Background
The etiopathogenesis of rheumatoid arthritis (RA) is complex and involves both environmental and genetic factors. Early diagnosis of RA is of importance for effective therapy to prevent irreversible damage. However, RA diagnostics are hampered by the unspecific clinical manifestations [
1]. One of the established clinically used RA markers is rheumatoid factor (RF), which represents a set of autoreactive antibodies against the immunoglobulin G (IgG) Fc fragment. According to authors of a recent meta-analysis, RF discriminates patients with RA from healthy subjects (HS) with only moderate sensitivity and specificity (69 and 85%, respectively) [
2]. Moreover, elevated RF has been detected not only in patients with RA but also in patients with other systemic autoimmune rheumatic diseases. RF was the most significant diagnostic marker associated with RA until the discovery of anticitrullinated protein/peptide antibodies (ACPA). Initially, the presence of ACPA was discovered to be particularly useful diagnostically: It was more specific, although less sensitive, than the presence of RF [
3]. Recent studies also suggested that ACPA positivity was predictive of RA months before the first clinical manifestations, and it was associated with more severe disease development and with better response to therapy with abatacept and adalimumab [
4,
5].
The Disease Activity Score (DAS) is one of the most commonly used tools to monitor disease activity in RA. C-reactive protein (CRP) is a sensitive but unspecific inflammation marker that is widely used for the 28-joint Disease Activity Score (DAS28) based on C-reactive protein definition in patients with RA and is in good agreement with DAS28 based on erythrocyte sedimentation rate [
6].
Increased cell-free nuclear circulating DNA (n-cirDNA) was detected in the blood plasma/sera of patients with certain disorders, such as cancer and autoimmune diseases, in particular patients with systemic lupus erythematosus (SLE) [
7‐
10]. Mitochondrial circulating DNA (m-cirDNA) was also elevated in patients with cancer and disorders associated with massive cell damage, such as acute ischemic stroke [
11], myocardial infarction [
12], trauma [
13], and severe sepsis [
14]. The biological role of n/m-cirDNA remains enigmatic in both health and pathology. Recently, attention has been focused on the characterization of a possible alternative form of cell-free nuclear and mitochondrial DNA acting as an autoantigen in triggering SLE [
15]. Accumulating data indicate that an increased amount of n/m-cirDNA in plasma can be associated with autoimmune pathogenesis, especially in SLE and Sjögren’s syndrome [
7,
9,
16,
17]. One research group used parallel genomic and methylomic sequencing for the comparison of plasma cell-free circulating DNA (cirDNA) and discovered a number of plasma DNA abnormalities in patients with SLE in contrast to HS [
18].
n-cirDNA was also found on the surface of blood cells—both erythrocytes and leukocytes. In patients with cancer, cell surface-bound DNA (csbDNA) demonstrated changes of concentration and composition due to accumulation of DNA molecules coming from cancer cells [
19,
20]. Our observations indicate a continuous exchange between circulating cell-bound and cell-free nuclear DNA (nDNA) pools in whole blood [
20]. Thus, the goal of this study was to investigate the occurrence of n/m-cirDNA in patients with RA and its association with disease activity. Accordingly, levels of n/m-cirDNA and n/m-csbDNA were determined in patients with RA to assess their association with RA development and to evaluate their potential as RA markers in combination with the routinely used RF, CRP, and ACPA.
Discussion
To our knowledge, this is the first study to simultaneously quantify circulating nDNA and mitochondrial DNA in blood plasma, as well as nDNA and mtDNA bound on the surface of blood cells of patients with RA. The study demonstrates significant differences of circulating cell surface-bound nDNA and cell surface-bound mitochondrial DNA levels in patients with RA compared with HS. Nuclear and mitochondrial DNA fragments were found at low levels in plasma/serum from HS and were elevated in patients with cancer and certain disorders associated with the increase of cell damage or death (autoimmune disorders, trauma, stroke) [
7‐
14]. The associated changes of mitochondrial DNA and nDNA in the blood suggest identical sources of their origin, such as probably cell death remnants along with complexes and vesicles secreted by living cells [
19,
28‐
31].
