Introduction

Rheumatoid arthritis (RA) is a common autoimmune heterogeneous joint disease of still unknown etiology. In RA inflammation starts in the synovial membrane and spreads further to surrounding tissues, leading finally to cartilage and bone destruction [1, 2]. Various genetic [3, 4] and epigenetic [5] factors have been found to be associated with RA, but also environmental factors such as cigarette smoke, and viral and bacterial infections play a significant role in development of the disease [6, 7]. The pathogenesis of RA is regulated by T- and B-cells. B-cells are involved in the production of autoantibodies such as rheumatoid factor (RF) [8] and anti-cyclic citrullinated peptide autoantibodies (ACPA) [9, 10]. T-cells produce a variety of pro-inflammatory cytokines, especially tumor necrosis factor (TNF), interleukin 6 (IL-6) as well as vascular endothelial growth factor (VEGF), which are involved in degradation of bone and cartilage within the joint [11]. The pathogenesis of the disease may also be related to IL-33. Extracellular IL-33 is a critical enhancer of TNF-induced RA synovial fibroblast activation [12]. IL-33 in experimental models of collagen-induced arthritis exhibits triggering properties, and the administration of interleukin 33 intensifies the disease process [13, 14].

One of the characteristic features of RA is oxidative stress [15], most likely induced and stimulated by inappropriate B- and T-cell activity. It is associated with a higher level of reactive oxygen species (ROS) production in RA patients as compared with healthy subjects. The main factor that increases the release of ROS in RA patients is overproduction of TNF [16]. TNF controls cell proliferation or cell death. The altered level of nuclear factor kappa-B (NF-κB) increases TNF-induced death, concurrent with sustained Jun N-terminal kinase (JNK) activation via the death response. JNK activation in NF-κB-deficient cells depends on the ROS, but the influence of ROS in stimulation of JNK is still unclear [17]. Moreover, TNF-α, by increasing the expression of arginase in endothelial cells during ischemia/reperfusion (I/R) injury, leads to a decrease in the availability of l-arginine for nitric oxidase synthase, thereby increasing the production of O₂^−•. Overproduction of O₂^−• impairs the NO-mediated vasodilation and favors endothelial dysfunction. TNF-α inhibitors significantly reduce inflammation in patients with chronic autoimmune diseases, improving endothelial function and reducing the risk of acute cardiovascular complications [18, 19]. ROS also contribute to the depolymerisation of hyaluronic acid, causing increased bone resorption and reduction of joint viscosity. The elevated ROS level was correlated with various clinical parameters of RA such as seropositive or seronegative status and DAS score [20].

The negative ROS effect in RA is not limited to joints. Excessive ROS production results in accumulations of oxidative DNA lesions in peripheral blood mononuclear cells (PBMCs) isolated from RA patients, as we demonstrated previously [21]. We have hypothesized that oxidative stress together with an impaired DNA damage response (DDR) to oxidative DNA lesions (limited to base excision repair (BER) pathway) could be responsible for increased incidence in RA patients of some diseases with a genetic instability background such as lymphoma and lung cancer [22]. Therefore, the aim of this study was to evaluate repair of oxidative DNA lesions and correlate it with common polymorphism of BER genes as well as their expression level, as the results from our previous studies suggest that polymorphism of DNA repair genes may have a phenotypic effects in repair of oxidative DNA lesions [23–26]. Therefore, we determined the level of oxidative DNA lesions and the kinetics of repair of DNA damage induced by tert-butyl hydroperoxide (TBH) in PBMCs of 30 RA patients and 30 healthy individuals. The metrics from the DNA damage and repair study were correlated with the genotypes of common polymorphisms of the key BER genes as well as their expression levels. DNA damage and repair were evaluated by alkaline single cell gel electrophoresis (comet assay), the genotypes of the polymorphism were determined by TaqMan SNP Genotyping Assay, and PrimeTime qPCR Assay was used to analyze the expression profile of genes related to BER.

