Kupffer cells: Orchestrators of liver physiology

Abstract

As the central metabolic organ, the liver coordinates fundamental biological processes through its specialized cellular architecture and regulatory networks, encompassing metabolism, immunity, and regeneration. Kupffer cells (KCs), the liver-resident macrophages, exhibit functional heterogeneity beyond classical polarization paradigms. Currently, multiple classification systems for KCs have been established utilizing distinct surface markers. However, there is no systematic theoretical framework for the classification of KCs. The strategic positioning of KCs within the hepatic Disse space enables intricate intercellular communication networks with neighboring hepatocytes for coordinated physiological regulation. Their functional plasticity critically regulates systemic iron and metabolic homeostasis, with KC-driven metabolic reprogramming directly influencing hepatic pathophysiology. Furtherly, KC activity shows spatiotemporal regulation by circadian rhythms and nutrient signals, reshaping the liver microenvironment to affect function. This review summarizes advances in liver macrophage biology, highlighting the classification challenges of KCs and their roles in hepatic physiology. Additionally, we discuss how circadian rhythms, aging, diet, and exercise dynamically influence KC functionality, which provides a framework to interpret their regulatory logic and dysfunction in disease.

1. Introduction

As the largest parenchymal organ in the human body, the liver accounts for approximately 2% of body weight, and plays a central role in key physiological processes such as metabolism, detoxification, and immune regulation.¹ The liver has a complex anatomical structure, composed of numerous repeating hepatic lobules, receiving oxygenated blood through the hepatic artery and nutrient-rich blood from the intestines via the portal vein.² The hepatic lobule contains various cell types, such as epithelial cells (hepatocytes and cholangiocytes), Kupffer cells (KCs), circulating monocytes, mesenchymal cells (hepatic stellate cells), and liver sinusoidal endothelial cells (LSECs).³

In healthy conditions, macrophages are the most abundant immune cells in the liver, accounting for around 20%–25% of non-parenchymal cells. Their functions are crucial for maintaining liver homeostasis and systemic metabolic balance.⁴ Hepatic macrophages not only regulate iron metabolism and glucose-lipid metabolism, but also play a key role in liver regeneration, tissue repair, and sensing and clearing intestinal pathogens. Under homeostatic conditions, liver resident macrophages are primarily composed of KCs, which are located in the liver sinusoids. Their superior anatomical position allows them to sense and respond to pathogens and metabolites entering the liver via the bloodstream.¹ KCs are in close contact with hepatic stellate cells (HSCs), hepatocytes, and LSECs, forming a complex intercellular network that maintains liver homeostasis through synergistic interactions. The properties of KCs are co-regulated by stellate cells, endothelial cells, and hepatocytes within their niche, thereby establishing liver-specific immune features.⁵

Therefore, understanding the mechanisms underlying the role of KCs in maintaining liver homeostasis, particularly their collaboration with other hepatic cells, provides a crucial foundation for developing novel strategies targeting macrophage regulation in liver disease treatment. In this review, we outline the major subtypes, origins, and heterogeneity of liver macrophages, particularly KCs with emphasis on their functions in modulating hepatic physiological processes and interacting with other liver cell populations.

2. Origin and heterogeneity of KCs

Since Elie Metchnikoff first proposed the concept of "macrophages" in the late 19th century, their origin and development have remained central topics in immunological research. Liver macrophages can be subdivided based on their origins into yolk sac-derived Kupffer cells (e.g., YS-KCs), bone marrow monocyte-derived Kupffer cells (e.g., moKCs/BM-KCs), and non-Kupffer resident macrophages (e.g., liver capsule-associated macrophages and lipid-associated macrophages).6, 7, 8

In the state of homeostasis, KCs represent the predominant subset of hepatic macrophages, originating from embryonic yolk sac progenitors. They are sustained by self-renewal mechanisms and operate independently of blood monocytes.⁹ Recent advancements in high-throughput single-cell sequencing have uncovered functional and phenotypic heterogeneity within KCs, notably distinguishing two subpopulations: KC1 (CD206⁻ESAM⁻) as the principal subset and KC2 (CD206⁺ESAM⁺) as the secondary subset.¹⁰ The identification of specialized hepatic macrophage subtypes has significantly enhanced our comprehension of the liver immune microenvironment. For instance, liver capsule macrophages (LCMs), located in the hepatic serosal layer, primarily function to combat peritoneal-derived bacterial infections.^1,11 LCMs express canonical macrophage markers (e.g., CD64 and F4/80) but lack expression of TIMD4 and CLEC4F.¹¹ Under inflammatory or hepatic injury conditions, bone marrow-derived monocytes migrate to the liver and differentiate into moKC. These cells exhibit elevated expression of CD11b and Ly6C, demonstrating enhanced pro-inflammatory capabilities compared to KCs.^8,12

