The molecular mechanism of neutrophil extracellular traps and its role in bone and joint disease

Abstract

As the body's first line of defense, neutrophils play an important role in the early stages of infection. Neutrophil extracellular traps (NETs), a novel way to kill pathogens, are released from activated neutrophils to trap and kill microorganisms and protect the body from invasion. However, studies have shown that NETs not only play a role in self-defense in vivo but also participate in some pathological processes. Current studies have found that excessive or abnormally activated NETs play a pathogenic role in a variety of diseases. NETs, in addition to killing pathogens during the pathology of sepsis, affect on coagulation function, and blood endothelium. Additionally, NETs have a wide range of effects in other inflammatory, immune, and other related diseases. NETs are involved in the pathology of atherosclerosis. NETs also play a role in systemic lupus erythematosus, diabetes mellitus, Alzheimer's disease, and tumors, but there are relatively few NETs studies on bone and joint diseases. This article discusses NETs, their formation, and their association with bone and joint disorders. New targets for the effective treatment of joint diseases may be identified by studying the relationship between NETs and bone and joint diseases.

1. Introduction

As the most numerous type of white blood cell in the human body, neutrophils account for 50%–70 % of the total number of white blood cells and are involved in the first line of immune defense against invading pathogens [1,2]. Neutrophils are the first immune cells to migrate to the site of the attack and can target microorganisms by a variety of mechanisms, including phagocytosis of pathogens, degranulation, cytokine production, and formation of neutrophil extracellular traps (NETs) [3].

Initially, researchers proposed that neutrophils release NETs to capture and kill pathogens after being stimulated by pathogens and some danger signals [4]. As an important part of innate immune stimuli [5], the process of NETs formation is called NETosis [6,7]. NETs is a highly concentrated reticular DNA complex that is released extracellularly by activated neutrophils in a variety of ways, characterized by chromatin fibers decorated with various granular proteins that are extrusion-like released into the extracellular space to eliminate pathogens [8]. Morphologically, NETs adopt depolymerized DNA as a skeleton to which histones and neutrophil granule proteins are attached [9]. Granule proteins include primary granule proteins such as myeloperoxidase (MPO), neutrophil elastase (NE), and cathepsin G, secondary granule proteins such as lactoferrin and gelatinase, and tertiary granules. NETs formation is influenced by many inducing factors, resulting in variable NETs production. Several pathogens, such as bacteria, fungi, viruses, some cytokines (IL-8, tumor necrosis factor α), ROS, LPS, chemical reagents (PMA), and other substances can significantly activate neutrophils and trigger the formation of NETs [10]. In addition, the release of NETs plays an important role in frustrated or ineffective phagocytosis. When encountering large biological structures that cannot be engulfed and internalized, NETs can capture and kill various pathogens and enhance the effectiveness of the innate response in coping with invading microbes [11,12]. However, although NETs were initially implicated in immune defense, subsequent studies have shown that excessive or abnormal NETs are involved in the onset and development of some diseases [2]. On the one hand, they are used as autoantigens to destroy the body's immune tolerance, leading to inflammation and autoimmune diseases, such as systemic lupus erythematosus (SLE) and ANCA-associated vasculitis [8,13]. On the other hand, the interaction between NETs and platelets promotes thrombosis, leading to a variety of pathological results [15]. In addition, most studies have shown that NETs are highly expressed in a variety of malignant tumors, and promote tumor invasion and distant metastasis through certain pathways [16]. However, recent developments in the field have provided some new insights that NETs also play a role in the pathogenesis of osteoarticular diseases.

Bone and joint disease is a common type of chronic condition that primarily affects middle-aged and elderly people. However, it is becoming increasingly common in younger people [14]. Its pathogenesis is mainly related to age, joint degeneration, abnormal metabolic function of cartilage tissue, long-term joint load factors, and other factors [15]. Some diseases are also related to autoimmune, such as rheumatoid arthritis, ankylosing spondylitis, and so on [16,17]. The symptoms of bone and joint diseases are mainly joint swelling, pain, and dyskinesia. Bone and joint diseases seriously affect the quality of life of patients and cause a huge economic burden to patients and their families, but the existing treatment modalities cannot completely improve the condition of patients [18].

Over the past few years, there has been more focus on bone and joint diseases. However, since these conditions are impacted by multiple factors and have a complicated pathogenesis, the current treatments are not very effective. Research has demonstrated that neutrophils, a type of immune cell, have a significant impact on maintaining bone health and balance. As a novel immune defense mechanism, NETs captures and destroys pathogens and helps reduce inflammation. However, when there are too many NETs, it can lead to inflammation and damage to the body's tissues. Current research indicates that NETs are closely linked to the onset and progress of joint diseases. Thus, this article examines the formation mechanism and role of NETs in the development of osteoarticular diseases. Furthermore, the article provides a summary of the factors that affect the development of NETs in osteoarticular diseases, analyzes potential anti-NET medications, and proposes novel therapeutic approaches for preventing and treating these conditions.

