Lipocalin-2 and intestinal diseases

Abstract

Dysfunction of the intestinal barrier is a prevalent phenomenon observed across a spectrum of diseases, encompassing conditions such as mesenteric artery dissection, inflammatory bowel disease, cirrhosis, and sepsis. In these pathological states, the integrity of the intestinal barrier, which normally serves to regulate the selective passage of substances between the gut lumen and the bloodstream, becomes compromised. This compromised barrier function can lead to a range of adverse consequences, including increased permeability to harmful substances, the translocation of bacteria and their products into systemic circulation, and heightened inflammatory responses within the gut and beyond. Understanding the mechanisms underlying intestinal barrier dysfunction in these diverse disease contexts is crucial for the development of targeted therapeutic interventions aimed at restoring barrier integrity and ameliorating disease progression. Lipocalin-2 (LCN2) expression is significantly upregulated during episodes of intestinal inflammation, making it a pivotal indicator for gauging the extent of such inflammatory processes. Notably, however, LCN2 derived from distinct cellular sources, whether intestinal epithelial cells or immune cells, exhibits notably divergent functional characteristics. Furthermore, the multifaceted nature of LCN2 is underscored by its varying roles across different diseases, sometimes even demonstrating contradictory effects.

Key Words: Lipocalin-2; Neutrophil gelatinase-associated lipocalin; Intestinal barrier; Immunity; Gastrointestinal diseases; Ferroptosis; Neutrophil extracellular traps

Core Tip: Lipocalin-2, also referred to as neutrophil gelatinase-associated lipocalin or 24p3, plays a pivotal role in diverse physiological processes and pathological injuries. In this review, we delineate its structure and the mechanisms underlying its action, elucidate its multifaceted interplay with the immune environment and its relevance to gastrointestinal disorders, thereby providing insights into potential avenues using targeted therapies directed to this multifunctional protein.

INTRODUCTION

Lipocalin-2 (LCN2), also referred to as neutrophil gelatinase-associated lipocalin (NGAL) or 24p3, was initially identified in neutrophils in 1994 by Kjeldsen et al[1]. The human LCN2 protein shares homology with the rat 24p5-associated protein and the mouse 24p3 protein, resulting in a similar tertiary structure despite lower amino acid sequence identity[2]. LCN2 has a highly conserved structure characterized by an eight-stranded β-barrel, forming a closed calyx structure in the antiparallel direction, which serves as its ligand-binding site. This closed calyx structure of LCN2 is notably polarized, enabling it to bind to larger and more hydrophobic ligands, thus facilitating a wide array of complex biological functions[3].

In this comprehensive review, our objective is to delve deeply into the intricate role of LCN2 in immune regulation, elucidating its multifaceted functions within the immune system. Additionally, we explore the existing body of literature concerning LCN2 and its influence on intestinal barrier function, revealing its intricate involvement in maintaining barrier integrity and regulating permeability. Moreover, we will critically analyze the diverse roles attributed to LCN2 in a spectrum of intestinal diseases, thereby shedding light on its intricate contributions to disease pathogenesis and progression. By synthesizing these insights, we aim to provide a nuanced understanding of the complex interplay between LCN2 and intestinal health, thereby offering valuable insights into potential therapeutic strategies targeting this multifunctional protein.

LCN2 AND ITS MECHANISM OF ACTION

LCN2 was initially isolated from neutrophils but has since been identified as being secreted by a variety of cell types, including adipocytes, hepatocytes, epithelial cells, macrophages, cardiomyocytes, and others[4-8]. In animals, LCN2 exists in various forms, such as monomers, homodimers, and heterodimers, each exerting distinct effects on cell proliferation, differentiation, and disease pathogenesis[9]. The functional diversity observed in different forms of LCN2 can be attributed to its ability to bind various ligands, forming complexes with unique functions[3]. For example, LCN2 is capable of binding to iron carrier proteins, and the presence or absence of iron within these complexes can profoundly alter their biological activity[10]. Moreover, LCN2 may interact with six distinct receptors, including NGAL receptor, low-density lipoprotein-related protein 2 (LRP2), LRP6, melanocortin 4 receptor (MC4R), MC1R, and MC3R[11]. However, the precise receptor-ligand interactions and the variable affinity of LCN2 for these receptors in different diseases or developmental processes remain incompletely understood. Furthermore, the downstream signaling pathways activated upon LCN2 receptor binding have not yet been fully elucidated. Further investigations into these aspects are crucial for obtaining a comprehensive understanding of LCN2-mediated cellular responses and their potential therapeutic implications in various pathological conditions. The crystal structure of LCN2 is demonstrated in Figure 1[12].

Figure 1 Crystal structure of lipocalin 2 with a closed calyx structure in the antiparallel direction generated via the AlphaFold protein structure database (AlphaFold Monomer v2.0 pipeline)[12].

The elucidation of the LCN2 receptor and its downstream signaling mechanisms remains an active area of research[11]. In this review, we aim to provide a concise overview of how LCN2 interacts with its receptors and elicits various downstream cellular functions. The first identified LCN2 receptor, also known as solute carrier family 22 member 17 (SLC22A17) or NGAL receptor[13], has been associated with endocytosis, although the specific substrates and transport mechanisms involved have yet to be fully elucidated[14]. In cancer, for example, tumor cells exploit the interaction between LCN2 and SLC22A17 to scavenge iron from the iron-deficient environment of the cerebrospinal fluid, promoting their own growth while simultaneously impairing macrophage function[15]. In addition to SLC22A17, LRP2 has been identified as another receptor for LCN2. LRP2 binds to LCN2 on the surface of proximal tubular epithelial cells, facilitating its endocytosis and subsequent retrieval from the glomerular filtrate. This process ultimately leads to the degradation of LCN2[16]. Notably, blocking the recycling of LCN2 through LRP2-mediated endocytosis represents a potential therapeutic approach for the treatment of iron overload conditions caused by genetic disorders or repeated blood transfusions[17]. In summary, while significant progress has been made in understanding the interactions between LCN2 and its receptors, further research is warranted to delineate the precise mechanisms underlying LCN2-mediated cellular responses. Such insights hold promise for the development of novel therapeutic strategies targeting LCN2 signaling pathways in various pathological conditions.

