The pathogenesis of RA is centered on inflammation and immune response. One of RA’s main clinical features is chronic synovitis, which progresses to cartilage and bone damage. Therefore, targeted analysis of these key genes, pathway molecules and immune infiltrations that are altered in disease will facilitate the discovery of valid and reliable biomarkers for early diagnosis, targeted personalized therapy and delayed disease progression, as well as facilitate the shift from a single-parameter model to a multi-parameter systemic model of traditional medical concepts[48, 49]. In this study, four knee synovial gene microarray expression data were downloaded from the GEO database. We mined these data to derive differentially expressed genes between RA synovial tissue and healthy controls. We found that 275 genes were significantly up-regulated, and 163 genes were significantly down-regulated among these differentially expressed genes. We expect that the hub genes, molecular pathways, and immune infiltration of inflammatory immune cells derived from the bioinformatics analysis of these differentially expressed genes will provide novel ideas and concepts for RA’s diagnosis and treatment. The results of this study are summarized below.
Analysis of molecular pathways that play a diagnostic and therapeutic role in RA
Through KEGG and GSEA analysis, The molecular pathways in the knee synovial membrane of RA have been primarily involved in the biological processes of chemotaxis, adhesion, proliferation, differentiation, activation, and regulation of inflammatory immune cells leukocytes, T cells, and monocytes. Such results also confirm that RA is a biological process with an intricate regulatory network involving various inflammatory immune cytokine interactions. Furthermore, signaling pathways such as NF-κB, FoxO, Toll-like receptors, PI3K, TGF-beta, MAPK, PPAR, AGE-RAGE, and cGMP-PKG are also involved.
The NF-κB pathway is activated and up-regulated in the synovial tissue of patients with RA. Using specific antibodies that bind to IL-1 and IL-17, blocking the NF-κB signaling pathway reduces serum inflammatory cytokine levels and decreases the expression of IL-1, IL-2, IL-6, IL-17, TNF-a, interferon-γ, and matrix metalloproteinase-3. Increasing the expression of IL-10 inhibits bone destruction, reduces histological damage, decreases the severity of arthritis, and has a therapeutic effect on RA[50, 51].
During RA, the FoxO signaling pathway is down-regulated. One experiment found that in RA patients’ peripheral blood, the expression of FoxO1 mRNA was significantly reduced. FoxO1 overexpression has induced apoptosis of RA synovial cells. Expression levels have been negatively correlated with the disease’s activity, which is consistent with our results[52].
A suitable blockade of Toll-like receptor (TLR) activation has been reported to have an inhibitory effect on RA development during disease progression[53, 54]. The TLR4 inhibitor TAK-242, which inhibits IL-6, MMP-1, and VEGF expression, reduces serum IL-6 and VEGF and has an inhibitory effect on the NF-κB pathway. The inflammatory status of joint tissues has significantly improved, thus controlling RA progression[55].
It has been shown that transforming growth factor (TGF) in synovial tissue maintains the knee joint’s normal physiological function, that increased expression of TGF in RA synovium may lead to abnormal synovial growth. That inhibition of the TGF signaling pathway reduces the survival and migration of synovial fibroblasts and attenuates pulmonary fibrosis in RA rats[56]. Betulinic acid (BA) inhibited the migration and invasion of RA synovial cells, suppressed vascular endothelial growth factor transcription and TGF. It also inhibited NF-3B inflammatory pathway-related protein expression, decreased IL-1 and IL-6 inflammatory mediator levels, and reduced RA symptoms in rats[57].
