Insights into the pathogenic roles and targeted therapy of neutrophil extracellular traps in inflammatory bowel disease

Abstract

Inflammatory bowel diseases (IBDs) are a group of relapsing intestinal inflammatory disorders characterized by a prolonged duration. Irregular immune responses toward gut microbial antigens play a pivotal role in the etiology and pathology of IBDs. Neutrophils, the most abundant responsive innate immune effector cells, and serve as the first line of defense against invading pathogens by degranulation, phagocytosis, generation of reactive oxygen species, and synthesis of chemokines and cytokines, thereby important in maintaining intestinal mucosal homeostasis. Neutrophil extracellular traps (NETs) are web-like extracellular chromatin structures consisting of DNA, histones, fibers, and neutrophil granule proteins formed and released by activated neutrophils. The state of neutrophils with NET formation is called NETosis, which includes suicidal, vital and mitochondrial NETosis. NETs can break down and eradicate pathogens thereby preventing them from uncontrolled spreading. Apart from the antimicrobial function, accumulative evidence has highlighted the pathogenic roles of NET components including neutrophil elastase, myeloperoxidase, and cell-free DNA, in inflammatory and autoimmune diseases and cancer. Excessive accumulation of NETs disrupt the intestinal mucosal barrier, amplify the inflammatory cascade, promote thrombosis and colitis-associated cancer. Herein, we review the formation of NETs, discuss NETs as a potential pathogenic mechanism in IBD, and therapeutic targets for IBD.

Key Words: Inflammatory bowel disease; Neutrophil extracellular traps; Mucosal immunity; Mechanism; Clinical translation

Core Tip: Neutrophil extracellular traps (NETs), while essential for pathogen clearance, act as a double-edged sword in inflammatory bowel diseases (IBDs). Excessive accumulation of NETs has been confirmed in IBD patients and colitis mouse model. NET components can exacerbate chronic intestinal inflammation, destroy intestinal mucosal barrier and aggravate tissue damage, promote thrombosis and colitis-associated cancer. Consequently, therapeutic strategies targeting NET formation or promoting NET degradation are emerging as promising avenues to restore intestinal mucosal homeostasis in IBD.

INTRODUCTION

Inflammatory bowel diseases (IBDs), encompassing Crohn’s disease (CD), ulcerative colitis (UC), and indeterminate colitis, are a group of chronic inflammatory disorders of the gastrointestinal tract with a relapsing-remitting course[1]. IBD has emerged as a worldwide health challenge, currently affecting > 6.8 million people globally. Although the etiology and pathology of IBD remain unclear, it likely arises from inappropriate immune reactions to the commensal gut microbiota in genetically predisposed individuals, triggered by environmental factors[2]. Both innate and adaptive immune responses contribute to gut mucosal inflammation in IBD patients.

Neutrophils are the most plentiful innate immune effector cells of the human immune system and are generally regarded as the primary responders during inflammatory events[3]. Hyperactivation or dysfunction of neutrophils triggers abnormal immune responses, resulting in a range of autoimmune and inflammatory diseases including IBD, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Recent studies have expanded our understanding of the role of neutrophils in pathogen elimination, immunoregulation and pathology of IBD. The deployment of neutrophils is regulated through three principal mechanisms: Phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs). Brinkmann et al[4] first reported the discovery of NETs in 2004. NETs are extracellular web-like structures composed of cytosolic and granule proteins that assemble on a scaffold of decondensed chromatin. NETs have been confirmed contribute to the pathogenesis of several immune-mediated diseases including SLE and RA[5,6].

The roles of NETs have dual characteristics[7]. One the one hand, NETs capture, inactivate, and destroy a wide range of microbes including bacteria, fungi, viruses and parasites, and are believed to hinder the spread of bacterial and fungal infections. On the other hand, dysregulated NET formation can exacerbate gut inflammation and trigger the pathogenesis of immune-related disorders. In this review, we discuss the formation of NETs and their protective vs harmful roles, especially their pathogenic mechanisms in IBD, the crosstalk between NETs and gut microbiota. NETs may serve as diagnostic and prognostic biomarkers in IBD. Targeting NET formation could offer a novel therapeutic approach for IBD.

MECHANISMS OF NET FORMATION, RELEASE, AND MIGRATION

NETs consist of decondensed chromatin adorned with histones, proteases, and granular and cytosolic proteins, which together form extracellular fibers extruding from neutrophils[4]. NETosis, a process distinct from apoptosis and necrosis, refers to the extracellular killing by neutrophils releasing NETs in response to inflammatory stimuli. Activated neutrophils seize and destroy invading microorganisms such as bacteria, fungi, or viruses, by unleashing NETs containing depolymerized chromatin and intracellular granule proteins.

To date, two main types of NETosis have been confirmed (Figure 1), suicidal (lytic) and non-suicidal (vital), which depends on the type of stimulus and differs in overall duration[8]. Suicidal NETosis is triggered by phorbol myristate acetate, antibodies bound to the Fc receptor, or cholesterol crystals. This process ruptures neutrophil membranes, releasing depolymerized chromatin, histones, and granule proteins into the extracellular environment. Suicidal NETosis depends on the activation of the Raf-mitogen-activated protein kinase-extracellular regulated protein kinases (ERK) signaling cascade, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent production of reactive oxygen species (ROS), and signaling mediated by receptorinteracting protein kinase and mixed-lineage kinase domain-like protein.

Figure 1 Two main types of neutrophil extracellular traps formation: Suicidal (lytic) and non-suicidal (vital). Suicidal state of neutrophils with neutrophil extracellular traps formation (NETosis) can be triggered by phorbol myristate acetate, antibodies bounding to the Fc receptor. The Raf-mitogen-activated protein kinase-extracellular regulated protein kinases pathway and nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species production are activated. Activation of peptidylarginine deiminase (PAD) 4 results in histone citrullination, followed by exocytosis of decondensed chromatin and enzymes like neutrophil elastase (NE) and myeloperoxidase (MPO). Vital NETosis can be triggered by Staphylococcus aureus infection recognized by Toll-like receptor (TLR) 2 or complement receptors, or by Escherichia coli directly via TLR4 or indirectly via TLR4-activated platelets. Activated PAD4 in turn translocates to the nucleus converting arginine to citrulline on histones, and resulting in chromatin depolymerization, along with its extracellular release including NE and MPO. NOX: Nicotinamide adenine dinucleotide phosphate-oxidase; Abs: Antibodies; FcR: Fc receptor; PMA: Phorbol myristate acetate; MERK: Mitogen-activated protein kinase; ERK: Extracellular regulated protein kinases; Ca²⁺: Calcium ion; ROS: Reactive oxygen species; PAD4: Peptidylarginine deiminase 4; MPO: Myeloperoxidase; NE: Neutrophil elastase; NETs: Neutrophil extracellular traps; S. aureus: Staphylococcus aureus; E. coli: Escherichia coli; LPS: Lipopolysaccharide; PKC: Protein kinase C; TLR: Toll-like receptor; MEK: Mitogen-activated protein kinase kinase.