The iron transport capacity of LCN2 has been the subject of extensive investigation[9], yet numerous functional and mechanistic controversies persist. Recent advancements in cutting-edge scientific techniques, such as X-ray diffraction, sequencing, and proteomics analysis, have offered insights into the molecular mechanisms underlying LCN2-mediated iron ion transport. However, the complexity of iron transport pathways is further complicated by the formation of ternary complexes involving LCN2, iron ions, and small molecular weight iron carriers, which are chelating compounds produced and secreted by bacteria to sequester iron. In this intricate process, iron ions can bind to small molecular weight iron carriers associated with LCN2, resulting in the formation of ternary complexes comprising LCN2, the iron carrier, and iron ions[18]. Upon interaction with cell surface receptors, these ternary complexes are internalized via endocytosis, facilitating the release of bound iron into the cells and subsequently increasing the intracellular iron concentration. Recent studies have also revealed an additional layer of complexity, wherein metformin, which is conventionally known for its antidiabetic properties, can function as an iron carrier. Metformin forms a complex with iron and LCN2, further diversifying the repertoire of LCN2-associated iron transport mechanisms[19]. Conversely, binary complexes of LCN2 with iron carriers can also be internalized by cells, thereby translocating intracellular iron to the extracellular compartment and consequently reducing the intracellular iron content. These findings underscore the multifaceted nature of LCN2-mediated iron transport pathways and highlight the need for further research to elucidate the precise mechanisms governing these processes. These insights not only deepen our understanding of iron homeostasis but also have potential implications for the development of novel therapeutic strategies targeting iron-related disorders[20].

As a secreted protein, LCN2 is widely expressed across various cell types, and its pivotal role in innate immunity was initially recognized[21]. During bacterial infections, there is a notable increase in the plasma level of LCN2, which is primarily attributed to its secretion by immune cells. The antimicrobial mechanism of LCN2 involves the formation of ternary complexes with iron carrier proteins produced by bacteria, which sequester iron ions, thereby reducing the concentration of free iron ions in the plasma. This process effectively hampers bacterial uptake of iron, consequently inhibiting bacterial growth[22]. Furthermore, LCN2 exerts its antimicrobial effects through various mechanisms. It dose-dependently downregulates toll-like receptor 4 signaling, targeting the Janus kinase/signal transducer and activator of transcription (STAT) pathway, thus impeding bacterial infection[23]; additionally, LCN2 can bolster the host defense against bacterial pathogens by stimulating macrophages to produce reactive oxygen species (ROS) and nitric oxide, which are potent antimicrobial agents[24]. Notably, studies by Wang et al[25] demonstrated that in an Escherichia coli-induced infection model, LCN2 knockout mice exhibited disrupted neutrophil maturation and impaired migratory capacity. These effects may be attributed to the reduced production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and chemokines such as monocyte chemoattractant protein-1[25]. Collectively, these findings underscore the multifaceted role of LCN2 in orchestrating the host immune response against bacterial infections, highlighting its importance as a potential therapeutic target in combating infectious diseases.

Indeed, LCN2 performs context-dependent functions on the basis of its cellular source and specific physiological or pathological conditions. In bacterial-induced inflammation, LCN2 secreted by neutrophils and hepatocytes primarily acts to combat infection, demonstrating an anti-infective role[26,27]. Conversely, during kidney injury, LCN2 production by macrophages and renal epithelial cells is induced by cytokines such as interleukin (IL)-10 and IL-1β, facilitating renal cell regeneration and mitigating kidney injury[28,29]. However, the role of LCN2 in cancer is multifaceted. While LCN2 production by neutrophils and macrophages can exacerbate malignancy and promote tumor progression and metastasis in certain tumors[7,30], conflicting findings suggest a potentially inhibitory role in macrophage function, with exacerbation of inflammatory injury and contributions to conditions such as ulcerative colitis (UC), thereby exacerbating intestinal damage[31,32].

In this review, we delve more deeply into the intricate interactions between LCN2 and immune cells, including neutrophils, macrophages, T cells, and B cells. We aim to elucidate how this communication evolves in response to the cellular state and the dynamic disease microenvironment, drawing upon the latest literature and advances in LCN2 research. By synthesizing these insights, we seek to elucidate the specific mechanisms underpinning the complex and often paradoxical functions of LCN2. Additionally, we aim to outline the progress made in future research directions, thereby providing valuable insights into the evolving landscape of LCN2-mediated immune modulation and corresponding implications for therapeutic interventions in various disease contexts. Figure 2 illustrates the regulation of LCN2 biosynthesis and summarizes the influencing factors of LCN2 expression alterations across various models.

