyl-p-aminophenol (APAP)] overdose is a leading cause of acute liver failure worldwide, chiefly due to its hepatotoxic effects. The pathogenesis of APAP-induced acute liver injury (ALI) involves complex interactions among various hepatic cell types, each playing a distinct role in the progression of the injury. Hepatocytes, the primary targets of APAP toxicity, undergo oxidative stress, mitochondrial dysfunction, and necrosis following the formation of the toxic metabolite N-acetyl-p-benzoquinone imine. Additionally, other hepatic cells and infiltrating immune cells responding to liver injury significantly contribute to the pathogenesis of APAP-induced ALI. This review synthesizes current mechanistic insights to offer a detailed understanding of the specific contributions of hepatic cells to APAP-induced liver injury, emphasizing potential therapeutic targets designed to reduce liver damage and enhance patient outcomes. Additionally, it identifies potential therapeutic targets within these cellular pathways that could be leveraged to alleviate liver damage and enhance clinical outcomes for patients affected by APAP overdose.

Key Words: Acetaminophen; Acute liver injury; Hepatocyte; Immune cell; Liver

Core Tip: This review provides a comprehensive overview of the cellular mechanisms underlying acetaminophen (APAP)-induced acute liver injury (ALI). It highlights the distinct yet interconnected roles of hepatocytes, Kupffer cells, monocyte-derived macrophages, hepatic stellate cells, liver sinusoidal endothelial cells, and adaptive immune cells in the initiation and progression of ALI. The review also explores how these cells contribute to both liver injury and repair, offering insight into the dynamic cellular crosstalk during APAP hepatotoxicity. By integrating recent findings, it identifies potential therapeutic targets and strategies that modulate cell-specific responses to improve clinical outcomes in drug-induced liver injury.

INTRODUCTION

The liver is an essential organ in the body, owing to its multiple functions in metabolism, detoxification, and overall homeostasis[1]. As the largest internal organ, it processes nutrients absorbed from the digestive tract, transforms them into vital chemicals required by the body, and controls a range of biochemical pathways involved in energy production, protein synthesis, and lipid metabolism[1,2]. Furthermore, the liver plays a crucial role in detoxifying harmful substances, producing bile to aid in digestion, and regulating blood clotting factors. Due to these critical functions, maintaining liver health is crucial for overall well-being.

The liver consists of two primary cell types: Parenchymal cells (mostly hepatocytes) and non-parenchymal cells (NPCs), each performing distinct roles in maintaining liver function and homeostasis. Hepatocytes are the primary functional cells of the liver, making up about 80% of the liver’s cellular population[3]. NPCs, comprising approximately 20% of the liver’s cellular population, include various cell types such as liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs). These cells contribute to the liver’s intricate functions, collaboratively maintain homeostasis, and respond to injury and disease[4]. Therefore, studying the functions of these diverse cell types in the liver, under both normal and disease conditions, is vital as it provides critical insights into how the liver upholds its essential functions and how disturbances can lead to various liver diseases.

This article offers a scoping review focused on acetaminophen (N-acetyl-p-aminophenol; APAP)-induced acute liver injury (ALI). The objective is to integrate mechanistic knowledge across hepatic cell types and to outline emerging therapeutic approaches.

APAP-INDUCED LIVER INJURY

The study of ALI is imperative due to its significant health impact, intricate pathophysiology, and the urgent need for effective treatments. ALI can result from various causes, including drug-induced liver injury (DILI), viral hepatitis, autoimmune diseases, toxins, and ischemia[5]. Each cause features unique pathophysiological mechanisms that contribute to liver damage. ALI can rapidly deteriorate into acute liver failure (ALF), a life-threatening condition with high mortality rates, and manifest severe symptoms like jaundice, coagulopathy, hepatic encephalopathy, and multi-organ failure[6]. There are currently limited therapeutic options for ALI, with liver transplantation often being the sole life-saving treatment for ALF[7]. Research in this field aims to identify new therapeutic targets to manage or reverse liver injury without resorting to transplantation. Animal models have played a crucial role in elucidating the pathophysiology of ALI and in evaluating potential therapeutic strategies. Among them, the APAP-induced liver injury model is particularly well established and clinically relevant.

APAP overdose represents the foremost cause of DILI globally and is a primary contributor to ALF in numerous countries[5,8]. APAP, the active ingredient in many over-the-counter analgesics, commonly causes unintentional poisoning or overdose. The majority of ingested APAP is safely excreted in the urine through phase II metabolic processes, mainly glucuronidation and sulfation[8]. However, a portion of APAP undergoes phase I metabolism, catalyzed by cytochrome P450 (CYP) enzymes including CYP1A2, CYP3A4, and CYP2E1, resulting in the creation of the toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive compound[5]. Under standard therapeutic dosing conditions (≤ 4 g/day in adults) in individuals with normal hepatic function and nutritional status[6], NAPQI is detoxified through conjugation with hepatic glutathione (GSH), resulting in the formation of stable compounds such as cysteine conjugates and mercapturic acid, which are then excreted[7]. In the setting of APAP overdose (≥ 5-10 g as a single ingestion in adults)[9], NAPQI production surpasses the detoxifying capacity of hepatic GSH, leading to GSH exhaustion. The unmetabolized NAPQI subsequently covalently binds to hepatic proteins, impairing their function and leading to hepatocellular death in the centrilobular region of the liver. This cascade of events compromises liver function and may progress to severe liver injury or failure[10].

