Crossroads: Pathogenic role and therapeutic targets of neutrophil extracellular traps in rheumatoid arthritis

Abbreviations

Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune disease dominated by multiple symmetrical arthritis [1,2], in which patients often experience symptoms of morning stiffness, swelling, and pain of the joints, which gradually progresses during recurrent episodes of prolonged disease to bone damage, joint deformity and functional disability [3,4]. In addition to joint damage, systemic inflammation can involve other organs, including the skin, eyes, blood vessels, heart and lungs in 40% of RA patients [5]. The global prevalence of RA is reported to be about 1%, with female patients being two to three times more likely to suffer than male patients, and the disability rate is also higher and increasing year by year, which seriously affects patients’ quality of life and social participation [6,7].

Although the exact etiology of RA is still not fully understood, most studies suggest that it may be closely related to environmental and genetic factors [8]. Previous studies have shown that autoimmune responses and inflammation are important pathophysiological bases for the development of RA [4]. Therefore, abnormal infiltration and activation of synovial membranes by different immune cells and an imbalance between pro- and anti-inflammatory cellular immune responses are crucial for the onset and progression of RA [9]. Neutrophils, as the most abundant circulating form of human leukocytes, are key effector cells in the innate immune system [10] as well as being considered the first line of defence during injury and infection. Neutrophils have been reported to be the most abundant cell type in the synovial fluid of RA patients [11]. Increasing evidence suggests that neutrophils are closely associated with RA synovial inflammation and cartilage damage, and may also be involved in the development of RA through mechanisms such as the release of immune mediators, respiratory burst, and apoptosis [12,13].

Neutrophil extracellular traps (NETs) are a specific defense mechanism of neutrophils, a large network of depolymerized DNA fibers and derived nuclear, cytoplasmic, and granule proteins released by that trap and kill pathogens [14]. Brinkmann et al. [15] first described and formally named the process NETosis, defining a new paradigm for antimicrobial innate responses. In addition to their role as host defense mechanisms, a large body of literature has demonstrated that NETs may also lead to toxic effects in the host. For example, a recent study reported the pro-tumourigenic activity of NETs in terms of their involvement in cancer immunoediting, progression, and metastatic spread [16]. The ability of NETs to induce autoimmunity has been widely suggested in a wide variety of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, antineutrophil cytoplasmic antibody-associated vasculitis, antiphospholipid syndrome, and type 1 diabete [17,18]. Thus, NETs often play a complex double-edged role in autoimmune diseases.

In this review, we first describe the mechanism of formation and the key pathogenic roles of NETs in RA (Fig. 1), then we show some key derived molecules, respectively, and accumulate evidence that NETosis is a potential biomarker for diagnosis, subtype identification, and risk detection. Finally, we discuss the targeting mechanisms of NETosis by various therapies, including conventional and biologic agents and traditional Chinese medicine, suggesting that NETosis has the potential to be a revolutionary precision medicine target for personalized treatment and subsequent drug development in RA.

Figure 1: The process of neutrophil extracellular traps (NETs) formation (NETosis) is involved in the pathogenesis and progression of RA in four main ways. (1) NETosis upregulates the production of inflammatory cytokines, amplifies joint inflammation, activates synoviocytes, and presents autoantigens to T cells, secretes BAFF to promote B cell proliferation, thus linking innate immunity with the modulation of adaptive immune responses; (2) components of NETs may act as a source of antibodies to guanosine-primed autoantigens; (3) NETs promote the production of anti-ammonocarbamylated protein (CarP) antibodies and upregulate RANKL production by FLS, which mediates osteoclast formation; (4) NETs are associated with periodontitis and smoking, two major susceptibility factors for RA. Abbreviations: ACPAs: anti-citrullinated peptide antibodies; BAFF: B-cell activating factor; CarPs: carbamoylated proteins; CitH3: citrullinated histone H3; FcR: Fc receptors; FLS: fibroblast-like synoviocytes; IFN-γ: interferon γ; IgG: immunoglobulin G; IL-6/8: interleukin 6/8; LPS: lipopolysaccharide; MHC : major histocompatibility complex; MPO: myeloperoxidase; NADPH: nicotinamide adenine dinucleotide phosphate; NE: neutrophil elastase; NETs: neutrophil extracellular traps; NOX: NADPH oxidase; PAD4: protein-arginine deiminase type 4; PMA: phorbol 12-myristate 13-acetate; ROS: reactive oxygen species; TLRs: toll-like receptors; TNF-α: tumor necrosis factor α.