Figure 2 Regulation of lipocalin-2 biosynthesis (schematic diagram). The factors influencing lipocalin 2 expression alterations across various models were summarized. TNFα: Tumor necrosis factor-alpha; LPS: Lipopolysaccharide; TRAD: Protein TraD; TRAF: Tumor necrosis factor receptor-associated factors; IAP: Inhibitor of apoptosis proteins; RIPK1: Receptor-interacting protein kinase 1; CARD9: Caspase-recruitment domain 9; IKK: IκB kinase; NLPR3: NLR-family pyrin domain-containing protein 3; IKB: Inhibitor of kB; NFKB: Nuclear factor kappa light chain enhancer of activated B; JAK: Janus kinase; SOC3: Suppressor of cytokine signaling 3; STAT1/3: Signal transducer and activator of transcription 1/3; S1P: Sphingosine-1-phosphate; S1PR: Sphingosine-1-phosphate receptor; H3K27m³: Histone H3 lysine 27 trimethylation; KDM6A: Lysine-specific demethylase 6A; LCN2: Lipocalin-2.

FUNCTIONS OF LCN2 IN IMMUNITY

In the intricate landscape of immune regulation, LCN2 has emerged as a central player, engaging in complex interactions with various immune elements. Not only is LCN2 expression tightly regulated by a diverse array of immune cells and cytokines, but it also exerts regulatory effects on the levels of numerous cytokines, thus intricately modulating immune cell function. The signaling pathways mediated by LCN2 drive immune cell function in a highly context-dependent manner, resulting in cell type-specific responses under different physiological and pathological conditions. For example, LCN2 may promote proinflammatory responses in certain immune cells while exerting anti-inflammatory effects in others, highlighting the nuanced and dynamic nature of its immunomodulatory functions. Understanding the intricate crosstalk between LCN2 and immune elements holds significant promise for revealing the underlying mechanisms governing immune dysregulation in various disease states. By elucidating the latest insights into LCN2-mediated immune modulation, future research endeavors can pave the way for the development of targeted therapeutic strategies aimed at restoring immune homeostasis and ameliorating disease progression.

LCN2 and neutrophils

Neutrophils constitute the highest percentage of immune cells in the body[33] and play pivotal roles in conditions such as sepsis, tumors, ischemia-reperfusion injuries, and other inflammatory processes[34-36]. The interaction between LCN2 and neutrophils has been documented since the last century, with LCN2 initially isolated from secreted granules of neutrophils[1]. Recent findings underscore the close relationship between LCN2 and neutrophil activation and function[37]. Moreover, LCN2 produced by neutrophils exerts diverse effects on various organs and tissues, largely contingent upon the physiological state of the disease[30].

In sepsis resulting from various injuries, inflammatory factors stimulate neutrophils to produce and secrete LCN2[38]. Neutrophil-derived LCN2 forms an immune complex with Ent, effectively controlling infection by chelating iron[39]. However, increased expression of neutrophil LCN2 in the early stages of sepsis-associated acute respiratory distress syndrome has been correlated with poor patient prognosis and may also induce iron-mediated cell death in cardiomyocytes, contributing to cardiac insufficiency[40,41]. In chronic rhinosinusitis with nasal polyps, the IL-17A/TNF-α-neutrophil-LCN2 axis exacerbates inflammatory progression by promoting IL-8 expression in nasal polyps[42]. Tissue-infiltrating neutrophils secrete elevated levels of LCN2, which induces iron-mediated cell death in adipocytes and exacerbates malignancy in patients[30]. Furthermore, neutrophil-derived LCN2 polarizes macrophages toward a “reparative” phenotype, promoting cardiac remodeling in patients with infarction[43]. Moreover, LCN2 produced by other tissues can influence neutrophil function. LCN2 serves as a migratory activator of neutrophils[44]; for example, alcohol upregulates LCN2 protein expression in hepatocytes via the IL-6/STAT3 pathway, leading to neutrophil recruitment in the liver and subsequent T-cell depletion[45]. These findings underscore the multifaceted role of LCN2 in regulating immunity across different physiological and pathological states, particularly in a neutrophil-dependent manner.

Neutrophil extracellular traps (NETs) represent a well-established form of neutrophil activation[46]. In bacterial infections, NETs generated by neutrophils can transport LCN2 to the site of inflammation and infection, exerting an anti-infective role. While knockdown of LCN2 in neutrophils does not impact NET production, antimicrobial efficacy is significantly diminished[27]. This phenomenon may be attributed to bacterial Ent production, which suppresses neutrophil ROS levels and NET formation via the iron-chelating activity against bacterial Ent-mediated iron sequestration[47]. Recent research has revealed the role of LCN2-metformin-iron complexes synthesized by renal parenchymal cells in exacerbating acute kidney injury by binding to the C-X-C chemokine receptor type 4 receptor on the surface of neutrophils, thereby inducing NETosis[19]. These findings suggest a potentially self-sustaining, mutually reinforcing interplay between LCN2 and NETs. For example, in psoriasis, LCN2 produced by keratinocytes stimulates NET production by neutrophils, thereby fueling skin inflammation via toll-like receptor 4/IL-36 crosstalk and the downstream MyD88/nuclear factor-kappaB (NF-κB) signaling pathway. Concurrently, NETs stimulate keratinocytes to increase LCN2 expression and secretion[48]. Furthermore, the combined assessment of NETs and LCN2 has emerged as a prognostic biomarker for various diseases. Elevated NETs and LCN2 are significantly associated with severe acute kidney injury induced by coronavirus disease 2019[49]. NETs and LCN2 contribute to pathological changes in ocular graft-versus-host disease[50], and they are significantly associated with the presence of peripheral artery disease[51]. Despite these findings, the precise mechanism governing the interaction between LCN2 and NETs remains elusive. Additionally, the mechanisms underlying the NET-mediated upregulation of LCN2 production and secretion, as well as the manner in which LCN2 promotes NET production, warrant further investigation.