Due to the cytotoxic effects of APAP, even its usage at recommended doses necessitates careful monitoring in individuals with pre-existing liver conditions. Chronic alcohol consumption heightens the risk of APAP toxicity by inducing CYP2E1, thereby increasing the production of the toxic metabolite NAPQI, which can exceed the liver’s detoxification capacity[11]. Beyond chronic alcohol use, Table 1 presents additional factors that modulate NAPQI production[10-16]. Additionally, patients with impaired renal function may also exhibit reduced APAP excretion, potentially leading to the accumulation of toxic metabolites in the body[17]. Furthermore, individuals with genetic metabolic disorders or diseases facing challenges in APAP metabolism is a significant concern[18]. Besides pre-existing liver diseases, environmental factors, and comorbid conditions can profoundly affect the metabolism and toxicological pathways of APAP, underscoring the importance of investigating diverse mechanisms to comprehend interindividual variability in APAP toxicity.

Table 1 Factors influencing N-acetyl-p-benzoquinone imine production.

Factor	Description	Ref.
CYP enzyme activity	CYP2E1, CYP1A2, and CYP3A4 are responsible for metabolizing APAP to NAPQI. Among these, CYP2E1 plays a predominant role and can be upregulated	[12]
Chronic alcohol consumption	Alcohol use results in induction of CYP2E1 and depletion of GSH, thereby raising NAPQI formation and diminishing detoxification capacity	[11]
Concomitant drug use	Drugs that induce CYP enzymes (e.g., phenobarbital) can increase the production of NAPQI	[13]
Genetic polymorphisms	Genetic polymorphisms in CYP genes can alter enzyme activity, influencing NAPQI formation	[14]
GSH levels	GSH conjugates NAPQI to facilitate detoxification. Insufficient GSH leads to the accumulation of NAPQI and subsequent toxicity	[10]
Fasting/malnutrition	Fasting depletes GSH reserves, thereby increasing susceptibility to NAPQI toxicity	[15]
Pre-existing liver disease	Pre-existing liver disease may modify CYP enzyme function and decrease GSH synthesis, resulting in impaired NAPQI elimination	[16]

CYP: Cytochrome P450; APAP: N-acetyl-p-aminophenol (Acetaminophen); NAPQI: N-acetyl-p-benzoquinone imine; GSH: Glutathione.

Given the central role of CYP enzymes in APAP biotransformation to its toxic metabolite NAPQI, there is significant interest and continued research into the specific CYP isoforms mediating this process. While APAP is predominantly metabolized by CYP2E1 in mice[19], both CYP2E1 and CYP3A4 contribute to its oxidative metabolism in humans, with CYP3A4 representing a relatively greater role[20]. These interspecies differences in the primary CYP enzymes necessitate careful consideration when extrapolating animal model data to human clinical scenarios. Furthermore, though transgenic “humanized” mouse models expressing CYP3A4 may enhance the translational relevance of preclinical studies[21,22], current therapeutic approaches remain suboptimal, especially for patients presenting outside the optimal intervention window. This underscores the critical need for advancing research into more effective therapies. Therefore, it is imperative to expand our knowledge of the cellular and molecular mechanisms underlying APAP-induced ALI, which may facilitate the development of focused therapeutic interventions. Design of strategies that specifically target cells most susceptible to APAP toxicity holds promise for achieving better clinical outcomes.

THE ROLES OF VARIOUS HEPATIC CELLS IN APAP-INDUCED ALI

Hepatocytes

The role of hepatocytes in ALI is fundamentally connected to their function in preserving the physiological operations of the liver. Loss or dysfunction of hepatocytes during ALI immediately disrupts these critical processes, leading to systemic metabolic imbalances, impaired detoxification capabilities, and, in severe cases, liver failure[7,10]. The extent of hepatocellular injury and the liver’s regenerative capacity are critical determinants of the clinical trajectory following APAP overdose. On a subcellular scale, APAP-induced hepatotoxicity triggers several intracellular stress responses, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades and endoplasmic reticulum (ER) stress[23-25], with mitochondrial dysfunction identified as a central mechanism responsible for hepatocyte demise. Mitochondrial dysfunction not only disrupts energy metabolism but also acts as a central integrator of oxidative stress and cell death signaling networks, making it a crucial focus for mechanistic investigation[26].

In hepatocytes, mitochondria serve not only as metabolic powerhouses responsible for adenosine triphosphate (ATP) production via oxidative phosphorylation but also as critical regulators of redox balance, lipid metabolism, and apoptotic signaling[27]. These organelles are particularly abundant and metabolically active in hepatocytes, which must sustain high levels of oxidative metabolism and detoxification reactions, particularly those involving CYP enzymes that localize to mitochondria and the ER[28]. Upon APAP overdose (Figure 1), the reactive metabolite NAPQI covalently binds to mitochondrial proteins, leading to sustained disruption of mitochondrial function, marked by impaired electron transport and excessive accumulation of reactive oxygen species (ROS)[29]. This redox imbalance activates the JNK signaling pathway through redox-sensitive mechanisms, leading to its translocation to mitochondria. The mitochondrial localization of phosphorylated JNK further amplifies oxidative stress and facilitates the generation of peroxynitrite, which contributes to the opening of the mitochondrial permeability transition (MPT) pore[30]. Subsequent collapse of mitochondrial membrane potential and cessation of ATP synthesis mark a critical turning point in mitochondrial failure. The opening of the MPT pore also disrupts the integrity of the mitochondrial outer membrane, resulting in the release of pro-death factors such as endonuclease G and apoptosis-inducing factor (AIF) into the cytosol[26]. These factors translocate to the nucleus, where they induce DNA fragmentation and ultimately trigger necrotic cell death.

