Rheumatoid arthritis (RA) is a chronic, progressive, and inflammatory autoimmune disease, which continues to cause global disability having a worldwide prevalence of 1% (1). The disease is classified primarily by clinical phenotype and predominantly associated with articular, extra-articular, and systemic complications including anemia, cardiovascular diseases, osteoporosis, fatigue, and depression (2, 3). There exists a considerable geographical and temporal variability as well as unpredictability based on severity and manifestations in the occurrence of RA within populations (4). RA is characterized by synovial inflammation [excessive infiltration and activation of neutrophils, mononuclear cells including T cells, B cells, plasma cells, and mast cells (5), as well as complement factors such as C5a at rheumatoid joints (6)], autoantibody generation (rheumatoid factor and anti-citrullinated protein antibody) (3) and degradation of bones and cartilage leading to bone deformity (5). The etiology or cause of RA remains elusive. However, various studies have illustrated a convoluted interplay between genetic factors and environmental exposures in most of the disease cases (3, 7). Unprecedented efforts have been made to identify multitude of factors that have foremost allusions in RA pathogenesis including pro-inflammatory cytokines (TNF-α, IL-6, IL-17, and IL-1) (8) as the orchestrator of vital processes during RA progression involving synovitis (9), angiogenesis (production of pro- angiogenic factors particularly VEGF) (10, 11), osteoclastogenesis (increased production of macrophage colony-stimulating factor and RANK ligand) (12), cartilage degradation (enhanced secretion of MMPs into synovial fluid at the joints) (9, 13), and acute-phase proteins production such as CRP (exacerbates RA-related tissue damage) (14).

On the genetic front, the strongest link in RA has been ascribed to the third hypervariable region of the HLA-DRβ chains (aa. 70–74) within MHC. The active epitope within the area, glutamine–leucine–arginine–alanine–alanine (QKRAA) or QRRAA has been found to be associated with multiple RA allied DR genes including DR1, DR4, and DR14 (15). The location of the epitope within HLA-DR determines the specificity of the peptides presented to CD4⁺ T-cells. However, the specific peptides that bind to DR proteins in RA patients have not yet been identified (6). The abundance of effector T cells and IL-17-producing Th17 cells in the synovial milieu emphasize the critical role of these cells in RA pathogenesis. The activation of T-cells through HLA-DR4 (present on APCs) mediated presentation of specific peptides during RA initiates an array of complex immune responses including T-cell oligoclonality, germinal center reactions, and B-cell hypermutation highlighting local antigen-specific T-cell-mediated B-cell responses (3). The appropriate interconversion between Th1/2/17/Treg phenotypes has crucial implication toward RA outcome (16). In particular, an imbalance between Th17 and Treg cells has an impact on local TNF levels in the synovial fluid that blocks the differentiation of Treg cells and along with IL-17, macrophage, and dendritic cells derived TGF-β and IL-1β, shifts the T-cell homeostasis toward inflammation (17). The importance of B-cells in RA have been highlighted in autoantigen presentation and cytokine production (IL-6, TNF, and lymphotoxin-β) (3). Apart from acting as the precursor for autoantibody-generating plasma cells, B-cells are the only antigen-presenting cells supporting the activation of autoreactive T-cells during RA (18).

Although several factors have been identified, yet there is a dearth of knowledge about the complex gene networks associated with the disease. Over the last two decades, DNA microarray technology allowed us to interrogate thousands of genes simultaneously enrooting the discovery of disease-relevant genes. Multiple microarray experiments have been conducted to identify potential gene signatures that are responsible for the pathogenesis of various diseases. The analyses of these experiments usually generate hundreds of differentially expressed genes (DEGs), making it difficult to generate a convincing transcriptome profile of a particular disease condition. To address this issue, we implemented a meta-analysis method to extract a global transcriptional profile of RA patients available on public databases. Our gene expression meta-analysis revealed some credible gene signatures that have shown relevance to RA condition. Finally, we went a step further to validate these gene signatures in RA patient samples obtained clinically. In conclusion, this meta-analysis approach of publicly available microarray data suggests us to generate a significant gene expression profile that might represent an early step toward enhancement in the detection, treatment, and prevention of RA.

