Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune condition characterized by inflammation of the joints and, in rare cases, other body parts (eyes, lungs, heart, blood vessels and skin).1 In RA, the immune system, which typically defends the body against infections, erroneously attacks the synovial membrane surrounding joints, thickening the synovium and destroying the joint's bone and cartilage. It usually affects the tiny joints in the feet and hands symmetrically, causing arthralgia, swelling, stiffness, and eventually joint degeneration and deformity if prolonged or untreated.2, 3 It predominately affects females aged 35 to 60 years compared to males in a ratio 4:1.4 RA above 65 years is known as elderly onset rheumatoid arthritis (EORA). As age progresses, the ratio of females to males also decreases to 2:1.5 The incidence and prevalence of RA vary based on geographical region, age and sex. The prevalence of RA was 208 cases per 100,000 people worldwide in 2020, accounting for 17.6 million cases.6 In India, the prevalence rate ranged from 0.28 to 0.7%, accounting for 13 million cases.7
Rheumatoid arthritis has a complicated aetiology, with several environmental, immunological, genetic and other factors (age, sex) influencing disease progression and gene expression. Long before the onset of clinical symptoms, genetic (MHC shared epitope alleles) and environmental factors (smoking, high sodium intake) might interact to cause adaptive responses associated with autoimmunity and induce the production of peptidyl arginine deaminase (PAD).8, 9 PAD enzyme modifies proteins post-translationally by converting arginine to citrulline, thereby changing the structure and function of the modified protein. The antigen-presenting cells process these modified citrullinated proteins presenting as foreign antigens to T-cells, thereby stimulating antibody or autoantibody or anti-cyclic citrullinated peptide antibodies (ACPA) production.10 Initially, immune cells are stimulated in the regions distant to synovial fluid and ACPA levels can be detected in the serum much before the onset of RA symptoms.11, 12 Over time, few individuals will ultimately develop immune-mediated inflammation primarily localized in the synovium. Within the synovial compartment, cytokines and chemokines (GM-CSF, IL-6, TNF) activate endothelial cells and attract immune cells, both innate (monocytes, mast and dendritic cells) as well as adaptative cells (Th-1, Th-17, B cells and plasma cells) into the compartment and activate fibroblast-like synovial cells (FLS). Proliferating in the synovium, cytokines are a major contributor to the severe inflammatory reaction that leads to cartilage loss and bone degradation. Progressive joint deterioration is caused by the migration of FLS from one joint to another.13, 14, 15
RA disease progression has been linked to its heterogeneity. Three types of synovial tissues-one with T cells, B cells, APC and MHC genes, second with stromal genes and third with mixed with both genes were observed.16 Gene profiling studies showed clear distinctions in gene expression between early RA (<12 months) and late RA (>5 years) in synovial tissue, indicating the existence of various pathophysiological mechanisms throughout the disease course.17, 18 Using microchip analysis, distinct molecular markers and biological processes, including host defences, stress responses, T-cell-mediated immunity, MHC class II-mediated immunity, stress responses, host defences and tumour suppressors, and MHC class II-mediated immunity were observed at different stages of the disease.19
Despite documented RNA heterogeneity, the application of treat-to-target techniques in RA is poor, with most studies showing increased treatments in almost 50% of patients diagnosed with moderate or high RA.20, 21 To comprehend the effectiveness of treatment and the course of the disease, it is essential to look into how interventions affect the expression of certain genes in patients with rheumatoid arthritis (RA). These interventions frequently cover a wide range of treatments, from new biologics or developing targeted medicines to more conventional meds like disease-modifying ones. The present systematic review aims to evaluate the effect of interventions on gene expression in patients with rheumatoid arthritis.
Materials and Methods
Literature search
Using PRISMA- systematic review and meta-analysis guidelines, the present study search to evaluate the effectiveness of interventions on gene expression in rheumatoid arthritis patients was conducted.22 The inclusion criteria for screening the research articles include randomized and observational studies to understand the association between interventions (medications) and gene expression in RA patients. The exclusive criteria include non-randomized studies, uncontrolled studies, laboratory studies and case reports. Literature searches are done only in English databases such as PubMed, Embase, and Google Scholar. Search was confined to a time period from 1995 to 2020. The main keywords for PubMed are rheumatoid arthritis, gene expression, gene profiling and interventions or therapeutic treatments. The pertinent medical subject headings (MeSH) used are (Arthritis, Rheumatoid"[Mesh] AND ("Gene Expression"[Mesh] OR "Gene Expression Profiling"[Mesh] AND "Interventions"[Mesh] OR "Medications"[Mesh] OR "Therapeutics"[Mesh]). To find other related studies, we thoroughly reviewed our options, selected the study with larger sample size or the most recent publication for our research samples, and then looked further into the publications' references to find comparable studies.