The PI3K/AKT signaling pathway is activated and up-regulated in RA. Shikonin(SKN) down-regulates PI3K and Akt’s expression, inhibiting the phosphorylation and gene-level signaling molecules such as ERK1/2, JNK1/2, and p38, and exerts anti-RA angiogenic effects[58]. Cinnamaldehyde (CA) can block PI3K/AKT signaling pathway and inhibit the proliferation and infiltration of rheumatoid synovial cells, which has potential therapeutic effects on RA[59]. Berberine inhibits inflammatory proliferation of synovial cells, suppresses DC activation, regulates Th17/Treg balance, and prevents cartilage and bone destruction by regulating various signaling pathways involved in inflammatory responses, including PI3K/Akt. Such molecular targets may explore new RA treatment targets[60].
In RA synovial tissue, MAPK signaling pathway activation expression is increased. Its activation plays a regulatory role in differentiating monocytes and cell survival. In mice, members of the receptor families PAQR11, progesterone, and AdipoQ regulate in vitro monocyte and macrophage differentiation. In vivo Paqr11 knockdown deletion inhibits monocyte differentiation, encourages cell survival, and delays RA progression[61]. Silencing of lncRNA para nuclear assembly transcript 1 promotes miR-129 and miR-204, inhibits the MAPK signaling pathway. Lipoxin A4 (LXA4), a MAPK signaling pathway p38 inhibitor, decreased p38 expression in mouse synovial tissues, reduced proliferation of synovial tissue and inflammatory cell infiltration, reduced proliferation of RA severity, and produced a protective effect in arthritic mice[62].
In various diseases, peroxisome proliferator-activated receptors (PPAR) are involved in normal physiological and pathological processes. Expression of PPAR is significantly lower than normal in RA synovial tissues. Down-regulation of PPAR promotes the expression of Cyclin D1, MMP1, and MMP9, and PPAR up-regulation may induce Wnt/β-catenin signaling activation, which plays a vital role in the synovial cells of RA. One study found that pioglitazone, a receptor agonist activated by peroxisome proliferators, promotes vasoprotective and anti-inflammatory effects in RA, significantly improves aortic elasticity, and decreases inflammation and activity of the disease[63, 64].
MiR-34a can activate the p53 signaling pathway and inhibit the abnormal growth of synovial tissue and inflammatory process in RA. Other studies have also found that mutations in the P53 signaling pathway, a traditional oncogene, may be associated with anti-rheumatic RA resistance[65].
AGE-RAGE signaling pathway and cGMP-PKG have not been reported in rheumatoid arthritis. Still, By activating NOx-1 and reducing SOD-1 expression, AGE/RAGE signaling has been shown to increase oxidative stress, thus promoting vascular calcification mediated by diabetes. And oxidative stress plays a key role in the synovial lesion process in RA, which needs to be further verified [66].
Natriuretic Peptides have been shown to cause inflammatory dissociation by activating the cGMP/PKG signaling pathway. By increasing intracellular cGMP levels, phosphodiesterases are activated while interfering with caspase-8 cleavage. It inhibits the activation pathway of inflammatory vesicles, thus acting as an anti-inflammatory and immunomodulatory agent. Therefore, we suggest that the signaling pathway of cGMP-PKG plays a significant role in RA and needs further research [67].
Analysis of Hub genes that play a diagnostic and therapeutic role in RA
We used STRING online database tool and R language to derive the top30 protein interaction networks, and Cytoscape software’s Cytohubba plug-in was also used, and ten genes were filtered out such as Ccr1, Ccr2, Ccr5, Ccr7, Cxcl5, Cxcl6, Cxcl13, Ccl13, Adcy2, and Pnoc. We then used the R language GOplot package to derive six hub genes associated with inflammatory immunity: Ccr2, Ccr7, Cxcl5. Cxcl6, Ccl13and Cxcl13,
Our study revealed that these hub genes are mainly chemokine and receptor-related genes. Chemokines are produced by synovial macrophages and exert chemotactic effects on inflammatory immune cells such as neutrophils, lymphocytes, and monocytes and are closely associated with vasculitis development. CCR, CXCL, and CCL, as essential components and receptors of the chemokine family, are closely related to RA development.