Peptidylarginine deiminase (PAD) 4 plays a pivotal role in the generation of NETs. The PAD enzymes catalyze the citrullination of arginine residues. Within the PAD family comprising PAD1-PAD4 and PAD6, PAD4 is predominantly found in hematopoietic cells like neutrophils[9], and uniquely contains a nuclear localization signal. Citrullination, a post-translational modification carried out mainly by the PAD enzymes, converts arginine to citrulline. By citrullinating histones, PAD4 weakens interactions between histones and DNA, and promotes NET formation[10]. In suicidal NETosis, activation of PAD4 results in histone citrullination, followed by exocytosis of decondensed chromatin and enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO), which together build the fibrous network of NETs. Hence, suicidal NETosis is characterized by co-expression of granule proteins such as MPO and NE, and chromatin, primarily citrullinated histone H3 (citH3)[11]. Suicidal NETosis occurs after several hours of stimulation.

In contrast, vital NETosis occurs within minutes upon stimulation in a ROS-independent way, which is demonstrated following microbe-specific molecular patterns recognized by host pattern recognition receptors, such as Toll-like receptors (TLRs)[12]. DNA-containing vesicles bud from the nuclear membrane, traverse the cytoplasm, and fuse with the plasma membrane, enabling NETs to be released from the cell without rupturing the plasma membrane. Vital NETosis can be initiated by Staphylococcus aureus infection through recognition by TLR2 or complement receptors, and by Escherichia coli either directly via TLR4 or indirectly through TLR4-activated platelets. DNA fragments and histones after chromatin depolymerization are released via exocytosis from live cells. Research on mitochondrial NETs is limited. Neutrophils release mitochondrial DNA into extracellular compartments, resulting in mitochondrial NET formation under specific stimulation such as granulocyte-macrophage colony-stimulating factor + lipopolysaccharide (LPS)/complement factor 5a[8]. Both suicidal and non-suicidal NETosis stimulate PAD4 activity; then PAD4 translocates to the nucleus converting arginine to citrulline on histones, which results in chromatin depolymerization, along with extracellular release of PAD4.

NETS IN HEALTH AND DISEASES

The protective roles of NETs in maintaining homeostasis and in IBD pathogenesis

Neutrophils are part of the first line of immunological defense and account for about 60%-70% of all leukocytes circulating in peripheral blood[13]. NETosis is a double-edged sword. Positively, NETs play a pivotal role in maintaining homeostasis through enhancing pathogen clearance, immune regulation, wound healing and immunothrombosis. NETosis enables neutrophils to trap and immobilize bacteria, fungi, and viruses, thereby enhancing pathogen clearance. Recent studies have highlighted that NETs play a crucial role in eradicating particular pathogens such as Citrobacter rodentium and restricting the widespread dissemination of bacteria during necrotizing enterocolitis (NEC)[14].

NETs promote neutrophil defense, enhance macrophage polarization, induce pyroptosis, and facilitate plasmacytoid dendritic cell differentiation which aid antiviral functions. NETs enhance cluster of differentiation 4⁺ T cell and B cell activation, although they may simultaneously compromise NK cell function. NETs neutralize anticoagulant factors and promote the extrinsic coagulation pathway, thereby enhancing pathogen elimination. NETs potentiate immunothrombosis through activating factor XII, binding von Willebrand factor, and triggering platelet activation via histones H3 and H4, thereby inhibiting tissue invasion by pathogens. Aggregated NETs containing diverse enzymes can degrade pro-inflammatory cytokines and chemokines, sequester NE to protect the extracellular matrix from proteolysis, which promote inflammation resolution and wound healing[15].

Cluster of differentiation 177⁺ neutrophil subset exhibits enhanced microbial killing ability through ROS production, antimicrobial peptide release, and NETosis. Conversely, it reduces the secretion of pro-inflammatory cytokines such as interleukin (IL)-6, IL-17A, interferon-γ, while increasing the production of reparative cytokines (IL-22, transforming growth factor-β), thereby facilitating tissue repair in IBD[16]. Triggering receptor expressed on myeloid cells-1 agonist promotes NET formation and IL-22 secretion by cluster of differentiation 177⁺ neutrophils, which enhances intestinal barrier integrity and promotes pathogen elimination in IBD models[17].

Conversely, sustained inflammation or persistent stimuli can provoke excessive NET formation, which in turn aggravates tissue damage and inappropriate inflammation. NETs contributes to the development of infectious diseases and noninfectious diseases, including, but not limited to RA, SLE, atherosclerosis, vasculitis, thrombosis, and cancer[18,19].

Overproduction of NETs results in the progression of intestinal infections, sepsis, NEC, intestinal ischemia-reperfusion injury, IBD, and gastrointestinal cancer. In the bacterial enteritis model, NETs promote the attachment of enteropathogenic Escherichia coli and Shiga-like Escherichia coli to the intestinal mucosa, through enhancing the microbial biofilm formation and function[20]. Zhan et al[21] have demonstrated that NETs aggravate organ injury caused by intestinal ischemia-reperfusion injury, while DNase I treatment markedly alleviated intestinal damage, suggesting a harmful role for NETs.

Intestinal dysfunction and lesions are frequently observed in septic patients. Infiltration of neutrophils and release of NETs were apparent in the intestine of a rat model with LPS-induced endotoxemia. In sepsis, exposure to LPS triggers PAD4 activation and NET release through the PAD-NETs-citH3 axis, which disrupts the permeability of pulmonary vascular endothelial cells (ECs)[22]. NEC is a severe intestinal disorder that primarily afflicts preterm infants. Elevated levels of circulating nucleosomes have been detected in premature infants with NEC, and NETs have been observed in the ileal tissue of human NEC cases. Using the NEC mouse model developed by Klinke et al[23], researchers reported that serum circulation of circulating free DNA (cf-DNA) correlated positively with clinical severity of NEC, and that NET markers were markedly higher in both the affected mice and human NEC samples compared with controls[23].

In addition to the intestinal diseases mentioned above, NETs are closely related to the proliferation and metastasis of gastrointestinal cancer. Elevated NET levels contribute to a poor prognosis of gastrointestinal tumors by promoting local growth of tumors and participating in tumor-related systemic injury. NETs can further drive tumor growth and metastasis by boosting the mitochondrial activity of cancer cells and awakening dormant tumor cells. PAD4-mediated NETs participate in the progression of digestive tumors, including gastric cancer, hepatocellular carcinoma (HCC), pancreatic cancer, and colorectal cancer (CRC)[24-28].

PAD4-driven NETs detected in the peripheral blood of gastric cancer patients showed higher relevance to tumor progression and metastasis, potentially due to an intensified epithelial-mesenchymal transition (EMT). In patients at advanced stages, neutrophil PAD4 is abnormally activated and more susceptible to NETosis[24]. CRC patients exhibit elevated PAD4-mediated NETs in tumor tissues and peripheral circulation, which are also associated with venous thrombosis[25]. In pancreatic cancer, disease-associated hypercoagulability is related to PAD4-mediated NET formation, thereby increasing the risk of thrombosis and mortality[26].

Higher levels of NETs have been observed in HCC patients and mouse models. LPS promotes NET formation via TLR4 signaling, thereby exacerbating hepatic steatosis and accelerating HCC development in alcohol-fed mice. Knockdown of PAD4 markedly reduces the number of hepatic tumor growths in the STAM mouse model[27]. PAD4-driven NET formation plays a crucial role in hepatic metastasis. NETs are plentiful in hepatic metastatic lesions of breast and CRCs, and circulating NET levels can serve as an early predictor of liver metastasis onset in breastcancer patients[28].

Opinions diverge on the functions of neutrophils and NETs in gastrointestinal cancer, as they appear to promote tumor progression or facilitate tumor eradication. This paradox may stem from the presence of distinct neutrophil subpopulations or from variations in cancer type and the surrounding microenvironment under investigation[29].

