Advances in research of neutrophil extracellular trap formation in osteoarticular diseases

Abstract

Neutrophil extracellular traps (NETs) have been the subject of research in the field of innate immunity since they were first described two decades ago. NETs are fibrous network structures released by neutrophils under specific stimuli, including DNA, histones, and a variety of granular proteins. NETs have been widely studied in the fields of infectious and immune diseases, and new breakthroughs have been made in the understanding of disease pathogenesis and treatment. In recent years, studies have found that NETs play an important role in the occurrence and development of osteoarticular diseases. This article reviews the progress in the research of NETs in common osteoarticular diseases such as rheumatoid arthritis, ankylosing spondylitis, gouty arthritis, osteonecrosis of the femoral head, osteoarthritis, and joint fibrosis, including the formation mechanism of NETs and its role in inflammation, joint destruction, pain and other pathological processes. The problems existing in current research are discussed, along with future research directions, to provide a reference for the in-depth study of osteoarticular diseases and the development of new treatment strategies.

Key Words: Neutrophil extracellular traps; Osteoarticular diseases; Inflammation; Fibrosis; Research progress

Core Tip: Neutrophil extracellular traps contribute to the development of inflammatory and immune diseases, and this particular mechanism opens up a new field for understanding the pathogenesis of osteoarthrosis. This article reviews the research progress of neutrophil extracellular traps in common osteoarticular diseases. In-depth study of them can help reveal the underlying pathophysiological processes of osteoarthrosis and provide a theoretical basis for the development of new diagnostic markers and therapeutic targets.

INTRODUCTION

Neutrophils are first responders to antimicrobial host defense and sterile inflammation and therefore play an important role in health and disease processes. Nearly 20 years after neutrophil extracellular traps (NETs) were first described as an alternative mode of killing pathogens, it has become clear that NETs also contribute to a large extent to sterile forms of inflammation[1]. NETs contribute to all forms of thrombosis, particulate-induced inflammation, autoimmune vasculitis, autoinflammatory diseases, and secondary inflammation caused by ischemic, toxic, or traumatic tissue injury. Recently, NETs have also been found to be an important component of the multiorgan complications of coronavirus disease 2019[2]. Osteoarticular diseases are a series of diseases that seriously affect human health and quality of life, including rheumatoid arthritis (RA), osteoarthritis (OA), ankylosing spondylitis (AS), and osteonecrosis of the femoral head (ONFH). Their pathogenesis is complex, involving multiple links such as immune response, inflammatory response, destruction and repair of cartilage, and bone tissue imbalance. Neutrophils, as an important part of the innate immune system, have long been considered to participate in anti-infective immunity, mainly through phagocytosis, degranulation, and other effects. However, the special mechanism of NETs discovered in recent years has opened up a new field for understanding the pathogenesis of osteoarticular diseases[3,4]. NETs can directly capture pathogens and interact with other immune cells and joint tissue cells through various pathways, promoting the continuation of inflammatory responses and the destruction of joint structures. In-depth study of NETs can help reveal the potential pathophysiological processes of bone and joint diseases, providing a theoretical basis for the development of new diagnostic markers and therapeutic targets.

STRUCTURE, FORMATION, AND CLASSIFICATION OF NETs

In 2004, Brinkmann et al[5] reported that human neutrophils, upon treatment with phorbol myristate acetate (PMA), experience a distinct form of cell death that differs from necrosis and apoptosis. In 1996, a Japanese research group also published a report on neutrophil responses to PMA treatment. Initially, it was uncertain whether this type of cell death led to the creation of extensive 3-dimensional structures made up of thin chromatin fibers and granular proteins within the cell[6]. NETs primarily consist of histones, with additional proteins such as granzymes and peptides. These include neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, leukocyte proteinase 3, lactoferrin, gelatinase, lysozyme C, calprotectin, neutrophil defensins, and antimicrobial peptides[7]. These structures are named NETs, and the specific cell death pathway is called NETosis[8].