Mechanisms for the Formation of NETs

In recent years, two different major pathways for the formation of NETs have been identified. The most well-known pathway is the traditional suicidal NETosis, which is a gradual process. Upon exposure of neutrophils to various stimuli such as fopperol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) [19,20], triggered by innate immune receptors in combination with downstream intracellular mediators [21], calcium is released from the endoplasmic reticulum of the neutrophils, which activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) via the protein kinase C (PKC) signaling pathway. Peroxisomes and mitochondria generate reactive oxygen species (ROS), which in turn activates myeloperoxidase (MPO) and mediates translocation of activated neutrophil elastase (NE) to the nucleus, collectively driving protein hydrolysis and cellular degradation [22]. At the same time, activated protein-arginine deiminase type 4 (PAD4) induces citrullination involved in the formation of citrullinated histone H3 (CitH3), which triggers chromatin decondensation [23]. Finally, this mixture of chromatin and granulin is formed and released into the extracellular space, where neutrophils undergo lysis and release NETs. The key to this process is the production of ROS by NOX during the “respiratory burst” of neutrophils, which is also known as the NOX-dependent pathway. The other pathway is the NOX-independent pathway, known as essential NETosis or non-lytic NETosis, where neutrophils release NETs without disrupting the nuclear or plasma membranes, are not accompanied by neutrophil death, and retain some of their regular cellular functions, such as cell phagocytosis and migration [24]. This NOX-independent NETosis is largely dependent on mitochondrial ROS (mROS) production, and after stimulation with LPS or C5a (protein fragments released from cleavage of the complement component C5), mitochondria in neutrophils act as ROS generators, with an influx of calcium carriers into the extracellular calcium, and inducing the production of mROS [25].

Involvement of NETosis in the Pathogenesis of RA

Neutrophils can participate in immunoinflammation of RA via the process of extracellular reticular traps (NETosis)

In the early stages of RA, large numbers of neutrophils pre-activated by immune complexes are recruited into synovial tissues and joints, and infiltrating neutrophils exhibit an ‘activated’ phenotype, including increased production of cytokines, chemokines, and ROS, delayed apoptosis and activation of NETs [26]. NETs has been reported to be detected in the synovial fluid of diseased joints as well as rheumatoid nodules in RA patients, and neutrophils in RA patients release more and faster NETs than in healthy subjects [27]. Excessive increase of NETs in synovial and peripheral blood neutrophils stimulates the induction of inflammatory factors such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, IL-17A, and interferon (IFN)-γ, further promoting the expansion of the inflammatory response and the formation of an “inflammatory storm” [28]. A large number of studies have confirmed the existence of an “inflammatory storm” in RA patients, which is an immune-inflammatory response characterized by a high release of pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines [29]. Pro-inflammatory cytokines such as IL-17A, TNF-α, and IL-8 are in turn strong inducers of NETosis in neutrophils [30], thus creating a vicious circle. Fibroblast-like synoviocytes (FLS) are known to be the main effector cells involved in the process of joint destruction [31]. FLS exposed to NETs exhibit pro-inflammatory, hyperproliferative and more than invasive properties, leading to pannus formation in chronic RA [32]. In addition, FLS promotes major histocompatibility complex (MHC) class II upregulation through the internalization of NETs and shares epitopes to present modified self-antigens to CD4 T cells [33]. Tillack et al. [34] first described that NETs mediate T cell proliferation and pro-inflammatory cytokine secretion by lowering its activation threshold. NETs also stimulate neutrophils to secrete IL-8 and B-cell activating factor (BAFF), promoting a vicious cycle between B cells and neutrophils [35]. In conclusion, NETs, as a specific structure and form of neutrophils, activate synoviocytes, upregulate pro-inflammatory cytokine production, amplify joint inflammation, and connect innate immunity with the modulation of adaptive immune responses involved in the inflammatory process of RA.