NETS IN IBD PATIENTS: INCREASED NET FORMATION AND POSITIVE ASSOCIATION WITH INTESTINAL INFLAMMATION OR DISEASE ACTIVITY

IBD is a chronic intestinal inflammatory disease characterized by the participation of both innate and adaptive immune mechanisms. NETs have dual functions in IBD, with both deleterious and protective effects. NETs playing a crucial role in protecting the intestinal mucosal barrier, supporting host defense, and facilitating the resolution of inflammation. Excessive NETosis also causes chronic inflammation and tissue injury. To further explain the relationship between NETs and IBD, many researchers have conducted both clinical and experimental investigations on UC and CD. Several studies have verified increased presences of NETs in blood, inflamed colonic mucosa, or stools in IBD patients (Table 1), characterized by elevated levels of proteins important for NET formation, including PAD4, MPO, NE, calprotectin, citH3, and cathepsin G. Several studies have stipulated that higher levels of NETs were associated with active disease[30-33].

Table 1 A summary of studies revealing increased presence of neutrophil extracellular traps in inflammatory bowel disease patients and dextran sulfate sodium-induced colitis.

Subjects	Sampling location	Detection method	Key findings	Region	Ref.
UC (n = 28), CD (n = 23), HCs (n = 12)	Peripheral blood	In vitro NET induction	(1) Sera from UC/CD enhanced patient/control NET formation in vitro; (2) IgG from PR3-ANCA-positive IBD enhanced control NET formation in vitro; and (3) DNase I decreased procoagulant activity in vitro	Harbin, Heilongjiang Province, China	He et al[30]
UC (n = 9), CD (n = 9), HCs (n = 12)	Colonic biopsies; peripheral blood	Immunohistochemistry, immunofluorescence, Western blot. In vitro NET induction, 3% DSS-induced colitis	(1) Over-expression of colocalized PAD4, NE, MPO, and citH3 in inflamed colon of UC; (2) IFX treatment diminishes NETs in UC; and (3) PAD4 inhibitor attenuates DSS induced-colitis in mice	Rome and Milan, Italy	Dinallo et al[31]
UC (n = 24), CD (n = 24), controls (n = 10, IC and HCs)	Peripheral blood, colonic mucosal biopsies	Immunofluorescence, ELISA, in vitro NET induction; 3.5% DSS-induced colitis	(1) Elevated cfDNA and MPO-DNA complexes in active UC or CD; (2) NET deposition in the colon, impaired NET degradation in the plasma of IBD; (3) Increased serum cf-DNA and enhanced NET formation in DSS-induced colitis in mice, attenuated by DNase perfusion or anti-Ly6G antibody; (4) NET degradation protects mice against DSS-induced colitis and colitis-associated tumorigenesis; and (5) NETs enhance the procoagulant activity in vitro. DNase blunt prothrombotic effects	Harbin, Heilongjiang Province, China	Li et al[32]
UC/CD (n = 51)	Peripheral blood	Immunofluorescence, ELISA. In vitro NET induction, 3.5% DSS-induced colitis, Western blot	(1) Increased MPO-DNA complexes in blood; (2) Enhanced NET formation in vitro; (3) NETs enhance procoagulant activity in vitro; and (4) DNase I protects against DSS-induced colitis	Harbin, Heilongjiang Province, China	Cao et al[33]
Pediatric IBD: UC (n = 6), CD (n = 6). Pediatric HCs (n = 2)	Biopsies from the small bowel and colon	Immunofluorescence	Increased, colocalized MPO, chromatin and histones, and NE in biopsies of pediatric IBD	Tel Aviv, Israel	Gottlieb et al[34]
UC (n = 14), CD (n = 11), HCs (n = 17). Others (n = 29, including 8 CA, 8 GCA and 13 IBS)	Fecal samples	Meta-proteomics: LC-MS	Increased NE, MPO, azurocidin, and cathepsin G in stool of UC and CD	Magdeburg and Hannover, Germany	Lehmann et al[35]
UC (n = 23), CD (n = 11), infectious colitis (n = 15), HCs (n = 25)	Peripheral blood neutrophils, sera, and colonic tissue	Immunofluorescence, Western blot; ELISA, PCR, flow cytometry; in vitro NET induction	(1) Increased, colocalized NE and citH3 in biopsies of active UC; (2) Increased MPO-DNA complexes in blood of UC; (3) Sera/ex vivo culture media from UC enhances control NET formation in vitro; and (4) Activation of REDD1/autophagy/NETs/IL-1β pathway in UC	Alexandroupolis, Greece	Angelidou et al[36]
UC (n = 10), HCs (n = 10)	Mucosal colon biopsies from noninflamed tissue	Proteomics (LC-MS); histology (light microscopy and confocal microscopy)	Increased lactoferrin, MPO, NE, calprotectin, neutrophil defensin 3 in colonic mucosa of UC	Silkeborg, Denmark	Bennike et al[38]

UC: Ulcerative colitis; CD: Crohn’s disease; HCs: Healthy control; IC: Indeterminate colitis; CA: Colon adenoma; GCA: Gastric carcinoma; IBS: Irritable bowel disease; IBD: Inflammatory bowel disease; NETs: Neutrophil extracellular traps; DSS: Dextran sulfate sodium; ELISA: Enzyme-linked immunosorbent assay; LC-MS: Liquid chromatograph-mass spectrometer; PCR: Polymerase chain reaction; IgG: Immunoglobulin G; PR3-ANCA: Anti-proteinase 3 antibody anti-neutrophil cytoplasmic antibody; PAD4: Peptidylarginine deiminase 4; MPO: Myeloperoxidase; NE: Neutrophil elastase; IFX: Infliximab; cfDNA: Circulating free DNA; citH3: Citrullinated histone H3; IL: Interleukin.

NETs were found in all six pediatric CD and UC patients. Immunofluorescence showed that activated neutrophils producing NETs were characterized by extracellular DNA, chromatin and histones co-localized with MPO/NE. In contrast, ileal and colonic biopsy specimens from the two healthy control children exhibited no evidence of NET formation[34]. Given the limited sample size and retrospective nature of this study, it preliminarily demonstrated the presence of NETs in IBD patients but not in healthy controls, although no clear clinical correlations were observed. Future research is needed to validate the association between NETs and clinical disease parameters of IBD in larger, multi-center cohorts.

In adult IBD patients, noninvasive liquid chromatography-mass spectrometry/mass spectrometry analysis revealed increased NET proteins, including MPO, NE, azurocidin, and cathepsin G in stool samples of patients with UC and CD[35]. Higher levels of NET proteins were found in the plasma and colonic tissues in patients with active IBD, particularly active UC[36]. NETs also showed a positive regulatory relationship with tumor necrosis factor (TNF)-α in UC patients. TNF-α spurs NET formation, and NETs subsequently facilitates the secretion of TNF-α. IBD patients with active disease show more NET release than those with quiescent lesions. Likewise, Angelidou et al[36] reported that NET production was elevated in patients with active UC compared to CD patients and healthy controls.