The CC Chemokine Receptor (CCR) is an integral membrane protein. The targeting of chemokines or chemokine receptors is a promising therapeutic strategy for the treatment of chronic inflammation. Small CCR1 and CCR2 molecule antagonists are effective at blocking inflammatory immune cell migration. Reduces the inflammatory response and decreases pain caused by chronic inflammation [68].
The knockdown of CCR5 and the application of PD98059 (mitogen-activated protein kinase 1 inhibitor) in rats resulted in a significant decrease in interleukin-6, metalloproteinase-1, metalloproteinase-3, and tumor necrosis factor levels. In addition to substantial reductions in JNK1, ERK1, p38, Cyclin protein, and Bcl-2, CCR5 may inhibit synovial cell activity, promote synovial cell apoptosis, and suppress the inflammatory response of synovial cells in RA rats via the MAPK pathway. For RA, it may provide a new therapeutic target[69].
The CCR7 chemokine receptor plays a key role in health and disease by directing immune cells’ migration, particularly DCs and T cells. Due to impaired CCR7 signaling, immune responses and misdirection of immune cells can lead to autoimmune diseases. [70, 71].
Inflammatory migration and erosive phenotypes have been observed in patients with RA. Identifying new pathways involved in various stages of the pathology of RA will provide valuable insights into different inflammatory immune cells’ mechanical behavior and strategies to suppress their activity. CCR7 is a marker of RA synovial fluid macrophages, and in early RA, monocytes infiltrate synovial tissue. By blocking CCR7/ CCL21 in synovial fluid, monocyte migration is prevented. It increases the number of M1 macrophages and raises the levels of IL-6 and IL-23. Increased transcription of M1 macrophage cytokines leads to the differentiation of primary T cells into Th17 cells, and M1 macrophage-driven Th17 polarization promotes osteoclast formation in RA during the aggressive phase of the disease. They are extending joint inflammation to bone erosion. It also induces neovascularization, further aggravating disease progression. CCR7/CCL21 is an exciting emerging RA therapy target, and obstructing its function reduces or eliminates RA immune-inflammatory infiltration. They play a major role in delaying or blocking the RA pathological process[72, 73].
For how CCR7 signaling directs cell migration and infiltration, we need to develop new tools to assess and monitor CCR7 signaling. The latest study identifies human CCR7 by novel nanotechnology. It interacts with CCR7 through bimolecular fluorescence
complementary recognition without interfering with G protein coupling and downstream signaling. Targeted analysis of the migration and invasion process of CCR7 [74].
Chemokine C-X-C motif chemokine ligand 5 (CXCL5) has functions such as chemotaxis of neutrophils, pro-angiogenesis, and involvement in inflammatory responses. Its pathological angiogenesis is highly correlated with RA activity[75].
CXCL13 is a known B cell chemokine that promotes B lymphocyte migration by interacting with its receptor CXCR5. CXCL13 and CCL20 act synergistically to increase B cell migration, leading to autoimmune inflammation, closely associated with disease activity [76, 77].
By recruiting monocytes and lymphocytes, CC motif chemokines are thought to be involved in RA’s pathogenesis. In both serum and synovial tissue, the expression of CCL13 is elevated in RA patients. Interestingly, tumor necrosis factor-alpha positively regulates CCL13 expression and inhibits synovial fibroblast apoptosis[78]. RA prevalence in women is higher than in men, and 17-estradiol mediates cellular activation signals in synovial fibroblasts via ERK-1/2, leading to uncontrolled apoptosis, increased matrix metalloproteinase-3 production, and excessive CCL13 production, leading to the progression of RA [79].