2. Mechanisms of NETs formation

NETs are fragile tissues composed of nuclear components and granules that in many cases trap and kill pathogens extracellularly [19]. The NETs formation can be triggered by a multitude of stimuli both in-vitro and in-vivo under different pathophysiological conditions [20]. Different stimuli lead to different types of NETs formation, and the same stimulus can also induce different types of NETs formation. Up to now, the formation of NETs is mainly divided into two types: (i) suicidal NETs: NETs are released when the nuclear membrane ruptures (Figuer 1); (ii) vital NETs: neutrophils are able to continue their immune function after NETs release [21] (Fig. 2).

Fig. 2.

Vital NETs.

2.1. Suicidal NETs

Suicidal NETs are the main pathway of the NETs generation of neutrophils. The morphological changes of neutrophils are involved in this process [19]. Activated neutrophils tend to flatten, lose nuclear lobules, their nuclear and granular membranes disassemble, histone degradation leads to chromatin depolymerization, and finally form NETs with loose structure, which is ejected from the cell and accompanied by the death of neutrophils [10,22]. Activated neutrophils increase NADPH oxidase to produce ROS via the protein kinase C (PKC)/rapidly accelerated fibrosarcoma (Raf)/MEK (MAPK/ERK kinase)/ERK signaling pathway and increased calcium influx when chemically stimulated by phorbol 12-myristate 13-acetate (PMA), immune complexes and certain microorganisms [5]. Chemical stimulation by phorbol myristate acetate (PMA), immune complexes, NADPH oxidases are a family of membrane-bound multiprotein enzymes that generate reactive oxygen species (ROS) for delivery to either extracellular or intracellular compartments [23]. The assembly of NADPH oxidase drives the generation of ROS. It has been documented that ROS subsequently disintegrates both the granule membranes and the nucleus [24]. Myeloperoxidase (MPO) is then activated and elastase (neutrophil elastase, NE) translocates to the nucleus, where it cleaves histones and facilitates chromatin decondensation [19]. At the same time, protein arginine deiminase type 4 (PAD 4) can translocate to the nucleus upon an increase in cytosolic calcium concentration, replace the positively charged histone arginine residue with neutral citrulline residue to promote histone H2A, H3, H4 citrullination, and reduce the electrostatic interaction between histone and DNA, thereby promoting nuclear decondensation [1,21]. Subsequently, the nuclear and granular membranes are degraded. The destruction of the nuclear membrane is one of the salient features of NETs formation. Studies have shown that activation of cyclin-dependent kinase (CDK) plays an important role in the signal transduction of NETs formation, which can pull neutrophils from the G0 phase back to the G1 phase, and CDK4 and CDK6 silencing blocked the release of NETs by blocking the translocation of elastase to the nucleus. The processes of generation, phagocytosis, and degranulation of ROS are not affected. This suggests that the cell cycle mechanism could play a significant role in promoting the disintegration of the nuclear membrane during NETosis [25,26]. After the rupture of the nuclear membrane [26], decoagulated chromatin and granule proteins are mixed in the cytoplasm and excreted from the cell through GSDMD pores or GSDMD-driven membrane rupture, leading to the death of neutrophils and form NETs [1,27]. This pathway involves ROS production induced by NADPH oxidase (NOX), which is a slow process that takes 3–4 h to complete.

Cathepsin G (cG) acts as a toxic protein on the NETs structure can form co-localization with citrullinated histone 3 to play a role in the formation of NETs [28]. Similarly, research indicates that the presence of protease 3 (PR3) on the structure of NETs can cause the production of PR3-specific ANCA autoantibodies, which in turn triggers the release of NETs from neutrophils [29,30].

Since the formation of NETs in this pathway mainly depends on NOX, some factors related to NOX can also affect the release of NETs. Neutrophil oxidative burst is necessary for host defense. The metabolic pathway plays a crucial role in the oxidative burst. This process involves a rapid rise in oxygen consumption and glucose uptake, resulting in increased NADPH levels through the pentose phosphate pathway. Phosphofructokinase-1 liver type (PFKL), a phosphofructokinase-1 isoform expressed in immune cells [31], has been shown to inhibit NOX-dependent oxidative burst, thereby inhibiting the generation of NETs, and in the process of the PKC kinase involved in the regulation of PFKL activity [32]. Moreover, proliferating cell nuclear antigen (PCNA) has also been shown to be involved in the formation of NETs in this

Pathway. As a scaffold protein present in the nucleus, PCNA has been shown to play a key role in neutrophil apoptosis [33]. Recent studies have shown that PCNA regulates NOX activation in neutrophils by interacting with p47phox, a key subunit of NOX. Upon stimulation, the binding of p47phox to PCNA could promote NADPH assembly and possibly regulate the NETosis [34]. Therefore, inhibiting PCNA is expected to be a potential anti-inflammatory strategy.