LCN2 and macrophages

Macrophages serve as crucial regulators of the immune system, and their functions extend beyond classical phagocytosis to include the extensively studied phenomenon of macrophage polarization[52]. The interplay between LCN2 and macrophages is intricate, with LCN2 potentially acting as a liaison between macrophages and other cell types or as a key mediator facilitating interactions with macrophages. Recent research suggests a close association between LCN2 and macrophage polarization, which is primarily mediated through the regulation of iron metabolism.

LCN2 expression in macrophages is typically induced by inflammation[21]. The systemic inflammatory cascade associated with sepsis induction also triggers LCN2 expression as a first-line response of the innate immune system[53]. During acute lung inflammation, the number of LCN2-positive macrophages significantly increases, leading to intracellular iron overload and the polarization of alveolar macrophages toward the M1 phenotype, exacerbating oxidative stress[54]. Studies on LCN2-deficient mice and macrophages have revealed their reduced resistance to Salmonella infection, as evidenced by increased bacterial loads; decreased levels of nitric oxide, TNF-α, and IL-6; and elevated levels of IL-10. LCN2 downregulates IL-10 expression and enhances the bactericidal function of macrophages by promoting macrophage iron efflux[55]. The iron-loading status and cellular source of LCN2 modulate its biological function during renal failure in sepsis. The secretion of iron-free LCN2 by renal tubular epithelial cells is associated with renal injury, whereas increased release of iron-conjugated LCN2 (hLCN2) by macrophages is linked to renal recovery[56]. This mechanism may involve hLCN2-induced activation of the integrative stress response, mediated by the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 pathway, which further upregulates the expression of SLC7A11 through phosphorylated eukaryotic translation initiation factor 2A and increased ROS generation, enhancing cellular resistance to iron-induced cell death[10]. Furthermore, IL-10-treated macrophages produce hLCN2 and simultaneously activate the LCN2 receptor SLC22A17[28]. This receptor-mediated mechanism provides iron to cells, promoting cell migration and matrix adhesion[57,58].

LCN2 production by macrophages is regulated by a variety of self-expressed proteins, which subsequently influence the functions of downstream tissues and organs. For example, macrophage-expressed KDM6A exacerbates diabetic retinopathy by promoting LCN2 expression and impairing glycolytic processes in photoreceptor cells through the removal of trimethyl groups from histone H3K27[59]. Additionally, macrophage-expressed caspase-recruitment domain 9 upregulates LCN2 expression via the NF-κB pathway, subsequently increasing matrix metalloproteinase 9 expression. This upregulation contributes to the deterioration of cardiac function and adverse myocardial remodeling after myocardial infarction, which is mediated by the interaction between LCN2 and matrix metalloproteinase 9[60]. Macrophages also exacerbate chemokine ligand 4-induced liver injury in an LCN2-dependent manner[61]. Furthermore, macrophage-derived LCN2 plays a pivotal role in renal fibrosis, leading to proteinuria and renal injury by modulating the salbutamol/chemokine ligand 5/IL4 pathway[62]. However, these findings contrast with those of Horckmans et al[43], who demonstrated that LCN2 production by neutrophils after infarction induces macrophage differentiation toward the highly phagocytic MertK-M2c phenotype, consequently enhancing cardiac healing. This discrepancy may be attributed to the different cellular sources of LCN2 in various disease models, which dictate the polarization of M0 macrophages toward either proinflammatory macrophages, exacerbating tissue damage[63], or reparative macrophages, promoting phagocytosis[31,43].

As LCN2 is a secreted protein expressed in multiple tissues, the interplay between macrophages and LCN2 produced by various tissues in different disease states has garnered considerable attention. For example, dysfunctional adipocytes secrete LCN2, promoting the inflammatory activation of macrophages[64]. TNF-α induces LCN2 expression and secretion in adipocytes in a NACHT, LRR, and PYD domains-containing protein 3-dependent manner, subsequently promoting the activation of the NACHT, LRR, and PYD domains-containing protein 3 inflammasome in macrophages[65]. LCN2 also plays a pivotal role in mediating communication between macrophages and other immune cells. It serves as a central mediator facilitating crosstalk between neutrophils and hepatic macrophages by inducing chemokine signaling, particularly through the downstream receptor C-X-C chemokine receptor type 2, thereby exacerbating steatohepatitis[44].

LCN2 has a complex relationship with macrophages in cancer[66]. Sphingosine-1-phosphate released from apoptotic cancer cells enhances the production of LCN2 by tumor-associated macrophages upon signaling through its receptor sphingosine-1-phosphate receptor 1. Subsequently, LCN2 released by tumor-associated macrophages promotes tumor growth by inducing the expression of the lymphangiogenic factor vascular endothelial growth factor-C[67]. In metastatic meningiomas, while LCN2 expression by macrophages and neutrophils remains relatively stable, there is a marked increase in LCN2 expression by cancer cells. This upregulation is mediated by cytokines such as IL-6, IL-8, and IL-1β in the tumor microenvironment, which induce LCN2 expression in cancer cells through STAT and NF-κB transcriptional promoters. Additionally, cancer cells acquire iron from the cerebrospinal fluid in an LCN2-dependent manner, facilitating their growth while concurrently inhibiting the iron uptake and iron-dependent functional activity of macrophages[15].