Figure 1 Hepatocellular mitochondrial injury and downstream death signaling triggered by acetaminophen overdose. Following acetaminophen overdose, N-acetyl-p-benzoquinone imine interacts with mitochondrial proteins, initiating oxidative stress and activation of phosphorylated c-Jun N-terminal kinase. These events induce mitochondrial permeability transition pore opening and result in the release of apoptosis-inducing factor and endonuclease G. Upon their translocation to the nucleus, these factors promote DNA fragmentation and necrotic cell death. AIF: Apoptosis-inducing factor; APAP: N-acetyl-p-aminophenol (Acetaminophen); JNK: C-Jun N-terminal kinase; MPT: Mitochondrial permeability transition; NAPQI: N-acetyl-p-benzoquinone imine; ROS: Reactive oxygen species. This figure was created using BioRender.

Given the central role of hepatocytes in APAP-induced liver injury, recent studies have focused on mitochondria-directed therapeutic strategies that target these organelles as both primary sites of damage and critical regulators of hepatocellular viability and metabolic homeostasis. In APAP-treated mice, the mitochondria-targeted antioxidant Mito-Tempo attenuated liver injury by scavenging mitochondrial oxidant stress and peroxynitrite, which in turn prevented Bax translocation, AIF release, and nuclear DNA fragmentation, while having no effect on upstream events such as APAP-protein adduct formation, GSH depletion, or JNK activation[31]. Consistent with this observation, Abdullah-Al-Shoeb et al[32] showed that delayed administration of Mito-Tempo significantly reduced oxidative stress, nitrotyrosine formation, and DNA fragmentation in both murine and 3D human hepatocyte models, without altering upstream events such as JNK activation or C/EBP homologous protein expression. These findings suggest that Mito-Tempo, while not altering the initial toxic events of APAP metabolism, exerts its protective effects by intervening at a critical downstream point in mitochondrial oxidant stress. This highlights the therapeutic potential of targeting mitochondrial dysfunction itself as an effective strategy to preserve hepatocyte viability in APAP-induced ALI. In a related strategy, mitoquinone mesylate (MitoQ), a mitochondria-targeted analog of coenzyme Q10, has also demonstrated protective effects against APAP-induced hepatotoxicity. A study in murine models revealed that MitoQ mitigates APAP-induced ALI in hepatocytes by suppressing lipid peroxidation through a ferroptosis suppressor protein 1 (FSP1)-dependent mechanism[33]. Despite its promising results, current evidence for the use of MitoQ in APAP-induced ALI remains limited to a single preclinical study. Further investigations are warranted to validate its therapeutic efficacy and to explore broader applications of mitochondria-targeted antioxidants in hepatocellular protection.

Beyond mitochondrial dysfunction, ferroptosis has recently gained increasing attention as a regulated form of cell death in APAP-induced hepatotoxicity, primarily driven by iron-dependent oxidative stress and lipid peroxidation in hepatocytes[34]. APAP metabolism generates excessive ROS and exhausts intracellular GSH stores, creating an oxidative environment that promotes ferroptosis[35]. In this setting, lipid peroxidation emerges as a critical process, as shown by the increased formation of malondialdehyde, the end-product of polyunsaturated fatty acid (PUFA) oxidation, and the generation of peroxynitrite, a potent reactive nitrating species resulting from the interaction of nitric oxide and superoxide[36,37]. These reactive metabolites not only serve as biochemical markers for membrane damage but also intensify oxidative stress, further promoting ferroptotic pathways in hepatocytes. The regulation of this ferroptotic process is heavily dependent on GSH peroxidase 4 (GPX4), a key antioxidant enzyme that inhibits lipid peroxidation by converting lipid hydroperoxides into non-toxic lipid alcohols[38]. Recent experimental studies have demonstrated that GPX4 is upregulated in hepatocytes via plant-derived flavonoids-induced Nrf2 activation, which in turn reduces lipid peroxidation and ferroptosis in APAP-induced liver injury[39,40]. While these findings underscore the central role of GPX4 in controlling ferroptosis, an emerging body of literature over the past five years has begun to highlight GPX4-independent mechanisms as additional regulatory layers in hepatocellular ferroptosis, especially under conditions of APAP-induced hepatotoxicity. Notably, growing evidence indicates that ferroptosis can still occur independently of GPX4, particularly in contexts where cells exhibit resistance to GPX4 inhibition[41]. A recent study further supports this notion by demonstrating that MitoQ alleviates APAP-induced lipid peroxidation and hepatocyte death through a mechanism that operates independently of GPX4 but is partially reliant on FSP1 activity[33].

Moreover, the SIRT1/NRF2/HO-1 pathway plays a significant role in mitigating ferroptosis during APAP-induced liver failure. Ulinastatin has been shown to attenuate lipid peroxidation and ferroptosis by influencing this pathway[42]. In parallel, mifepristone (RU486) has recently been recognized in ferroptosis studies, exhibiting hepatoprotective properties in the context of APAP-induced hepatotoxicity[43]. At the mechanistic level, RU486 raises intracellular GSH concentrations and enhances NRF2 activity, leading to increased expression of detoxifying enzymes including GST. This newly described antioxidant-detoxification pathway promotes conjugation of 4-hydroxynonenal, a reactive lipid peroxidation product, helping to reduce ferroptotic cell damage. In addition to redox-regulatory processes, enzyme- driven lipid remodeling pathways have emerged as important in regulating ferroptosis during APAP-induced hepatotoxicity. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a central regulator of ferroptosis; it mediates the incorporation of PUFA into phospholipids, increasing their susceptibility to peroxidation and thus facilitating ferroptosis. Ji et al[44] found that ACSL4 silencing decreases lipid peroxidation and protects tissues from ferroptotic injury. ACSL4 is upregulated early in APAP-induced hepatotoxicity and is associated with heightened oxidative stress and hepatic damage[33]. By blocking ferroptosis with ferrostatin-1, a well-established ACSL4 inhibitor, APAP-induced hepatotoxicity and lipid peroxidation can be markedly reduced[35]. Additionally, the concurrent use of ferroptosis inhibitors and antioxidants that replenish GSH may offer enhanced protection against ACSL4-driven injury and improve clinical outcomes in ALI[45].