NETs as a chromatin-containing structures, the nuclear chromatin protein DEK can also affect chromatin organization to regulate NETs formation. Mechanistically, DEK regulates chromatin structure in the nucleus and can directly enter the nucleus to affect chromatin structure and cell function [35,36]. In this respect, DEK could be involved in the early events of NETs formation. In addition, DEK can affect NETs formation by acting as a scaffold [37]. However, recent studies have shown a novel signaling pathway for generating NETs.When neutrophils are exposed to LPS or Gram-negative bacteria activates the casepase-11-mediated non-canonical inflammasome pathway, followed by the combined action of GSDMD and caspase-11 to drive nuclear permeabilization, chromatin relaxation, and chromatin fragmentation. And plasma membrane disruption which in turn releases NETs. Mechanistically, GSDMD pores in the nuclear envelope allow nuclear envelope disruption and access of caspase-11 to chromatin, where caspase-11 performs a similar function to NE, mediating histone cleavage and inactivation then causing NETs production [38].

2.2. Vital NETs

Vital NETs are a rapid process. This process differs from NADPH-dependent NETosis, which requires intracellular calcium influx and is regulated by mitochondrial ROS and small conductance potassium channel 3 (SK3) [24]. During this process, the formation of the NETs did not require the lysis of the neutrophil or even the rupture of the plasma membrane [10,21]. The nuclear DNA-filled vesicles were extruded intact into the extracellular space, where they ruptured and released chromatin [39]. Vital NETs are an oxidant-independent process. Cells do not die immediately, and still retain activity and phagocytosis. This pathway is mostly induced by bacterial infection, and the process is more rapid, about 5–60 min [40].

The stimulator represented by this pathway is calcium ionophore A23187. Calcium ionophore A23187 mediates the translocation of PAD4 into the nucleus by causing calcium influx and mitochondrial ROS production, which leads to citrullination of histone proteins and leads to a nuclear depolymerization process [41,42]. Additionally, a recent study has demonstrated the collaborative effect of PAD4 and calpain-1 in the formation of NETs induced by calcium ionophores. This collaboration can result in nuclear depolymerization, and the process is influenced by mitochondrial ROS. This discovery shows a connection between the protein citrullination process, which is carried out by PAD4, and calpain-1 proteolysis. This suggests that calpain-1 is involved in the pathway of NETs formation. Additionally, it proposes a new NOX-independent mechanism of PAD4 in the formation of NETs [43]. Additionally, LPS, a Gram-negative bacterial stimulus, can also induce this rapid NETosis was promoted by TLR4 on platelets, which promoted the neutrophil release of nuclear DNA to form NETs in a manner independent of NADPH oxidase and maintain their integrity under flow conditions and engulf bacteria in the circulation, whereas conventional NETs form dependent on NADPH oxidase under PMA stimulation [24,44]. Moreover, both TLR2 and complement are involved in rapid NETosis induced by S. aureus in vivo [45]. Yousefi and colleagues also described this novel pathway that neutrophils release NETs generated from mitochondrial DNA after pre-treatment with GM-CSF and subsequent stimulation with LPS or complement factor 5a, a process that does not result in lytic cell death [45,46]. A complete block of mitochondrial DNA release from neutrophils can be observed when diphenyleneiodonium (DPI), an inhibitor of ROS, is applied [46]. This phenomenon also indicates that ROS are required for neutrophils to release mitochondrial DNA.

In addition to the formation mechanism of several NETs mentioned above, recent studies have also shown that autophagy is also involved in the spontaneous formation of NETs. Guo et al. demonstrated that autophagy has a synergistic relationship with the formation of NETs, which can modulate the release of NETs in a variety of ways. Autophagosomes can be observed in neutrophils under the stimulation of PMA and LPS [33]. Membrane nucleation is an essential step in the pathway of autophagy that can influence vesicle formation and thereby NETs formation. Autophagy can also prevent NETs formation through the inhibition of oxidative respiratory bursts and the blockade of histone citrullination. mTOR is an important node in the regulation of autophagy, and the mTOR inhibitor rapamycin greatly enhances the formation of NETs [47].

Fig. 1. Suicidal NETs. Suicidal NETs is a slow-progressing (lasting a few hours) form of cell death, distinct from necrosis and apoptosis. PMA, phorbol 12-myristate 13-acetate; MSU, monosodium urate; PAD4, protein arginine deiminase type 4; MPO, myeloperoxidase; NE, neutrophil elastase; Cit, citrullination.

Fig. 1.

Suicidal NETs.

Fig. 2. Vital NETs. Vital NETs release NETs at a very rapid rate (5–60 min) and are not dependent on cell death. S. aureus, Staphylococcus aureus; TLR4, toll-like receptor 4; TLR2, toll-like receptor 2; mtROS, mitochondrial ROS; Ca5R, complement component 5a receptor.

2.3. NETs in bone and joint disease

The prevalence of bone and joint diseases is increasing year by year along with the increasing number of elderly people in society. The pathogenesis of common bone and joint diseases is mainly affected by a variety of factors such as inflammatory response, proliferation of chondrocytes, and differentiation of osteoblasts and osteoclasts [48]. Neutrophils are produced by myeloid precursor cells in the bone marrow and are controlled by a variety of cytokines, mainly G-CSF, which have been shown to express and secrete inflammatory mediators that directly or indirectly affect bone marrow mesenchymal stem cells, osteoblasts and osteoclasts and thus participate in the pathogenesis of bone and joint diseases [49]. Mechanistically, excessive NETs production by stimulated neutrophils lead to an inflammatory response, which in turn may lead to excessive activation of osteoclasts and consequent bone loss. Most bone and joint diseases involve NETs release that is generated by NOX-dependent pathways. Here, we discuss the role of NETs in several bone and joint disorders to provide potential therapeutic targets for disease treatment.