Furthermore, during the progression of liver disease, specific KC subpopulations emerge. These primarily consist of a transitional macrophage population derived from monocytes, which partially acquire KC characteristics, often as a consequence of the depletion of the resident KC pool. For instance, in drug-induced acute liver injury, the originally stable KC community undergoes dynamic changes. Single-cell RNA sequencing has identified a significant expansion of a KC subpopulation expressing TREM2 and C1q. These cells are localized at the periphery of necrotic areas, where they function to clear cellular debris and contribute to tissue repair.¹³ In the context of alcohol-associated cirrhosis (AC), several distinct macrophage subpopulations have been identified, including C1Q⁺, S100A8⁺, APOE⁺, TNF⁺, and VSIG4⁺ macrophages, each exhibiting a unique gene expression signature. Among these, the C1Q⁺ macrophage population is notably expanded in patients with severe alcoholic hepatitis (sAH). Their transcriptomic profile displays a hybrid phenotype, combining features of both resident KCs and disease-associated macrophages. They exhibit high expression of macrophage receptor with collagenous structure (MARCO) but low expression of TIMD4, indicative of a transitional phenotype between circulating monocytes and tissue-resident macrophages. These cells also demonstrate potent phagocytic capacity.¹⁴

The polarization state of KC determines their functional characteristics. Conventionally, KC are classified into classically activated M1-type and alternatively activated M2-type based on their polarization states. M1 macrophages exhibit a pro-inflammatory phenotype, rapidly obtaining energy through glycolysis and producing high levels of inflammatory cytokines and nitric oxide (NO).¹⁵ In contrast, M2 macrophages demonstrate anti-inflammatory and tissue-repair properties, relying on fatty acid oxidation and mitochondrial oxidative phosphorylation to sustain their functions.^6,15 Macrophage polarization is dynamically regulated by multiple factors. Under different dietary conditions, KCs display distinct polarization patterns: high-fat diet (HFD) promotes M1 polarization, whereas fasting or intermittent fasting favors M2 polarization.16, 17, 18 Additionally, physiological factors including hormonal levels and circadian rhythms can significantly influence macrophage phenotypes.^19,20

However, this M1/M2 classification model has been found to be inadequate. Recent advances in single-cell sequencing have revealed more refined classifications.¹⁷ For instance, macrophages can be subdivided into pro-inflammatory and immunoregulatory subsets using biomarkers such as MARCO and other genetic markers.^5,21 Notably, KCs in specific microenvironments exhibit high expression of ferroportin (FPN), primarily involved in erythrocyte metabolism and iron recycling.²² It should be noted that single-cell analyses require careful consideration of technical artifacts, including signal contamination from adjacent cells. To overcome these limitations, an integrated approach combining RiboTag technology, single-nucleus RNA sequencing, and high-resolution imaging flow cytometry is recommended for comprehensive characterization.²³ Table 1 summarizes the classification, function, and distribution of liver macrophages.^10,12,23, 24, 25, 26, 27

Table 1.

Classification, function, and distribution of liver macrophages.

Class	Markers	Functions	Spatial distribution	Reference
KC1 (Nature, 2024)				24
MP1	CD206– ESAM– MARCO–	Pro-inflammation	Widely distributed
MP2	CD206– ESAM– MARCO+	Inhibiting neutrophil adhesion Immune tolerence	Periportal vein zones
KC2 (Nature, 2024)	CD206+ ESAM+ MARCO–	–	–
Long-term resident KC	FOLR2+ VSIG4+ CLEC4F+ (∗) CLEC2+ (#) TIMD4+ (∗)	–	Periportal and middle zones (mouse) Middle zone (human)	25
moKC	TIMD4–(∗)	–	At PV and CV expressing Ccr2 and Chil3
Non-KC macrophages
Liver capsule macrophages and CV CD207+ macrophages	CD207+ CX3CR1+	–	–
BD-LAM	GPNMB+ CD68+VSIG4–	LPS tolerance Protective role in NAFLD	PV peritubular bile ducts
KC1 (Immunity, 2021)	CD206– ESAM–	–	–	10
KC2 (Immunity, 2021)	CD206+ ESAM+	Promoting diet-induced hepatic steatosis	–
Inflammatory macrophages	MARCO– CD74+	Pro-inflammation	Pericentral	23, 26
Non-inflammatory macrophages (KC)	MARCO+ CD5L+ VSIG4+	Immune tolerance	Periportal
YS-derived KC	MARCO+ TIMD4+ CD163+	Ac-LDL uptake	–	12
BM monocyte-derived KC	MARCO– CLEC4F+	Bacterial phagocytosis	–
KC	CD68+ (#) CD14+ (#) CD64+ (∗) F4/80+ (∗)	Lipid and iron homeostasis	–	27
Liver capsule macrophages and CV macrophages	CLEC4F+ (∗) VSIG4+ (∗) CX3CR+ (∗) CD207+ (∗)	–	Liver capsule and CV
BD-LAM	VSIG4– TREM2+ GPNMB+ SPP1+ FOLR2–(#)CD141+ (#) CD26+ (#) CLEC4F–(∗)	–	Close proximity to bile ducts

Abbreviations: Ac-LDL, acetylated low density lipoprotein; BD-LAM, lipid-associated macrophage at the bile duct; BM, bone marrow; CV, central vein; KC, Kupffer cell; LPS, lipopolysaccharide; MARCO, macrophage receptor with collagenous structure; moKC, monocyte-derived KC; NAFLD, non-alcoholic fatty liver disease; PV, portal vein; YS, yolk sac.