Numerous stimulators of NETosis have been identified, such as bacteria and their components, fungi, protozoa[9], viruses, activated platelets, complement-related peptides, autoantibodies, interleukins, hydrogen peroxide, urate crystals, cigarettes, and ion carriers[10-12]. The molecular events involved in PMA-induced NETosis and the formation of NETs have been extensively studied. PMA directly binds to protein kinase C, triggering calcium release from intracellular stores and activating the Raf-MEK-ERK pathway[13]. A polymeric nicotinamide adenine dinucleotide phosphate oxidase complex forms on the phagocyte membrane downstream to produce reactive oxygen species. The serine protease NE is mobilized alongside MPO and transported to the nucleus to process core histones[14]. Elevated intracellular calcium levels also activate peptidyl arginine deiminases 4 (PAD4)[15,16], PAD4 mediates the post-translational deimination of histone arginine residues, converting them into citrulline moieties through hydrolytic removal of an imino group. This citrullination modification diminishes the positive charge on histones, thereby attenuating their electrostatic interaction with the negatively charged DNA backbone. Consequently, the chromatin structure becomes less compact, facilitating transcriptional activation or other chromatin-templated processes[17]. These molecular events reduce the compression of chromatin. Thus, after about 2 hours, PMA-stimulated neutrophils lose the heterochromatic region of the nucleus and the characteristic nuclear lobules. As a result, the nucleus depolymerizes and expands. The nuclear membrane breaks down into vesicles and granules, and mitochondria break down. The cytoplasm and nucleoplasm fuse with each other, and eventually the cell membrane ruptures, releasing the cell contents and unfolding in the extracellular space to form NETs[11,18]. The proposed mechanism is illustrated in Figure 1[19].

Figure 1 Schematic diagram of suicidal NETosis. 1: Receptor-mediated stimulus detection initiates the signaling cascade; 2: The Raf/MEK/ERK pathway activation and elevated calcium levels trigger gp91phox phosphorylation, enabling nicotinamide adenine dinucleotide phosphate oxidase assembly and reactive oxygen species generation; 3: Reactive oxygen species-dependent mechanisms facilitate nuclear translocation of azurophilic granule components (elastase and myeloperoxidase), inducing chromatin decondensation and nuclear morphological changes; 4: Nuclear envelope disintegration allows mixing of decondensed chromatin with cytoplasmic proteins; 5: Plasma membrane rupture results in the release of chromatin-protein complexes as extracellular traps. Created by Figdraw, ID: SPUAUc244c. TLR: Toll-like receptor; ROS: Reactive oxygen species; PKC: Protein kinase C; PAD4: Peptidyl arginine deiminases 4; NE: Neutrophil elastase; MPO: Myeloperoxidase.

This sequence of events is known as suicidal NETosis. Neutrophils can reportedly release their nucleus partially or entirely without compromising the cell membrane, allowing the anucleated cytoplasm to remain motile and capable of engulfing bacteria[20]. Co-culturing neutrophils with Staphylococcus aureus, results in rapid DNA release, accompanied by significant nuclear expansion and vesicle formation. These vesicles carry DNA that merges with the cell membrane, discharging their contents without cell dissolution. In vivo imaging revealed that neutrophil behavior relies on Toll-like receptor 2 (TLR2) and complement component C3[21]. Neutrophils remain viable and mobile within tissues post-nuclear DNA release, albeit with an altered crawling pattern compared to those retaining their nucleus. This form of DNA release, where neutrophils remain viable, is termed viable NETosis (Figure 2)[19].

Figure 2 Schematic diagram of vital NETosis. 1: Pathogen recognition via surface receptors triggers cellular activation; 2: Nuclear lobulation is lost, accompanied by chromatin decondensation; 3: The nuclear envelope undergoes sequential disassembly - outer and inner membranes separate and vesiculate; 4: Nuclear-derived vesicles containing stretched chromatin filaments accumulate in the cytoplasm; 5: Cytoplasmic granules migrate toward the intact plasma membrane while maintaining membrane integrity; 6: Focal plasma membrane rupture enables extracellular DNA release, while selective granule exocytosis deposits antimicrobial proteins onto the expelled chromatin network. Created by Figdraw, ID: USWOWe861a. C. albicans: Candida albicans; S. aureus: Staphylococcus aureus; CR3: Complement receptor 3; TLR2: Toll-like receptor 2.