Selection and screening
The screening approach consists of research articles performed separately by two researchers. Research documents were screened in the first step based on the title and abstract. Based on the inclusion and exclusion criteria, the selected research documents were thoroughly reviewed and articles not meeting the inclusion criteria were excluded from the study. The research articles were excluded if the study was about rheumatoid arthritis gene profiling for diagnostics or rheumatoid arthritis therapeutics not targeted towards gene expression. Finally, the researcher independently retrieved pertinent data from the included studies using a pre-designed data-collecting form. Any differences in the data collected were sorted out through discussion. The primary contents of the data collection form include the title of the research article, author name, year of publication, study design, indication, inclusion and exclusion criteria of the selected article, sample size, study population, methodology, gene expression analysis methodology, expressions analyzed, targeted interventions, outcomes and conclusion of the study.
ROB analysis
For randomized studies, the ROB were assessed using JBI critical appraisal tools.23 The subsequent queries were addressed to evaluate the bias.
Was proper randomization employed for allocating patients to therapeutic groups?
Were the treatment group blinded from allocation?
At the initial stages, were the treatment groups comparable?
Were participants concealed of their treatment regime?
Were physicians or nurses administering treatment blinded of the treatment assignment?
Did outcome assessors concealed from the treatment assignment?
Apart from the relevant intervention, were the treatment groups treated similarly?
Was follow-up done to the end, and if not, were the variations in follow-up between the groups sufficiently explained and examined?
Were the patients or participants assigned randomly to a group examined?
Were the treatment groups' outcomes evaluated identically?
Were results measured accurately?
Was relevant statistical analysis performed?
Did the study's conduct and analysis consider any modifications from the conventional RCT design, such as individual randomization and parallel groups, and was the trial design appropriate?
The overall rating for each question was rated as Yes (implies high quality), No (indicates quality or particular criteria not fulfilled) and Unclear (suggests particular criteria in the selected paper not accurate) (Table 1 Suppl).
Results
Literature search and study characteristics
A summary of the systemic review search strategy with inclusion and exclusion articles was presented in a flowchart (Figure 1). From English databases, 1970 studies were retrieved, 40 duplicates were discarded. After the initial article title and abstract screening, 1890 were excluded. Further, in the full-text analysis, 32 non-relevant articles were excluded, and 8 research articles were selected for the final study. All the selected studies were randomized controlled studies.24, 25, 26, 27, 28, 29, 30, 31 Among these, one of the study was a randomized controlled non-placebo study,25 4 were randomized double-blinded placebo-controlled trials,26, 29, 30, 31 1 study was stratified randomized27 and 1 was randomized open-label study.28 The cumulative sample size of the present study was 557. Table 2 shows the study characteristics of the selected studies.
Gene expression analysis analytical techniques
Molecular techniques commonly used in the selected studies were quantitative reverse transcriptase PCR (qRT-PCR) and microarray techniques. 25, 26, 27, 28, 29, 31 Immunohistochemical analysis was conducted by.26, 30 Quantitative cytokine analysis was performed using integrated optical density.30 Anti-TNF blockage was quantitively analyzed using dynamic contrast MRI.29 Whole blood or peripheral blood mononuclear cells were commonly used to analysize the gene expression 25, 26, 27, 28, 29, 30, 31 whereas 2 studies used synovial tissue for their studies.24, 25, 26
Interventions and their outcomes
Traditional herbs like Ginger (1500 mg/daily) were supplemented to active RA patients to study immunity factors and intermediate gene expression. After 12 weeks of intervention, an increase in transcription factor (FoxP3; GATA3) and antiinflammatory factors PPAR-γ whereas a decrease in inflammatory factors such as T-bet, RORγt and NFκB was observed.31 Methotrexate was able to decrease the expression of pro-inflammatory cytokines (IL-12A; IL-6)27 and in combination with prednisone was significantly able to reduce serum TNF-α levels in active RA patients.25 Similarly, infliximab therapy showed respondents had high Ktrans and low B-cell expression compared to non-respondents. However, a study showed that only anti-TNFα therapies like infliximab and MTX were insufficient in rheumatoid arthritis treatment.29 Also, only anti-CCR2 antibodies targeting chemokine CCR2 receptor present on monocytes and T-subset cells were found not to decrease synovial inflammation in active RA26 whereas rituximab (RTX), a chimeric monoclonal anti-CD20 antibody targeting CD-20 found the B-cell surface was found to downregulate B-cell gene expression. RTX can also used in diagnostic differentiation between responders and non-responders based on the signature of upregulation of NF-kB genes and downregulation of interferon genes.28 Mavrilimum, a human monoclonal antibody targeting the α-subunit of GM-CSF receptor, was reported to decrease the T-cell and myeloid gene expression and indirectly suppress IL-2 α receptor and IL-17/IL-22mRNA expression.30 Lastly, gold therapy singly or combined with methylprednisolone acetate decreased endothelial leukocyte adhesion molecule 1 expression levels and synovial blood vessels (Table 3).24