^∗, mouse; ^#, human; unmarked, shared by human and mouse.

3. Metabolic sentinels of the liver: KCs at the homeostasis nexus

As the metabolic regulatory hub, the liver plays an irreplaceable role in modulating various metabolic processes, particularly iron metabolism. Serving as the central regulator of systemic iron balance, the liver maintains iron homeostasis through synthesis and secretion of iron regulatory factors such as hepcidin.¹⁷ Furthermore, the hepatic tissue microenvironment undergoes dynamic adaptation in response to physiological states, including dietary patterns, physical activity, circadian rhythms, and aging. Throughout these adaptive processes, KCs demonstrate remarkable metabolic flexibility and phenotypic plasticity, enabling prompt responses to systemic changes and effective regulation of hepatic metabolic and inflammatory responses.²⁸

3.1. Iron metabolism

Iron is an essential element required for maintaining various vital biological processes, playing pivotal roles in oxygen transport, mitochondrial respiration, nucleic acid synthesis, and host defense mechanisms.²⁶ The hepcidin, synthesized and secreted by the liver, can regulate systemic iron reserves by binding to FPN, the principal cellular iron exporter, thereby inhibiting iron efflux from cells.¹⁰

In the liver, hepatocytes are responsible for iron storage and hepcidin secretion, while KCs primarily participate in systemic iron metabolism through phagocytosis of senescent or damaged erythrocytes.^18,29 KC-mediated phagocytosis is strictly regulated by erythrocyte senescence levels, with phagocytic mechanisms being initiated only when erythrocytes reach specific senescence thresholds.³⁰ Healthy erythrocytes evade unintended phagocytosis through CD47-SIRPα receptor interactions between erythrocyte surfaces and KCs. In contrast, senescent erythrocytes expose phosphatidylserine (PS) on their surfaces, enabling recognition and clearance via PS receptors (TIM4, AXL, and MERTK) highly expressed on KCs.³¹ LSECs facilitate this process by capturing damaged erythrocytes through PS-dependent mechanisms utilizing stabilin-1 and stabilin-2, subsequently transferring them to KCs to enhance erythrophagocytic efficiency.³² During phagocytosis, KCs internalize erythrocytes to form phagosomes, which subsequently fuse with lysosomes to create phagolysosomes. Within phagolysosomes, erythrocytes and their hemoglobin undergo degradation, with liberated heme being transported to the cytoplasm via HRG1 for subsequent metabolic processing.³³

Beyond erythrophagocytosis, KCs also internalize hemoglobin-haptoglobin complexes via the CD163 receptor, or sequester circulating free heme through the low-density lipoprotein receptor-related protein (LRP) receptor.^34,35 Additionally, KCs can acquire heme from the hemopexin (Hpx) complex, a non-hemoglobin heme-binding protein.³⁶ The iron released into the cytoplasm is partially stored within ferritin, establishing a dynamic iron reservoir. During cellular iron demand, KCs mobilize stored iron through the nuclear receptor coactivator 4 (NCOA4)-dependent ferritinophagy pathway.^31,37,38 The remaining iron is exported as ferrous iron (Fe²⁺) through FPN, the sole known mammalian iron exporter.³⁹ Extracellular Fe²⁺ is oxidized to ferric iron (Fe³⁺) by copper-containing oxidases (e.g., ceruloplasmin), then bound to transferrin (TF) for systemic circulation, facilitating erythropoiesis and other physiological processes.^22,40 Furthermore, KCs regulate their iron export through hepcidin, a hepatocyte-derived iron-regulatory hormone, thereby contributing to hepatic iron metabolism. Hepcidin inhibits macrophage iron release into plasma by binding to FPN through two mechanisms: blocking FPN's extracellular conformational opening and inducing internalization of the hepcidin-FPN complex under high TF conditions.^39,41,42 Hepcidin expression is modulated by multiple tissue microenvironment signals including hypoxia, TF saturation, paracrine factors, and inflammatory status.²² Hypoxia-inducible factors (HIFs), along with HSCs-derived hepatocyte growth factor (HGF) and cholangiocyte-produced epidermal growth factor (EGF), enhance KC iron release by suppressing hepatocyte hepcidin expression.^40,43 Under iron-replete conditions, LSECs sense iron overload and secrete bone morphogenetic protein 6 (BMP6), which upregulates hepatocyte hepcidin production to modulate KC-mediated hepatic iron homeostasis.⁴⁴

Studies have revealed that KCs also participate in systemic iron metabolism through alterations in their polarization states. On one hand, intracellular iron levels influence macrophage phenotypes. Elevated iron concentrations activate pro-inflammatory M1 macrophages while suppressing anti-inflammatory M2 macrophages, thereby modulating the metabolic status of KCs.²⁸ On the other hand, research demonstrates that under inflammatory conditions, KC polarization shifts regulate hepcidin synthesis in hepatocytes, consequently affecting cellular iron efflux. Excess iron generates reactive oxygen species (ROS) and lipid peroxidation products (e.g., 4-hydroxy-2-nonenal) via the Fenton reaction, driving KC polarization toward the pro-inflammatory M1 phenotype and triggering substantial interleukin-6 (IL-6) secretion.⁴⁰ KC-derived IL-6 upregulates hepcidin synthesis and secretion in hepatocytes through the IL-6-IL6R-JAK-STAT signaling pathway, which further inhibits iron export from KCs, ultimately reducing plasma iron levels.⁴⁴