NETS AND DISEASES

Although NETs have antibacterial activity and help address inflammation[22], they also induce diseases in a variety of contexts[23], including arthritis[24,25], systemic lupus erythematosus[26], antiphospholipid antibody syndrome[27,28], small vasculitis, and psoriasis[29-31]. In cancer, neutrophils affect health through a variety of mechanisms, and NETs has been demonstrated to promote tumor metastasis[1,32-34]. In the cardiovascular system, NETs play a role in atherosclerosis and venous thrombosis[35]. Another important disease associated with the pathophysiology of NETs is sepsis. In this process, the release of NETs has been shown to increase the risk of venous thromboembolism. Studies have also shown that high rates of NETs are associated with the severity of sepsis[36]. The presence of NETs in laboratory sepsis models induces multiple organ damage[37]. In lung diseases, NETs play an important role in chronic inflammation, including cystic fibrosis[38] and chronic obstructive pulmonary disease[39]. Overall, from inflammation to thrombosis to tumors and fibrosis, NETs can perform basic functions when they are produced in the right place and at the right time, but can have serious effects[40] when their production or clearance is not adequately controlled.

NETs and RA

RA is the most widely studied disease among osteoarticular diseases. RA is an autoimmune disease that affects 0.5%-1% of the world's adult population[41]. As an autoimmune disease, RA is characterized by the presence of specific autoantibodies, such as anti-citrullinated protein antibodies[42]. RA is characterized by synovial inflammation, cartilage damage, and bone damage. These injuries cause severe joint dysfunction and disability[43], causing severe economic and social burdens, significantly affecting patients’ quality of life. Neutrophils are the most abundant cells in joints during the early stages of RA or during the onset of acute disease, suggesting that these cells may be involved in tissue damage that occurs in the disease[44]. High levels of NETs have been observed in synovial fluid of RA patients and in different experimental models of RA[45]. Treatment of arthritic mice with DNase or knockout of PAD4, a key enzyme involved in histone citrullination and NETs production, reduced the severity of experimental arthritis[46]. At the same time, NETs can also produce new autoantigens, such as citrulline histone, which can enhance the autoimmune response of RA[47]. These are enough to illustrate the important role of NETs in the pathogenesis of RA.

In the pathology of RA, synovial inflammation and pannus, and cartilage and bone damage are major manifestations, and each aspect has been specially studied. Inflammation-related receptors and ligands have been demonstrated to be involved in the relationship between NETs and RA. Navrátilová et al[40] and other studies have shown that interleukin-40 (IL-40) is upregulated in RA and decreased after B-cell depletion. IL-40 is a newly discovered B-cell-related cytokine, which is related to humoral immune response and B-cell homeostasis. The correlation of IL-40 with autoantibodies, chemokines, and NETosis markers suggests that it is involved in the development of RA. IL-40 upregulates the secretion of chemokines and matrix metalloproteinase-13 in synovial fibroblasts, suggesting that it plays a role in the regulation of inflammation and tissue destruction in RA[48]. Other related studies have also shown that inflammatory factors such as IL-6, IL-33, IL-1, and tumor necrosis factor-α and NETs form positive feedback, prolong neutrophil action, enhance the inflammatory and invasive phenotypes of synovial fibroblasts, and maintain inflammation without remission[16,49,50]. Inflammation-associated receptor ligands, such as TLRs and endothelial protein C receptors, have also been shown to influence the disease or severity of RA[51,52]. The role of NETs in the pathogenesis of RA is shown in Figure 3[53].

Figure 3 The role of neutrophil extracellular traps in the pathogenesis of rheumatoid arthritis. In rheumatoid arthritis, circulating factors promote excessive neutrophil extracellular traps formation. This overproduction results in citrullinated histones (including citH2A, citH2B, and citH4) that serve as modified autoantigens. Within rheumatoid arthritis joints, these citrullinated neoepitopes trigger adaptive immune responses, driving the production of neutrophil extracellular trap-targeting autoantibodies. This self-perpetuating cycle of autoantigen presentation and autoantibody generation contributes to chronic synovial inflammation. Created by Figdraw, ID: IYTII99622. NETs: Neutrophil extracellular traps; IL: Interleukin; TNF: Tumor necrosis factor; MMP: Matrix metalloproteinase; ACPA: Anti-citrullinated protein antibody; RA: Rheumatoid arthritis.