NETs as a source of antibodies to citrullinated autoantigens

Autoantibodies, including rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs), are diagnostic markers of RA according to the 2010 American College of Rheumatology/European League Against Rheumatic Diseases mandated classification criteria for RA [36]. It has been claimed that ACPAs are present in the serum of more than two-thirds of RA patients, several years before the onset of clinical symptoms, compared to the classical and well-known RA-specific antibody RF [37,38], which are widely used for diagnosis and prognosis prediction. It has been demonstrated that histones and other NETs-associated proteins can be citrullinated by PAD enzymes (especially PAD4), providing a source of citrullinated proteins for the immune system and facilitating the production of ACPAs [39]. In turn, ACPAs can perpetuate inflammation by stimulating the formation of NETs and promoting inflammation in RA [40], thus creating a vicious cycle. As observed by Okamoto [41], NETs residues are associated with localized ACPA production and levels as well as inflammatory factors such as IL-1β and IL-6. It has been documented that NETs are positively correlated with ACPA in the sputum of susceptible individuals and first-degree relatives of RA patients, which may be associated with the onset or early stages of the disease [42]. In addition, Felty syndrome is a severe form of RA, and Pratesi et al. [43] further proposed that citrullinated H4 from activated neutrophils and NETs is the target of the antibody ACPA in Felty syndrome, all of which further elucidates the role of NETs in RA.

NETs mediate osteoclast formation

Anti-carbamoylated protein (CarP) antibodies have been reported to be significantly associated with bone destruction, joint erosion, and disease activity in RA patients, and are emerging as a biomarker for RA [44,45]. NETs release carbamoylated proteins (CarP), which leads to the production of antibodies to CarP and antibodies to carbamoylated NETs proteins (cNET) production, which promotes osteoclast formation and activation [46]. At the same time, NETs are internalized by FLS and upregulate FLS production of RANKL, which promotes osteoclast formation by CD14 monocytes [47]. In addition, NE in NETs can directly degrade cartilage components and expanded FLS and macrophages can further contribute to joint damage [48].

NETs are associated with susceptibility factors for RA

Periodontitis and smoking have been documented as susceptibility factors for RA [49,50]. It has been reported that expression of PADase homologs by Porphyromonas gingivalis induces the production of citrullinated antigens and enhances NETosis in patients with periodontitis, which triggers pathophysiology associated with RA [51]. Kaneko et al. [52] found that compared to control individuals, RA patients with periodontitis had significantly higher serum levels of CarP and NETs, which correlated with disease severity. Oliveira et al. [53] were even more direct in pointing out that enhanced localized formation of NETs in periodontitis may be an early event in RA, and that periodontal treatment also leads to circulating significant changes in NETs levels. Several studies have confirmed the strong association between cigarette smoking and increased risk of RA, especially in ACPA-positive patients [54,55]. Cigarette smoking can induce ACPA in the lungs by nicotine-inducing NETs and citrulline formation in a PAD4-dependent manner [56]. Lee et al. [57] demonstrated that nicotine is a potent inducer of NETosis, which may accelerate arthritis progression on a murine model of collagen-induced arthritis. Proteomic analysis of bronchial biopsies and synovial biopsies also identified the same citrullinated protein targets in the lungs of RA patients and RA joints, suggesting that the lungs are a possible origin of ACPA [58]. Demoruelle et al. [59] found that in individuals at risk of developing RA individuals with high levels of NETs in sputum were strongly associated with antibody reactivity such as ACPA, further supporting the link between NETs and the development of ACPA in the lungs.

NETosis-Related Molecules and Derivatives

Interleukin 8 (IL-8)

IL-8, also known as CXCL8, is a CXC-type pro-inflammatory chemokine that plays an important role in neutrophil activation and recruitment to sites of inflammation. It activates and induces neutrophils to undergo migration to the site of inflammation while promoting their mass infiltration, thus facilitating the formation of NETs at their focal sites [60]. The data of Shu et al. [61] confirmed that the formation of NETs is induced by IL-8 and proposed that the process is associated with the mitogen-activated protein kinase and nicotinamide adenine dinucleotide phosphate pathways. Shang et al. [62] found that exosomes stimulate the expression of IL-8 expression which in turn promotes the formation of tumor-associated NETs. Abrams et al. [63] developed a new assay for NETs and found that IL-8 levels are closely related to their formation, and that inhibition of IL-8 therapy significantly attenuates NETosis and thereby reduces organ damage and mortality, as demonstrated in a mouse model of sepsis. In conclusion, numerous studies have confirmed that IL-8 is a strong inducer of NETosis.