The etiology of RA like most of the immune-mediated disorders is multifactorial, which is influenced by genetic, environmental, hormonal, and immunological factors. However, genes, gene networks associated with RA onset and progression are not clearly defined. Our meta-analysis of RA gene expression data identified a robust meta-gene profile comprising 33 DEGs, which showed a consistent and significant up-or downregulation across all the data sets. To further understand the significance of these meta-genes in RA pathogenesis, we performed a GO functional and pathway enrichment analysis, which led to the identification of novel gene signatures that went unnoticed in previous RA studies. These genes include PLCG2, HLA-DOB, HLA-F, EIF4E2, and CYFIP2, which have been previously implicated in inflammation, antigen presentation, hypoxia, and apoptosis. Interestingly, expressions of these genes were also found to be significantly upregulated in PBMCs of clinical RA patients. It has been well established that the severity of RA is majorly dictated by the production of an array of pro-inflammatory cytokines in the synovial joint particularly cytokines like TNF and IL-1β, which triggers multiple cellular and immunological interactions as a result of excessive inflammation. Although biological therapies against TNF and other pro-inflammatory cytokines offer momentary clinical benefits, they are often associated with severe side effects (60). Therefore, it is decisive to identify and quantify combination of other genes, which might be helpful in controlling the massive inflammatory responses occurring during RA progression. Our meta-analysis study exemplifies the involvement of few inflammatory genes (PLCG2, AIM2, and ALOX-5), which might act as novel candidates for controlling inflammation during the disease condition (Figure 2B). Additionally, we have identified a previously unnoticed inflammatory gene, PLCG2 (phospholipase C-gamma 2), which has been found to be upregulated in all the microarray data sets as well in the clinical RA patients (Figures 2B and 3A). PLCG2, a transmembrane signaling enzyme, catalyzes the conversion of membrane phospholipid, phosphatidylinositol 4,5 bisphosphate into second messenger molecules inositol trisphosphate (IP3) and diacylglycerol (DAG) using calcium as the cofactor, thereby activating NLRP3 inflammasome. A gain of function mutation in PLCG2 gene is associated with phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID), dominant inherited disorder, and auto-inflammation (61), thereby suggesting that PLCG2 might be a potential therapeutic target for controlling inflammation during RA pathogenesis. In consent with previous studies that have highlighted the role of AIM2 (absent in melanoma 2) and ALOX-5 (arachidonate 5-lipoxygenase) genes in RA pathogenesis, our study also demonstrated the enhanced expression of these genes in RA patients as compared to HCs (Figures 3B,C). AIM2 is known to induce the formation of caspase-1-activating inflammasome, thereby controlling the proteolytic maturation of pro-inflammatory cytokines IL-1β and IL-18 (62). Conditional deletion of AIM2 in RA mice model shows reduced inflammatory responses suggesting the role of AIM2 in controlling inflammation during RA (62). ALOX-5 is expressed basically in mature myeloid cells and developing/memory B-lymphocytes and is responsible for the generation of excessive leukotrienes (LTB4, LTC4, LTD4, and LTE4) in the synovial fluid of RA patients (63). Inhibitors of ALOX5 gene dampen TNF-α-induced NF-κβ, thereby abrogating synovial inflammation (63). ALOX5 in coordination with 5-LO-activating protein (FLAP) leads to the synthesis of LTA4, one of the critical intermediates of leukotrienes synthesis pathway (64). The long association of human leukocytic antigen (HLA) is confirmed in patients positive for rheumatoid factor. Appropriate antigen presentation or alteration in peptide affinity play a crucial role in promoting autoreactive adaptive immune responses (3). Intriguingly, our study for the first time has documented gene signatures HLA-DOB and HLA-F, which were found to be upregulated across all RA patient’s microarray data sets as well as in RA patients’ PBMCs (Figures 3D,E). HLA-DOB, a B-cell lineage, MHC-II-related molecule has been reported to have strong immunogenicity for human T-cells, one of its identified CTL epitopes HLA-DOB_232–240 acts as a resilient immunotherapeutic candidate for targeting multiple myeloma (65). HLA-F, a non-classical MHC-I molecule, is currently enigmatic of all the HLA molecules; hence, its precise function remains elusive. Recent studies have demonstrated enhanced HLA-F expression in cancer stroma in the case of both breast and gastric cancers (66). HLA-F on B-cells was found to induce immune tolerance in tumor cells by interacting with inhibitory receptor ILT-2 and ILT-4 of the natural killer or CTLs, thereby blocking their cytotoxicity for the tumor cells (67). These findings categorically depict the immunological relevance of HLA gene signatures in multiple cancers; however, their abundance in RA patients and correlation with RA onset and progression needs to be completely deciphered.

Angiogenesis is one of the critical events in the perpetuation of RA (68). One of the important signals required for triggering angiogenesis during RA is hypoxia, which is created majorly as a result of synovial hyperplasia (10). Our meta-analysis study for the first time has identified EIF4E2 gene (eukaryotic initiation factor 4E2) encoding eIF4E2 protein, an mRNA cap binding homolog of eIF4E, which was found to be upregulated across all RA microarray data sets considered in our meta-analysis study as well as through RT-qPCR in RA patients (Figure 3F). eIF4E2 was found to be an inhibitor of translation in normal cells (69), while it binds with HIF-2α-RBM4 complex and drives the translation of proteins under hypoxic (low oxygen tension) conditions. Cancer cells exploit the eIF4E2-mediated protein synthesis to sustain hypoxic conditions and consequently grow to significant sizes (70). Perhaps, eIF4E2 might play a critical role in driving hypoxia-induced angiogenesis during RA, hence can act as a valuable pharmacological candidate for controlling RA. Further, our study also depicts the involvement of previously unnoticed genes such as protein kinase C-β, PRKCB (a negative regulator of BCR signaling) and cytoplasmic FMR1-interacting protein 2, CYFIP2 (inducer of p53 mediated apoptosis), which were also upregulated in all data sets and clinical RA patients (Figures 3G,H). CYFIP2 gene has a p53 responsive element that confers binding of p53 as well as the activation of some heterologous reporter. Inducible expression of CYFIP2 is responsible for caspase activation and cellular apoptosis (71). Although these novel immune signatures are abundantly expressed in RA patients, which categorically suggests the involvement of these gene/pathways in RA pathogenesis, still their functional relevance in terms of gene expression and disease outcome need to be deciphered. Nevertheless, identification of these novel gene signatures might provide a unique prospective to define novel molecular diagnostic candidates and ascertain potential pharmacological targets for the development of therapeutic interventions against RA.

All authors were involved in drafting the article or revising it critically for important intellectual content. All the authors read and approved the final version of the manuscript to be published. NK had full access to all of the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. NK, SA, and JG participated in study conception and design. SA and JG were involved in the acquisition of data. SV and SN were responsible for recruitment and clinical evaluation of RA patients. NK, SA, and JG participated in analysis and interpretation of data and manuscript writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.