Expanding the current understanding of ferroptosis regulation, recent evidence has identified ferritinophagy as a distinct mechanism that exacerbates APAP-induced liver injury through iron release. Liang et al[46] demonstrated that ferritinophagy via nuclear receptor coactivator 4 (NCOA4)-mediated autophagic degradation of ferritin is a critical contributor to APAP-induced hepatotoxicity. In vivo and in vitro evidence indicates that ferritinophagy-derived labile iron promotes protein nitration and mitochondrial dysfunction, rather than classical lipid peroxidation, thereby driving hepatocyte injury. Although based on a single preclinical study, these findings position NCOA4 as a promising therapeutic target to mitigate iron-mediated mitochondrial damage in APAP-induced ALI, highlighting the need for further investigation into ferritinophagy-specific interventions.

Beyond iron-related pathways, pyroptosis has emerged as a distinct form of inflammatory cell death in hepatocytes, primarily triggered by infections or cellular damage. This process is characterized by an intense inflammatory response, predominantly mediated by the activation of inflammatory caspases[47]. A central mediator of pyroptosis is the NLR family pyrin domain containing 3 (NLRP3) inflammasome. In response to stimuli such as mitochondrial damage or microbial components, NLRP3 assembles with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and pro-caspase-1 to form the inflammasome complex, leading to caspase-1 activation and pyroptotic cell death[48]. Once activated, caspase-1 cleaves pro-interleukin (IL)-1β and pro-IL-18 into their active, mature forms[49]. The subsequent secretion of IL-1β and IL-18 exacerbates liver inflammation by recruiting and stimulating immune cells, thereby worsening secondary injury in APAP-induced ALI[48]. As a result, these cytokines are commonly assessed as reliable indicators of inflammasome activation and pyroptotic processes in research models. Interestingly, a recent study demonstrated that inhibiting the NLRP3 inflammasome mitigates pyroptosis, subsequently protecting against APAP-induced ALI and enhancing liver recovery through increased hepatocyte proliferation[50]. When the NLRP3 inflammasome is indirectly activated via the stimulator of interferon genes (STING) pathway, it precipitates a cascade of amplified inflammatory responses. The link between STING, a cytosolic protein that activates interferon and inflammatory pathways, and the NLRP3 inflammasome has recently gained significant attention in acute hepatotoxicity and liver fibrosis models[51,52]. Emerging evidence suggests that an APAP overdose activates the STING pathway in the liver, promoting inflammatory responses[52]. This activation further intensifies inflammation via stimulation of the NLRP3 inflammasome, while treatment with MCC950, a selective NLRP3 inhibitor, significantly reduces APAP-induced hepatotoxicity and inflammation. Nevertheless, these findings have largely been derived from experimental models of APAP-induced ALI, and their translational relevance to human pathology remains to be fully established. Further investigation is warranted to determine whether targeting the STING-NLRP3 axis offers a viable therapeutic strategy in clinical settings.

Given the critical role of hepatocytes in maintaining liver function, targeting regulated cell death pathways is essential for preserving hepatic integrity during APAP-induced ALI (Figure 2). Interventions aimed at modulating these pathways have demonstrated beneficial effects in preclinical studies, including enhanced hepatocyte survival and reduced tissue damage. However, clinical translation remains a significant challenge due to the complexity of liver pathophysiology and the limited availability of selective, well-characterized pharmacological agents. Moreover, most current findings are based on experimental models, and their applicability to human disease requires further validation. As such, there remains a critical unmet need to develop clinically viable therapies that not only prevent hepatocellular injury but also facilitate liver regeneration in the setting of ALF.

Figure 2 Targetable cell death and protective mechanisms in acetaminohphen-induced hepatotoxicity. This figure provides an overview of the principal pathways involved in hepatocellular injury and protective responses in acetaminophen [N-acetyl-p-aminophenol (APAP)]-induced acute liver injury. Mitochondria-targeted antioxidants, such as Mito-Tempo and mitoquinone mesylate, safeguard hepatocytes by decreasing mitochondrial oxidative stress. Ferroptosis, which results from lipid peroxidation and is regulated by glutathione peroxidase 4, can be mitigated by inhibitors like Ulinastatin and mifepristone. Ferritinophagy, a process mediated by nuclear receptor coactivator 4 that leads to ferritin degradation, also promotes iron-dependent mitochondrial injury. Pyroptosis, initiated by activation of the NLR family pyrin domain containing 3 inflammasome and further enhanced via the stimulator of interferon genes pathway, can be reduced by using the selective inhibitor MCC950. Collectively, these mechanisms represent essential therapeutic opportunities aimed at maintaining hepatocyte viability in APAP-induced liver damage. AIF: Apoptosis-inducing factor; APAP: N-acetyl-p-aminophenol (Acetaminophen); FSP1: Ferroptosis suppressor protein 1; GPX4: Glutathione peroxidase 4; GSH: Glutathione; HO-1: Heme oxygenase-1; MitoQ: Mitoquinone mesylate; NCOA4: Nuclear receptor coactivator 4; NLRP3: NLR family pyrin domain containing 3; NRF2 Nuclear factor erythroid 2-related factor 2; ROS: Reactive oxygen species; SIRT1: Sirtuin 1; STING: Stimulator of interferon genes. This figure was created using BioRender.