2.4. Rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by chronic systemic inflammation. The primary joint manifestations include synovial swelling and pain, cartilage damage, and periarticular bone erosions, as well as extra-articular manifestations such as rheumatoid nodules and vasculitis [50,51]. RA is a multifactorial disease generally thought to be related to the genetic, infectious, immune system, and other factors. RA serum contains multiple autoantibodies, among which the formation of anti-citrullinated protein antibodies (ACPAs) is considered to be the key link to RA pathogenesis [52]. ACPAs are highly specific for RA and can be detected 3–5 years before the onset of joint symptoms [53,54]. Recent studies have shown that NETs, as a novel anti-inflammatory mechanism, are closely related to ACPAs [54]. Citrullination is a post-translational modification in which arginine is enzymatically converted to citrulline by peptidyl arginine deiminases (PADs) [51]. Many studies have shown that blood neutrophils in RA have an aberrant, activated phenotype [55]. Neutrophils may be involved in RA pathology and have the greatest cytotoxic potential of all cells involved in RA pathology, releasing ROS and degrading enzymes [56]. Neutrophils in RA show an enhanced ability to form NETs. On the one hand, in the absence of microbial stimulation, in the pro-inflammatory environment of RA characterized by specific autoantibodies and increased pro-inflammatory cytokines, neutrophils showed increased production of ROS and cytokines and delayed apoptosis [52,54]. At this time, neutrophils in the synovium of RA patients are activated, promoting the production of NETs. On the other hand, activated neutrophils undergoing NETosis contribute to the generation of citrullinated autoantigens through the liberation of enzymatically active PAD isoforms, causing the body to mount an autoimmune response [57]. Thus, the inflammatory environment of RA may promote the occurrence of NETs. In turn, NETs as one of the sources of citrullinated autoantigen, and significantly enhance the inflammatory response of fibroblast-like synoviocytes (FLS) and lead to the production of inflammatory molecules such as IL-6, IL-8, and adhesion proteins, which have the potential to form a vicious cycle in susceptible individuals, thus causing the continuation of the inflammatory response and the production of specific antibodies, leading to joint destruction and disease aggravation [51]. In addition, antibodies to carbamylated proteins (anti-CarP), also known as antihomocitrulline antibodies (AHCPA), have recently been described in RA. Anti-CarP antibodies recognize homocitrulline, a modification structurally similar to citrulline that is also generated in proinflammatory environments [58]. Since carbamoylation requires the presence of cyanate esters (CN⁻), when MPO is released from neutrophils in the inflammatory environment, this enzyme converts thiocyanate to cyanate, allowing more carbamylation to occur [59,60]. Studies have confirmed that NETs can be used as a source of carbamoylated autoantigens, and RA patients can produce autoantibodies against the new carbamoylated antigens in NETs to mediate autoimmune responses [51]. The presence of this autoantibody is associated with the severity of bone erosion. Of the NETs proteins identified, 2/3 are the target of RA autoantibodies, further confirming that the abnormal production of NETs plays a critical role in RA [61]. At the same time, it has been shown that inflammatory cytokines and RA antibodies can induce the formation of NETs and exacerbate the severity of RA. In addition to the RA-related autoantibodies mentioned above, recent studies have identified a novel mechanism in which the presence of NE in NETs can degrade cartilage components directly in the synovium and simultaneously amplify the inflammatory pathways in FLSs and macrophages, further promoting joint damage [13]. In addition, NETs may further enhance the citrullination of cartilage protein by inducing FLSs to release PAD2. A recent study showed that anti-NET antibodies (ANETA) occur in RA patients and may be used as biomarkers for RA. However, the specific mechanism and specificity of ANETA on RA need to be further studied [62]. In summary, there have been many studies on the pathogenesis of NETs and RA, but the role of NETs in RA etiology and the understanding of the mechanisms leading to strong autoimmune responses still need to be further elucidated.