3.2. Nutritional and exercise interventions

In recent years, an increasing number of studies have focused on the effects of different dietary states on organismal metabolism. The liver, as a key organ for metabolic regulation, changes its local environment in response to dietary shifts. KCs, which highly express lipid-related transcription factors (Table 2), can adapt to these changes through their strong metabolic and functional plasticity.45, 46, 47, 48 They play an important role in controlling glucose and lipid metabolism. During periods of food shortage, the body initiates various metabolic pathways to ensure energy supply and cellular repair. During fasting and intermittent fasting, KCs exert anti-inflammatory and reparative effects through metabolic reprogramming and phenotypic transformation, aiding in the restoration of hepatic homeostasis. KCs can promote fatty acid oxidation and ketogenesis in hepatocytes via the tumor necrosis factor (TNF) signaling pathway, thereby maintaining ketone body levels and regulating overall glucose and lipid metabolism balance.¹⁸ Intermittent fasting can activate the macrophage migration inhibitory factor (MIF) and AMP-activated protein kinase (AMPK) signaling pathways, regulating autophagy and apoptosis, which helps to clear excess lipids and damaged organelles, thereby reducing hepatic lipid accumulation.⁴⁷ Additionally, intermittent fasting also reduces the expression of pro-inflammatory factors (such as TNF-α, IL-6, and MCP-1) induced by the HFD, while preserving the levels of anti-inflammatory factors (such as IL-10 and TGF-β1).⁴⁸

Table 2.

Metabolic transcription factors expressed in Kupffer cells.

Key factors	Target genes	Function	Reference
LXRα	ABCA1 and ApoE	Promoting lipid metabolism	45
RXRα	ABCG1, CYP27A1, and NTCP	Regulating cholesterol transport	46
PPARδ/β	STAT6	Promoting M2 polarization	47
PPARγ	NF-κB	Promoting M2 polarization	48

Abbreviations: ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; ApoE, apolipoprotein E; CYP27A1, sterol 27-hydroxylase; LXRα, liver X receptor alpha; NF-κB, nuclear factor-kappaB; NTCP, sodium taurocholate co-transporting polypeptide; PPAR, peroxisome proliferator-activated receptor; RXRα, retinoid X receptor alpha; STAT6, signal transducer and activator of transcription 6.

Although the health benefits of fasting and intermittent fasting are significant, modern dietary habits often run counter to fasting patterns. The consumption of large amounts of high-sugar and high-fat foods, especially excessive nutrients, often places an additional burden on the liver. Overconsumption of nutrients can alter the liver's metabolic functions, thereby significantly changing the hepatic microenvironment. In contrast to the anti-inflammatory phenotype under fasting conditions, excessive nutrient intake induces KCs to switch to a pro-inflammatory phenotype, a mechanism associated with the synergistic effects of free fatty acids and microbial signals. In the HFD, KCs promote the production of ROS and pro-inflammatory factors (such as TNF-α and IL-6) by sensing free fatty acids (FFAs) and bacterial lipopolysaccharide (LPS) signals, exacerbating inflammation and lipid accumulation.49, 50, 51, 52 Additionally, KCs activate stellate cells by secreting fibrogenic factors (such as TGF-β and PDGF), accelerating extracellular matrix (ECM) deposition and fibrosis progression. Beyond HFDs, excessive fructose intake also affects KC function through unique metabolic pathways. When fructose intake is excessive, the resident KC population significantly decreases, while promoting the recruitment of transitional monocytes.⁵³ KCs regulate anti-inflammatory and repair functions through the pentose phosphate pathway (PPP) in fructose metabolism, but the accumulation of fructose metabolism byproducts (such as acylcarnitines and diacylglycerols) drives the expression of pro-inflammatory factors, exacerbating metabolic imbalance.⁵³ A study revealed that nutritional overload (e.g., elevated glucose and other metabolite concentrations) or certain microbial products, including LPS, can induce KCs to produce IL-1β, which suppresses fibroblast growth factor 21 (FGF21) expression in hepatocytes.⁵⁴ As an endocrine hormone, FGF21 promotes adipose tissue lipolysis, hepatic ketogenesis, and circulating β-hydroxybutyrate levels. Inhibition of FGF21 in hepatocytes may consequently lead to hepatic lipid accumulation.⁵⁴

However, dietary factors are not the only elements influencing liver metabolism. In addition to diet, another critical factor in lifestyle—exercise—also profoundly affects the function of liver immune cells, particularly KCs. Research indicates that moderate exercise can regulate the function of KCs, improve the liver's immune response, and promote the restoration of metabolic homeostasis. Preoperative exercise (4-week aerobic training) induces metabolic reprogramming through the tricarboxylic acid (TCA) cycle, transforming the phenotype from pro-inflammatory to anti-inflammatory. Furthermore, during exercise, myocytes release a significant amount of high mobility group box 1 (HMGB1) signaling molecules, which can promote the expression of immunoresponsive gene 1 (IRG1) in KCs through Toll-like receptor 4 (TLR4), significantly increasing the level of itaconate in KCs. As an anti-inflammatory metabolite, itaconate effectively inhibits the release of pro-inflammatory cytokines (e.g., IL-1β and TNF-α) and promotes the production of anti-inflammatory cytokines (e.g., IL-10 and IL-1Ra), thereby ameliorating the hepatic microenvironment.⁵⁵