Furthermore, circulating plasma/serum nDNA levels were reported to be elevated in patients with different systemic autoimmune disorders. In a recent study, Bartoloni et al. detected significant changes of n-cirDNA levels in 48 patients with SLE and 44 patients with Sjögren’s syndrome compared with control subjects [
10]. Also, Chen et al. [
9] demonstrated n-cirDNA increases in SLE; however, two earlier reports showed no changes in SLE compared with healthy individuals [
7,
32]. Patients with systemic sclerosis developed increased n-cirDNA levels only in the case of active disease [
8]. These controversial data could be the result of different protocols of blood sample processing and n-cirDNA extraction and analysis.
In our study, the plasma n-cirDNA level in patients with RA was increased, which is in accordance with two earlier reports [
33,
34]. In contrast, the level of n-csbDNA was decreased in patients with RA compared with HS. Earlier, we detected decreased levels of n-csbDNA in patients with breast, lung, and prostate cancer, which was not observed in patients with benign tumors [
19,
22]. Multiple reasons can be responsible for the decreased binding of n-cirDNA to cell surfaces in patients with RA, including the characteristics of circulating DNA-protein complex modification as well as disease-induced changes in the composition and amount of the blood cell surface proteins and plasma proteins. Recently, a link between circulating cell-free DNA levels and neutrophil extracellular trap (NET) formation was established in a number of autoimmune conditions, including RA [
35]. NETs are extruded by polymorphonuclear neutrophils (PMNs) and consist of chromosomal DNA complexed with antimicrobial peptides and proteases. Earlier studies suggested that RA-derived PMNs were more prone to undergo NETosis and that components of NETs, including circulating DNA, could contribute to the generation of autoantigens [
35].
Recent reports showed increased plasma levels of total microparticles (MPs) in patients with as RA, SLE, and Sjögren’s syndrome compared with healthy donors [
36,
37]. MPs are known to contain fragmented nDNA, and thus the increase of plasma n-cirDNA levels may be a result of systemic MP release due to constant cytokine stimulation in steroidal anti-inflammatory drugs [
38]. Nielson et al. evaluated the putative role of MPs in SLE as circulating antigenic targets and carriers of autoimmune complexes [
37]. Zhong et al., using protein G sepharose bead adsorption of plasma to isolate antibody-bound DNA [
34], reported an association of elevated n-cirDNA with higher DNA-binding antibody levels in the plasma of patients with RA. They concluded that the content/composition of DNA-bearing complexes in the circulation of patients with RA differed from that in a control group [
34].
This study demonstrates an increase of cell surface-bound mitochondrial DNA in patients with RA compared with HS, whereas plasma m-cirDNA in patients with RA was not modulated. Researchers in several recent studies reported elevated levels of plasma m-cirDNA, too, and discussed their diagnostic significance in breast, prostate, bladder, renal cell, and testicular germ cell cancers [
39‐
42]. Significantly elevated levels of plasma m-cirDNA have been found in patients with other clinical conditions associated with massive cell damage, such as acute ischemic stroke [
11], trauma [
13], and severe sepsis [
14]. Interestingly, extracellular mitochondrial DNA was shown to be recognized by immune cells as a danger signal and to trigger an inflammatory response, such as in cases of posttraumatic shock [
43]. Mitochondrial DNA possess immunomodulatory properties of bacterial DNA as a result of mitochondrial endosymbiotic origin. Probably owing to its proinflammatory potential, m-cirDNA in plasma was recently demonstrated as a potent predictor of posttraumatic systemic inflammatory response syndrome, multiple organ dysfunction syndrome [
44,
45], and intensive care unit patient mortality [
46]. Despite the accumulating evidence of cell-free mitochondrial DNA-induced immune deregulation, there is only one study on m-cirDNA in autoimmune disorders. Hajizadeh et al. described increased m-cirDNA levels in plasma and synovial fluid of patients with RA [
47]. Those authors proposed the involvement of m-cirDNA in joint inflammation by activating immune cells to produce proinflammatory cytokines. However, the mechanisms leading to autoreactivity induction by mitochondrial DNA remain elusive. Mitochondrial DNA released into the extracellular milieu and circulation under certain conditions has potential to trigger autoreactivity because of being totally CpG-unmethylated [
48,
49]. A recent study on DNase II-knockout mice demonstrated that the mice developed symmetrical erosive polyarthritis. This indicates that cell-free nDNA mitochondrial DNA itself can be directly involved in the etiology of RA [
50,
51]. In these mice, the ability of macrophages to degrade DNA from erythrocyte precursors and apoptotic cells was severely reduced, and immune cells produced large quantities of proinflammatory cytokines, RF, and antinuclear antibodies.