Myeloperoxidase (MPO) and neutrophil elastase (NE)

MPO and NE are well-defined markers of neutrophil infiltration [64]. MPO is one of the most abundant proteins in neutrophils, accounting for up to 5% of the cell’s dry weight, and is normally stored in nitrogenophilic granules and released in response to stimulation of neutrophils. Metzler et al. [65] demonstrated that the process of NETs formation requires high local concentrations of MPO products, while pharmacological inhibition of MPO delays and reduces the formation of NETs. It has been reported that abundant MPO is detected in the inflamed synovium of RA patients [66], which leads to further neutrophil recruitment, amplification of inflammation, and FLS proliferation [67]. In addition, MPO also interacts with vascular endothelial cells interacting to increase endothelial permeability, which is also a key process during inflammation [68]. NE, a protein hydrolase stored in the neutrophilic granules of neutrophils, derives its name from its ability to degrade the extracellular matrix protein elastin, as well as being an important source of extracellular killing of bacteria and degradation of virulence factors by NETs [69]. Studies have shown that NE can induce cell proliferation and activate a variety of cytokine and chemokine signaling pathways [70]. Conversely, inhibition of NE signaling inhibits the formation of NETs, reduces Nets-mediated vascular damage, and mitigates the production of inflammatory cytokines [71]. Muley et al. [72] also found that NE can promote knee injury in mice through PAR2-dependent activation of the p44/42 MAPK pathway.

Peptidyl arginine deiminase 4 (PAD 4)

The PAD gene encodes a PAD protein that converts arginine residues to citrulline. Interestingly, many pathological and genetic studies have identified the gene encoding the PAD4 enzyme (PADI4) as a susceptibility gene for RA, which has recently been identified as an important risk factor for people at high risk of RA, especially in North American populations [73]. Turunen et al. [74] found that PAD4 was abundant in the synovial tissue of RA and involved in the citrullination of fibrin, which was closely related to tissue inflammation. The PAD4 enzyme catalyzes the deimination of arginine residues to produce the main antigenic target of ACPA in RA, considered a key enzyme in the pathogenesis of RA [75]. PAD4-mediated citrullination has also been found to be involved in monoclonal ACPA-induced tenosynovitis, as well as a key mechanism of pain and bone loss [76]. Jonsson et al. [77] found that the ability of serum to activate PAD4 was associated with early RA patients’ ACPA and RF positivity and disease flares. Gómez-Bañuelos et al. [78] identified associations between various subtypes of anti-PAD4 and clinical outcomes in RA including imaging joint damage, rheumatoid nodules and interstitial lung disease. Similarly, anti-PAD4 antibodies were found to recognize RA patients with higher levels of radiographic damage and bone erosion and lower levels of anti-PAD4 antibodies were associated with lung involvement in RA patients in a cohort by Palterer et al. [79]. This is supported by another finding that PAD4 antibodies are present in sputum and saliva of some RA patients [80], while a meta-analysis by Zhang et al. found that RA patients carrying anti-PAD4 antibodies had a higher likelihood of DAS28, ESR, swollen joint count (SJC), as well as of developing interstitial lung disease and pulmonary fibrosis [81]. In conclusion, the above evidence suggests that the PAD4 enzyme and anti-PAD4 antibodies are involved in three important features of RA: citrullination, lung involvement, and bone destruction, and thus could be potentially useful diagnostic biomarkers of RA and important therapeutic target candidates.