2.5. Ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis characterized by lesions of the sacroiliac joints and axial skeleton. It affects mainly young people, with a male predominance, and has a strong association with HLA-27 [63]. AS is characterized by focal changes in the subchondral bone marrow and adjacent soft tissues, causing inflammatory bone erosions, abnormal repair processes, and bone ankylosis [64,65]. Waist, back, and hip pain, and joint swelling are the primary symptoms. In more serious cases, deformity of the spine may be observed. AS can also be accompanied by extra-articular manifestations, often presenting as acute anterior uveitis (AAU), psoriasis, and inflammatory bowel disease (IBD) [66]. Recent experimental and clinical evidence has shown that AS-derived neutrophils exhibit enhanced spontaneous NETs extrusion [67]. At the same time, the molecular mechanisms and signaling elements involved in NETs formation are also increased in AS neutrophils and can be used as potential disease-active markers of AS. Neutrophils from patients with AS were characterized by increased formation of NETs carrying bioactive IL-17 A and IL-1β [64]. Mechanistically, inflammatory factors IL-17 expression is positively regulated by IL-1β. NETs are a major source of IL-1β and IL-17 and play an essential regulating role in the inflammation and bone formation associated with AS. In active AS, NETs released by neutrophils may further participate in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin-17 A (IL-17 A), which may lead to bone loss [64]. RNA sequencing in an experimental spinal arthritis model also showed that genes associated with neutrophil function and NETs formation were significantly up-regulated [68]. Previous studies have shown that osteocytes are highly sensitive to IL-17, it can stimulate MSCs to produce factors that regulate osteocyte activity and are involved in osteogenesis and bone remodeling, inhibits matrix production of osteoblasts and chondrocytes and leads to joint injury [69,70]. Thus, neutrophils play a central role in the initiation and progression of inflammation in AS by expressing interleukin-17 and forming NETs. Furthermore, the increased bioactivity of IL-17 in AS may be related to the dense structure of NETs [71]. Moreover, other studies have shown that the immune complex formed by P30 and its specific antibodies in the serum of AS patients induces the high-level release of NETs after neutrophil stimulation [65]. Previous studies have reported that patients with AS have high levels of antibodies to a 30 kDa protein from Salmonella typhimurium (p30). Neutrophils respond to P30 in immune complexes by releasing NETs and pro-inflammatory cytokines, which in turn are involved in maintaining chronic inflammatory responses in disease [65,72]. This evidence has shown that NETs may be involved in the pathogenesis of AS.

2.6. Idiopathic osteonecrosis of the femoral head

Osteonecrosis of the femoral head (ONFH), also known as avascular osteonecrosis of the femoral head, refers to the damage or interruption of blood supply to the femoral head, which leads to the death of bone marrow components and bone cells and subsequent tissue repair, and then leads to the structural change and collapse of the femoral head, eventually causing hip pain and dysfunction. According to different causes, it can be divided into traumatic femoral head necrosis and non-traumatic femoral head necrosis. It is well known that the use of corticosteroids and excessive alcohol consumption are the main risk factors for non-traumatic femoral head necrosis [73,74]. Corticosteroids can induce platelet activation and aggregation, leading to impaired blood flow around the femoral head, inadequate blood supply, and then femoral head necrosis [75]. Alcohol may impair the osteogenic differentiation of MSCs and promote their adipogenesis [76]. Lipid peroxidation is known to cause membrane damage. By affecting infiltration, lipid peroxidation induces arteriolar degeneration and arteriolosclerosis, which ultimately leads to femoral head ischemia [77]. In addition, direct cytotoxicity of lipid peroxidation by alcohol and its metabolites such as acetaldehyde and free radicals may further damage ischemic osteocytes, leading to irreversible injury and eventual femoral head necrosis [77,78]. The necrosis of the femoral head is a sterile inflammatory change. Studies indicate that neutrophils, which exist during osteolysis and at neighboring sites of inflammation, are the most important cells to infiltrate in this process [79]. In patients with nontraumatic ONFH, NET-forming neutrophils can be found in small vessels around the femoral head through the co-localization of citrullinated histone H3 (NETs marker), MPO, and DAPI (DNA marker) [74]. It has been demonstrated that platelets and alcohol can enhance the ability of neutrophils to form NETs and participate in the pathogenesis of femoral head necrosis. The interaction between platelets and neutrophils is involved in the recruitment of neutrophils to inflammation [80,81]. P-selectin is the major molecule mediating platelet-leukocyte interaction, which is recognized by P-selectin glycoprotein ligand (PSGL)-1 on the leukocyte surface [82,83]. Mouse experiments have shown that the combination of P-selectin and PSGL-1 can be an inducer of NETosis [82]. Previous studies have shown that platelet-driven NOX-independent NETosis can be observed in the presence of some classic platelet agonists such as thrombin, ADP, collagen, arachidonic acid, and several TLR ligands. In the absence of platelet activation, NETs formation does not occur [84]. In turn, the structure of NETs can serve as a scaffold for platelet adhesion, activation, and aggregation, thereby promoting intravascular coagulation and thrombosis [[85], [86], [87]]. Electron microscopy showed that NETs can also bind directly to platelets in vitro [88]. In addition, activated platelets also can present HMGB1 to neutrophils and for NETs formation [80]. On the other hand, after human alcoholism, the NETs inducers (endotoxin and bacterial DNA) in the circulation increase significantly [89]. After alcohol treatment, the release of extracellular DNA and NE from neutrophils was significantly increased. Although some studies have shown that drinking alcohol can destroy the oxidative burst and phagocytosis of neutrophils, the mechanism of NETs formation and clearance of neutrophils after excessive drinking is still unclear [89]. However, some research has shown that the use of acute alcohol treatment before PMA stimulation of neutrophils can result in reduced extracellular DNA release and fewer NETs released [89]. This result shows that alcohol can destroy the oxidative rupture of neutrophils, but in some cases, it can also inhibit the formation of NETs in neutrophils. Finally, recent studies have found that after the neutrophils forming NETs are injected into WKY rats, the neutrophils forming NETs can enter the surrounding tissues of the femoral head, the osteocytes of the femoral head are ischemic, and the expression of HIF-1a in the tissues is increased [74]. Based on the above conclusions, the formation of NETs may be involved in the local blood flow disturbance and ischemia of the femoral head, which is related to the occurrence of non-traumatic ONFH.