LCN2 and T lymphocytes

Orabona et al[68] were the first to report that T lymphocytes can upregulate LCN2 expression in response to IL-9 stimulation. This regulatory relationship is mediated by STAT3, and the upregulation of LCN2 expression occurs gradually, reaching its peak 36-72 hours after stimulation. Additionally, as mentioned earlier, tumor cells can activate downstream LCN2 expression via STAT3, resulting in T-cell depletion and facilitating hepatocellular carcinoma metastasis[45]. In colorectal cancer (CRC), CD4⁺ T cells induce apoptosis by producing LCN2, thereby reducing the concentration of intracellular iron ions in T cells and compromising their antitumor capabilities. Concurrently, LCN2 overexpression promotes cholesterol metabolism and accelerates tumor metastasis. Moreover, iron transferred from T cells enters the tumor microenvironment, promoting tumor cell proliferation[69]. This finding parallels the role of LCN2 in macrophages[7], although further investigation is needed to determine whether the underlying mechanism is identical.

Silencing LCN2 expression has been shown to inhibit tumor proliferation, whereas reducing LCN2 expression enhances RSL3-induced ferroptosis in acute T lymphoblastic leukemia[70]. Inhibition of phosphoglycerate mutase 1 exerts antitumor effects by inducing energy stress and ROS-dependent protein kinase B inhibition. Consequently, this downregulates LCN2 expression in tumor cells, promoting ferroptotic cell death and CD8⁺ T-cell infiltration in hepatocellular carcinoma cells[71]. However, previous studies have presented contrasting findings, where Gprc5a^-/-Lcn2^-/- mice exhibited a heightened ability to promote lung adenocarcinoma proliferation. Additionally, these mice presented a decreased abundance of CD4⁺ T cells and increased levels of protumor inflammatory signals[72]. This apparent contradiction could stem from variations in the systemic and local tissue effects of LCN2 or differences in the source of LCN2. Further investigations are warranted to elucidate the underlying mechanisms driving these observations.

An intricate immune dialog between T cells and macrophages is orchestrated by LCN2. In the context of Mycobacterium tuberculosis infection, LCN2-mediated modulation of ROS levels culminates in the downregulation of major histocompatibility complex class I molecules in macrophages, subsequently attenuating CD8⁺ T-cell activation during the infection process[73]. Furthermore, LCN2 assumes multifaceted roles in tandem with T cells. Notably, Furutani et al[72] demonstrated the therapeutic potential of a novel proresolving lipid mediator mimetic, 3-oxa-PD1n-3, which was administered via the intrathecal injection of docosapentaenoic acid. This intervention effectively curtails LCN2 production in cutaneous T-cell lymphomas, thereby mitigating pruritus through the modulation of synaptic transmission[74]. Nevertheless, the intricacies of these interactions necessitate deeper mechanistic elucidation.

LCN2 and B lymphocytes

B lymphocytes play pivotal roles in orchestrating immune responses through their diverse effector functions, including the production of immunoglobulins for antigen neutralization, the modulation of immune responses, and antigen recognition and presentation to stimulate other effector cells. Despite their central importance in immune regulation, the direct interaction between LCN2 and B lymphocytes remains relatively understudied in current research endeavors[75]. Expanding our understanding of this potential interaction could offer valuable insights into the nuanced interplay between LCN2 and the adaptive immune system, warranting further investigation in future studies.

Recent studies have shown that inhibiting B-cell activation can reduce the secretion of LCN2 and slow the progression of lupus nephritis[76]. Moreover, limiting B-cell activation has been found to decrease LCN2 secretion, thereby alleviating renal ischemia-reperfusion injury[77]. Additionally, symptomatic patients with B-cell-derived non-Hodgkin’s lymphoma exhibit higher levels of LCN2 than normal individuals do[78]. Despite these significant findings, none of these studies have explored the direct interaction between LCN2 and B cells. Investigating these interactions should be a priority for future research to better understand their roles in disease pathogenesis and potential therapeutic interventions.

In future studies, investigating the iron-carrying status of LCN2 and its cellular origin is imperative. Additionally, LCN2 produced by the same cells in different diseases may manifest diverse roles. Taking a holistic approach, we should explore the intricate crosstalk between LCN2 and immune elements, with a focus on the disease context. This comprehensive analysis will be pivotal in unraveling the role of LCN2 in immune regulation. The mechanisms of LCN2 Interaction with cells are demonstrated in Figure 3.

Figure 3 Mechanisms by which lipocalin-2 interacts with cells (schematic diagram). The mechanisms by which lipocalin-2 complexes exert their effects on cells have been summarized. SLC22A17: Solute carrier family 22 member 17; ROS: Reactive oxygen species; ATG4B: Autophagy-related gene 4B; LC3B: Light-chain 3B; ER: Endoplasmic reticulum; elF2α: Alpha-subunit of eukaryotic translational initiation factor 2; ATF4: Activating transcription factor 4; CHOP: C/EBP homologous protein; Keap1: Kelch-like ECH-associated protein 1; NrF2: Nuclear factor erythroid 2-related factor 2; VEGF: Vascular endothelial-derived growth factor; CXCR4: C-X-C chemokine receptor type 4; LCN2: Lipocalin-2; ARE: Antioxidant response elements.

LCN2 AND GASTROINTESTINAL DISEASES

Gastrointestinal diseases encompass a range of multifactorial triggers marked by compromised barrier function and heightened permeability of the gastrointestinal mucosa. These conditions often manifest symptoms such as digestive and absorption dysfunction, along with bacterial overgrowth, culminating in damage across multiple tissues. The impacts of LCN2 on gastrointestinal diseases are likely intricate and influenced by various factors, including the underlying cause of gastrointestinal damage and the microenvironment surrounding the affected tissues.

