Introduction

COVID-19 which is caused by SARS-CoV-2 is a very common cause of an excessive pro-inflammatory response. The Similarity between cytokine hyperactivation in COVID-19 and in rheumatoid arthritis (RA), a chronic autoimmune disease was reported. RA is characterized by T cell hyperactivation and synovial inflammation ( 1 ). Many viruses, including respiratory viruses, are associated with arthritis-like symptoms and joint pain. Studies showed that COVID-19 results in muscle and joint pain in 44% of infected individuals ( 2 ).

Furthermore, individuals with inflammatory arthritis may experience a relapse of symptoms due to infections ( 3 ). Consequently, the development of RA or its attacks may be triggered/enabled by SARS-CoV-2 infection. Limited data is currently available and it is not yet proven that SARS-CoV-2 infection and the development of RA are directly related ( 4 ). When SARS-CoV-2 infection occurs, it could lead to increased release of inflammatory mediators such as C-X-C motif chemokine ligand 10 (CXCL10), interleukin (IL)-17, and tumor necrosis factor-alpha (TNF-α). These mediators are implicated in initiating osteoclast genesis and reducing osteoblast differentiation and proliferation, which ultimately decreases bone density ( 5 ). In RA patients, insufficient expression of CTLA-4 in T reg cells due to the methylation of the CTLA-4 promotor's DNA at the NF-AT binding site occurs. Such an event makes Treg cells unable to activate the kynurenine pathway, which triggers the development of RA ( 6 ).

While SARS-CoV-2 vaccines are generally safe for healthy individuals, a small percentage of people may experience rare side effects, including autoimmune disorders ( 7 ). Vaccines primarily aim to prevent infection, with clinical trials proving their safety and efficacy. However, real-life data have reported adverse effects, possibly due to short study durations and the urgent need to start immunizations. Reports showed that immune-mediated disease attacks or new inflammatory diseases post-vaccination ( 8 ) . Some reports listed the development of autoimmune disorders after SARS-CoV2 vaccination, including autoimmune glomerulonephritis ( 9 ), autoimmune rheumatic diseases ( 7 ), and autoimmune hepatitis ( 10 ). So, the aim of the study was to study the potential role of SARS-COV-2 infection in the progression of RA by evaluating CTLA-4 gene expression in rheumatoid arthritis patients.

Materials and Methods

Sample Collection: Blood samples (2 ml) were obtained from 61 diagnosed rheumatoid arthritis patients and 40 control subjects (pre-infection without developing RA) at the Biological Pharmaceutical Unit of Al-Basrah Teaching Hospital between September 2023 and December 2023. The patients were diagnosed by the physician (arthrologist) based on clinical features mainly warm tender joints. Laboratory diagnosis was further supported by the clinical part including: Rheumatoid factor (RF), Anti-citrullinated Peptide Antibody (ACCP), and Erythrocyte Sedimentation Rate (ESR).

RNA extraction and cDNA synthesis: Using the Solarbio (Cat No. R1200, China) following the manufacturer's instructions, total RNA was isolated from whole blood. According to the manufacturer's instructions, cDNA was then synthesized from total RNA using the Universal RT-PCR Kit (M-MLV, free Taq polymerase) purchased from Salorbio, Cat No: RP1105, China.

Real Time PCR for Genes: The specific primers of CTLA-4 For.5′-CTACCTGGGCATAGGCAACG-3′ Rev.5′-CCCCGAACTAACTGCTGCAA-3′ ( 11 ) and GAPDH For.5′-CTTTTGCAGACCACAGTCCATG-3′ Rev. 5′-TTTTCTAGACGGCAGGTCAGG-3′( 12 ) were used. The kit from SolGent Co., Ltd. (cat. No. 34014, Korea) was employed to prepare the Real Time PCR experiment. SYBER green master mix (10 µl), Forward and reveres primers (100 pmol), nuclease-free water (5 µl), and cDNA template (7 ng/µl) were mixed to prepare the PCR reaction mixture. It was first denatured for 10 minutes at 95 °C, followed by 35 cycles of denaturing for 10 seconds at 95 °C, annealing for 30 seconds at 60 °C, extension for 30 seconds at 72 °C, and a final extension step of 5 minutes at 72 °C.

Statistical Analysis : The t-test (two sample unequal variance) was used to statistically analyze the data at P ≤0.05.