Circulating neutrophils of UC patients produced NETs when stimulated with TNF-α. Consistently, a decrease in NET-related proteins and diminished NET formation were seen in patients receiving infliximab and anti-TNF-α treatment[31]. UC patients with corticosteroid resistance or unresponsive to cyclosporine A (CsA) showed increased neutrophil accumulation in the colon compared with those treatment-responsive individuals, accompanied by elevated ROS levels, increased NE, and uncontrolled T-cell proliferation[37]. NET formation was found in the inflamed colon tissues of IBD patients, and in noninflamed tissue of UC patients. Bennike et al[38] analyzed NETs in UC patients. Endoscopic mucosal colon biopsies were obtained from non-inflamed areas in 10 UC patients and 10 healthy controls, followed by high-throughput gel-free quantitative proteomics and histological examination. Among 5711 different proteins identified and quantified with proteomics, 46 proteins were significantly different in abundance between UC and controls. Eleven proteins with increased abundance included MPO, NE, lactoferrin, calprotectin, and neutrophil defensin 3 in UC biopsies, which were associated with neutrophils and NETs. Biopsy specimens confirmed that the abundance of calprotectin and lactotransferrin showed a strong correlation with the extent of tissue inflammation. These results indicated that, even in well-treated UC patients, these proteins were clinically quiescent, the innate immune system remained activated, and chronic inflammation persisted in morphologically normal colonic tissue. Dinallo et al[31] also revealed increased PAD4 levels in the colon biopsies of UC patients compared to normal colon tissue, indicating the prognostic and therapeutic significance of NET-associated markers in the colonic tissue of UC patients and the potential of targeted treatment using selective PAD4 inhibitors.

UC is characterized by barrier deterioration. Several overlapping NET-related differentially expressed genes, which are strongly related to barrier dysfunction, have been revealed in UC by integrated bioinformatics analysis based on the Gene Expression Omnibus database. Further enrichment and correlation analysis indicated that DDIT4/IL-1β NETs might trigger macrophage-driven phagocytosis, causing barrier dysfunction in UC[39].

Angelidou et al[36] have investigated how the disease microenvironment influences the release of NETs by studying peripheral blood neutrophils, sera, and colonic biopsy specimens collecting from patients with active UC, active CD, infectious colitis or healthy controls. They observed that the inflammatory environment of UC triggered the generation of autophagy-driven NETs decorated with bioactive IL-1β and tissue factor. The level of REDD1 in gut neutrophils was related to their autophagic activation and the NET-driven inflammation in UC. Their work highlighted that the activation of the REDD1/autophagy/NETs/IL-1β pathway played a pivotal role in the initiation and spread of intestinal inflammation of UC.

Proteomic and metabolomic studies of colon tissue from CD patients revealed increased NE expression compared to healthy individuals, and showed significant differences in calprotectin and metabolic protein abundance[40]. Significantly higher levels of nucleosomes, cf-DNA, and NET formation (MPO-DNA complexes) were found both active UC or CD patients, compared to patients with inactive UC or CD[33]. Overexpression of NET-associated proteins, including colocalized PAD4, MPO, NE, and citH3 was detected in the inflamed colon tissues of UC patients compared to CD patients or normal controls. Higher levels of IL-1β and tissue factor thrombin were detected in NETs isolated from the colonic tissue and blood of these patients, and their production was linked to autophagy induced by the REDD1 protein[36].

Collectively, an increased presence of NETs has been confirmed in the blood, inflamed colonic mucosa, or stool samples of patients with UC and CD. Further studies are needed to elucidate the exact mechanisms of NETs in the pathogenesis of IBD.

PATHOGENIC MECHANISMS OF NETS IN IBD

Increased presence of NETs and activated pathways in colitis model

In mice with dextran sulfate sodium (DSS)-induced colitis, NETs were induced and inhibition of NET release attenuated colitis as well as colitis-associated tumorigenesis[31-33]. NETs play important roles in the onset and progression of colitis, through facilitating inflammatory cell infiltration, triggering cytokine secretion, inducing tissue damage and disruption of the intestinal barrier function (Figure 2). NETs also promote thrombotic tendency and colitis-associated CRC (CAC) (Figure 3).

Figure 2 Pathogenic mechanisms of neutrophil extracellular traps in the pathogenesis of inflammatory bowel disease. In ulcerative colitis (UC) patients, increased REDD1 from gut neutrophils triggers the generation of autophagy-driven neutrophil extracellular traps (NETs) decorated with bioactive interleukin-1β and tissue factor, exacerbates the inflammatory environment of UC. In dextran sulfate sodium-induced colitis mouse model to mimic human ulcerative colitis, Intestinal neutrophil-derived peptidylarginine deiminase (PAD) 4 induces mitochondrial creatine kinase 1 citrullination and intestinal epithelial cells apoptosis, leading to exacerbation of mucosal inflammation. NETs activate the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (STING) pathway to induce barrier dysfunction. PAD4-induced recruitment of TBK1 by STING subsequently activates downstream Interferon regulatory factor 3 and nuclear factor kappa-B signaling pathways, leading to the production of type I interferon and inflammatory cytokines. IECs: Intestinal epithelial cells; IBD: Inflammatory bowel disease; IL-1β: Interleukin-1β; TF: Tissue factor; DSS: Dextran sulfate sodium; ROS: Reactive oxygen species; PAD4: Peptidylarginine deiminase 4; CKMT1: Mitochondrial creatine kinase 1; NETs: Neutrophil extracellular traps; cGAS: Cyclic guanosine monophosphate-adenosine monophosphate synthase; cGAMP: Cyclic guanosine monophosphate-adenosine monophosphate; NF-κB: Nuclear factor kappa-B; IκB: Inhibitor of nuclear factor kappa-B; IRF3: Interferon regulatory factor 3; IFN: Interferon.

Figure 3 Neutrophil extracellular traps constitute a central component in thrombosis formation and colitis-associated colorectal cancer. Neutrophil extracellular traps promote the thrombotic tendency in inflammatory bowel disease. The molecular mechanisms included in colitis-associated colorectal cancer: Mutations on protein p53 result in its over-expression and the turbulence of anti-tumor signaling. MyD88-dependent Toll-like receptor 4 alterations trigger and amplify a pro-inflammatory environment through nuclear factor kappa-B pathway. Cyclooxygenase-2 up-regulates prostaglandin E2 production, which in turn promotes angiogenesis and modulates immune responses through phosphatidylinositol 3-kinase (PI3K) signaling. The PI3K/protein kinase B axis stimulates many cytokines and facilitates glucose transporter 4 migration, thereby enhancing metabolic activity in T helper (Th) 2 and Th17 lymphocytes. TF: Tissue factor; NETs: Neutrophil extracellular traps; IBD: Inflammatory bowel disease; CAC: Colitis-associated colorectal cancer; TLR: Toll-like receptor; PGE2: Prostaglandin E2; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; NF-κB: Nuclear factor kappa-B; COX-2: Cyclooxygenase-2.

Significantly increased concentrations of circulating cf-DNA and enhanced NET formation were observed on days 4 and 6 following 3.5% DSS initiation. Increased NETs release and deposition were also found in colonic tissues from mice through western blotting and immunofluorescent staining. Administration of DNase protected mice from DSS-induced colitis characterized by decreased inflammatory cytokines and MPO levels, indicating DNase downregulated neutrophil infiltration and induced NET degradation during DSS-induced colitis. Administration of DNase also mitigated the accelerated thrombus development and platelet activation[32].