Today, the most informative RA-specific serological marker is the occurrence of autoantibodies to citrullinated proteins. According to recent reports, the autoimmune response against citrullinated proteins develops in correlation with autoreactive antibodies against the self IgG Fc fragment (RF). The combination of these two markers enables higher accuracy of RA diagnostics [
52]. In accordance with earlier observations [
53], the present study demonstrated a positive correlation of ACPA with RF levels in patients with RA. In contrast, we found a negative correlation of ACPA with m-csbDNA and n-csbDNA levels. These data suggest that ACPA/RF, on one hand, and m-csbDNA, n-csbDNA, on the other hand, could be independent circulating markers of RA development and that their combination may provide a powerful diagnostic tool.
According to our results, the combination of two circulating markers (m-csbDNA + n-csbDNA) had a sensitivity of 84% and specificity of 89% for RA diagnosis in patients with different disease activity levels and stages. The combination of the routinely used ACPA, RF, and CRP resulted in 90% sensitivity and 94% specificity. Addition of the two circulating DNA markers to ACPA (ACPA + m-csbDNA + n-csbDNA) showed the highest power for the discrimination of RA from HS (97% sensitivity and 98% specificity). This resulted in an improved LR− of the blood-based test (0.19 versus 0.03).
Our study, in accord with earlier reports, indicates that RF and CRP levels are dependent on disease stage and activity. In contrast, ACPA, m-csbDNA, and n-csbDNA changes are found not only in end-stage RA but also in recent-onset/established RA stages and are not associated with disease activity; therefore, these parameters look promising as diagnostic markers. One inclusion criterion for patients with RA in our study was a uniform therapeutic treatment with MTX and etoricoxib along with folic acid cotherapy. Etoricoxib belongs to the class of nonsteroidal anti-inflammatory drugs acting as cyclooxygenase 2 inhibitors with no reported influence on DNA synthesis [
54]. MTX is a disease-modifying antirheumatic drug that produces measurable cytotoxic effects in cancer therapy when administered in short courses at very high doses (up to 1000 mg). The induced depletion of folate-dependent thymidine and purine residues, and thus antagonism of DNA synthesis as well as cell-cycle arrest at S
1, are well established. However, these are not the same mechanisms by which low-dose MTX exerts its therapeutic effect in RA [
55]. Evidence derived from clinical practice does not support the purine-pyrimidine antagonism hypothesis in RA treatment. The concomitant administration of folate once per week is believed to reduce the incidence of treatment-related adverse effects as well as liver function abnormalities and does not result in a loss of clinical benefit [
56]. Smolenska et al. reported that a single-dose MTX treatment in patients with RA reduced the concentration of uric acid and hypoxanthine in whole blood for 1 day and then returned to the pretreatment levels within 2 days without any concomitant change in blood adenosine levels [
57]. However, the influence of MTX in combination with folic acid on whole-blood purine levels was not studied. The present study reveals circulating DNA changes associated with RA development in patients who received a uniform treatment. To our knowledge, this treatment should not influence disease-associated circulating DNA changes. The folate cotherapy should abrogate MTX’s supposed effect on folate-dependent purine-pyrimidine synthesis and its putative influence on cirDNA levels. Spearman’s rank-order correlation test demonstrated no correlation of circulating DNA levels with the duration of therapy ranging from 3 months to 5 years in our study.
To clarify the question whether circulating DNA level is changed in patients in response to a different therapy, we studied the group of patients with active disease who did not respond adequately to MTX and therefore were treated with the B-cell-depleting biological agent rituximab. It is of interest that m-csbDNA levels in this group of patients showed a tendency to be decreased compared with the group of MTX/etoricoxib-treated patients. One can propose this change to be associated with the positive response to rituximab; however, this effect should be validated in a larger group of patients. Further studies evaluating cirDNA as a biomarker of RA therapy efficiency in differentially treated patients can provide information on therapeutic effects on cirDNA in RA.
A limitation of our study is the low number of patients with early RA as well as that a group of high-risk persons should have been examined. Future investigations are warranted to assess the value of m-csbDNA and n-csbDNA for an early noninvasive diagnostic test in combination with ACPA. An additional point that needs further evaluation is the specificity of the combination of m-csbDNA, n-csbDNA, and ACPA regarding the discrimination of RA from other autoimmune diseases.