LCN2 and autoimmune enteropathy

Autoimmune enteropathies are a group of rare and incurable conditions that include inflammatory bowel diseases (IBDs) [Crohn’s disease (CD); UC], intestinal Behçet’s disease, and others. LCN2 plays a pivotal role in the pathogenesis of these disorders. Research has confirmed elevated LCN2 expression in intestinal Behçet’s disease, but the corresponding specific mechanisms of action have yet to be clarified[79]. Current research has focused primarily on the interaction between LCN2 and IBD.

IBDs, which represent a relatively prevalent subset of gastrointestinal autoimmune conditions, are characterized by a propensity for pan-enteric involvement[80]. The pathological manifestations of IBD arise from a vicious cycle driven by an imbalance between intestinal mucosal damage and mucosal immunity[81]. Numerous studies have identified LCN2 as an associated gene in IBD[82], highlighting its potential utility as a diagnostic marker of CD[83]. The expression levels of LCN2 are correlated with disease activity and immune infiltration in UC[84,85], and LCN2 has emerged as a marker for UC-associated carcinoma[86]. Additionally, some studies suggest its involvement as a potential mediator of iron-induced cell death[87]. Furthermore, elevated levels of LCN2 have been consistently observed in the feces of IBD patients, suggesting that LCN2 is a promising noninvasive screening indicator[88]. The increased expression of LCN2 in UC is believed to be regulated by proinflammatory cytokines such as IL-17A, IL-22, and TNF-α, potentially via TNF-α-mitogen-activated protein kinases-NADPH oxidase 1 axis-derived ROS[84,89].

IBD often coincides with alterations in the composition of the intestinal microbiota[90]. LCN2 may modulate IBD by influencing the intestinal microbiota. Previous studies have demonstrated that LCN2 exerts protective effects against early-onset colitis and tumor formation by modifying the composition of the intestinal microbiota, particularly Lachnospiraceae and Alistipes spp.[91]. Additionally, LCN2 can promote cell migration, contributing to mucosal regeneration and tissue repair[92]. LCN2 deficiency has been associated with alterations in the microbiota-derived metabolome, characterized by reduced production of short-chain fatty acids and short-chain fatty acid-producing microorganisms, thereby exacerbating the intestinal inflammation induced by a high-fat diet[93]. However, conflicting findings have emerged regarding the role of LCN2 in IBD progression. For example, some studies suggest that LCN2 may aggravate IBD by binding to Ent and reducing myeloperoxidase activity[94]. Moreover, a lack of suppressor of cytokine signaling 3 in myeloid cells has been shown to increase LCN2 expression in neutrophils, exacerbating dextran sulfate sodium-induced colitis. Conversely, suppressor of cytokine signaling 3 has been implicated in preventing immune system activation in IBD[95]. Although LCN2 is widely recognized as a marker gene for IBD, its mechanistic involvement in IBD and the inconsistencies in its functional roles warrant further investigation.

LCN2 and infectious diseases of the gastrointestinal tract

Infectious diseases affecting the gastrointestinal tract encompass a spectrum of conditions induced by bacterial and viral pathogens, commonly presenting with symptoms such as abdominal pain, diarrhea, and vomiting. Among its initially recognized functions[21,22], LCN2 exhibits potent antimicrobial properties. During bacterial enteritis, LCN2 levels notably increase[96]. However, in cases of enteritis stemming from viral infections, LCN2 levels remain largely unchanged. Consequently, LCN2 has emerged as a valuable tool for discerning the etiology of pathogen-induced gastrointestinal inflammation[88]. Elevated expression of LCN2 in intestinal epithelial cells is facilitated by IL-17/IL-22 signaling following bacterial infection. Notably, a deficiency in LCN2 promotes the proliferation of Salmonella typhimurium, an enterobacterium, within the inflamed gut milieu. This exacerbates susceptibility to further bacterial invasion[97], exemplified by heightened susceptibility to Escherichia coli infection[98].

The bacteriostatic action of LCN2 primarily stems from its role in restricting bacterial access to and utilization of iron. By facilitating internal iron export in macrophages, LCN2 increases the production of proinflammatory mediators while concurrently suppressing IL-10 levels, thereby fostering host-mediated bacterial clearance and elimination. Furthermore, LCN2 orchestrates intercellular interactions within the immune milieu. Deficiencies in LCN2 compromise neutrophil chemotaxis and migration and disrupt the normal secretion of inflammatory cytokines by macrophages[25], thereby compromising intestinal barrier integrity[99]. However, this protective effect is limited to infections caused by Gram-negative bacteria that rely exclusively on iron carriers susceptible to LCN2 binding. Conversely, LCN2 has no discernible effect on bacteria, such as Staphylococcus aureus, which employ alternative iron acquisition mechanisms unaffected by LCN2[21]. LCN2 secreted by colonic epithelial cells confers anti-inflammatory effects by augmenting phagocytic activity and bolstering bacterial clearance[100]. Moreover, in LCN2-deficient mice, alterations in the composition of the ileal intestinal flora manifest, notably marked by a surge in injury-associated segmented filamentous bacteria. This surge correlates closely with mucosal cell layer injury, provoking an antimicrobial response within the intestinal epithelium and influencing helper T cell polarization[101].