Disruption of the intestinal mucosal barrier is a hallmark of IBD. Studies in murine colitis models have confirmed that NET-associated proteases can cause barrier damage directly or indirectly. NE can directly destroy key epithelial tight junction proteins like occludin, zonula occludens-1, and adherens junction protein E-cadherin, contributing to increased epithelial permeability[41]. Matrix metallopeptidase 9 degrades type IV collagen which is a major component of the basement membrane, leading to the destruction of the anchoring and support structure for epithelial cells[42].

MPO induce programmed necroptosis of epithelial cells through catalyzing excessive oxidants production and stimulating mitochondrial dysfunction, which can further amplify inflammation[43]. Neutrophils can also amplify the inflammatory response by establishing complex positive feedback loops. Granulocyte colony-stimulating factor (G-CSF) plays a key role in promoting neutrophil production and release. Neutrophil-derived IL-1β induces production of G-CSF and granulocyte-macrophage-CSF from epithelial and stromal cells, which further accelerate neutrophil production and release, and inhibit neutrophil apoptosis through the Janus tyrosine kinase-signal transducer and activator of transcription (STAT) and phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) pathways, enhancing their pro-inflammatory functions. NE can activate pro-IL-1β in macrophages, further amplifying IL-1 signaling[41].

As a universal DNA sensor, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) can be stimulated with cytosolic DNA, which in turn activates the downstream signaling effector stimulator of interferon genes (STING) to induce inflammatory cytokines. STING recruits TANK-binding kinase 1, subsequently activating downstream interferon regulatory factor 3 and nuclear factor kappa-B (NF-κB) signaling pathways, thereby triggering intestinal inflammation[44]. Recent research indicates that an excessively active cGAS-STING pathway is associated with intestinal inflammation. cGAS has been identified as the DNA sensor for NETs, thereby triggering downstream immune cells to produce type I interferon[45]. Sun et al[46] proved that NETs disrupted intestinal barrier integrity in DSS-induced UC by activating the cGAS-STING pathway. Activation of the cGAS-STING pathway was mitigated by blocking NET formation through PAD4 genetic knockout in DSS-induced chronic colitis. STING deficiency ameliorated the clinical colitis index, intestinal inflammation, and barrier dysfunction, implying that targeting cGAS-STING to regulate NET-mediated intestinal inflammation may offer a novel approach for UC treatment.

PAD4, which is abundantly expressed in neutrophils and essential for NET formation, plays a critical role in the development of IBD through modulating intestinal epithelial cell (IEC) plasticity via mitochondrial creatine kinase (CKMT) 1 citrullination[47]. During NET formation, PAD4 is released from neutrophils via extracellular vesicles and subsequently taken up by IECs. Within IECs, PAD4 facilitates the citrullination of CKMT1 at the R242 site, which contributed to reduced CKMT1 protein levels and increased IECs apoptosis during colitis, leading to a compromised mitochondrial homeostasis and impaired intestinal barrier integrity. PAD4 deficiency mitigated colonic inflammation and restored intestinal barrier function in a DSS-induced colitis mouse model. Clinical studies have shown that PAD4 levels are elevated, CKMT1 undergoes increased citrullination, and CKMT1 expression is reduced in IBD patients, indicating that PAD4 could serve as a therapeutic target for IBD.

Sequential or parallel pathways may exist in the aforementioned mechanisms. Mitochondrial damage and ROS production are key triggers for cGAS-STING pathway activation. Therefore, PAD4-induced CKMT1 upregulation compromises mitochondrial homeostasis, further exacerbating intracellular ROS levels and activating the cGAS-STING pathway. Activation of cGAS-STING can upregulate the NOD-like receptor thermal protein domain associated protein 3 inflammasome, promoting the maturation and secretion of IL-1β and an interferon response. This may further stimulate the expression of REDD1, forming a positive feedback loop of “oxidative stress/mitochondrial damage-DNA stress sensing-inflammation-cell death/autophagy”. Further studies are needed to deeply explore the crosstalk between distinct pathogenic pathways.

Research on NETs in CD has mostly been in basic experimental investigations. In a mouse model of CD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), the colonic tissues showed elevated expression of lymphocyte antigen 6G, citH3, and PAD4. Neutrophils exhibited an enhanced ability to generate NETs in vitro, together with disruption of the intestinal mucosal barrier and apoptosis of epithelial cells[48]. Suppressing NET formation markedly reduced the colitis severity and tissue inflammation, with modulating the levels of both pro- and anti-inflammatory cytokines.

In non-IBD diseases, Wilson et al[49] confirmed that NETs directly promote the differentiation of the pro-inflammatory T helper (Th) 17 cell subset, establishing a feedback loop between innate and adaptive immune systems that intensifies autoimmunity. More specifically, as the major protein component of NETs, histones stimulate the differentiation and activity of Th17 cell directly via a TLR2/MyD88-dependent pathway, leading to phosphorylation of STAT3 and subsequent induction of RAR-related orphan receptor (ROR) γt. A histone inhibitor ameliorated this effect. They demonstrated that histones can directly bind to TLR2 on T cells, then trigger the activation of STAT3 and RORγt, which subsequently enhances cytokine production by Th17 cells. In a study of atherosclerosis, Tsourouktsoglou et al[50] demonstrated that DNA, histones, and citrullination enhance NET-mediated inflammation by modulating localization and activation of TLR4. Similar immunoregulatory mechanisms may exist in IBD, therefore further studies are needed.

Taken together, NETs exacerbate intestinal inflammation and colonic tissue damage during IBD. Thus, targeting NETs, such as using DNase to hasten the breakdown of overly abundant NET release and accumulation, could provide therapeutic advantages for patients with IBD.

Interactions between gut microbiota and NETs

IBD patients show marked shifts in the composition and metabolites of gut microbiota, which are closely correlated with disease severity. Decreased bacterial diversity such as Firmicutes, reduced short-chain fatty acids (SCFAs), especially butyrate, depletion of butyrate-producing taxa such as Faecalibacterium prausnitzii, along with disturbed immunoregulatory properties of gut microbiota have been demonstrated in both CD and UC[51,52]. Increased Proteobacteria in IBD, and higher abundance of adhesive-invasive Escherichia coli in CD, have been reported, which all contribute to increased intestinal permeability and inflammation[53]. Butyrate inhibits infiltration of neutrophil, limits formation of NETs, restores mucosal equilibrium through regulating leukocyte activation, innate immunity, and oxidative stress, which are characterized by enhanced regulatory T cell differentiation[54].

The crosstalk between NETs and gut microbiota is a key focus in IBD, especially in UC research. Gut microbiota dysbiosis disrupts neutrophil homeostasis. Adherent-invasive Escherichia coli exacerbates antibiotic-associated intestinal dysbiosis, NET formation and oxidative damage[55]. Clostridium difficile often appears as a co-infection in acute flare-ups of UC, which can exacerbate intestinal inflammation and lead to poor prognosis. In mice with DSS-induced colitis, colonization by Clostridium difficile altered the microbiota profile, mainly characterized by reduced abundance of Prevotellaceae UCG-001 and Muribaculaceae. This shift results in robust neutrophil infiltration and NET generation[56]. Excessive accumulation of NETs leads to inflammatory microenvironment which in turn causes further disruption of gut mictobiota.

Collectively, a bidirectional interplay exists between NETs and gut microbiota in UC pathogenesis. UC-associated gut microbiota dysbiosis facilitates a pro-inflammatory microenvironment, leading to heightened NET formation and disease pathogenesis. NETs in turn modulate gut microbiota composition.