Carmona et al[54] demonstrated experimentally and in animal models that NET-produced elastase disrupted the cartilage matrix and induced fibroblast-like synovial cells to release membrane-bound peptidyl arginine deiminase 2. The cartilage fragments are then citrullinated, internalized by fibroblast-like synovial cells, and then presented to antigen-specific CD4⁺ T cells. The immune complex containing the citrullinated cartilage component can activate macrophages to release proinflammatory cytokines. HLA-DRB1*04:01 transgenic mice immunized with NETs produced autoantibodies against citrullinated cartilage proteins and showed enhanced cartilage damage. Inhibition of NET-derived elastase can salvage NET-mediated cartilage damage. These results suggest that NETs and NE play a fundamental pathogenic role in promoting cartilage injury and synovial inflammation. Strategies targeting NE and NETs may play a therapeutic role in RA and other diseases associated with inflammatory joint injury. NETs mediate cartilage injury in RA and enhance the immunogenicity of cartilage components (Figure 4). Schneider et al[55] have shown that NETs mediate bone erosion in RA by enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation. The level of NETs in synovial fluid of RA patients is higher than that of patients with OA. Inhibition of NETs with DNase or PAD4 deletion alleviates bone loss in arthritic mice. Similarly, NETs enhance RANKL-induced osteoclast generation, which is dependent on TLR4 and TLR9 generation, and increased osteoclast uptake in vitro.

Figure 4 Schematic representation of the role of neutrophil elastase in synovial cartilage integrity in rheumatoid arthritis. 1: Neutrophils in rheumatoid synovium exhibit dysregulated NETosis; 2: Neutrophil-derived elastase within neutrophil extracellular traps degrades cartilage matrix, producing immunogenic fragments; 3: Membrane-bound peptidyl arginine deiminase 2 is liberated from fibroblast-like synoviocytes (FLSs) through elastase-mediated cleavage; 4: The liberated peptidyl arginine deiminases 2 catalyzes citrullination of cartilage fragments, which are subsequently internalized by FLSs; 5: FLSs present these citrullinated peptides to autoreactive CD4⁺ T cells; 6: Triggering B cell differentiation and production of autoantibodies targeting citrullinated proteoglycans; 7: The resulting immune complexes stimulate macrophage activation and secretion of proinflammatory mediators; 8: These cytokines further prime neutrophils for elastase release, establishing a self-amplifying inflammatory cascade that perpetuates cartilage destruction. Created by Figdraw, ID: TAASU77e88. NETs: Neutrophil extracellular traps; IL: Interleukin; TNF: Tumor necrosis factor; MMP: Matrix metalloproteinase.

NETs and AS

AS is a chronic systemic disease that is the most common type of chronic inflammatory spondyloarthritis. The global prevalence of AS is generally estimated to be between 0.1% and 1.4%[56]. The onset of AS occurs most often in the third decade of life, and is higher in men than in women. The uncontrolled evolution of AS results in severe impairment of spinal mobility and bodily function, which adversely affects patients' quality of life. Similar to other autoimmune diseases, the etiology of AS remains unclear, although it is thought to involve a combination of genetic and environmental factors that produce chronic inflammation. The main genetic risk factors can be attributed to the human leukocyte antigen-B27 gene[57]; however, other non-major histocompatibility complex genes or genetic regions are also associated with susceptibility to AS, including IL-23, IL-27, and Fc receptors[58]. Environmental factors are also thought to play an important role in the activation and exacerbation of AS. Infections caused by bacterial pathogens such as Yersinia, Shigella, Salmonella, Campylobacter, and Chlamydia are thought to be the main environmental factors associated with the development of AS.