Mitogen-activated protein kinases (MAPKs)

A series of protein kinases in the MAPK signaling pathway, including c-Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK) and p38 kinase, are known to promote the early production of inflammatory cytokines [82]. Currently, it has been documented that MAPKs are the upstream signaling pathways of NETs in a variety of diseases. Dömer et al. [83] first determined that Nets-induced neutrophil activation occurs through pathways involving phosphorylation of protein kinase B (Akt), ERK1/2, and p38. Ji et al. [84] found that anggranin prevents neutrophil recruitment and NETosis by inhibiting the activation of nuclear factor (NF)-κB and MAPK signaling pathways in LPS-induced acute lung injury. Zhou et al. [85] confirmed that cannabinoid receptors play a key role in neutrophil chemotaxis and NETosis in aseptic liver inflammation, which is related to the ROS/p38 MAPK signaling pathway. Jati et al. [86] found in rats with gouty arthritis induced by monosodium urate that piperine regulates the immunosuppression of NLRP3 inflammasome through MAPK/NF-κB, reducing inflammatory symptoms and improving gouty caused by NETosis. In conclusion, we believe that the MAPK family is one of the key regulatory pathways of NETs expression.

Application in the Diagnosis and Treatment of RA

Some scholars have proposed that neutrophil activation and NETs derivatives in the plasma of RA patients have become promising diagnostic markers for monitoring RA. NETs derivatives including plasma cell free nucleosomes, MPO, NE and cathepsin G were closely related to the severity of RA. Pérez-Sánchez et al. [87] evaluated 40 patients with RA and 75 healthy individuals and found the elevated levels of NETs degradation products, such as circulating free DNA, free nucleosomes, NE, and MPO complexes in patients with RA, which may have diagnostic potential for disease activity and atherosclerosis as well as for the assessment of therapeutic effectiveness in RA. Sur Chowdhury et al. [88] measured Nets-derived free nucleosomes in RA serum by receiver operating characteristic (ROC) curve, showing diagnostic value with area under ROC > 97%, sensitivity of 91%, and specificity of 92%. de Bont et al. [89] first reported that antibodies against NET (ANETA) were detectable in baseline RA patients (22%–69%) and that serum ANETA levels in RF-positive RA patients were significantly higher than in negative RA patients. This evidence suggests that quantitative testing of NETs and their derivatives may be a useful complementary tool for identifying individuals at risk and monitoring patients with RA.

Therapeutic RA Targeting Strategies Related to NETosis

Elimination and inhibition of NETosis

As previously stated, dysregulated NETosis is closely implicated in the pathogenesis of RA. Therefore, targeting specific steps or products of NETosi might be a viable strategy for developing drugs and therapies for RA (Table 1). DNase I was the earliest described direct degrader of NETs [90]. Studies have shown that DNase-1 cuts and fragments DNA and destroys the skeleton of NETs [91]. Kolaczkowska et al. [92] also demonstrated that DNase not only effectively removed DNA carried by NETs, but also significantly inhibited the proteolytic activity of NE. At present, DNase I targeted destructive NETs have been used in the treatment of a variety of diseases, such as systemic lupus erythematosus [93], ischemia-reperfusion [94], malignant tumors [95], etc. Wang et al. [96] developed a DNase-functionalized hydrogel that demonstrated significant efficacy in a mouse model of CIA by continuously degrading NETs with new drug targets to reduce inflammatory responses in RA. In addition, some drugs designed to indirectly inhibit the formation of NETs may also be used to treat and relieve RA. Wan et al. [97] reported that dexamethasone can significantly reduce Staphylococcus aureus-induced NETosis by modulating toll-like receptors (TLRs), primarily TLR2 and TLR4. Kaundal et al. [98] conducted a study on 103 RA patients and found that MTX treatment was associated with the reduction of ROS production and CD177 expression, indirectly inhibiting NETosis, which may be one of the mechanisms of MTX treatment of RA. Kawaguchi et al. [99] demonstrated that injection of the PAD inhibitor chloramidine (CI-amidine) reduced protein citrullination and IL-6 in a mouse model of glucose-6-phosphate isomerase-induced arthritis (pGIA). A novel oral selective inhibitor of PAD4, JBI-589, prevents the formation of NETs and PAD4-dependent citcitline, reducing the severity of arthritis in mouse models [100]. Ruiz-Limón et al. [101] reported that after 6 months of treatment with the IL-6 R inhibitor tocilizumab, MPO and NE in RA patients decreased along with suppressed NETosis. Ascomycin and cyclosporin A can reduce IL-8-induced NETs by inhibiting the calcineurin pathway, and have been widely used in the treatment of rheumatoid arthritis, asthma, etc., [102,103]. Prostaglandin E2 (PGE2) is the most abundant prostaglandin in the human body and has been shown to have anti-inflammatory effects. Zhao et al. [104] reported that an emerging infusion therapy for gingival-derived mesenchymal stem cells (GMSCS) in recent years can improve inflammatory arthritis by inhibiting NETosis through the PGE2-PKA-ERK signaling pathway. From this perspective, pharmacological interventions or patient management are currently being developed to target inhibition ofNETosis at several levels: inhibiting the activation of inducible cytokines such as IL-8, targeting the intracellular processes of NETosis, and facilitating the removal or neutralization of NETosis-derived products such as MPO and NE.