NETs facilitate a prothrombotic state and progression toward CAC in IBD

NETs are a key element in the progression and prothrombotic state of IBD. IBD patients exhibit heightened procoagulant activity (PCA), although the precise mechanisms remain incompletely understood. He et al[30] reported that elevated phosphatidylserine (PS) exposure on microparticles (MPs) and cells plays a critical role in elevating thrombotic risk. Additionally, serum from patients with UC and CD, as well as media from ex vivo culture of inflamed UC mucosa, can induce a markedly higher spontaneous NET formation. They also found that neutrophils obtained from IBD or non-IBD subjects had similar NET formation capacity when stimulated with IBD sera. Higher PS exposure on circulating MPs, blood cells, as well as the ECs isolated from IBD patients was found when incubated with patients’ sera, which facilitated fibrin formation. Blocking of PS with lactadherin markedly reduced the activity of procoagulant enzyme complexes, thereby decreasing the PCA of MPs, blood, and ECs. In the active phase of IBD, a systemic environment primes circulating neutrophils to release NETs, enhancing the coagulation process, which could be abolished by DNase I. Their study revealed a link between hypercoagulable state and PS exposure or NETs, and demonstrated that patients with active IBD display increased NET release, which contributes to the hypercoagulable state[30].

CAC refers to CRC arising from chronic IBD. Abnormalities in neutrophil count and function significantly contribute to initiation, development, and relapse of IBD, and to its progression toward CAC. IBD patients show an elevated risk for developing CRC. While the progression from colitis to CRC generally takes 16-21 years, CAC patients with a history of IBD develop malignancy about 7.7 years earlier than those with sporadic CRC[57]. Compared with sporadic CRC cases, CAC patients with a history of IBD experience poorer prognosis and elevated mortality[58]. Neutrophils exhibit diverse functions in IBD, particularly by generating ROS and NETs, which are implicated in the pathogenesis of CRC. ROS generated by neutrophils can facilitate tumor metastasis, angiogenesis, and foster immunosuppression within the tumor microenvironment. Several molecular mechanisms have been demonstrated in CAC. MyD88-dependent TLR4 alterations trigger and amplify a pro-inflammatory environment through the NF-κB pathway. The PI3K/AKT axis stimulates many cytokines and facilitates glucose transporter 4 translocation, thereby enhancing metabolic activity in Th2 and Th17 lymphocytes. cyclooxygenase-2 upregulates prostaglandin E2 production, which in turn promotes angiogenesis and modulates immune responses through PI3K signaling[59].

CRC patients have elevated levels of NETs in blood and tissue samples. NETs facilitate tumor progression and metastasis indirectly through stimulating angiogenesis, which involves EMT-related cell migration, matrix-metalloproteinase-mediated degradation of basement membrane proteins, and capture of CRC cells. Increased MPO in NETs also leads to oxidative stress in epithelial cells, exacerbating DNA damage and raising the probability of mutational events[60].

Overall, excessive neutrophil infiltration in IBD leads to accumulation of ROS and NETs, aggravates inflammation and facilitates tumorigenesis through inducing aberrant cancer metastasis, angiogenesis, and immunosuppression.

NETs as diagnostic and prognostic biomarkers in IBD

A case-control study including a total of 100 patients with CD or UC and 100 healthy controls has indicated circulating NETs as diagnostic and prognostic markers for IBD[61]. Levels of circulating NET markers such as cfDNA, MPO-DNA complexes, and citH3 were significantly higher in the active IBD patients compared to the inactive IBD and healthy controls. Besides, all the three circulating NET markers showed positive correlation with inflammatory cytokines including IL-1β, IL-6, and vascular endothelial growth factor. Furthermore, cfDNA [odds ratio (OR) = 1.045], MPO-DNA (OR = 1.084), and CitH3 (OR = 2.871) have been identified as independent risk factors for IBD based on multivariate analysis. IBD patients with higher NET levels showed more frequent relapses. Collectively, NET biomarkers may serve as effective diagnostic and prognostic tools, enabling early intervention and improved long-term management of IBD.

Despite the diagnostic potential of these NET-associated proteins, lack of consideration of heterogeneity of IBD, standard detection methods, and relevant clinical research limit their use in clinical practice at the current stage. Future studies involving multi-center, large-sample cohorts distinguishing UC and CD, along with more standard detection method may identify more robust and stable NET biomarkers which can better define pathogenic subsets across UC and CD, and different disease stages.

NETs as a therapeutic target for translation in IBD

Guidelines for treating adult IBD have been provided by several organizations, including the American Gastroenterological Association and the European Crohn’s and Colitis Organization[62-64]. Conventional treatments are concentrated on targeting factors in adaptive immunity, aiming at controlling symptoms and achieve mucosal healing through pharmacotherapy. Conventional therapeutic strategies include the combined application of aminosalicylates, corticosteroids, immunomodulators, and biologics, with surgical resection if necessary. However, these non-specific anti-inflammation strategies have many side effects, limited effectiveness when used over a prolonged period, and patients have a high recurrence rate.

Recent progress in biologics for moderate to severe IBD has not only reshaped treatment strategies but also altered the perspective of IBD therapy. Biologics, such as IL inhibitors, integrin inhibitors, Janus kinase inhibitors, and antisense oligonucleotides, each influence IBD pathogenesis in distinct ways. Early use of biologics or combination therapies may benefit some patients; especially those with medically refractory IBD or extraintestinal manifestations. However, 30%-50% of patients experience insufficient outcomes under current therapies, with substantial side effects or lack of personalized approaches. These call for better treatment strategies. Emerging options for IBD management include novel monoclonal antibody therapy, apheresis, and modulation of the intestinal microbiome[65].

Researchers have identified many NET-related proteins and structures in blood or intestinal biopsy samples from UC and CD patients by utilizing proteomics and confocal microscopy analysis. Modulating the innate immunity, especially NET formation, and boosting drug delivery to inflamed intestine are promising therapeutic directions for IBD.

Potential targets to regulate NETs for IBD treatment are proposed in Table 2. PAD accelerates the post-translational conversion of peptidylarginine to peptidylcitrulline through a calcium-dependent, irreversible reaction and mediates the effects of proinflammatory cytokines. Levels of PAD are elevated in both mouse and human colitis. Chloramidine, a PAD inhibitor, induces apoptosis of inflammatory cells in vitro and in vivo, and suppressed colitis in a DSS-induced mouse model through reducing the clinical manifestations of colitis; both prophylactically and after the onset of disease. Chumanevich et al[66] validated PADs as therapeutic targets for managing IBD, and proposed chloramidine as a candidate therapy.

Table 2 Therapeutic targets: Inhibition of neutrophil extracellular traps to ameliorate progression of inflammatory bowel disease.