LCN2 expression was markedly elevated in patients with Helicobacter pylori (H. pylori)-infected gastritis, whereas quercetin treatment led to decreased LCN2 expression in H. pylori-infected GES-1 cells. Mechanistically, SP1 was found to bind to the LCN2 promoter, thereby facilitating its transcription. Quercetin intervention mitigated M1 macrophage polarization via modulation of the SP1/LCN2 axis and subsequently attenuated H. pylori-induced apoptosis and inflammatory injury in gastric epithelial cells[102]. However, LCN2 has also been demonstrated to exert a proinflammatory effect in cases of low-grade intestinal inflammation[103].

LCN2 and intestinal tumors

The association between LCN2 and CRC has been extensively investigated in the context of intestinal tumors. As early as 1996, studies revealed high expression of LCN2 in colon cancer, including precancerous lesions[104]. Through various bioanalytical and proteomic approaches, researchers have consistently identified LCN2 as a potential biomarker for CRC development[105-108], which can be used in the diagnosis of CRC[109]. Its expression levels hold promise for CRC diagnosis and may serve as indicators for predicting metastasis and poor prognosis[110,111]. Notably, a recent study demonstrated correlations between LCN2 gene expression and the abundance of CRC-associated bacteria, such as Lactococcaceae and Veronica spp.[112].

In primary CRC, LCN2 mediates apoptosis by modulating TNF-α apoptosis-inducing ligand and its cell-surface-expressed death receptors 4/5. Silencing LCN2 expression leads to the upregulation of death receptor 5 expression, enhancing cancer cell sensitivity to chemotherapy via the p38 mitogen-activated protein kinases/C/EBP homologous protein pathway[113]. Deletion of the PKP3 gene results in increased LCN2 levels, rendering colon cancer cells resistant to 5-fluorouracil[114]. Conversely, LCN2 has been reported to inhibit the progression of duodenal tumors while promoting the growth of small intestinal tumors[115]. These conflicting findings underscore the need for further in-depth exploration of the functions of LCN2 in organisms.

In vitro experiments using various colon cancer cell lines have revealed differential expression levels of LCN2, which are correlated with variations in migration and invasion capacities[116]. Elevated LCN2 expression in CRC is often associated with a greater risk of distant metastasis. Kim et al[117] demonstrated that LCN2 negatively modulates epithelial-mesenchymal transition in CRC cells by modulating glucose metabolism, with reduced LCN2 expression enhancing the migratory and invasive potential of cancer cells. Further investigations indicated that mesenchymal stem cells engineered to overexpress LCN2 could target and deliver LCN2 to tumor sites, effectively suppressing liver metastasis in colon cancer[118]. Recent studies have confirmed that LCN2, which acts upstream of the NF-κB/Snail pathway, significantly inhibits epithelial-mesenchymal transition induction and metastasis both in vitro and in vivo by attenuating NF-κB/Snail pathway activation[119]. The outcomes and mechanisms of LCN2 in different organ and diseases are demonstrated in Table 1.

Table 1 Lipocalin-2 expression and its impact on disease pathogenesis.

Organ/site	Disease	Outcome	Mechanism	Ref.
Eye	Retinal inflammation	Overexpression of LCN2 in RPE cells manifested protection from cell death	LCN2 induced expression of antioxidant enzymes HMOX1 and SOD2 in RPE cells and inhibited the cytotoxic effects of H2O2 and lipopolysaccharide	[120]
	Age-related macular degeneration	In RPE cells, elevated LCN2 expression disrupts autophagy, enhances inflammasome activity, induces oxidative stress, triggers ferroptosis, and exacerbates cellular damage, thereby impacting iron homeostasis	LCN2 forms a complex with ATG4B, designated as LCN2-ATG4B-LC3-II, modulating ATG4B activity and LC3-II lipidation to regulate autophagy	[121]
Brain	Cerebral ischemia/reperfusion injury	After stroke in mice with middle cerebral artery occlusion, astrocyte LCN2 levels surged within 24 hours. Mice lacking LCN2 had smaller infarcts and better neurocognitive recovery poststroke	24p3R inhibition reduced pyroptosis and pro-inflammatory cytokine secretion via LCN2, an effect reversed by Nigrostatin-induced NLRP3 activation. Pyroptosis in astrocytes was exacerbated by rLCN2 in Lcn2-/- mice	[122]
	Cancer brain metastasis	LCN2 drives neuroinflammation, promoting brain metastasis in melanoma and breast cancer	Signals from the primary tumor activate astrocytes, which attract immunosuppressive granulocytes to brain metastases, a process that increases LCN2 levels. Targeting LCN2 genetically reduces neuroinflammation and brain metastasis	[123]
	Neurodegenerative diseases	LCN2 in astrocytes can shift their state from neurotoxic to neuroprotective, possibly preventing neuronal death and halting neurodegenerative disease progression	Activated astrocytes secrete LCN2, which binds to the 24p3R receptor in an autocrine loop, further activating astrocytes through the NF-κB pathway	[124]
Kidney	Active lupus nephritis	Biomarker	LCN2 is highly secreted by renal tubular epithelial cells in response to various renal injuries, leading to elevated levels in blood and urine, and can serve as a biomarker for multiple kidney injuries and prognosis	[125]
	Acute kidney injury			[126]
	Kidney transplant			[127]
Liver	NASH	In patients with NASH, LCN2 expression was significantly associated with inflammation and fibrosis	LCN2 serves as a central mediator in hepatic stellate cell activation during leptin-deficient obesity, via the α-SMA/MMP9/STAT3 pathway, worsening NASH	[128]
			SREBP-1c mediates LCN2 expression, linking to NASH pathogenesis. SREBP-1c deletion downregulates LCN2, alleviating NASH severity. It also couples iron and lipid metabolism via LCN2, impacting NASH development	[129]
	Hepatocellular carcinoma	Elevated LCN2 expression promotes hepatocellular carcinoma cell proliferation	Targeting PGAM1 suppresses hepatocellular carcinoma growth by inhibiting LCN2 and PD-L1 expression through an energy stress/ROS-mediated reduction in AKT activity, thereby enhancing CD8+ T-cell infiltration	[71]
Pancreas	Pancreatic ductal adenocarcinoma	Overexpression of LCN2 markedly inhibits pancreatic cancer cell adhesion and invasion	LCN2 diminishes pancreatic cancer cell adhesion and invasion by partially suppressing focal adhesion kinase tyrosine-397 phosphorylation and inhibits angiogenesis by impeding VEGF production	[130]
		Lcn2 deficiency in PDAC mouse models retarded weight gain, adiposity, and tumor progression and enhanced survival	Pancreatic stromal cells, devoid of LCN2 synthesis, express the LCN2 receptor SLC22A17. LCN2 binding to SLC22A17 on cancer-associated fibroblasts stimulates secretion of inflammatory mediators and MMP9, enhancing immune cell infiltration and tumor progression in the pancreatic cancer microenvironment	[131]
Artery	Atherosclerosis	LCN2 enhances HDL metabolism and facilitates macrophage reverse cholesterol transport	LCN2 enhances HDL metabolism and attenuates atherogenesis by inhibiting Nedd4-1-mediated SR-BI ubiquitination at lysines 500 and 508	[132]
Bone	Osteoporosis	LCN2, acting as a downstream factor, induces iron overload in osteocytes, promoting their apoptosis	Repin1 knockdown reduced LCN2 expression, mitigating the toxic effects of intracellular iron overload	[133]
Other	Chronic stress	Peripheral and central inhibition of the LCN2 pathway significantly alleviated CRS-induced behavioral deficits	LCN2’s anxiogenic effects are linked to its modulation of neuronal activities in the medial prefrontal cortex. Under CRS, hepatic tissues are identified as the main source of serum LCN2, released via the vagal efferent pathway from the dorsal motor vagal nucleus	[134]