NETosis participates in Chinese medicine treatment

In recent years, TCM has attracted more and more attention due to its advantages such as safety and fewer adverse reactions [105]. Using herbs and their extracts to treat RA has been practiced in Asian countries for thousands of years, whose efficacy has been confirmed by clinical applications and experimental studies [106]. Emodin accelerates apoptosis and inhibits autophagy and NETosis by reducing IL-6, IFN-γ, and TNF-α in adjuvant arthritis in mice [107]. Andrographolide inhibits autophagy-dependent NETosis by accelerating neutrophil apoptosis, while significantly reducing the phosphorylation of p38 MAPK and ERK1/2 [108]. Triptolide (TP) has shown potential as a treatment for RA by reducing neutrophil recruitment and down- regulation of TNF-α and IL-6 expression, and TP can also inhibit autophagy and NETosis in neutrophils [109]. Polydatin (PD) significantly inhibited the formation of NETs in myelo-derived neutrophils in RA patients as well as reduced the deposition of NETs in the ankle of CIA mice [110]. Compound Simiaoyong’an Decoction effectively inhibited arthritis in CIA mice by promoting neutrophil apoptosis, reducing ROS production and NETosis [111]. In conclusion, the above studies provide references and ideas for exploring new drug-target strategies for RA (Table 2).

Conclusion and Prospect

In conclusion, in this paper, we discussed the multiple mechanisms of NETs in the pathogenesis of RA. Neutrophils participate in the immunoinflammation of RA in the form of NETosis. NETs may also be an important source of autoantigens, mediate the formation of osteoclasts, and are closely related to RA susceptibility factors. These may provide more insights into the occurrence and development of RA. In addition, the presence of NETs and their related components may provide potential biomarkers and indicators, and further studies of inhibition of NETosis processes and disruption of interactions between various stimuli will help identify new therapeutic targets and strategies. Nevertheless, experimental studies are still needed here to evaluate the efficacy and value of various NETosis-derived product assays as biomarkers for predicting RA and to assist in assessing drug efficacy. Given the differences in immune system and physiologic functions between mouse models and humans, more clinical studies are needed in the future to confirm the efficacy of various drugs or therapies that inhibit aberrant NETosis or related molecules.

Acknowledgement: We take thankful pleasure in acknowledging the unsparing assistance of all participants.

Funding Statement: This work was supported by grants from the National Traditional Chinese Medicine Inheritance and Innovation Project Fund (Development and Reform Office [2022] 366), National Key Discipline of Traditional Chinese Medicine (Traditional Chinese Medicine [2023] No. 85), the Ministry of Science and Technology National Key Research and Development Program Chinese Medicine Modernization Research Key Project (2018YFC1705204), National Nature Fund Program (82074373, 82274490, 82205090), and Anhui Provincial Laboratory of Applied Basis and Development of Internal Medicine of Modern Traditional Chinese Medicine (2016080503B041).

Author Contributions: The authors confirm contribution to the paper as follows: study conception and design: Yang Li and Jian Liu; data collection: Yuedi Hu, Chengzhi Cong; analysis and interpretation of results: Qiao Zhou, Yiming Chen; draft manuscript preparation: Yang Li; final approval of the manuscript: Jian Liu. All authors reviewed the results and approved the final version of the manuscript.

Availability of Data and Materials: The datasets generated during the current study are available from the corresponding author on reasonable request.

Ethics Approval: Not applicable.

Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.

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