Macrophages

KCs: As resident hepatic immune cells, KCs act as the first line of defense by swiftly recognizing and eliminating pathogens or toxins that enter the liver[53,54]. During ALI, these cells detect signals from damaged hepatocytes and release cytokines and chemokines, modulating the inflammatory response[55]. While this inflammation is essential for clearing damaged tissue and promoting liver regeneration, excessive inflammation can further exacerbate liver damage. Thus, KCs play a crucial role in balancing the inflammatory response during liver injury.

To investigate the role of KCs and macrophages, two widely used compounds in depletion studies are clodronate and gadolinium chloride. Clodronate, a bisphosphonate compound, targets KCs for depletion. When delivered as liposome-encapsulated clodronate, it is preferentially taken up by macrophages, inducing apoptosis due to its intracellular toxicity[56]. Furthermore, studies have demonstrated that repeated pre-treatment with liposomal clodronate (LC) mitigates ALI in models of APAP-induced hepatotoxicity[57]. LC pre-treatment in these models does not affect APAP bioactivation but enhances hepatic resistance to oxidative stress by upregulating the levels of glutamate-cysteine ligase, a rate-limiting enzyme in the synthesis of GSH. Although the regulation of KCs using LC exhibits protective effects in mitigating ALI, its influence on liver recovery can differ based on the timing and mechanics of the repair process. Nguyen et al[58] reported that LC-induced depletion of KCs delayed liver recovery by diminishing the expression of C-X-C motif chemokine receptor 2 (CXCR2) 24 hours after APAP treatment. Remarkably, alanine aminotransferase levels, elevated from KC depletion at 24 hours after APAP treatment, recovered to baseline within 48 hours, paralleling the trend in CXCR2 expression. These findings underline the necessity of examining liver damage over several time points and the dual role of KCs in both mitigating ALI and facilitating recovery.

Unlike clodronate, gadolinium trichloride (GdCl₃) does not directly eliminate KCs but rather inhibits their activity, which is beneficial in models of liver injury[59]. Zhao et al[60] demonstrated that modulating KC activity with GdCl₃ treatment reduced hepatotoxicity in models of APAP-induced ALI by suppressing the detrimental role of macrophage-inducible C-type lectin. In experiments on APAP-induced ALF, either GdCl₃ or clodronate-mediated depletion of KCs showed that Toll-like receptor (TLR) 4 expression in KCs was inversely related to the expression of pro-inflammatory cytokines like IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α)[61].

Meanwhile, studies employing Cre recombinase transgenic mice targeting the C-type Lectin Domain Family 4 Member F (CLEC4F) have been extensively utilized. For instance, Clec4f-Cre mice were used to induce macrophage-specific deletion of IL-4rα following ALI incidents such as ischemia-reperfusion, to investigate its impacts on liver injury and repair[62]. Furthermore, in APAP-induced ALI models, the Clec4f-Cre system effectively revealed that a KC-specific deficiency in the copper metabolism MURR1 domain 10 impeded the ability of KCs to eliminate dying or apoptotic cells[63]. Notably, Jiang et al[64] recently showed that CLEC4F is detectable in human liver samples and colocalizes with the KC marker CD68, suggesting CLEC4F may serve as a marker for investigating human KCs in APAP-induced ALI.

Overall, these observations highlight the complex functions of KCs in both the development and resolution of APAP-induced ALI. As both defenders and possible mediators of hepatic injury, KCs serve as a key component within the hepatic immune network. Continued research into the temporal, mechanistic, and context-dependent roles of KCs in both animal models and human tissue is critical to advancing our understanding of their dual roles and to guide the development of therapies that target liver inflammation and regeneration.

Monocyte-derived macrophages: Unlike resident macrophages, monocyte-derived macrophages (MoMFs) are recruited to sites of injury or infection in response to chemotactic signals, where they perform adaptive immune functions[65]. Given that macrophages can exhibit diverse functions based on microenvironmental cues, the concept of M1 and M2 macrophage polarization has been introduced[66]. In the early stages of ALI, M1-type polarization predominates and adopts a pro-inflammatory role, secreting cytokines such as TNF-α, IL-1β, and IL-6, thereby amplifying the immune response and recruiting additional immune cells to the site of injury[6,67]. As the inflammatory phase transitions into the resolution and healing phase, MoMFs adopt an anti-inflammatory, tissue-reparative phenotype[67]. Holt et al[68] reported that macrophages activated after APAP-induced ALI are likely contributing to resolving inflammation and promoting tissue repair through efferocytosis by phagocytizing apoptotic cells. Additionally, activation of TLR signaling induces an increase in chemokine (C-C motif) ligand 2 (CCL2), which significantly contributes to monocyte infiltration into the liver. Puengel et al[69] demonstrated that monocyte infiltration and accumulation of MoMFs in the liver with ALI can be suppressed by CCR2 and CCR5 antagonists. Given recent findings that CCR2 inhibition alleviates APAP-induced ALI[70], the intrahepatic accumulation of MoMFs regulated by CCR2 is likely a critical target in macrophage-mediated therapeutic strategies.

HSCs

HSCs are liver-specific mesenchymal cells situated in the perisinusoidal space between LSECs and hepatocytes, known as the space of Disse[71]. Under normal conditions, HSCs remain quiescent, primarily contributing to the storage of vitamin A-containing metabolites[72]. Following liver injury, HSCs become activated and differentiate into collagen type I-producing myofibroblasts, a key event in the onset and progression of hepatic fibrosis[73]. Given the central role of HSC activation in hepatic fibrosis, efforts to counteract these fibrogenic processes have been explored. For instance, intraperitoneal administration of gliotoxin for HSC depletion demonstrated significant alleviation of diet-induced steatosis and hepatic fibrosis in mice[74]. Furthermore, a study utilizing HSC-specific lecithin-retinol acyltransferase-Cre transgenic mice to deplete HSCs emphasized that HSCs should be considered a primary cellular target for antifibrotic therapy[73].