Agent	Target molecule/function	Effect	Disease/animal model	Ref.
Cl-amidine	PAD inhibitor	Inhibit colon inflammation; stimulate apoptosis of inflammatory cells in vitro and in vivo	DSS-induced mouse model	Chumanevich et al[66]
Butyrate	Suppresses neutrophil migration and NETs production	Ameliorate mucosal inflammation	DSS-induced murine colitis	Li et al[69]
Cyclosporine A	Inhibit NETs via the regulating PPP	Reduce ROS-dependent NETs release via downregulating PPP and cellular ROS levels by decreasing G6PD activity directly by activating the P53 protein	DSS-induced colitis mouse model	Xu et al[70]
Hydroxychloroquine	Inhibit NET formation	HCQ inhibit the activation of MEK/ERK/ROS signaling pathway	DSS induced colitis	Jiang and Zhang[74]
Thymopentin	Inhibit NET formation	Reduced DAI, weight loss and colon shrinkage; decreased MPO, NE, citH3, PAD4, dsDNA	TNBS-induced colitis mouse model	Cao et al[79]
PAD4 genetic deficiency	Down-regulate cGAS-STING pathway	Inhibit NET formation, protect the intestinal barrier, alleviate intestinal inflammation	DSS-induced colitis mice	Sun et al[46]
CXCR1 and CXCR2	Promote UC and NET formation through neutrophil chemotaxis and PAD4-mediated pathways	Higher expression of CXCR1/CXCR2 in colon tissues of UC patients	UC; summary data from a genome-wide association study and FinnGen	Xv et al[71]
Hydrogen sulfide	Inhibit NET formation	Inhibit the expression of NF-κB and HMGB1; elevate the expression of anti-inflammatory mediator UCHL-1	TNBS-induced colitis rat model	Török et al[80]

PAD4: Peptidylarginine deiminase 4; NETs: Neutrophil extracellular traps; PPP: Pentose phosphate pathway; cGAS: Cyclic guanosine monophosphate-adenosine monophosphate synthase; STING: Stimulator of interferon genes; UC: Ulcerative colitis; ROS: Reactive oxygen species; G6PD: Glucose-6-phosphate dehydrogenase; HCQ: Hydroxychloroquine; ERK: Extracellular regulated protein kinases; MEK: Mitogen-activated protein kinase kinase; DAI: Disease activity index; MPO: Myeloperoxidase; NE: Neutrophil elastase; citH3: Citrullinated histone H3; dsDNA: Double-stranded DNA; NF-κB: Nuclear factor kappa-B; HMGB1: High-mobility group box 1; TNBS: 2,4,6-trinitrobenzene sulfonic acid; DSS: Dextran sulfate sodium.

Intestinal homeostasis depends on the ingenious balance between host and gut microbiota. Abnormal immune responses triggered by antigens originating from the gut microbiota are regarded as the initial events that lead to the chronic, relapsing intestinal inflammation observed in IBD. Changes in the composition of the gut microbiota and its metabolite profile also participate in the pathogenesis of IBD[67].

SCFAs are metabolites produced by gut microbes when they ferment indigestible dietary fiber, and abundant evidence have shown that SCFAs exert anti-inflammatory effects and help maintain epithelial barrier integrity[68]. Li et al[69] have found that butyrate markedly reduces the production of pro-inflammatory cytokines, chemokines, and calprotectin by neutrophils in IBD patients. In addition, butyrate inhibited the migration of neutrophils and the formation of NETs in CD and UC patients in vitro studies. In DSS-induced murine colitis, oral administration of butyrate significantly ameliorated mucosal inflammation by suppressing the secretion of proinflammatory mediators and the formation of NETs, while simultaneously enhancing neutrophils’ phagocytic and ROS-dependent bactericidal functions, primarily through a histone deacetylase-mediated pathway. Further clinical investigations also revealed that butyrate treatment significantly reduces ROS generation and NET release in neutrophils isolated from IBD patients. Hence, butyrate limits neutrophil functions and alleviates intestinal inflammation in IBD, highlighting its potential as a novel therapeutic strategy.

Therapies that target NETs are currently being investigated in animal models of IBD. CsA, now used as a rescue therapy for acute severe UC, mitigates colitis by suppressing NET formation via regulating the pentose phosphate pathway (PPP). Enrichment of NETs was elevated in moderate-to-severe UC patients compared to healthy controls. In a DSS-induced colitis mouse model, CsA reduced NETs expression in the colon. A decrease in cellular ROS, cellular ribulose 5-phosphate and NADPH/NADP⁺ related to the PPP, along with intracellular glucose-6-phosphate dehydrogenase (G6PD) activity were confirmed in the LPS + CsA group. Higher expression of p53 protein was found in the LPS + CsA group compared with the LPS group. CsA suppresses ROS-dependent NETs release via downregulating the PPP and decreasing cellular ROS levels through direct activation of p53 that reduces G6PD activity[70].

Xv et al[71] examined the association between NET-associated genes (NRGs) and UC by analyzing summary statistics from a genome-wide association study (12366 cases vs 33609 controls) together with FinnGen (8279 cases vs 261098 controls). The Bayesian method was used to assess the evidence supporting one of five exclusive hypotheses (H0-H4). H4 refers to the scenario where the association with both traits is present and the causal variant is shared by both traits. The default prior probability was set at 1 × 10^-5, which means that single nucleotide polymorphisms (SNPs) within the given genomic region are significantly associated with NRG expression and UC risk. SNPs located within 500 kb upstream and downstream of NRGs were used to perform colocalization analysis to obtain a higher statistical power. The significance threshold for colocalization was set at a posterior probability of H4 > 0.80. They screened out seven NRGs linked to an increased risk of UC. Among these NRGs, expression of ITGB2 showed positive correlation with higher UC risk, while higher expression of CXCR1, CXCR2, IRAK4, MAPK3, SIGLEC14, and SLC22A4 were inversely associated with UC risk. Colocalization analysis confirmed the association between CXCR1/CXCR2 and the risk of UC. Higher expression of CXCR1/CXCR2 in colonic tissues of UC was confirmed using immunohistochemical analysis. CXCR1/CXCR2 promoted UC and NET formation through neutrophil chemotaxis and PAD4-dependent pathways, based on gene set enrichment analysis and correlation analysis. Their study identified CXCR1 and CXCR2 as potential NET-related treatment targets in UC through multi-method argumentation, offering novel perspectives on how NET formation is regulated during the development of UC.

Traditional Chinese medicine has shown therapeutic potential in gastrointestinal disorders, including ulcers and inflammation[72]. Hydroxychloroquine (HCQ), forsythiaside A (FA), Coptis chinensis (Huanglian) and its active component berberine, all alleviate DSS-induced colitis in mice[73]. HCQ alleviates DSS-induced colitis by inhibiting the formation of NETs. HCQ decreases ROS generation and blocks activation of the mitogen-activated protein kinase kinase (MEK)/ERK signaling cascade in vitro. HCQ inhibits the MEK/ERK signaling cascade, which can be reversed with MEK agonist C16-PAF, leading to the enhancement of ROS generation and NET formation[74]. FA alleviated colitis by inhibiting PAD4-mediated NET formation[75]. FA reduced intestinal inflammatory cytokines, elevated the levels of the tight junction protein, and alleviated gut microbiota dysbiosis.

Yang et al[76] demonstrated that Coptis chinensis-derived extracellular vesicle-like nanoparticles (Cc-ELNs) delivered microRNA-5106 and inhibited NET formation by re-establishing zinc balance to alleviate colitis. Intraperitoneal administration of Cc-ELNs in DSS-induced colitis mice selectively targeted the inflamed intestine, reduced neutrophil recruitment, and inhibited NET formation through delivering miR-5106 and downregulating Slc39a2 expression to restore zinc homeostasis. Furthermore, Cc-ELNs suppressed pyroptosis in IECs, and promoted proliferation of IECs and intestinal stem cells by suppressing NET formation. Their findings highlight the potential of traditional Chinese medicine, especially plant-derived nanoparticle-based therapies by suppressing NET formation. The enhanced permeability and retention effect, coupled with the compromised vascular endothelium and elevated interstitial fluid pressure, may facilitate the extravasation and preferential accumulation of nanoparticles in pathological sites, ultimately leading to the selective enrichment of Cc-ELNs in inflamed colons. Moreover, Cc-ELNs could possess surface ligands capable of actively binding to receptors that are overexpressed in inflamed intestinal tissues. Further investigations are required to pinpoint the specific ligands and their corresponding targets.