Results

CTLA4 Gene expression analysis

Due to the importance of CTLA4 gene in the immune response and the development of immune diseases, CTLA4 gene expression in both RA positive and negative samples was quantified using qPCR relative assay with SYBR green master mix After subtracting the signal of GAPDH as the housekeeping gene, CTLA4 expression was higher in RAI samples than in the control sample while lower in the RAV samples compared with control (A). After normalizing the control sample to 1 it was shown that the CTLA4 expression was reduced to about 25% in RAV group while the RAI expressed the gene to about 0.7 compared to C group. Figure (B).

Figure (1).Gene expression of CTLA-4 in RAV and RAI compared to the control group. Gene expression was measured using RT-PCR. The t-test was used to determine the statistical significance, showing * P = 0.045 that the difference is statistically (P > 0.01).

Discussion

One of the most important factors that can cause a failure in the T-cell function is the expression of the CTLA-4 by Tregs which transduces inhibitory signals and maintains an immune response homeostasis. CTLA-4 haplotype absence could cause autoimmune diseases, multiorgan invasion by lymphocytes as well as peripheral B cells, and immunoglobulin deficiency ( 13 , 14 ). It was also found that Single‐nucleotide polymorphisms in CTLA‐4 have been correlated with some autoimmune diseases including, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and type 2 diabetes ( 15 , 16 , 17 ). The results found upregulated gene expression in RAI samples compared to the control. The results are well supported by the conclusion of Jury et al., that high levels of CTLA-4 in FOXP3- T cells from patients with systemic lupus erythematosus (SLE) compared to healthy people. The suggested mechanism is that FOXP3- T cells in (SLE) patients are unable to control the activation and proliferation of effector T cells ( 18 ). Furthermore, the literature supports that CTLA-4 gene is significantly highly expressed in SLE patients compared to controls ( 19 ). A study was conducted in Egypt on Type 1 diabetes (T1D) patients and compared with controls they found a significant increase in CTLA-4 (+49 A/G), especially in younger people and females ( 20 ). A possible mechanism that could be involved in developing RA could be the decrease in Mitogen-activated protein kinase (MAPK) phosphorylation and an increase in Small mothers against decapentaplegic 2/3 (Smad2/3) nuclear localization. Overexpression of the cytoplasmic domain of CTLA-4 (cdCTLA 4) transforms naive T-cell preferred to Foxp3+ T cells under Th17 differentiation circumstances ( 21 ).

However, the results disagree with Liu et al who found that CTLA-4 is low in severe aplastic anemia (SAA) patients ( 22 ). Lower levels of sCTLA-4 are present in RA patients in the quiescent stage of the disease compared to those in the activation stage. However, this study differs in age categories, severity of the disease, and time of diagnosis ( 23 ).

On the other hand, CTLA4 gene expression was low in RAV samples compared with control which open the door for evaluating the exact mechanisms that new fashion vaccination is working through. However, an increasing body of evidences reported adverse effects of the vaccination such as myocarditis, and pericarditis ( 24 ).

Conclusion

COVID-19 seems to have an important role in the development of the RA disease, as during its occurrence immuno-change disorders can happen. This could be not very surprising since evidences correlated different viral infections with joint pain and inflammation. The study provided evidence about the mechanism underlying the development of RA by assessing the relative and fold change expression, one of the obvious influences of which is the significant changes in the gene expression of CTLA-4 gene, which in turn could be involved in the development of the RA. The increased expression of CTLA-4 was reduced by vaccination which could explain the importance of vaccination to lessen the adverse effects of SARS-CoV2 infection.

Acknowledgment

I acknowledge all members of the Biological Pharmaceutical Unit at Al-Basrah Teaching Hospital, and the Central Laboratory, College of Medicine, University of Basrah

Consent for publication

Each patient signed a form has the following statement: I understand that my participation is voluntary and that I am free to withdraw at any time, without giving a reason and without cost. I understand that I will be given a copy of this consent form.

Limitations

It is not easy to generalize the results of this study due to the small sample size. The mechanisms underlying the observed gene expression changes require further investigation. Finally, the study did not explore the potential confounding factors such as comorbidities or genetic variability among patients.

Conflicts of interest

The authors declare that there is no conflict of interest.

Ethical Clearance

This work is approved by The Research Ethical Committee.

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