In the context of APAP-induced ALI, HSCs are known to play diverse roles. While they initially contribute to tissue repair, prolonged activation can result in chronic fibrosis and ultimately impair liver function. Therefore, the activation of HSCs and the metabolic changes that follow are critical in determining whether liver damage in ALI will progress or resolve. Reduction of activated HSCs through gliotoxin treatment exacerbated APAP-induced ALI and inhibited hepatocyte proliferative capacity by increasing the number of CD45-positive cells, which are associated with severe liver damage[75]. Furthermore, early activated HSC-derived paracrine factors (HSC-CM) were demonstrated to decrease hepatocellular death and downregulate systemic inflammation in APAP-treated mice[76]. These findings were further validated by in vitro assessments, which showed that HSC-CM ultimately alleviates APAP-induced ALI via the phosphoinositide 3-kinase/Akt signaling pathway.

Contrary to this hepatoprotective role, a study using transgenic mice found that HSC depletion alleviated APAP-induced ALI by modulating the expression of interferon beta (IFN-β) and reducing oxidative stress through the IFN-β/IFN regulatory factor 1 signaling pathway[77]. Moreover, interactions between HSCs and immune cells, such as KCs, amplify the inflammatory responses, further exacerbating liver damage[78]. Early activated HSCs promote the release of extracellular vesicles, which induce the secretion of the pro-inflammatory M1 phenotype in KCs[79]. Given these various roles, targeting the interactions between HSCs and other hepatic cells could offer a strategic approach for managing ALI progression or ALI-associated liver regeneration.

LSECs

LSECs, specialized endothelial cells positioned between blood and hepatocytes, play a prominent role in maintaining overall liver homeostasis[80]. Their distinct morphology, characterized by fenestrae and the absence of a basal lamina, sets LSECs apart from other endothelial cell types in the body[81]. This structure not only facilitates molecular exchange but also supports liver regeneration and immune tolerance[80].

Damage to LSECs during ALI can impair the liver’s ability to maintain vascular integrity and may lead to increased oxidative stress, further exacerbating hepatocyte injury[80]. Furthermore, impaired LSEC function disrupts their role in immune tolerance, which can lead to an excessive inflammatory response[82]. Therefore, the health and integrity of LSECs are pivotal in modulating the balance between inflammation and tissue repair during ALI. Recent study has reported that both human and murine LSECs express CYP2E1, albeit at lower levels than hepatocytes, and demonstrate significant upregulation following ethanol treatment[83]. CYP2E1 is involved in metabolizing APAP, ultimately generating the toxic intermediate NAPQI[5]. These series of findings collectively suggest that LSECs may modulate hepatotoxicity in APAP-induced ALI via their involvement in CYP2E1 expression.

LSECs express a variety of specific proteins that reflect their distinct structural and functional roles within the liver. One such protein, von Willebrand factor (VWF), a platelet-adhesive glycoprotein localized to LSECs, has been reported to have significantly elevated plasma levels in patients with ALF[84]. Moreover, a deficiency of VWF in mice has been shown to accelerate recovery from APAP-induced ALI[85]. These findings indicate that VWF expression in LSECs is closely linked to the progression of ALI. Similarly, stabilin (STAB) is a multifunctional scavenger receptor predominantly expressed in LSECs and the spleen, where it plays a critical role in homeostatic and clearance functions[86]. A recent study demonstrated that the STAB2 promoter allows LSEC-specific transgene expression via lentiviral vector delivery, facilitating long-term expression through induction of immune tolerance[87]. Despite the cytoprotective functions of STAB2, its role in ALI remains unexplored. Therefore, targeting LSEC-specific proteins offers a promising therapeutic strategy for ALI, as it enables precise modulation of liver-specific immune responses and protective functions. This strategy could potentially minimize systemic side effects and enhance liver repair mechanisms.

Granulocytes in the liver

The recruitment of granulocytes during ALI is critical for the immune response. Hepatocyte-derived inflammatory mediators stimulate chemotactic signaling following an injury[88]. These signals are pivotal in directing granulocytes, especially neutrophils, from the bloodstream to the injury site[88,89]. Neutrophils are initial responders that eradicate bacteria and damaged cells[90]. They achieve this by releasing ROS and proteolytic enzymes, which contribute to protecting the tissue from further harm[91]. Supporting this, Chauhan et al[92] reported that the deficiency in C-type lectin-like receptor-2, a platelet activation receptor, enhances recovery from APAP-induced ALI. This improvement results from increased hepatic neutrophil accumulation and elevated phagocytic activity, driven by heightened TNF-α production. Furthermore, research indicates that controlling neutrophil activation is essential to prevent excessive liver damage from APAP[93]. Although these findings emphasize the multifaceted functions of neutrophils in ALI, it must be acknowledged that neutrophil properties vary between species. Substantial differences exist between human and murine neutrophils in both their maturation profiles and expression of cytokines[94]. For example, in a comparative study assessing neutrophil activation during recovery from APAP overdose, both humans and mice showed increased neutrophil activation, yet CD11b upregulation was restricted to neutrophils in mouse peripheral blood[95]. These observations likely reflect interspecies differences and variation in the kinetics and severity of injury, highlighting the importance of careful interpretation when translating findings from murine models to human ALI.

Eosinophils play a larger role in regulating immune responses and promoting the resolution of inflammation[96]. Research has shown that infiltrating eosinophils protect the liver in APAP-induced ALF by releasing IL-4/IL-13 in a cyclooxygenase-dependent manner, using eosinophil-specific IL-4 and IL-13 knockout mice[97]. Also, studies with various toxins causing ALI, including APAP, revealed that IL-33 induces the release of IL-4 from eosinophils, thus driving the production of potent eosinophil chemoattractants[96]. These results collectively underscore the distinct roles of neutrophils and eosinophils in the immune dynamics of ALI, highlighting the therapeutic potential of specifically targeting these cells to aid liver recovery.