2.7. Gout

Gout is a chronic disease caused by monosodium urate (MSU) crystals becoming deposited in the joints. Hyperuricemia is the main contributor to the deposition of MSU in the joints and the pathogenesis of gout [90]. Gout flares are usually characterized by acute, intermittent, severe pain from arthritis and are self-limiting [91]. Innate immune response to MSU crystals is responsible for acute severe joint inflammation in gouty arthritis [92]. MSU inflammasome in monocytes and macrophages, and then activate cysteine protease 1 (caspase-1) [93]. The latter converts the inactive precursors of interleukin-1β (IL-1β) and interleukin-8 (IL-8) into active, mature cytokines that are rapidly released outside the cell. IL-1β and IL-8 can activate several pro-inflammatory signaling pathways [94]. The massive production of these pro-inflammatory factors and cellular chemokines leads to the intensification of neutrophil infiltration and the recruitment of other immune cells, resulting in the amplifying effect of the inflammatory cascade [91,95]. Mechanistically， NETs is involved in gouty disease mainly by the NOX-dependent pathway and is associated with spontaneous remission of gouty arthritis and gouty stone formation. Animal models and in vitro experiments show that MSU deposited in the joint cavity can activate the formation of NETs [96]. Interleukin-1 (IL-1) may play a key role in the production of NETs, as the IL-1 receptor antagonist anakinra reduces NETs production [97]. Quantitative analysis of real-time imaging shows that physical interaction between neutrophils and crystals is required to trigger NETs formation [98]. At the site of inflammation, neutrophils ingest MSU crystals, which trigger ROS production and further lead to the formation of NETs. With the formation of NETs, DNA and histone within the neutrophils overflow, and NETs, along with a variety of inflammatory factors, trigger the body's immune system and cause severe inflammatory responses [99,100]. Hence, NETs can cause damage to the surrounding tissues by enhancing the proinflammatory response, trigger the acute attack of gout, and affect its chronic process. Additionally, Mitroulis et al. studies have shown that peripheral and synovial neutrophils in patients with gout inflammation can form NETs in an autophagy-dependent manner [97]. Conversely, research has confirmed that NETs also have a significant impact on the regression of arthritis in gout. The uptake of MSU crystals by neutrophils results in the release of a variety of cytokines, including inflammatory mediators (such as TNF-α and IL-6), neutrophil attractants (such as IL-8), and activators (such as CCL3 and CXCL10), leading to higher neutrophil densities. After a certain threshold, a large number of NETs form an aggregated NET (aggNET) [99]. In the late stage of gouty arthritis, gouty stones appear in joints and other places. The gouty stone gradually penetrates into the bone, which is the main cause of bone destruction in the joints of patients with gouty arthritis. Studies have shown that the composition of gouty stones is very similar to that of NETs [101]. At that time, aggNET will tightly encapsulate MSU crystals, capture and degrade some cytokines and pro-inflammatory mediators by its own protease, destroy the recruitment and activation of neutrophils, and promote the regression of inflammation in gouty arthritis [101,102]. In addition, neutrophils recruited to the site of inflammation can also induce the production of anti-inflammatory transforming growth factor-β1 (TGF-β1) through the production of NETs. At the same time, the production of TGF-β1 inhibits the oxidative burst of neutrophils and reduces the production of NETs [103]. It is well known that TGF-β1 is a classical anti-inflammatory factor in the regulation of autoimmune diseases. It is essential for the initiation and inhibition of inflammatory diseases. TGF-β1 is considered to be an important anti-inflammatory factor in the spontaneous remission of gout [104]. It appears that neutrophil NET formation has a dual effect in gout: on the one hand, after the formation of too many NETs causes aggregation, it is easy to form gouty stone, which can cause bone erosion and induce chronic inflammation; on the other hand, NETs can embed and clean MSU crystals to protect the body.

2.8. Potential Anti-NETs therapeutics

Although NETs are important as anti-infectives in the innate immune response, the imbalance of NETs clearance can lead to excessive tissue damage and pathology. The formation of NETs are closely associated with the onset and development of many diseases. Inhibition of NETs formation has emerged as a potential therapeutic target to slow disease progression [105,106] (Table 1).

Table 1.

Different pathways for inhibiting NET formation.