Material and methods

Results

Differences in DReff between RA patients and controls

PBMCs isolated from RA patients were more sensitive to TBH as compared to healthy patients (Figure 1 A; median RA = 13.515; 25% = 10.090; 75% = 13.515 vs. 6.350; 25% = 3.685; 75% = 8.540; Mann-Whitney rank sum test p < 0.001). Moreover, the RA group has more subjects in groups classified as low efficient and inefficient DNA repair as compared with the control (Figure 1 B and Table I). Similarly, we observed more people with inefficient DNA repair in the RA group treated with DMARDS (Table I).

Figure 1

A – Peripheral blood mononuclear cells (PBMCs) show greater sensitivity to tert-butyl hydroperoxide (TBH) at 7 µM for 15 min as scored by DNA damage. PBMCs were isolated from 30 healthy controls (white box) and 30 rheumatoid arthritis (RA) patients (grey box). B – Distribution of individual DNA repair efficiency in 30 healthy controls (white box) and RA patients. Data are presented as median. Differences between groups were analyzed using the Mann-Whitney rank sum test. ***p < 0.001

Table I

Efficiency* of repair of DNA lesions induced by TBH (the lower the value, the better)

DReff	Range	Controls (n = 30)	RA (n = 30)	RA no DMARDS	RA DMARDS
Group 1Highly efficient DNA repair	< 16.04	16	2	2	0
Group 2 Efficient DNA repair	16.05–22.54	10	3	3	0
Group 3 Low efficient DNA repair	22.55–29.03	3	12	4	8
Group 4 No repair	> 29.04	1	13	0	13

RA – rheumatoid arthritis.

* Efficiency was calculated as follows: The DNA damage measured immediately after exposure to TBH was set as 100% of DNA damage. Next, the percentage of repaired DNA after 60 min was measured. The individual DNA repair efficiency was obtained by subtracting this value from 100.

Discussion

We observed an association between RA occurrence, impaired DNA repair of oxidative DNA lesions in PBMCs, polymorphism of the UNG gene and expression of key BER genes – UNG, MUTYH and NEIL3. Therefore, our results suggest that the genetic variations within BER genes as well as epigenetic factors may be linked with RA by the modulation of the cellular response to oxidative stress, and this polymorphism may be a useful additional marker in this disease and/or an environmental indicator of oxidative stress. Because of oxidative stress, mainly 8-oxo-7,8-dihydroguanine (8-oxoGua) and 2,6-diamino-4-hydroxy-5-formamido-pyrimidine were formed in DNA, which arise as a result of attaching to the guanosine ring at the C8 position of the hydroxyl radical and their further transformations in redox reactions [30]. These DNA lesions are the main substrate for the BER pathway and are removed with the help of MUTYH and NEIL3 DGs. MUTYH is responsible for the removal of incorrectly substituted adenine and 2-hydroxyadenine paired with 8-oxo-dG, whereas NEIL3 exhibits a broad substrate recognition spectrum and can excise both oxidized pyrimidines and purines except 8-oxo-G [31]. A few reports have been published describing the effect of altered expression of BER genes in RA. Most of them associated the PARP1 gene with the progression of inflammation in RA (reviewed in [32]). The second concerns the MUTYH gene. The observed serum level of this gene was 8.8% higher in RA than in healthy people [33]. Our research clearly indicates that patients with RA have a reduced expression level of genes encoding two important DGs involved in repair of oxidative DNA lesions in PBMC compared to the control group. Decreased gene expression may result in ineffective or complete lack of repair, and our study is in line with this hypothesis. Unrepaired damage can lead to formation of double-stranded DNA strand breaks (DSBs) as a result of further cellular metabolism [34]. DSBs are among the most serious forms of DNA damage, often resulting in cell death, aging or mutations. The latter can lead to the development of cancer, which accounts for the significant number of RA patients who develop cancer. The mutagenic effects of oxidative DNA damage are well recognized and associated with DSBs. For example, GC → TA and CC → TT are signature mutations for oxidative DNA lesions, and they have been observed in the p53 tumor suppressor or the ras oncogene gene in various cancers.