T cells or B cells

The preclinical model of APAP-induced hepatotoxicity is an established system for studying immune-mediated liver injury. Although hepatocytes are the primary targets of the toxic metabolite NAPQI, the immune system, particularly T cells and B cells, plays a pivotal role in both exacerbating and mitigating liver damage[98]. Indeed, the complex interaction between innate and adaptive immune responses significantly affects the progression and resolution of APAP-induced ALI[6].

Among the components of the adaptive immune system, T cells and B cells serve as key mediators, contributing to both inflammatory damage and tissue repair through diverse mechanisms. Kim et al[99] proposed that regulatory T cells (Tregs) mitigate liver damage by releasing anti-inflammatory cytokines such as IL-10, thereby modulating inflammatory responses. The depletion of Tregs exacerbates liver injury, underscoring their protective role and emphasizing the significance of adaptive immune responses in controlling liver inflammation and promoting tissue repair during APAP toxicity. Conversely, other T cell subsets, particularly γδ T cells, contribute to the inflammatory process by activating neutrophils, thereby aggravating hepatotoxicity[7]. KCs release cytokines such as IL-23, which activate γδ T cells and enhance neutrophil activity, consequently contributing to further liver damage. Furthermore, emerging evidence suggests that other T cell subsets, including natural killer and natural killer T cells, may also play roles in mediating liver damage through their interactions with immune cells and hepatocytes, although their precise functions are still being investigated[7].

Although B cells are less frequently discussed in the context of APAP toxicity compared to T cells, their role in immune responses during liver injury is increasingly recognized. B cells may contribute to both the inflammatory phase and tissue repair by interacting with other immune cells to regulate the balance between tissue damage and recovery[100]. Evidence from some studies suggests that B cells play a role in adaptive immunity, particularly during the later stages of recovery[98]. Collectively, the roles of T cells and B cells in APAP-induced liver toxicity underscore the critical importance of the adaptive immune system in both exacerbating and mitigating liver damage. These findings suggest that modulating these immune pathways could provide promising therapeutic strategies for the treatment or management of APAP-induced ALI.

THERAPEUTIC APPROACHES

Conventional therapeutic approaches

N-acetylcysteine (NAC) remains the primary treatment for APAP-induced ALI, functioning predominantly by restoring hepatic GSH levels. These levels are profoundly depleted during the metabolic detoxification of NAPQI, which is the highly reactive and toxic metabolite derived from APAP[8]. GSH is critical in counteracting NAPQI-induced toxicity by neutralizing reactive metabolites and protecting hepatocytes from oxidative injury, ultimately supporting liver function and limiting cellular damage[101].

The effectiveness of NAC for treating APAP overdose hinges largely on both the timing of administration and appropriate dosing. Administering NAC within 8 to 10 hours after APAP ingestion is associated with significant reductions in hepatocellular injury and mortality. Established clinical protocols recommend intravenous doses of 100-150 mg/kg and oral doses of 70-140 mg/kg, with both approaches providing equivalent efficacy when started within the optimal therapeutic window[102,103]. Delayed initiation of therapy beyond 24 hours significantly reduces the hepatoprotective effect of NAC and is often linked to progression to ALF and adverse clinical outcomes[102,104]. Such challenges in managing late-presenting cases underscore the critical need for more effective and flexible therapeutic strategies to extend the treatment window.

To address the shortcomings of NAC in treating APAP-induced ALI, researchers have been exploring numerous alternative therapeutic strategies to enhance the efficacy and broaden the treatment window. A prominent approach involves the use of fomepizole (4-methylpyrazole), which is chiefly recognized as an alcohol dehydrogenase inhibitor and is clinically indicated for the treatment of methanol or ethylene glycol poisoning[105]. In the setting of APAP toxicity, its protective effect arises not from its traditional mechanism of action, but from its ability to broadly inhibit CYP-dependent oxidative biotransformation of APAP into the toxic metabolite NAPQI[106]. Evidence indicates that, fomepizole substantially decreases hepatic injury during APAP overdose by inhibiting CYP2E1-catalyzed production of the reactive NAPQI metabolite[107]. Furthermore, several case reports have described hepatoprotective effects resulting from empirical fomepizole administration in APAP overdose cases, supporting its role as an adjunctive agent for APAP toxicity[108,109]. Importantly, given the infrequent adverse effects observed during its use for toxic alcohol poisoning, fomepizole is often incorporated as an additional therapy. Additionally, clinical investigations have started to examine the direct influence of fomepizole on APAP metabolism in human subjects. Kang et al[110] performed a crossover study in healthy adults to assess the modulation of APAP metabolism by fomepizole (ClinicalTrials.gov identifier: NCT03878693). The findings demonstrated that fomepizole administration after a single oral dose of 80 mg/kg APAP led to decreased urinary and plasma concentrations of oxidative APAP metabolites (APAP-cysteine and APAP-NAC). More recently, a randomized controlled trial (ClinicalTrials.gov identifier: NCT05517668; currently recruiting) has commenced to determine the efficacy and safety of combined fomepizole and NAC therapy in patients with APAP overdose.