Category	Mechanisms regulating NETs	Reference
Immunomodulatory	CsA by downregulating NFAT and inhibiting the calcineurin pathway and then regulating NETs formation. Rapamycin inhibits fMLP-driven NETs formation by disrupting autophagosome formation.	[105] [107]
Anti-platelet	APC can reduce platelet adhesion to NETs by cleaving and detoxifying extracellular histones. Thrombomodulin limits the procoagulant response and inhibits LPS-induced NETosis. Anti-HMGB1 antibody reduces NETs formation by decreasing the interaction between TLR4 and HMGB1.	[108] [109] [110]
Anti-Inflammatory	PGE2 can activate the CAMP-PKA pathway and inhibit PMA-induced NETs formation. Aspirin inhibits NF-κB and regulates NETs formation. Antibiotics affect neutrophil activation and migration and play a role in regulating NETs.	[111] [112] [113,114]
Regulatory factors	AntagomiR-155 can reduce the levels of PAD4 and DNA histone complexes in PMN-stimulated neutrophils, and then regulate the generation of NETs. Cl-amidine as an inhibitor of PAD4, reduced NET-neutrophils. PA-dPEG24 can inhibit the activity of myeloperoxidase and reduce neutrophil NETs production. DPI inhibits the formation of NETs by inhibiting the production of NADPH oxidase and ROS. NAC regulates the production of ROS and affects the generation of NETs.	[115] [116] [117,118] [119,120] [121,122]
Nucleases	DNase degrades NETs by destroying their backbone. Staphylococcus aureus degrades NETs by converting NETs to deoxyadenosine.	[123] [124]
Cell cycle	Negative regulation of cell cycle regulators blocks cell cycle proteins and inhibits NETs production. Abemaciclib inhibition interrupts the cell cycle process and thus inhibits NETs release.	[26] [125]

2.9. Immunomodulatory

Because excessive or inappropriate NETosis can trigger the production of autoantibodies and cause organ damage in autoimmune diseases, some immunosuppressants have been used to alleviate rheumatoid arthritis, systemic lupus erythematosus (SLE), and other immune diseases [106]. Cyclosporine A (CsA) is a widely used immunosuppressive drug that can cause reversible inhibition of immunocompetent lymphocytes and reduce the activity of the patient's immune system [105]. The mechanism of action of CsA is that it binds to cytophils, leading to the downregulation of nuclear factor activated T-cell (NFAT) transcription factors, inhibition of the calcineurin pathway, and subsequent inhibition of NETs formation. Previous studies have shown that the treatment of neutrophils with CsA reduces ROS production [126,127]. In addition, data from a study showed that treatment with either CsA or ascomycin significantly reduced NETosis in a dose-dependent manner [128]. Furthermore, rapamycin can inhibit fMLP-driven NETs formation by disrupting autophagosome formation [107]. HIF-1α (hypoxia-inducible factor 1α) has also been reported to affect NETs by rapamycin through mTOR-dependent pathways [129].

2.10. Anti-platelet

The evidence presented shows that platelets can bind to neutrophils and activate them to release NETs under the stimulation of bacteria, viruses, or traditional agonists [130]. Therefore, NETs formation may be reduced by inhibiting this interaction with antiplatelet therapy. Activated protein C (APC) is a serine protease produced by the zymogen protein C via enzymatic cleavage by thrombin, which may play a role as an anticoagulant and anti-inflammatory molecule in disease. Previous studies have shown that APC can cleave and detoxify extracellular histones and reduce platelet-NETs binding by inhibiting neutrophil death [108]. Pre-treatment of neutrophils with APC prior to the induction of NETosis resulted in the inhibition of platelet adhesion to NETs. Thrombomodulin is a transmembrane glycoprotein mainly expressed by endothelial cells that is essential for hemostasis. It was found to limit procoagulant responses and inhibit LPS-induced NETosis [109]. Activated platelets express HMGB1 and present it to neutrophils. Exposure of neutrophils to HMGB1 promotes the formation of NETs through interaction with TLR4, so the use of anti-HMGB1 antibodies can reduce the formation of NETs. Therefore, the pathological phenomenon caused by NETs can be alleviated by inhibiting the interaction between platelets and NETs [80,110].

2.11. Anti-inflammatory

Some common bone and joint diseases usually show aseptic inflammatory changes and inflammatory cytokines can be seen around the lesions. Studies indicate that the components of NETs up-regulate the production of pro-inflammatory cytokines and increase the inflammation of the joints [131]. Because an inflammatory microenvironment is an important part of the NET formation, the application of some anti-inflammatory drugs can affect the formation of the NETs structure [111]. Prostaglandins (PGs) play a key role in the production of inflammatory responses [132]. PGE2 is a potent inflammatory regulator and plays an important anti-inflammatory role in some cases. A recent study found that PGE2 activated the cAMP-PKA pathway via EP2 and EP4 and inhibited PMA-induced NET formation in vitro [112]. In another study, PGE2 was shown to inhibit autophagy-induced NET release. In addition, aspirin can also inhibit NF-κB (an inflammatory transcriptional regulator that promotes NETosis), which acts to inhibit the formation of NETs [111]. Aspirin has also been demonstrated in a study of acute lung injury caused by endotoxin; when mice were pretreated with aspirin, the result showed that the formation of vascular NETs was reduced and the degree of lung injury was significantly reduced [133]. Some antibiotics, such as azithromycin, chloramphenicol, and gentamicin, have been shown to regulate NETs by affecting neutrophil activation and migration [113,114].