Recent studies have shown that there may be a correlation between NETs and the occurrence and development of AS[59]. Zambrano-Zaragoza et al[59] have shown that the release of NETs may be an important source of persistent inflammation in AS. Van Tok et al[60], based on the serum of nine AS patients, confirmed for the first time that NET formation is involved in AS inflammation and inflammation-driven bone overformation, a major disease feature. Papagoras et al[61] showed clinically and experimentally that neutrophils from AS patients are characterized by the formation of NETs carrying biologically active IL-17A and IL-1β. NETs from IL-17A-rich patients mediate the differentiation of bone marrow mesenchymal stem cells into osteoblasts. IL-1β positively regulates the expression of neutrophil IL-17A. Blocking IL-1β signaling on neutrophils using anakinra, or using DNase-I against NETs, would disrupt IL-17A NET-driven osteogenesis (Figure 5). These findings suggest a novel role for neutrophils in AS-related inflammation. IL-1β promotes IL-17A expression on NETs, providing an additional therapeutic target for AS.

Figure 5 The role of neutrophil extracellular traps in the pathogenesis of ankylosing spondylitis. The interleukin-17 (IL-17)/neutrophil extracellular traps axis directs mesenchymal stem cell osteogenesis, while IL-1β (from both serum and neutrophil extracellular traps) further stimulates neutrophilic IL-17 secretion. This reciprocal interaction establishes a pro-osteogenic inflammatory microenvironment that perpetuates both NETosis and aberrant bone formation. Created by Figdraw, ID: WRRPRa9680. NETs: Neutrophil extracellular traps; IL: Interleukin; MSCs: Mesenchymal stem cells; AS: Ankylosing spondylitis.

NETs and gouty arthritis

The relationship between NETs and the onset and remission of gouty arthritis has been controversial. Gout is a common metabolic disease. Recent studies have shown that the incidence of gout has increased worldwide, affecting 1%-6.8% of the population and placing a heavy burden on the healthcare system[62]. Gout is characterized by the deposition of monosodium urate (MSU) crystals in joints or other extra-articular tissues, leading to self-limiting inflammation. Hyperuricemia is the most important factor in the deposition of MSU crystals. When hyperuricemia occurs, a persistent increase in serum uric acid levels leads to supersaturation of uric acid in the body and deposition in tissues[63].

Previous studies have suggested that NETs have a significant impact on the onset and remission of gouty arthritis[64]. Aggregated NETs may secrete a large number of enzymes, degrade inflammatory factors and chemokines, and cause spontaneous remission of gouty arthritis. Therefore, the role of NETs in gout has attracted the attention of scholars. However, Vedder et al[65] showed that the level of serum NETs in gout patients was higher than that in the normal control group, but it was not related to the disease activity and inflammation of gout, indicating that NETs may be one of the causes of gout attacks. Reber et al[66] showed that in the absence of neutrophils, the MSU crystal capture process was not affected, and it was possible that other types of cells produce extracellular traps and affect the development of gout. Gout represents a prototypical crystal-driven autoinflammatory disorder wherein MSU crystal deposition in articular structures initiates a cascade of pathogenic events. These birefringent crystals serve as potent danger signals that are phagocytosed by innate immune cells, triggering nucleotide-binding domain, leucine-rich repeat and pyrin domain-containing protein 3 inflammasome activation through a distinctive mechanism involving: (1) Rapid intracellular sodium accumulation; (2) Osmotically-driven water influx; and (3) Critical potassium efflux, collectively culminating in IL-1β maturation and release. The inflammatory milieu is further amplified by neutrophil-derived mediators (IL-8, tumor necrosis factor-α, and IL-6) that establish a chemotactic gradient for additional leukocyte recruitment while simultaneously promoting NETs generation. Notably, extruded NETs components may become structurally incorporated into developing tophi, as evidenced by the close association between NETs constituents and these characteristic gouty lesions. This NET-tophus interplay potentially contributes to the chronic granulomatous response observed in advanced disease (Figure 6)[19,67]. One of the reasons why NETosis is thought to be a mechanism of spontaneous resolution of gout is that tophi share characteristics with aggregated NETs. Aggregated NETs may be the basis of tophi and have an impact on the evolution of gout. However, it is unreasonable to simply speculate that aggregated NETs and NETosis are mechanisms of spontaneous resolution of gout, and further research on the composition, function, and mechanism of MSU crystal-induced NETs is required[68].