However, Adcy2 and Pnoc genes have not been reported in RA studies. Adenylate cyclase 2 (Adcy2), a member of adenylate cyclase class B, plays a crucial role in promoting phosphorylation, glycogen synthesis, and catabolism. By studying the anti-migratory effect of adenylate cyclase-related protein 1 in pancreatic cancer cells, cyclic adenosine monophosphate was found to be a second messenger regulating the migration and invasion of pancreatic cancer cells. Its elevation prevented the migration and invasion of pancreatic ductal carcinoma cells [80]. We speculate that Adcy2 may play a role in inflammatory immune cell migration and infiltration in RA and merits further investigation.
The prepronociceptin (Pnoc) gene encodes bioactive peptides involved in sensory, emotional, cognitive, and neurogenesis. The interaction between these active peptides and the corresponding receptors can modulate inflammation and immune diseases. Silencing the gene reduces inflammatory factor levels, oxidative stress, and glial fibrillary acidic protein expression. Conversely, overexpression of this gene reverses the changes in the above biochemical indicators[81]. Considering that it can regulate inflammation and immunity, we hypothesized that this gene might play an important role in RA’s inflammatory response and potential therapeutic target.
Analysis of immune infiltrating cells that play a diagnostic and therapeutic role in RA
To further investigate the RA synovium role of inflammatory immune cell infiltration, we performed immune infiltration analysis on a combined dataset of four databases by the CIBERSORT algorithm. We found that CD8 T cells, Plasma cells, Monocytes, follicular helper T cells, gamma delta T cells, and M0 macrophages were significantly elevated in RA. In contrast, CD4 memory resting T cells, regulatory T cells (Tregs), M1 Macrophages,
activated NK cells, resting Dendritic cells, resting Mast cells, Eosinophils, and activated Mast cells were significantly reduced in the RA synovial tissue.
Plasma cells (PC) are also known as antibody-secreting cells. With antigen-presenting cells and Th cells, mature B cells become activated B cells after receiving antigen stimulation. They then differentiate into plasma cells, which synthesize different kinds of immunoglobulins and secrete them.β-ARR2 has been found to be a crucial protein mediating the desensitization and internalization of inflammatory and immune responses involving G protein-coupled receptors. Its deletion inhibits the endocytosis of Toll-like receptor 4 (TLR4) on B lymphocyte membranes while activating the NF-κB signaling pathway, increasing plasma cell differentiation and antibody production and exacerbating arthritis symptoms[82]. Myeloid suppressor cells derived from granulocytes secrete exosomes, transport different bioactive molecules, and play a regulatory role in immune cells. It also promotes the secretion of IL-10 from splenic B cells, reduces the proportion of plasma and follicular helper T cells and serum IgG levels, and mitigates rheumatoid arthritis symptoms in mice[83]. It is evident that plasma cells play an essential role in RA and deserve further study.
In RA, CD8 + CD28-T cells are significantly elevated and may be associated with T-lymphocyte homeostasis dysregulation. There is also an increase in their supracellular expression of programmed death receptor-1 (PD-1). When regulating T lymphocytes, PD-1 plays a key role. Although PD-1 induces immunosuppression of effector T cells in synovial inflammation, its expression is up-regulated in synovial inflammation, closely correlating with the severity of its disease [84]. Another study found that CD8 + T cells are involved in the pathogenesis of RA through the release of pro-inflammatory and cytolytic mediators and that targeting lactate dehydrogenase remodels CD8 + T cells and changes the metabolism of glucose and glutamine, thereby reducing the adipogenic, migratory and proliferative capacity of CD8 + T cells while losing the ability to induce a pro-inflammatory phenotype in B cells [85].
One of the potential mechanisms promoting RA is the imbalance in the ratio of M1 macrophages, which produce pro-inflammatory factors, to M2 macrophages, which produce anti-inflammatory factors.M2 macrophages may have a positive role in delaying the development of RA. In contrast, M0 macrophages may have a potential role in the immune imbalance associated with RA pathogenesis. Our results found a significant increase in the proportion of M0 macrophages and a substantial decrease in the proportion of M1 macrophages in RA’s synovial tissue. The common knowledge contradicts this that M1 macrophages should be significantly up-regulated in the synovium of RA. It has been found that M0 macrophages can differentiate into M1 and M2 macrophages under certain circumstances, and these paradoxical phenomena and whether M0 macrophages are converted to M1 macrophages in the inflammatory setting in RA deserve further investigation [86].