Nontoxigenic Gram-negative Bacteroides fragilis, a human colon symbiote, has been proposed as a probiotic in treating colitis. Outer membrane vesicles (OMVs) released by Gram-negative bacteria play a crucial role in host-microbe interactions. OMVs contain adhesins, proteases and sulfatases, which facilitate their interaction with host cells and enable them to enter cells via micro-vesicle-mediated endocytosis, lipid-raft-dependent endocytosis, and clathrin-dependent endocytosis[77]. Yang et al[78] explored the underlying mechanisms. They demonstrated that Bacteroides fragilis-derived OMVs (Bf^OMVs+) delivered miR-5119 and alleviated DSS-induced colitis by targeting programmed death protein ligand 1 to inhibit neutrophil recruitment, suppress gasdermin D-mediated NET formation, and promote proliferation of ISCs[78].

In a mouse model of TNBS-induced colitis to mimic CD, thymopentin (TP5) ameliorated weight loss, disease activity index, and colon shrinkage in experimental colitis via inhibiting NETs, characterized by decreased CitH3, PAD4, MPO, NE, and double-stranded DNA levels. Neoseptin 3, a specific NET agonist, markedly counteracted the effect of TP5[79].

Török et al[80] have investigated the impact of hydrogen sulfide (H₂S) treatment on NET formation and inflammatory mediator expression in a TNBS-induced experimental rat colitis. They confirmed that H₂S exerted anti-inflammatory effects by inhibiting NET formation and the expression of NF-κB and high-mobility group box 1 (HMGB1). Decreased expression of PAD4, citH3, MPO, and inflammatory regulators including NF-κB and HMGB1 were reduced by H₂S. Administration of H₂S donor also upregulated expression of ubiquitin C-terminal hydroxylase L1, which is a potential anti-inflammatory mediator. Their findings indicated that H₂S treatment may achieve an anti-inflammatory effect through suppressing NET formation, suggesting a novel therapeutic strategy for IBD.

The classical methods of drug delivery include parenteral, oral, and rectal routes, among which, oral delivery remains the most widely used. Oral pharmaceutical formulations are designed to attain systemic absorption. Nonetheless, targeting the inflamed intestine locally could provide benefits compared with systemic treatment for IBD. Rectal administration is effective for patients with inflammation limited to the distal colon. However, rectal administration becomes less appropriate when inflammation spreads to other intestinal segments. Since IBD can affect any region of the gastrointestinal tract, delivering drugs directly to the inflamed sites could enhance the treatment effectiveness while minimizing systemic adverse effects.

Current trends in drug-delivery strategies for IBD encompass nanoparticle-mediated drug delivery systems, enteric-coated microneedle pills, lipid-based vesicular systems, prodrug approaches, hybrid drug delivery platforms, and biologic drug delivery systems[81]. Higher levels of ROS at the inflamed site have been demonstrated in IBD. Redox-responsive nanoparticles have been explored as a promising approach for treating IBD. Hence, strategies combining NETs with a ROS-responsive drug delivery platform that specifically targets gut inflammation may achieve more specific and efficacious treatment for IBD.

Exercise may influence the composition of the gut microbiota[82]. Regular exercise constitutes a key lifestyle approach for UC patients[83]. Epidemiological research indicates that engaging in regular exercise lowers the risk of UC by enhancing intestinal barrier integrity and maintaining gut microbiota homeostasis. Gut microbiota dysbiosis and dysregulated immune responses play important roles in the pathogenesis of IBD. Fecal microbiota transplantation (FMT) from healthy donors is a promising intervention to reconstruct the gut microbiota in IBD[84]. In a recent study, FMT from voluntary exercised mice showed protective effects in DSS-induced colitis, marked by strengthened intestinal barrier function, prevention of gut microbiota dysbiosis, and inhibition of NET formation[85]. Their study highlights the protective effect of the gut microbiota against colitis and suggests that FMT derived from exercise, together with targeted NET inhibition, could serve as a promising therapeutic approach for UC.

Research gaps and challenges in targeting NETs for treatment

Despite the promising evidence to support the aforementioned strategies in targeting NETs to treat IBD, several research gaps and challenges remain to be addressed. First of all, most studies are preclinical, mainly based on animal models such as DSS-induced mouse colitis. Lack of clinical validation limits the translation of these strategies into evidence-based therapies. Although studies have confirmed that microRNA-5106 delivered from Cc-ELNs and miR-5119 delivered from Bf^OMVs can alleviate DSS-induced colitis, these studies were conducted in animal models. Before clinical application, more research is needed to improve drug delivery efficiency to inflamed intestinal sites, optimize dosage, and evaluate in vivo stability and long-term safety. At the same time, the precise mechanisms underlying the crosstalk between NET formation, gut microbiota, metabolites are still not fully elucidated. Causal relationships between gut microbiota and NETs remain unclear. Utilizing multi-omics approaches to unravel the complex interactions between NETs and gut microbiota, to identify immune pathways involved are important in further research.

Second, the risk of immunosuppression and infection must be squarely addressed. Inhibition of NET formation is effective in reducing inflammation, while it may affect the essential host-defense functions of neutrophils, contributing to an increased susceptibility to bacterial or fungal infections. Future work must focus on developing localized delivery systems or shorter treatment regimens to minimize systemic exposure and reduce the risk of infection.

Finally, more rigorous randomized controlled trials should be conducted to evaluate the efficacy and safety of traditional Chinese medicine in UC patients, especially compared to conventional therapeutics. Addressing these gaps will facilitate the development of novel integrative treatment strategies for IBD.

CONCLUSION

NETs play a pivotal role in maintaining homeostasis by enhancing pathogen clearance, immunoregulation, wound healing, and immunothrombosis. The balance between NET formation and clearance is essential for homeostasis; when disrupted, excessive NETs can lead to inflammatory cytokine infiltration and disease development. Studies have demonstrated elevated NET formation in the blood, feces, and intestinal mucosa of IBD patients, which correlates positively with disease activity. NETs participate in IBD pathophysiology, ranging from intestinal inflammation and tissue damage to gut microbiota dysbiosis, and they also increase the tendency for thrombosis and CAC. Circulating NET-associated molecules, such as cfDNA, MPO-DNA complexes, and citH3, may serve as diagnostic and prognostic markers for IBD. However, due to the heterogeneity of IBD, varying disease stages, lack of standard detection methods and multi-center cohort validation, these markers have yet to be widely adopted in clinical practice. Potential therapeutic targets for regulating NETs in IBD have been discussed. Nevertheless, most studies remain preclinical, primarily based on animal models, and lack clinical validation. The precise mechanisms underlying the crosstalk between NET formation, gut microbiota, and metabolites are still not fully elucidated. Additionally, the risks of immunosuppression and infection must be taken into consideration. Drug delivery efficiency to inflamed intestines, dosage optimization, as well as in vivo stability and long-term safety require further exploration. Consequently, a more thorough investigation of the functional roles of NETs in IBD is crucial for deepening our understanding of IBD pathogenesis and uncovering potential diagnostic biomarkers or therapeutic targets.