Levels of base excision repair proteins in CD4 ⁺ T cells in patients with systemic lupus erythematosus

Abstract

Objective

Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4 ⁺ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease.

Methods

From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively.

Results

In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI.

Conclusion

In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.

Systemic lupus erythematosus (SLE) is a multisystem involved autoimmune disease characterized by lymphocyte infiltration and autoantibody overproduction ^{[ 1]}. Immunocomplex formation and complement activation cause serious inflammation, leading to tissue damage ^{[ 2]}. However, the SLE pathogenic mechanism remains unknown. Our preceding research ^{[ 3]} found that DNA demethylation participated in SLE pathogenic mechanism and growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is also a DNA repair-related protein ^{[ 4]}. Rai, et al ^{[ 5]} found that Gadd45a interacts with activation-induced cytidine deaminase (AID), apolipo-protein B RNA-editing catalytic component (Apobec), and methyl-CpG-binding domain protein 4 (MBD4), which contribute to DNA demethylation in zebrafish embryos. In HEK293 cells, thymine DNA glycosylase (TDG) can interact with AID and Gadd45a, which promotes DNA demethylation ^{[ 6- 7]}. AID, TDG, and MBD4 all take participate in base excision repair (BER) process. BER is a primary pathway in DNA repair of base damage caused by oxidation ^{[ 8]}.

Many previous studies ^{[ 9- 10]} suggested that the abnormality of BER may take part in the underlying mechanism of lupus development. Thus, in this study, we aim to investigate BER protein expression in CD4 ⁺ T cells in patients with SLE, including AID, TDG, and MBD4, and analyze the correlations between BER proteins and lupus disease activity.

1. Subjects and methods

1.1. Patients and controls

Twelve SLE subjects [age (38.25±11.33) years] who met at least 4 standards in the classification of SLE listed by American College of Rheumatology ^{[ 11]} were recruited from outpatient and inpatient departments of Second Xiangya Hospital, Central South University between January 2019 and September 2020. We used SLE disease activity index (SLEDAI) to quantify lupus disease activity ^{[ 12]}. Patient demographics are given in Table 1. And we recruited 12 healthy controls from the medical personnel in Second Xiangya Hospital. We obtained the signed consent from each patient. The human sample research was evaluated and endorsed by the Human Ethics Committee of Second Xiangya Hospital (No.SADISD 20190220).

1.2. Cell separation

We drew about 50 mL of venous peripheral blood from each subject and then immediately conserved with heparin. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation. We isolated CD4 ⁺ T cells via positive selection using Miltenyi beads (Miltenyi, Germany) according to the manufacturer’s instructions. We incubated the separated cells with CD4 antibody (BD, US) and detected the cells by flow cytometry. The cell purity was higher than 95%.

1.3. RNA isolation and real-time RT-PCR

We extracted the total RNA from CD4 ⁺ T cells using TRIzol agentia and synthesized cDNA using PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). And the cDNA formed by reverse transcription of SLE patients and healthy controls was used as an amplification template to amplify target genes and GAPDH via real-time RT-PCR with a SYBR Premix Ex Taq Kit (Takara, Japan). A LightCycler96 Real time PCR System (Roche, Switzerland) was applied to detect the transcription levels of AID, TDG, MBD4, and GAPDH. We quantified mRNA expressions with a TB Green Premix Ex Taq (Takara, Japan). The 2 ^-ΔΔCt method (ΔCt=Ct_{BER proteins} -Ct_GAPDH; ΔΔCt =ΔCt_SLE-ΔCt_{healthy controls}) was used to determine the value of every cDNA, and these values were then standardized to GAPDH. The primers are exhibited in Table 2.