Monocytes are attracted to pro-inflammatory mediators in response to inflammation or tissue injury, differentiate toward macrophages, activate macrophages, and the Paqr11 gene regulates monocyte-to-macrophage differentiation. Knockdown of Paqr11 in mice inhibited monocytes’ differentiation and delayed RA progression [61]. Tumor necrosis factor-alpha and IL-6 were used in other experiments to induce osteoclasts with the ability to resorb bone matrix to stimulate peripheral blood monocytes. The expression of IL-1β, IL-12, TNF alpha, and MMP3 has been significantly increased in tumor necrosis, factor-alpha, and IL-6-induced osteoclasts. RA and other joint destruction-related inflammatory arthritis may involve these monocytes with the capacity to resorb bone matrix[87].
Dendritic cells (DCs) are the nexus of innate and acquired immunity, and increased expression of pro-inflammatory cytokines, chemokines, and adhesion molecules was found in DCs by culturing RA synovial tissue in vitro. Hydroxychloroquine (HCQ) is one of the most commonly used immunosuppressive agents in treating RA. HCQ inhibits DC maturation and migration to lymph nodes, decreases CXCR4 expression and interferon-α secretion, and DC activation is involved in RA’s pathogenesis. HCQ protects against RA by blocking TLR9[88]. TARM1 is a member of the leukocyte immunoglobulin-like receptor family. Its expression is elevated in the joints of a mouse model of RA. It is an important stimulus for dendritic cell maturation, which may be a good target for the treatment of autoimmune diseases[89].
Mast cells are tissue-resident innate immune cells involved in the pathogenesis of many autoimmune diseases; they are mainly present in RA’s synovial tissue. Their activation is associated with the amelioration of inflammation. However, a growing body of proof indicates that mast cells can act as modifiable immune cells with pro-and anti-inflammatory functions. Synovial mast cells are a potential research hotspot for this subtle, conflicting, and exciting hypothesis[90].
Substance P (SP) is a pro-inflammatory substance, and activated synovial mast cells rapidly degrade SP in RA while downregulating SP-mediated synovial cell activation. On the other hand, SP activates inflammatory mediators induced by synoviocytes, suggesting a dual regulation of SP-mediated synovial mast cell inflammation in RA[91]. It has been found that gene expression of synovial mast cells is negatively correlated with disease activity. When IL-33 activates synovial mast cells, they produce an immunomodulatory phenotype and inhibit monocyte activation[92]. However, a study found that mast cells are important targets of Toll-like receptor ligands and immune complexes. Mast cells greatly enhance the inflammatory response of synovial tissue in RA through these pathways’ combined activation[93].
Eosinophils have a pro-inflammatory role in asthma. However, recent studies have shown that eosinophils have a pro-soluble effect in RA. After induction of eosinophil asthma, arthritis subsided, and joint tissue was protected in mice. Single-cell RNA sequencing approaches identified a specific subpopulation of regulatory eosinophils in the joints, distinct from inflammatory eosinophils in the lungs. The reduction of eosinophils instead eliminated the beneficial effects of asthma on arthritis. Such a result is consistent with our study that concluded a significant down-regulation of eosinophils in RA[94].
The anti-inflammatory effect of eosinophils was also confirmed in another experiment. Eosinophil treatment reduced iNOS, TNF-α, IL-6, and IL-12 levels, while Arg1, transforming growth factor-β, IL-10, and IL-13, increased while inhibiting MAPK signaling polarizing M2 macrophages, exerting anti-inflammatory and reducing RA symptoms[95]. The double-edged role of these eosinophils needs to be studied in depth.