Figure 6 The neutrophil extracellular traps alleviate inflammation caused by monosodium urate crystals. 1: Monosodium urate crystals nucleate in joints either through de novo precipitation or release from existing tophi, subsequently being phagocytosed by antigen-presenting cells; 2: Intracellular crystal dissolution creates osmotic stress - sodium influx drives water entry, critically reducing potassium concentration below the threshold required for nucleotide-binding domain, leucine-rich repeat and pyrin domain-containing protein 3 inflammasome assembly; 3: Inflammasome activation triggers interleukin-18 (IL-18) and IL-1β release, establishing a chemokine gradient for neutrophil recruitment; 4: Infiltrating neutrophils amplify inflammation through proteinase 3-mediated pro-IL-1β processing while simultaneously engaging in crystal clearance via NETosis; 5: The resulting neutrophil extracellular trap (NET) structures serve dual roles: Physically encapsulating monosodium urate crystals to limit their bioavailability and forming aggregated NETs that proteolytically degrade inflammatory mediators; 6: Paradoxically, persistent aggregated NET deposition may nucleate new tophi, completing the pathogenic cycle. Created by Figdraw, ID: UAWAYbf41e. AggNETs: Aggregated neutrophil extracellular traps; NLRP3: Nucleotide-binding domain, leucine-rich repeat and pyrin domain-containing protein 3; DC: Dendritic cells; MSU: Monosodium urate; IL: Interleukin; TNF: Tumor necrosis factor.

NETs and OA

OA is a widespread degenerative joint disease that can lead to severe damage to joint cartilage and bone. The progression of OA is closely associated with aging, joint damage, muscle atrophy around the joints, synovial inflammation, subpatellar fat pad degeneration, and meniscus degeneration. Approximately 60% of older people in the world are affected by OA, resulting in a significant decrease in their quality of life[69]. The process of OA has been thought to be influenced by multiple mechanisms of cell death, such as apoptosis, necrosis, and ferroptosis[70], but the contribution of NETs to OA is unknown. In the current literature, the correlation between NETs and OA has been studied[16,71]. OA almost always appears as a control group to study the relationship between NETs and autoimmune diseases, such as RA and AS. Luan et al[72] identified and analyzed NET-related genes in OA through bioinformatics and experimental verification. Kyoto encyclopedia of genes and genomes pathway enrichment results show that cytokine receptors are most closely related to cytokines, osteoclast differentiation, and mitogen-activated protein kinase signaling pathway in OA. During progression of OA, the interaction of cytokine-cytokine receptors may lead to activation of the mitogen-activated protein kinase signaling pathway, thereby promoting the differentiation of osteoclasts. Increased activity of such osteoclasts can promote the destruction of articular cartilage and bone. Model analysis revealed that TLR7 is significantly enriched in NET formation. TLR7 is also significantly associated with neutrophil degranulation and the neutrophil granule components pathway. Therefore, the authors speculate that TLR7 influences the OA process by activating NET formation. The effect of TLR7 on OA was confirmed by using mouse OA models, human OA samples, and cell experiments. The article cites a small dataset, and the results may have some bias. Larger sample sizes and sequencing are needed for future studies.

Xu et al[73] conducted a study on causal associations between circulating immune cells and OA, and showed a positive relationship between spine OA and the count of circulating neutrophils (odds ratio: 1.104, 95% confidence interval: 1.032-1.181, P = 0.0039, positive false discovery rate = 0.0233). Rai et al[74] demonstrated that the expression of IL-33 was increased in OA cartilage, and IL-33 increased the expression of IL-6, tumor necrosis factor-α, TLRs, and matrix metalloproteinases and favored phenotypic conversion towards the M1 phenotype, while IL-37 and blocking the IL-33 receptor ST2 showed opposite effects. Overall, the results suggest that blocking IL-33 and increasing IL-37 act synergistically to attenuate inflammation and might serve as potential therapeutics in OA. From Tang et al’s research results[49], it can be seen that in RA, IL-33 and NETs promote each other to form a positive feedback, and the role of NETs in OA should be further explored.