3.3. Circadian regulation and aging

The function and metabolism of KCs are not only directly influenced by dietary status but also regulated by the circadian clock. Core circadian genes (e.g., BMAL1, REV-ERBα, and PER/CRY) are expressed in KCs, where they participate in modulating TLR expression, KC polarization states, and cytokine secretion, thereby directly affecting KC functionality.⁴⁹ For instance, genetic ablation of Bmal1 in KCs promotes glycolytic pathways, consequently accelerating M1 macrophage polarization.⁵⁰ Furthermore, diurnal rhythms regulate KC functions by altering their population dynamics and proteomic profiles. Studies indicate elevated daytime expression of TLR4 signaling components (e.g., Myd88, Nf-κb, and Irak4) in KCs compared to nighttime levels.⁵¹ Conversely, circadian rhythms indirectly modulate KC activity via hepatocyte-derived factors. Hepatocytes rhythmically secrete mediators like colony-stimulating factor 1 (CSF1), which governs KC proliferation and activation.⁵¹ Although hepatocyte-specific deletion of core clock components REV-ERBα/β doesn't affect KC-intrinsic clock genes, it disrupts developmental and metabolic rhythms, including CSF1 signaling, ectonucleotide pyrophosphatase/phosphodiesterase 6 (Enpp6) gene expression, and C22:4 lipid level variations.²⁰ These findings suggest circadian regulation of macrophage homeostasis likely involves metabolic crosstalk between hepatocytes and KCs. While the importance of circadian rhythms in physiological regulation is increasingly recognized, current study exploring their impact on KCs remains limited, warranting further investigation.

The impact of aging on KCs manifests through multifaceted dimensions, including quantitative expansion, alterations in gene expression profiles, and diminished biological activity. Research has demonstrated upregulation of pro-inflammatory regulatory genes such as TNF, CCL2, CXCL2, and CXCL10 in senescent liver KCs, concurrent with downregulation of anti-inflammatory mediators including HP, CHIL3, and IL-18.⁵² Furthermore, aged livers exhibit increased KC population density and elevated RNA expression of the inflammatory cytokine IL-6.⁵³ Augmented IL-6 secretion by KCs stimulates hepatic hepcidin production, which subsequently impairs KC-mediated iron efflux, leading to iron deposition and consequent reduction in biological activity.⁵⁶

Beyond alterations in inflammatory cytokine expression, aging modulates hepatic metabolic homeostasis through KC-specific regulation of metabolic genes such as forkhead box O1 (FOXO1) and arachidonate 15-lipoxygenase (ALOX15). FOXO1 plays a pivotal role in transcriptional control of gluconeogenic enzymes, notably glucose-6-phosphatase (G6Pase) and pyruvate dehydrogenase kinase 4 (PDK4).⁵⁵ During senescence, murine hepatic FOXO1 activity is markedly potentiated, driving excessive gluconeogenesis and lipid metabolism dysregulation, which exacerbates insulin resistance and promotes hepatic steatosis.^52,57

The attenuated regenerative capacity of the aged liver may primarily stem from its heightened susceptibility to fibrosis development, which exacerbates hepatic inflammation and injury. On one hand, increased CXCL2 secretion by KCs during hepatic aging promotes neutrophil migration to senescent livers through the CXCL2-CXCR2 axis. These infiltrating neutrophils subsequently stimulate neutrophil extracellular trap (NET) formation via TNF-α and IL-1β secretion, thereby aggravating hepatic damage.⁵⁸ Conversely, aging is associated with diminished pericentral endothelium-derived C-kit expression (a receptor tyrosine kinase). This C-kit deficiency or functional impairment may disrupt LSEC homeostasis and enhance the recruitment of KCs and neutrophils via CXCR4 signaling pathways, ultimately intensifying hepatic inflammation.⁵⁹ Furthermore, LSECs in healthy livers actively suppress HSC activation. Age-related alterations in LSEC homeostasis compromise this inhibitory function, leading to HSC activation and subsequent impairment of hepatocyte regeneration.⁵⁹

4. KCs: key cell in hepatic regeneration and tissue repair

The liver is the only organ capable of ensuring its mass remains in balance with body weight through a regenerative mechanism. Unlike the damage adaptation mechanisms of other solid organs (such as the lungs, kidneys, and pancreas), the liver can restore its function to 100% through complete regeneration.⁶⁰ This process requires complex intercellular collaboration, in which liver macrophages (particularly KCs) play a crucial role in tissue repair and the maintenance of regeneration.61, 62, 63, 64, 65