1.4. Western blotting

Total proteins from CD4 ⁺ T cells were isolated using RIPA buffer and detected the concentration with a Pierce BCA Protein Assay (Thermo Fisher Scientific, US). Then we performed gel electrophoresis experiments and transferred the samples to PVDF membrane. We used 5% defatted milk to block the membrane. Primary antibodies used included: Anti-AID (1꞉150; Abcam, UK), anti-TDG (1꞉900; Abcam, UK), anti-MBD4 (1꞉200; Abcam, UK), and β-actin (1꞉2 000; Thermo Fisher Scientific, US). We visualized the blots using Developer (Paison, Shanghai, China), and exposed them to X-ray films, then quantified band densities using Image-Pro Plus software. Densitometry was used to normalize protein quantification to β-actin. The ratio of the optical density value of the AID, TDG, and MBD4 proteins to the β-actin protein was used as the relative expression amount of the above protein.

1.5. Statistical analysis

All data were processed using SPSS 25.0 statistical analysis software, and the measurement data were expressed as mean±standard deviation ( $\overline{x}$ ± s). Measurement data were analyzed with the unpaired Student’s t-test (meeting normal distribution and homogeneous variance), otherwise, the Mann-Whitney U test was used. Spearman correlation test was used to analyze the correlations of the mRNA and protein levels of the BER proteins with SLEDAI. P<0.05 was considered a statistically significant difference.

2. Results

2.1. Expressions of AID, TDG, and MBD4 in SLE CD4 ⁺ T cells

Compared with the healthy controls, the mRNA and protein expressions of AID in SLE CD4 ⁺ T cells were significantly elevated (1.00±0.43 vs 3.37±2.12, P=0.003; 0.35±0.25 vs 0.63±0.28, P=0.022; Figure 1A, 2A, and 2C); TDG protein expression was obviously elevated (0.52±0.23 vs 0.88±0.44, P=0.017; Figure 2A, 2D); and no obvious difference was found in mRNA expression of either TDG or MBD4 between healthy controls and SLE patients (1.00±0.23 vs 1.06±0.69, P=0.776; 1.00±0.43 vs 1.00±0.27, P=0.984; Figure 1B, 1C). Compared with healthy controls, MBD4 protein expression was visibly reduced in CD4 ⁺ T cells in SLE subjects (0.90±0.21 vs 0.38±0.12, P<0.001; Figure 2B, 2E).

Figure 1. Relative mRNA expression levels of AID, TDG, and MBD4 in CD4 ⁺ T cells measured by real-time RT-PCR ( n=12).

A: Comparison of the AID mRNA expression levels in CD4 ⁺ T cells between SLE patients and healthy controls; B: Comparison of the TDG mRNA expression levels in CD4 ⁺ T cells between SLE patients and healthy controls; C: Comparison of the MBD4 mRNA expression levels in CD4 ⁺ T cells between SLE patients and healthy controls. AID: Activation-induced cytidine deaminase; TDG: Thymine DNA glycosylase; MBD4: Methyl-CpG-binding domain protein 4; SLE: Systemic lupus erythematosus.

Figure 2. Relative protein expression levels of AID, TDG, and MBD4 in CD4 ⁺ T cells measured by Western blotting ( n=12).

A, C: Comparison of the AID protein expression levels in CD4 ⁺ T cells between SLE patients and healthy controls; A, D: Comparison of the TDG protein expression levels in CD4 ⁺ T cells between SLE patients and healthy controls; B, E: Comparison of the MBD4 protein expression levels in CD4 ⁺ T cells between SLE patients and healthy controls. SLE: Systemic lupus erythematosus.

2.2. Correlations of the mRNA and protein levels of the BER with lupus disease activity

The mRNA and protein levels of AID were positively correlated with SLEDAI ( r=0.615, P=0.033; r=0.900, P=0.001, respectively; Figure 3A, 4A). No obvious correlation was found between the mRNA level of TDG and SLEDAI, while the protein expression of TDG was positively correlated with SLEDAI ( r=-0.124, P=0.702; r=0.642, P=0.024, respectively; Figure 3B, 4B). The mRNA and protein levels of MBD4 were negatively correlated with SLEDAI ( r=-0.713, P=0.009; r=-0.615, P=0.033, respectively; Figure 3C, 4C).