Beyond pharmacological repurposing strategies, natural products with hepatoprotective properties have gained significant attention for their potential role in mitigating APAP-induced ALI. Silymarin, extracted from the milk thistle plant, has been extensively studied for its ability to scavenge free radicals, stabilize cellular membranes, and prevent lipid peroxidation, offering comprehensive protection to liver cells[111]. Preclinical studies have consistently shown that silymarin delivers potent hepatoprotective effects in APAP-induced liver toxicity in mice by modulating oxidative stress and enhancing antioxidant enzyme activity[111,112]. Similarly, curcumin, a polyphenolic compound derived from turmeric, exhibits robust anti-inflammatory and antioxidant properties by modulating key signaling pathways, including the extracellular signal-regulated kinase pathway[113]. Intraperitoneal administration of curcumin has significantly alleviated APAP-induced liver toxicity in mice[114]. Recent advancements in developing curcumin nanoparticles have improved its low solubility and bioavailability, with studies indicating that curcumin nanoparticles provide superior hepatoprotective effects compared to pure curcumin[115,116]. Despite these promising findings, the current body of evidence is largely limited to experimental models, and the clinical applicability of these natural compounds remains uncertain due to challenges such as poor bioavailability, variability in formulation, and limited human trials. Consequently, their future in clinical practice remains speculative until more robust human data are available.

Cell-based therapies

In recent years, cell-based therapies have emerged as a promising approach for treating APAP-induced ALI, particularly when conventional treatments such as NAC are inadequate. Inspired by the liver's remarkable regenerative capacity, therapies utilizing stem cells and other cell types aim to enhance liver regeneration and repair[117]. Experimental models of ALI have shown promising results for cell-based therapies. For instance, in an animal model of ALI, the injection of activated syngeneic primary myeloid macrophages reduced hepatocyte necrosis and neutrophil infiltration, decreased circulating proinflammatory factors, and stimulated the proliferation of hepatocytes and endothelial cells[6]. Mesenchymal stem cells, when injected into a mouse model of APAP-induced ALI, alleviated hepatocyte necrosis by secreting hepatocyte growth factor[118].

Hepatocyte transplantation is being explored as an alternative approach to treat hepatotoxicity resulting from APAP overdose. This method involves isolating hepatocytes from a healthy liver and administering them to a damaged liver to replace lost cells and restore liver function[119]. Transplanted hepatocytes can integrate into the recipient’s liver tissue, performing essential liver functions and aiding hepatic recovery. One study demonstrated that hepatocytes transplanted from healthy mice into the portal vein of mice with APAP-induced ALI successfully engrafted into the liver and spleen, though no significant reduction in alanine aminotransferase levels was observed[120]. Another study using encapsulated immortalized human hepatocytes transplanted into mice effectively mitigated liver damage[121]. Viswanathan et al[122] reported that hepatocytes transplanted via the peritoneal cavity in a mouse model of ALF facilitated DNA damage repair and promoted liver regeneration, underscoring the therapeutic potential of this innovative cell-based approach. However, hepatocyte transplantation faces significant challenges. As hepatocytes are derived from donor tissue, they may be recognized as foreign by the recipient’s immune system, leading to immune rejection[123]. Immunosuppressants are commonly used to manage this response, but prolonged use can compromise overall immune function. Furthermore, ensuring the survival and functionality of transplanted hepatocytes in compromised liver tissue continues to be a critical concern. Ongoing research is concentrating on strategies to enhance cell survival, integration, and functionality, vital for the success of hepatocyte transplantation as a therapeutic option.

CHALLENGE AND PERSPECTIVES

APAP-induced ALI continues to be a significant clinical challenge due to its complex pathophysiology involving diverse cellular populations. The dynamic interplay among hepatocytes, immune cells including KCs, LSECs, and HSCs orchestrates the progression of liver injury and repair (Figure 3). Gaining a comprehensive understanding of these interactions is critical for advancing the diagnosis and treatment of ALI.

Figure 3 Overview of hepatic cell-type-specific responses in acetaminophen-induced acute liver injury. This schematic depicts the coordinated actions of hepatic cell populations under both homeostatic conditions and following acetaminophen [N-acetyl-p-aminophenol (APAP)]-induced acute liver injury. An APAP overdose induces mitochondrial oxidative stress in hepatocytes, stimulates activation of hepatic stellate cells, elicits pro-inflammatory responses from Kupffer cells and monocyte-derived macrophages, and causes dysfunction of liver sinusoidal endothelial cells. The roles of adaptive immune cells, including T and B cells, are highlighted in the modulation of inflammation and tissue repair. This comprehensive illustration underlines the importance of multicellular dynamics governing both the progression and resolution phases of APAP-induced liver injury. APAP: N-acetyl-p-aminophenol (Acetaminophen); CCL2: Chemokine (C-C motif) ligand 2; HSCs: Hepatic stellate cells; IL: Interleukin; KCs: Kupffer cells; LSECs: Liver sinusoidal endothelial cells; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor-alpha; VWF: Von Willebrand factor. This figure was created using BioRender.

Despite recent advances in understanding cell-type specific roles in ALI, several crucial knowledge gaps remain. The precise molecular signals controlling the transition from injury to repair are not fully understood. Furthermore, the variability in responses among different patient populations underscores the need for studies on genetic and epigenetic factors influencing cellular behavior in ALI.

To address these challenges, innovative approaches such as single-cell RNA sequencing, advanced imaging techniques, and organ-on-a-chip models hold promise. These technologies have the potential to unravel the intricate cellular networks involved in ALI, offering unprecedented insights into the spatiotemporal dynamics of liver injury. Moreover, integrating these findings with systems biology approaches could pave the way for the development of personalized medicine strategies to mitigate ALI risk and improve patient outcomes.

CONCLUSION

In conclusion, a deeper investigation into the diverse roles of cellular populations in APAP-induced ALI is imperative for devising effective therapeutic interventions. By bridging existing knowledge gaps and leveraging cutting-edge technologies, future research has the potential to revolutionize our understanding and management of this pervasive clinical problem. Through these efforts, the ultimate goal of reducing the global burden of DILI can be realized.