Following liver injury, KCs undergo significant phenotypic changes, exhibiting specialized functions with regenerative properties and releasing a large number of regeneration-related factors, such as IL-6 and HGF. These factors effectively promote liver repair and regeneration by enhancing hepatocyte proliferation and survival. Studies have shown that KCs enhance hepatocyte survival and promote hepatocyte proliferation through the IL-6-STAT3 pathway and the DP1-Wnt2-β-catenin pathway, while also inducing the expression of anti-apoptotic genes Mcl-1 and Bcl-2, thereby improving hepatocyte adaptability to the injury environment.66, 67, 68, 69 A recent study discovered that perivenous macrophages can regulate hepatocyte proliferation in distant zones through glutamate-mediated paracrine signaling, thereby promoting liver regeneration.⁷⁰ Additionally, KCs undergo significant metabolic reprogramming during the regeneration process, exhibiting dynamic shifts between the M1 and M2 phenotypes. The M1 phenotype primarily participates in the early pro-inflammatory response, while the M2 phenotype plays a role in the later anti-inflammatory and repair phases. Mesenchymal stem cells can alleviate inflammation and accelerate liver injury repair by inhibiting M1 polarization and promoting M2 polarization.⁷¹

In addition to enhancing their own functions through phenotypic transformation, KCs further promote liver repair and regeneration through interactions with other liver cells. During liver regeneration, KCs collaborate with other liver cells to regulate the composition ratio of the liver macrophage pool. Firstly, KCs attract monocytes to the damaged site by releasing monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and induce LSECs to express intercellular adhesion molecule-1 (ICAM-1), which interacts with monocytes to promote vascular sprouting and repair.⁷² Activated HSCs recruit monocytes to the injury site by secreting chemokines (such as CCL2, CCL4, and CX3CL1), and activated LSECs and HSCs further promote monocyte recruitment by upregulating adhesion molecules and endothelial migration-related receptors.^5,73 Once these monocytes enter the liver, they receive signals from LSECs, including the Notch ligand DLL4 and TGF-β, as well as signals from HSCs, such as TNF-α and IL-1β, which activate the KC-specific transcriptional program.⁷⁴ During this process, monocytes upregulate the expression of liver-associated transcription factor inhibitor of DNA-binding 3 (ID3) under the regulation of hepatocytes, while LSECs and HSCs synergistically promote liver X receptor alpha (LXRα) expression through the Notch-BMP pathway.⁵ The newly formed monocyte-derived KCs receive signals from LSECs, including TGF-β, DLL4, and LXR ligands, maintaining their specific phenotype and function.⁷⁵ As the injury repair progresses, CD68⁺ KCs replenish the original cell population through local proliferation, while CD11b⁺ monocytes gradually transform into typical F4/80⁺ KCs. Ultimately, these new KCs play multiple roles in liver regeneration, including synthesizing ECM components (such as collagen) to support tissue structure reconstruction, and ensuring the restoration of complete liver function by regulating hepatocyte proliferation and ECM remodeling at the end of regeneration.⁶⁰

As liver regeneration progresses, KCs not only regulate their own composition ratio but also optimize the regenerative environment of the liver through various mechanisms. Under physiological conditions, HSCs are the primary source of the ECM, maintaining a quiescent state in the liver and secreting ECM proteins to sustain liver homeostasis.^76,77 LSECs, as the most abundant non-parenchymal cells in the liver, support liver regeneration by coordinating vascular growth and repair, and secrete NO to maintain the quiescent state of KCs.^78,79 However, during the initial phase of liver injury, the existing KCs are rapidly depleted, and their depletion simultaneously activates HSCs and LSECs. KCs secrete pro-inflammatory factors such as IL-1β, TNF-α, and CCL2, as well as TGF-β, which promote the proliferation and activation of HSCs, leading to the release of more collagen and ECM components, thereby remodeling the tissue structure.⁸⁰ Additionally, KCs express vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), enhancing the proliferation of LSECs and promoting liver regeneration.⁸¹

5. KCs: a pivotal role at the nexus of host defense and inflammatory regulation

As the largest solid organ in the human body, the liver serves not only as the central hub for metabolism and detoxification but also as a key player in immune defense, responsible for capturing and clearing bacteria that enter the hepatic circulation to maintain tissue homeostasis.⁸² KCs, the largest population of tissue-resident macrophages, play a pivotal role in bridging innate and adaptive immune responses. They are responsible for the rapid recognition and clearance of exogenous particles and immune-reactive substances deemed harmful to the body, forming the first line of defense against bacteria, microbial debris, and endotoxin originating from the gastrointestinal tract.