Figure 3. Correlations of the relative mRNA levels of AID, TDG, and MBD4 with SLEDAI.

A: Correlations between the relative mRNA levels of AID and SLEDAI; B: Correlations between the relative mRNA levels of TDG with SLEDAI; C: Correlations between the relative mRNA levels of MBD4 and SLEDAI. AID: Activation-induced cytidine deaminase; TDG: Thymine DNA glycosylase; MBD4: Methyl-CpG-binding domain protein 4.

Figure 4. Correlations of the relative protein levels of AID, TDG, and MBD4 with SLEDAI.

A: Correlations between the relative protein levels of AID and SLEDAI; B: Correlations between the relative protein levels of TDG with SLEDAI. C: Correlations between the relative protein levels of MBD4 and SLEDAI. AID: Activation-induced cytidine deaminase; TDG: Thymine DNA glycosylase; MBD4: Methyl-CpG-binding domain protein 4.

3. Discussion

In recent years, we have seen some interesting developments in the pathogenesis of SLE, including metabolic disorders, signaling, and biochemical defects in immune cells, and impaired repair of DNA damage ^{[ 13]}. Every day, each of our cells faces at least 10 ⁴ DNA damage, and their effective repair is an indispensable biological process to maintain cell survival and function ^{[ 14]}. The genomic integrity is maintained through a network of DNA damage responses and repair pathways, which is a comprehensive signaling process that can determine the capacity of cells to repair DNA damage, mutations or apoptosis ^{[ 15]}. The diversity of DNA damage types requires a variety of DNA repair pathways. Generally, the main DNA repair pathways remain conservative, containing nucleotide excision repair (NER), BER, mismatch repair (MMR), double-strand break repair (DSBR), and directly reverse multifarious forms of DNA damage ^{[ 16]}. DNA repair substantially removes DNA damage, cleavages, and base mismatches, and ultimately restores DNA to its original form ^{[ 16]}.

BER is a primary pathway in DNA repair of base damage caused by oxidation ^{[ 8]}. In the BER system, deamination of CpG by AID involves active DNA demethylation ^{[ 17]}. AID deaminates 5-methylcytosine (5-mC) to form U, which causes G/T mismatches ^{[ 18- 19]}. Both TDG and MBD4 are primary G꞉T glycosylases that excise the uracil and thymine generated by deamination and mispaired guanine ^{[ 20- 23]}. AID, TDG, and MBD4 all play crucial roles in DNA demethylation. In other research, it has also demonstrated that in contrast to healthy controls, the AID expression was obviously higher in PBMCs from SLE subjects ^{[ 24]}. Jiang, et al ^{[ 25- 26]} found that in contrast to AID wild-type mice, the levels of lupus pathogenic antibodies were visibly decreased, the progress of lupus nephritis was delayed in AID heterozygous MRL/lpr mice, and the lupus nephritis did not occur in AID-deficient MRL/lpr mice. Our study demonstrated that compared with healthy controls, the mRNA and protein expressions of AID were visibly elevated in SLE CD4 ⁺ T cells. The mRNA and protein expressions of AID were positively correlated with SLEDAI. These above results indicated that AID may take part in the pathogenic mechanism of SLE.