NETs and ONFH

The relationship between ONFH and NETs has been less studied. ONFH is defined as osteonecrosis caused by nontraumatic ischemia of the femoral head. This is a refractory disease that can lead to femoral head collapse and hip dysfunction, which can lead to reduced quality of life. Although its pathogenesis has not been revealed, glucocorticoid use and prolonged heavy alcohol consumption are risk factors for ONFH[75]. A survey by Kubo et al[76] showed that in patients with idiopathic ONFH, 51% were associated with glucocorticoids, 31% with alcohol, and 3% with both. Glucocorticoid-induced platelet activation contributes to local blood flow disturbances in the femoral head. Both activated platelets and alcohol induce NETs. To determine the relationship between NETs and ONFH, Nonokawa et al[77] assessed the presence of NETs in the femoral heads of surgically resected ONFH and OA patients by immunofluorescent staining and validated the result using a rat model. The results showed that NETs were present in the small vessels around the femoral head of ONFH patients, but not in OA patients. At the same time, intravenous NETs can enter the tissues around the femoral head and may induce femoral head ischemia. Studies suggest that NETs are involved in the development of ONFH, and this promote the understanding of the pathophysiology of ONFH.

Lee et al[78] conducted an integrative analysis of genes related to osteonecrosis of the femoral head. In their study, rs693 and rs1042031, which are polymorphisms of ApoB, each showed high significance in the dominant models. Their mean odds ratios were over 2.5, indicating that if apolipoprotein B did not function normally, the ONFH incidence could be increased. These results indicated that lipid metabolism disorders could lead to adipogenesis in the femoral head, which is one of the main causes of steroid-induced ONFH. Warnatsch et al[79] showed that in a murine model of atherosclerosis, cholesterol crystals acted as both priming and dangerous signals for IL-1β production. Cholesterol crystals triggered neutrophils to release NETs. NETs primed macrophages for cytokine release, activating T helper-17 cells that amplify immune cell recruitment in atherosclerotic plaques. Therefore, dangerous signals may drive sterile inflammation, such as that seen in atherosclerosis, through their interactions with neutrophils. These studies show that abnormal lipid metabolism is closely related to ONFH and can induce NETs, indicating a potential relationship between ONFH and NETs.

NETs and fibrosis

Scar adhesion and limited joint movement are common problems after osteoarticular disease. The relationship between NETs and organ tissue fibrosis has also gradually attracted attention[80,81]. Epidural fibrosis after spinal surgery is a difficult clinical problem, and fibrotic tissue often causes complications such as low back pain, which seriously affects the quality of life of patients[82]. Jin et al[83] found a potential link between NETs and the development of epidural fibrosis. In a laminectomy mouse model, they found that NETs in wound tissue increased the expression of α-smooth muscle actin and fibronectin in macrophages. Conversely, the use of DNase reduced the severity of epidural fibrosis. Although there is a lack of research on the role of NETs in scarring, current studies suggest that NETs are potential targets for treatment. Further research on NETs could lead to new strategies for treating scarring and improving patients’ quality of life. Table 1 summarizes the roles and mechanisms of NETs in various osteoarticular diseases.

Table 1 Role and mechanism of neutrophil extracellular traps in different osteoarticular diseases.

Ref.	Disease	Mechanism	Role of NETs	Species
Schneider et al[55]	RA	NETs/TLR/RANKL/osteoclastogenesis	Bone erosion	Mouse/cell
Carmona et al[54]	RA	NETs/NE/PAD2/CD4+ T cells	Cartilage damage and synovial inflammation	Mouse/cell
Song et al[53]	RA	ACPA	Autoimmune diseases	Human/mouse/cell
Papagoras et al[61]	AS	IL-1β/IL-17/NETs/MSCs/bone-forming cells	Inflammation and neoosteogenesis	Mouse/cell
Maueröder et al[67]	Gout	MSU/NLRP3/ IL- 1β/NETs	Inflammation	Mouse/cell
Luan et al[72]	OA	TLR7/MAPK/NETs	Cartilage and bone damage	Human/mouse/cell
Nonokawa et al[77]	ONFH	Activated platelets/NETs	Microangiopathy	Human/rat
Jin et al[83]	Fibrosis	HMGB1/NETs/RAGE (macrophages)/NF-κb/Smad3	Increased α-SMA and fibronectin expression	Mouse