LCN2: Lipocalin-2; RPE: Retinal pigment epithelium; HMOX1: Heme oxygenase 1; SOD2: Superoxide dismutase-2; ATG4B: Autophagy-related gene 4B; NLRP3: NACHT, LRR, and PYD domains-containing protein 3; NF-κB: Nuclear factor-kappaB; NASH: Nonalcoholic steatohepatitis; α-SMA: Alpha-smooth muscle actin; MMP9: Matrix metallopeptidase 9; STAT3: Signal transducer and activator of transcription 3; PGAM1: Phosphoglycerate mutase 1; PD-L1: Programmed death-ligand 1; ROS: Reactive oxygen species; AKT: Protein kinase B; VEGF: Vascular endothelial growth factor; SLC22A17: Solute carrier family 22 member 17; PDAC: Pancreatic ductal adenocarcinoma; HDL: High-density lipoprotein; CRS: Chronic restraint stress.

The status of LCN2 in relation to intestinal barrier function remains to be further elucidated. LCN2 has been shown to protect intestinal barrier function against invasion by Escherichia coli[99]. In CD and UC, LCN2 acts as a proinflammatory cytokine, exacerbating destruction of the intestinal barrier[87]. The precise relationship between LCN2 and gastrointestinal diseases remains incompletely understood, particularly concerning its involvement in complex conditions such as intestinal fistula, intestinal ischemia/reperfusion injury, and other forms of intestinal damage. A comprehensive exploration of the mechanisms underlying the actions of LCN2 in intestinal injury is essential for gaining insights into its roles in various diseases. Additionally, future research should focus on investigating drug therapies targeting LCN2 to explore potential therapeutic options for these conditions.

CONCLUSION

A wealth of literature on LCN2 has highlighted its utility as an indicator of disease activity and prognosis in bacterial infections and IBD, owing to its heightened expression during the acute phase. Initially recognized as an anti-infective protein secreted by neutrophils for the chelation of bacterial ectoferric iron, the role of LCN2 has since been extensively investigated in various diseases. However, differing cellular sources of LCN2 may involve specific mechanisms within distinct disease microenvironments, yielding varying or even conflicting results. This variability may be attributed to the iron-carrying state of LCN2, necessitating further research to elucidate its immunomodulatory role in diverse scenarios.

Moreover, recent studies have shed light on the ability of LCN2 to bind other small molecules, subsequently binding iron ions. For example, the LCN2-metformin-iron complex has been implicated in exacerbating renal ischemia-reperfusion injury[19]. These findings suggest that LCN2 may interact with conventional small molecule drugs to yield unforeseen effects, suggesting intriguing avenues for future research. Investigations of whether LCN2 can serve as a drug carrier to deliver therapeutics to damaged sites or function in vivo with the aid of cellular carriers (e.g., mesenchymal stem cells) or drug carriers (e.g., hydrogels and chitosan) represent promising future directions. However, the specific intracellular mechanisms of LCN2 action remain incompletely understood, and the interaction between LCN2 and the intestinal flora warrants further elucidation. Therefore, unraveling the potential cellular and molecular mechanisms of LCN2 in various diseases constitutes a vast area for future exploration.

ACKNOWLEDGEMENTS

We express our gratitude to Dr. Xin-Yu Wang and Dr. Cheng-Nan Chu for providing assistance in the design of the study.