The finding suggesting the potential involvement of UNG at the genetic and epigenetic levels in the inefficient repair of oxidative lesions in RA is exciting. UNG is the primary glycosylase involved in the removal of uracil (U) from DNA. The involvement of UNG in the removal of oxidative DNA lesions is minimal in contrast to other glycosidases; however, it plays a very important role in promoting lymphocyte development. During lymphocyte development, two molecular processes in DNA occurred – V(D)J recombination and class-switch recombination (CSR). Both are important for the formation of a wide range of lymphocyte B antigen receptors and heavily depend on somatic hypermutation (SHM). SHM is conducted in cooperation with BER and MMR systems and UNG plays a key role in this process by recognizing and processing U. Imbalance of UNG level increased the risk of developing B-cell lymphomas. However, it is not only carcinogenesis that should be addressed, as UNG is also likely involved in development of autoimmune diseases. The involvement of UNG in autoimmune diseases is complex, and loss of UNG or changes of its activity via SNP can affect RA progression in two ways. Firstly, it can be responsible for severely impaired CSR and antibody affinity maturation via SHM [35]. Secondly, it can induce apoptosis in B cells. Apoptosis results in release of DNA fragments outside the cell. DNA fragments can stimulate innate inflammatory responses via activation of the stimulator of interferon genes adaptor protein (reviewed in [36]). This can produce a feedback loop, as inflammation induces production of ROS that in turn generate oxidized DNA lesions, which – if not repaired – lead to apoptosis.

These findings confirm those of our earlier research [37], and also suggest that SNPs in DNA repair genes may be additional genetic factors associated with RA and contribute to the etiology, pathogenesis and outcome of RA. However, the groups were too small to enable a final conclusion, and further studies are required to address the question.

Although our study has the character of basic research, clinical aspects can be derived from it. The antioxidant potency of the host should be increased in order to avoid the effects of oxidative stress at the DNA level in people with reduced DNA repair efficiency. This can be done with an antioxidant-rich dietary intervention, because such a diet ameliorates the state of oxidative stress and improves the antioxidant potency [38, 39]. In addition, we believe that a closer understanding of the genetic background of RA in association with the clinical phenotype will allow for future personalized treatment of RA.

Our study has some limitations. First of all, the influence of the drugs taken by the patient cannot be neglected. MTX and GCS have a plethora of effects on RA patients. MTX but not GCS was associated with a slightly higher level of endogenous DNA damage compared with RA patients without treatment, as we reported previously [21]. However, considering the research hypothesis of this article, it is irrelevant whether the observed alterations in the activity of DNA repair processes and their association with genotype or gene expression alterations are due to RA phenomena or therapy. Nonetheless, we are more convinced of the former option, since MTX and GCS are drugs which are generally considered safe, and no association between treatment with these drugs and cancer development has been demonstrated. Regardless, our study had mainly a preliminary character, and these results need confirmation in larger cohorts outside the Central European populations, as the main limitations of the study are its single-center design, sample size and ethnicity.

In conclusion, impaired repair of oxidized DNA lesions in RA patients may accelerate PBMC aging and contribute to the RA phenotype and association of RA with cancer development. Genetic and epigenetic factors contribute to this phenomenon, but epigenetic factors that lead to lower expression of BER genes need to be identified. We excluded the possibility of a different methylation pattern in RA patients as an epigenetic factor. Thus, we must consider the two latter options: histone code and miRNA expression. The last one is particularly interesting, as some miRNAs are being considered as biomarkers for RA, but they need validation in a real-world clinical setting (reviewed in [40]). A potential link between RA and ineffective repair of oxidative DNA damage may be miRNA-155, as it is associated with the occurrence of both phenomena [41, 42].