KCs highly express various pattern recognition receptors (PRRs), including TLRs, C-type lectin receptors (e.g., the mannose receptor), and NOD-like receptors (NLRs). This enables the precise identification of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), facilitating rapid sensing and response to foreign substances such as bacteria, microbial debris, and endotoxins. Upon receptor activation, KCs coordinate immune responses by releasing key cytokines, including TNF-α, IL-1, IL-6, IL-10, and IL-12.⁸³

In addition to PRRs, KCs express a variety of complement receptors. Research has revealed that the complement receptor of the immunoglobulin superfamily (CRIg) can function as a pattern recognition receptor independently of complement factors, directly recognizing Gram-positive bacterial lipoteichoic acid (LTA) and mediating the direct binding and capture of Staphylococcus aureus by KCs.⁸² Furthermore, anaphylatoxin receptors (C3aR and C5aR) and complement receptors 1, 3, and 4 (CR1, CR3, and CR4) are primarily involved in the clearance of senescent or apoptotic red blood cells, thereby maintaining microcirculatory homeostasis.⁸⁴

Recent studies have further uncovered that the immune function of KCs is modulated by metabolites derived from the gut microbiota. Pan et al.⁸⁵ discovered that indole-3-propionic acid (IPA), produced by gut microbiota, is crucial for maintaining KC volume and phagocytic capacity. In a mouse model of heparanase deficiency, the abundance of IPA-producing Lactobacillus intestinalis in the gut was reduced, leading to a decrease in KC volume, impaired ability to capture bacteria from the bloodstream, and consequently, an increased risk of sepsis.⁸⁵

Beyond pathogen clearance, KCs are also important mediators of immune tolerance in the liver. They possess the capacity for dual MHC antigen presentation: presenting endogenous antigens to CD8⁺ cytotoxic T cells via MHC class I molecules, and presenting exogenous antigens to CD4⁺ helper T cells via MHC class II molecules.⁸⁶ Under physiological conditions, KCs exhibit a tolerogenic phenotype, characterized by low surface expression of MHC class II molecules and co-stimulatory molecules (e.g., CD80/CD86), which effectively prevents excessive immune responses against gut-derived or self-antigens and avoids immune-mediated damage.⁸⁷ However, upon stimulation by foreign substances, KCs can upregulate both MHC class I and II molecules to initiate immune responses. Activated T cells can differentiate into various subsets, including T-helper 1 (Th1), Th2, Th17, and Treg cells. Among these, Th1/Th17 cells promote hepatocyte injury by secreting IL-2, interferon-gamma (IFN-γ), and IL-17, whereas Th2/Treg cells exert anti-inflammatory effects through IL-10 and TGF-β, demonstrating the dual role of KC-mediated T cell regulation in inflammation.⁸⁶

In addition to this dual role in regulating T cells, the polarization plasticity of KCs themselves exhibits a similar dichotomy. Classically activated M1-type KCs highly express TNF-α, IL-6, IL-12, and inducible nitric oxide synthase (iNOS), driving pro-inflammatory responses. In contrast, alternatively activated M2-type KCs upregulate anti-inflammatory mediators such as IL-10 and the IL-1 receptor antagonist, which function to suppress the progression of inflammation. This distinct polarization mechanism further underscores their dual role in the modulation of inflammatory responses.⁸⁷

In summary, KCs serve a complex and pivotal role in host defense and maintenance of inflammatory homeostasis through multiple mechanisms, encompassing pathogen recognition and clearance, regulation of immune cell activation and differentiation, and bidirectional modulation of inflammatory responses, thereby functioning as a central regulator of hepatic immune homeostasis.

6. Conclusions and future perspectives

As the first line of defense in liver immunity, KCs interact with other liver cells to regulate and coordinate immune defense functions upon bacterial infection. For example, LSECs release chemokines and bind to the glycocalyx, establishing a chemokine gradient within the liver. This gradient guides KCs to migrate to the periportal region of the liver lobule, thereby enhancing the efficiency of bacterial capture.⁸⁸ Additionally, KCs can mediate the release of itaconate, an immunomodulatory metabolite, through the SLC13A3 membrane protein to enhance the antibacterial capacity of hepatocytes.⁸⁹

On the other hand, KCs are crucial sensors and regulatory hubs for hepatic metabolic homeostasis. In iron metabolism, KCs recognize and phagocytose senescent or damaged red blood cells, recycle Fe²⁺, and oxidize it to Fe³⁺ through copper-containing oxidases. The latter binds to transferrin and enters systemic circulation.^22,40 Meanwhile, LSECs sense systemic iron stores and secrete BMP6, which regulates the expression of hepcidin in hepatocytes, thereby indirectly influencing the iron homeostasis function of KCs.⁴⁴ Additionally, LSECs can capture and sequester damaged red blood cells and deliver them to KCs via stabilin-1 and stabilin-2, promoting erythrocyte clearance.³² In terms of glucose and lipid metabolism, KCs sense excessive FFAs and bacterial LPS in the liver through surface TLRs, secrete TNF signals, and regulate the lipid metabolism processes in hepatocytes.⁹⁰ Furthermore, during liver regeneration and tissue repair, KCs communicate with hepatocytes and HSCs through the secretion of cytokines (e.g., IL-6), while also regulating the repair process and immune status through metabolic reprogramming (Fig. 1).

Fig. 1.