2.12. Inhibition of regulatory factors

The mechanism of NETs release into the extracellular space is mediated by NOX, PAD4 and the expression of ROS, MPO, NE, cG and PR3, making inhibition of these regulatory factors a potential therapeutic target. It is known that miR-155 is a type of microRNA that is essential for the regulation of the immune system. In a study, it was observed that simulated miR-155 transfection increased the protein level of PAD4 and the expression of DNA histone complexes in PMA-activated neutrophils, whereas transfection of antagomiR-155 decreased the levels of PAD4 and DNA histone complexes in PMA-stimulated neutrophils. This study suggests that miR-155 controls the formation of PAD4-dependent networks [115]. Another study found that Cl-amidine, an inhibitor of PAD4, reduced NET-neutrophils and rescued wound healing in diabetic mice [116]. Peptide Inhibitor of Complement C1 (PIC1), a myeloperoxidase activity inhibitor, and its derivative PA-dPEG24 have been shown to inhibit the formation of human neutrophil NETs induced by PMA, MPO, or immune complex-activated human serum [117,118]. Another study investigated the impact of DPI on ROS surrounding NETs. It was found that the addition of DPI could reduce the release of extracellular DNA and prevent the formation of NETs. DPI is a hypoglycemic drug that can inhibit the production of ROS by inhibiting NADPH oxidase [119,120]. As an antioxidant, N-acetylcysteine (NAC) may also affect NETs formation by regulating ROS production [121,122].

2.13. Nucleases

Since NETs use DNA as their backbone, the application of Deoxyribonuclease (DNase) can destroy the structure of NETs. DNase degrades NETs released by neutrophils in a concentration-dependent manner, thereby reducing the body's damage and inflammatory response [123]. In addition, staphylococcal nuclease secreted by Staphylococcus aureus can degrade NETs [124]. It has been reported that Staphylococcus aureus evades immune defenses by converting NETs to deoxyadenosine. Therefore, these enzymes may open up new avenues for treatment in the future [134].

2.14. Cell cycle

CDK4/6 are fundamental drivers of the cell cycle and are required for the initiation and progression of a wide range of malignancies [135]. Since CDK plays an important role in signaling the formation of NETs, CDK interacting protein/kinase inhibitor protein (cip/kip), a family of negative cell cycle regulators that block the formation of NETs at several different points, is the prototype of p21^cip1 (p21) [125]. In addition, Some CDK4/6 inhibitors may block the release of NETs, such as abemaciclib, trilaciclib.

2.15. Concluding remarks

For a long time, bone and joint diseases have affected the quality of life of modern people. As a multifactorial disease, although there have been exact surgical treatment and drug treatment, it has not improved the patient's condition from the root. Related studies have shown that NETs may be involved in the pathogenesis of bone and joint diseases. NETs as a double-edged sword of immunity, on the one hand, they clearly show an important function in host defense. On the other hand, they have been found in many autoimmune diseases, inflammatory diseases, and thrombosis, and there are a variety of studies. In this review, we describe the pathological relationship between NETs and RA, AS, ONFH, and gout to provide a theoretical basis for future pathological mechanism research and clinically targeted therapy. NETs, as an important barrier to infection for the innate immune system, also provide a different perspective for understanding some diseases. While much research remains to be conducted before the findings can be translated into clinical treatment, our belief is that NETs are involved in the pathogenesis of some bone and joint diseases, and further research will provide opportunities for the diagnosis and treatment of bone and joint diseases.

2.16. Perspectives

• ·Bone and joint diseases seriously affect the quality of life of patients and pose a major challenge to public health. The pathogenesis of related diseases is complex. Although there are relevant surgical and drug treatments, the treatment time is long and there are many adverse reactions.
• ·As a special mechanism, NETs act as a double-edged sword in the human body. As a product of activated neutrophils, an autoantigen, and a mediator of the expression of related inflammatory factors, NETs play a certain role in the pathogenesis of bone and joint diseases.
• ·Treating bone and joint diseases through the related pathways of NETs is a novel approach that deserves to be investigated, but so far there are few relevant studies. Elucidating the mechanism and role of NETs in bone and joint diseases may lead to promising new strategies in the treatment of bone and joint diseases.

Funding

This research was supported by the National Natural Science Foundation of China Youth Fund Project (Grant no. 82101956), the Natural Science Foundation of Shandong Province (Grant no. ZR2021QH159), the Traditional Chinese Medicine high-level Talents Cultivation Project of Shandong Province, the Major Science and Technology Innovation Project of Shandong Province (2022CXGC020510, 2021SFGC0502), the Academic Promotion Project of Shandong First Medical University (2019QL003), Shandong Province Traditional Chinese Medicine Science and Technology project (M − 2022253), the Shandong Provincial Central Government Guides Local Science and Technology Development Fund Projects (YDZX20203700002055).

Data availability statement

No data was used for the research described in the article.

CRediT authorship contribution statement

Mengting Xiang: Writing – original draft, Methodology. Meng Yin: Writing – review & editing, Writing – original draft. Siwen Xie: Writing – original draft. Liang Shi: Validation, Funding acquisition. Wei Nie: Writing – original draft. Bin Shi: Visualization, Validation. Gongchang Yu: Writing – original draft, Validation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.