NETs: Neutrophil extracellular traps; RA: Rheumatoid arthritis; TLR: Toll-like receptor 2; RANKL: Receptor activator of nuclear factor kappa-B ligand; NE: Neutrophil elastase; PAD: Peptidyl arginine deiminases; ACPA: Anti-citrullinated protein antibody; AS: Ankylosing spondylitis; OA: Osteoarthritis; ONFH:; MSCs: Mesenchymal stem cells; IL: Interleukin; NLRP3: Nucleotide-binding domain, leucine-rich repeat and pyrin domain-containing protein 3; MSU: Monosodium urate; MAPK: Mitogen-activated protein kinase; α-SMA: Alpha-smooth muscle actin; HMGB1: High mobility group box 1; NF-κb: Nuclear factor kappa-B.

CONCLUSION

It is becoming increasingly clear that the study of NETs has revealed new insights into the pathogenesis of many diseases. However, there is currently little research on the impact of NETs on OA, ONFH, and bone and joint fibrosis. The pathogenesis of these diseases is still unclear, and treatment methods are limited. First, there is currently a lack of uniform and standardized methods for the detection of NETs. Markers used in different studies to detect NETs, such as cellular free DNA, MPO-DNA complexes, and citrullinated histones, vary in sensitivity and specificity, making it difficult to accurately compare and analyze research results. Second, the complex interaction network between NETs and other cells and molecules in OA has not been fully elucidated. For example, the interaction between NETs and synovial cells, chondrocytes, osteoblasts and other cells involves crosstalk of multiple signaling pathways, and the detailed molecular mechanisms and regulatory methods need to be further studied. At the same time, we see that NETs trigger different results in different diseases under inflammatory environments. In RA, NETs cause osteoporosis and bone destruction, while in AS, NETs cause osteogenesis, which is almost opposite, indicating that the local microenvironment has a huge impact on the role of NETs. Research on targeting NETs for OA is still in its infancy. Although some drugs that inhibit the formation or degradation of NETs have shown some efficacy in vitro or in animal models, they still need a large number of clinical trials to verify their clinical application.

Current research shows that NETs have a bright future in the diagnosis and treatment of bone and joint diseases; however, there are still many challenges in specific clinical applications. The current research focuses on cell or animal experiments, and the studies in humans only involve the detection of cytokines or NETs components in the blood, joint fluid, synovium, and cartilage tissue. Observational or interventional experiments on human NETs mainly focus on tumors, thrombosis, coronavirus disease 2019, and cardiovascular diseases. Duan et al[84] reported that the NETs index can predict the incidence of deep incision infection after laparotomy. Ibrahim et al[85] reported the role of NETs in diabetic foot ulcers, showing that in the ulcer group, the serum NETs level is increased, and the NETs level can predict the amputation rate. Given the role of NETs, enhancement of NETs is critical for the function of eradicating pathogens in the early stages of infectious disease. Conversely, for aseptic inflammation, such as osteoarticular diseases, it is advantageous to inhibit the formation of NETs. When considering inhibition of NETs, more attention should be paid to modulating the formation of NETs rather than eliminating NETs that have already formed. Dynamically modulating NETs levels in vivo to maintain homeostasis is an exciting avenue of research.

Future research needs to combine multidisciplinary collaborative research, and integrate knowledge and technology in immunology, cell biology, biochemistry, and clinical medicine, to more deeply analyze the complex role of NETs in bone and joint diseases. In view of the diversity of the roles of NETs, it is still unclear that they are proinflammatory or anti-inflammatory, and osteogenic or bone-breaking, and that they play a starting or maintenance role in disease. Thus, it is necessary to study NETs in different diseases by specialty and stage. In addition, the impact of local microenvironment on NETs needs to be further studied. Finally, in terms of treatment, in addition to targeting NETs to degrade or inhibit their synthesis, exercise has become a popular topic in reversing the role of NETs[86,87], and may be a new direction in the study of osteoarthrosis, such as frozen shoulder.