The role of Kupffer cells(KCs)in liver functions. (A) KCs and liver homeostasis. KCs regulate liver homeostasis through cellular adaptation and intercellular interactions. Iron metabolism: when systemic iron levels are excessively high, liver sinusoidal endothelial cells (LSECs) upregulate the secretion of bone morphogenetic protein 6 (BMP6), which in turn activates the expression of hepcidin in hepatocytes. Additionally, excess iron ions (Fe²⁺/Fe³⁺) generate ROS via the Fenton reaction, directly promoting the polarization of KCs toward the pro-inflammatory M1 phenotype. This process also enhances IL-6 expression in KCs, further stimulating hepcidin synthesis in hepatocytes. Hepcidin binds to ferroportin (FPN), inhibiting the release of iron from KCs into the circulatory system. Diet and exercise: under the high-fat diet, KCs produce large amounts of IL-1β and pro-fibrotic factors (TGF-β and PDGF). Among these, IL-1β suppresses the expression of FGF21 in hepatocytes, thereby impairing adipose tissue lipolysis and leading to hepatic lipid accumulation. Meanwhile, TGF-β and PDGF activate hepatic stellate cells (HSCs), accelerating ECM deposition. Exercise training, however, can mediate KC phenotypic switching through TCA cycle metabolic reprogramming, elevating itaconate levels and promoting a shift from a pro-inflammatory to an anti-inflammatory phenotype. This process is accompanied by enhanced secretion of anti-inflammatory cytokines (IL-10 and IL-1Ra). Aging and circadian regulation: in the aging microenvironment, overexpression of ALOX15 in KCs leads to excessive 13-HODE production, which activates the SREBP1 pathway in adjacent hepatocytes through paracrine signaling to promote hepatic lipogenesis. Additionally, circadian clocks modulate KC proliferation and function via temporal regulation of hepatocyte-derived factors such as colony-stimulating factor 1 (CSF1). Liver injury and regeneration: when liver injury, KCs release IL-6 and HGF to promote hepatocyte proliferation and survival. Additionally, KCs can secrete pro-inflammatory factors (IL-1β, TNF-α, and CCL2) and TGF-β to activate HSCs, stimulating collagen and ECM synthesis. Moreover, KCs can express VEGF and MMPs to synergistically enhance LSEC proliferation, establishing a regenerative microenvironment. (B) KCs act as both sensors and signal integrators. As sensors, KCs directly respond to senescence-associated secretory phenotype (SASP) factors, dietary components, and liver injury signals. In the aging liver, SASP factors drive the upregulation of pro-inflammatory genes and iron accumulation in KCs. The high-fat diet activates pro-inflammatory responses in KCs via the TLR4 pathway and stimulates the secretion of fibrogenic factors (e.g., TGF-β), which in turn activates HSCs. Upon liver injury, KCs undergo a significant phenotypic shift, releasing a plethora of regeneration-associated factors that promote hepatocyte proliferation and survival. They also facilitate interactions between LSECs and monocytes to promote angiogenesis and vascular repair. As signal integrators, KCs receive and transduce environmentally derived signals that have been pre-processed by other cell types. In iron metabolism, upon sensing iron overload, LSECs secrete BMP6, which regulates hepcidin expression in hepatocytes, thereby indirectly inhibiting iron efflux from KCs. The circadian rhythm system orchestrates KC proliferation and function through the rhythmic secretion of CSF1 by hepatocytes. Furthermore, aerobic exercise induces intrinsic metabolic reprogramming within myocytes, which subsequently modulates the phenotypic polarization of KCs. Abbreviations: ALOX15, arachidonate 15-lipoxygenase; ECM, extracellular matrix; FGF21, fibroblast growth factor 21; HGF, hepatocyte growth factor; 13-HODE, 13-hydroxyoctadecadienoic acid; IL, interleukin; MMPs, matrix metalloproteinases; PDGF, platelet-derived growth factor; ROS, reactive oxygen species; SREBP1, sterol regulatory element-binding protein 1; TCA, tricarboxylic acid; TGF-β, transforming growth factor-beta; TLR4, Toll-like receptor 4; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

In recent years, the development of single-cell sequencing technology has revealed the high heterogeneity of KCs, making the traditional M1/M2 polarization classification method inadequate for comprehensively describing their functional diversity. In addition to the classical M1 (pro-inflammatory) and M2 (anti-inflammatory) types, several unique macrophage subtypes have been discovered. For example, CXCL4-stimulated M4, oxidized phospholipid-activated Mox, angiogenesis-associated M (Hb), and heme-induced Mhem macrophages.91, 92, 93, 94 Furthermore, under different anatomical microenvironments in the liver, KCs appear to exhibit distinct immune and metabolic regulatory functions.^10,12,21,24, 25, 26 This suggests that the classification of KCs still requires further refinement, and there is a need for in-depth exploration of their dynamic functions under various physiological and pathological conditions.

Although advancements in single-cell technologies have deepened our understanding of the heterogeneity and functions of KCs, many questions remain unanswered. For instance, how do KCs dynamically detect changes in various metabolites in vivo? How do KCs precisely regulate liver metabolism under different feeding states? Future research should focus on developing a more comprehensive classification system, integrating high-resolution imaging, single-cell sequencing, and metabolomics technologies to further elucidate the operational patterns and mechanisms of KCs in both homeostasis and pathological conditions.

Authors' contributions

Qin Cai: Writing – original draft, Writing – review & editing, Conceptualization. Yusi Chen: Writing – review & editing, Conceptualization. Li Tang: Writing – review & editing, Funding acquisition, Conceptualization, Supervision.

Declaration of competing interest

The authors declare that there is no conflicts of interest.