Currently, there is no relevant research showing the expression of TDG in SLE patients. The TDG gene is hypermethylated in the malignant plasma cell neoplasia multiple myeloma (MM) cell lines compared with normal human plasma cells, resulting in a lower TDG expression and a reduced DNA repair efficiency ^{[ 27]}. In the Human Protein Atlas database, the TDG protein nuclear expression in melanoma maintains a medium-to-high expression and high level is related to adverse outcomes. TDG plays crucial roles in DNA demethylation. Current research ^{[ 28]} showed several possible mechanisms may underlie active DNA demethylation. First, methylated CpG dinucleotides are directly removed by nucleotide excision repair pathway. Second, 5-mC is directly removed by BER pathway or 5-mC is deaminated by deaminase to form U, which causes G/T mismatches. Third, active DNA demethylation also involves oxidative demethylation. Ten-eleven translocation (TET) proteins can oxidize 5-mC to various forms of compounds, including 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-CaC). TDG can excise 5-fC and 5-CaC, which triggers subsequent BER ^{[ 29- 30]}. Researches ^{[ 7, 31]} in HEK293 T cells showed that Gadd45a can activate expression of methylated reporter genes by cooperating with TDG and TET proteins. Demethylation mechanisms in T cells in patients with SLE have always been hot topics in pathogenesis research of lupus erythematosus. Zhao, et al ^{[ 32]} noticed an elevated expression of 5-hmC in CD4 ⁺ T cells in patients with SLE combined with high level of the TET family proteins. Wu, et al ^{[ 33]} found that NaCl induces DNA hypomethylation and enhances hydroxymethylation levels with increased TET2 and TET3 expressions in CD4 ⁺ T cells in patients with SLE. Our study demonstrated that compared with healthy controls, the TDG protein expression in SLE CD4 ⁺ T cells was elevated and the relative protein level of TDG was positively correlated with SLEDAI. Based on the above results, TDG may participate in SLE pathogenic mechanism, and we need further study to explore whether it is involved in the autoimmunity via accelerating the demethylation process of T cells.

Through the study on MBD4, we found that in CD4 ⁺ T cells in patients with SLE, the MBD4 protein expression is visibly lower than healthy controls. Balada, et al ^{[ 34]} reported increased MBD4 mRNA level in CD4 ⁺ T cells in patients with SLE. However, other studies ^{[ 35- 36]} reported opposite results, indicating that, the MBD4 level is lower in CD4 ⁺ T cells in patients with SLE than that in healthy controls. The decreased MBD4 protein expression indicated that MBD4 is involved in DNA demethylation ^{[ 36]}. Our research also illustrated that the mRNA and protein levels of MBD4 are negatively correlated with SLEDAI. The above results indicated that the decreased MBD4 expression may take part in the pathogenic mechanism of SLE.

In our study, there was no significant difference between TDG and MBD4 mRNA expression of patients and controls, while the TDG and MBD4 proteins levels of patients were more and less than controls, respectively. There may be several reasons for this situation. First, gene expression is consisted of transcription and translation levels, that is, mRNA and protein levels. During the process of gene expression, there is a time and space gap between transcription and translation ^{[ 37]}. Second, after transcription, there will be post-transcription processing, translation, and posttrans-lational modification. Hence, the transcription level and translation level are not completely consistent ^{[ 37]}.

We investigate BER protein expression including AID, TDG, and MBD4 in CD4 ⁺ T cells in patients with SLE and analyse the correlations between BER proteins and lupus disease activity, but we did not study how these proteins participate in DNA demethylation mechanism. Further investigates would focus on whether BER proteins coordinate with Gadd45a to promote DNA demethylation in CD4 ⁺ T cells in patients with SLE.

Overall, the results showed that BER elements are probable in connection with SLE pathogenic mechanism. Our further study will focus on the demethylation mechanism through BER pathway in CD4 ⁺ T cells in patients with SLE.

Funding Statement

This work was supported by the National Natural Science Foundation of China (81472881).

Conflict of Interest

The authors declare that they have no conflicts of interest to disclose.

AUTHORS’CONTRIBUTIONS

ZHOU Xingyu Conducted the sample collection, cell separation, RNA isolation, RT-PCR, Western blotting and statistical analysis, drafted and revised the manuscript; WU Xiaoqi Conducted the sample collection, cell separation, RNA isolation, RT-PCR, and Western blotting; DENG Min, QIU Yueqi, and ZHOU Shengnan Helped with the sample collection; LI Yaping Designed the study, helped with statistical analyses, and revised the manuscript. All authors read and approved the final manuscript.

Note

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/2022121655.pdf