Table 1
Suplementary ROB analysis of the included studies
Table 2
Study characteristics of the present study
Table 3
Interventions and outcomes of the included 8 studies
ROB analysis
Randomized studies RoB analyses showed that the included research publications had sufficient qualitative standards. Nevertheless, two studies' participants or physicians or outcome analyzers were not blinded to the treatment28, 30 and two studies were unclear in the blinding strategy applied.24, 31
Discussion
The molecular fingerprints underpinning human disease are represented by gene expression profiles, which can predict an outcome with the highest likelihood of success. Due to the heterogeneous nature of rheumatoid arthritis, research into putative RA biomarker genes may lead to the discovery of new therapeutic targets for rheumatoid arthritis susceptibility.33 The present study has revealed that a range of therapy modalities, such as biological and targeted therapies and disease-modifying anti-rheumatic medications (DMARDs), might affect the expression of specific genes in patients with active RA and also help in the differentiation of respondents and non-respondents to the specific therapy.
DMARDs agreement and disagreement with other studies
Cost-effective DMARDs such as methotrexate singly or in combination with prednisone were able to decrease serum TNF alpha levels as well as decrease the IL6 and IL-12A mRNA expression without any use of corticosteroids.25, 27 Although MTX is still used as the first line of therapy for RA treatment, MTX therapy alone is not sufficient to treat active RA. Studies showed in due course, the MTX therapy was discontinued due to secondary infections, gastrointestinal toxicity and hepatotoxicity as it also suppresses immunoglobulin production.34 Also, studies could not identify any biomarkers or prediction models that could have predicted the response of MTX based just on baseline measures.35
Biological and targeted therapy agreement and disagreement with other studies
Biological therapy-anti CCR2 antibodies are insufficient for the active RA treatment due to low expression and redundancy of the CCR2 chemokine receptor. In addition to CCR2, monocytes synthesize other CCR chemokine receptors (CCR1, CCR5); thus, CCR2 blockage can be bypassed by the activity of other receptors. Combinational antibody therapy for all chemokine receptors must be developed for effective treatment.26, 36 Previous studies on anti-TNF α using infliximab were not consistent as each reported a different set of gene expressions either due to differences in techniques used like microarray, q-RT PCR or sample selected, thus making it difficult to distinguish between responders and non-responders.37 A study conducted by MacIsaac et al., 2014 employed dynamic contrast enhance MRI to obtained a pre-treatment whole blood gene expression signature, which was correlated with a shift in the imaging biomarker Ktrans. The signature was linked to improved Ktrans in patients receiving infliximab but a decline in Ktrans in the placebo group.29 However, further studies are required using Ktrans as an end biomarker in identifying respondents and non-respondents to anti-TNF alpha therapy. GM-CSF blockage therapy using the monoclonal antibody mavrilimumab was effective in RA treatment by suppressing the chemokine and interleukin genes involved in inflammation.30 No adverse effect was detected, and clinical studies are going on in the commercialization of mavrilimumab treatment.38
Herbal therapy
Ginger was found to decrease the expression of inflammatory genes (T-bet and RORγt genes) and upregulation of antiinflammatory genes (FoxP3, PPAR-γ). Also, the DAS-28 score decreased significantly on 12 weeks of therapy.31 Herbal medicines can serve as a substitute for allopathic pharmaceuticals in the treatment of RA patients, providing an alternate means of addressing the limitations of current therapeutic approaches. However, Ginger should be used cautiously by those on anticoagulants. There were currently no suggested safe and efficient dosages available.
Conclusion
Targeted interventions can significantly impact gene expression in active RA patients and help in early diagnosis and prognosis of RA disease. The present study makes it rather evident that gene expression profiling has a lot of promise for comprehending the biology of RA. Nevertheless, individual variation in treatment responses and gene expression profiles highlights the complexity of RA, prompting continuing study to elucidate the precise effects of interventions on gene expression and generate more personalized and successful therapeutic options.
