Rheumatoid arthritis (RA) is a chronic autoimmune disease intricately linked to inflammation. Recent statistics reveal a global prevalence of RA as high as 0.24%, with an annual incidence ranging from 20 to 45 per 100,000 individuals (1). In existing guidelines, the diagnosis of RA is contingent upon clinical manifestations, physical examination findings, as well as serological and imaging results (2). Regrettably, while most patients with RA exhibit positive laboratory test results (rheumatoid serum factor and ACPA), approximately one-third of RA cases yield negative findings (3). Furthermore, no single pathological laboratory discovery or imaging method has been demonstrated to definitively diagnose RA, posing significant challenges to its diagnosis and subsequently influencing treatment and prognosis. Moreover, numerous studies emphasize the importance of early diagnosis, prompt intervention, timely treatment initiation, and, if necessary, immediate referrals for RA patients (4–6). Despite advancements, RA remains incurable, and disease-modifying anti-rheumatic drugs (DMARDs) represent the primary treatment option (7). Nevertheless, prolonged DMARD use is often associated with adverse effects, including gastric ulcers, vomiting, heartburn, or gastrointestinal bleeding (8, 9). Hence, the imperative for dependable biomarkers persists to facilitate early diagnosis, accurate prognosis, and treatment efficacy assessment.

Cuproptosis represents a distinct form of programmed cell death distinguished by the intracellular accumulation of copper, leading to subsequent disruption in mitochondrial lipid peroxidation and instability of Fe-S cluster proteins (10). The conversion of copper ions (Cu2+) to Cu by ferredoxin 1 (FDX1) facilitates the lipid acylation of mitochondrial proteins, thereby inducing the overproduction of crucial enzymes linked to the tricarboxylic acid (TCA) cycle. This mechanism plays a regulatory role in fundamental biological pathways, including the maintenance of redox balance, iron metabolism, oxidative phosphorylation, and the modulation of aberrant cell proliferation (11). Moreover, we observed that individuals diagnosed with active rheumatoid arthritis showed elevated levels of serum copper, exhibiting a negative correlation with hemoglobin levels, an ancillary disease marker, and a positive correlation with erythrocyte sedimentation rate and morning stiffness (12). Notably, a study proposed that serum copper levels could potentially serve as an indicator of erosive activity in RA (13). Numeric studies have proposed a multifaceted relationship between cuproptosis and RA (14–16). For example, Zhao et al. suggested that cuproptosis-related genes (PDHA1, PDHB, CDKN2A, and DLAT) were closely involved in the onset and development of RA (14). Jiang et al. (15) proposed a cuproptosis-related diagnostic model incorporating immune infiltration in RA. Hu et al. (16) developed a novel cuproptosis-related gene signature for RA. However, the current literature on the relationship between cuproptosis and RA remains relatively limited, highlighting the potential for cuproptosis to emerge as a new therapeutic target.

In this study, unveiling the ramifications of copper homeostasis imbalance on RA would significantly enhance our comprehension of its pathogenesis and facilitate the quest for efficacious agents. Cuproptosis-related genes are intricately associated with imbalance in copper homeostasis. To elucidate the repercussions of copper homeostasis imbalance on mitochondria in RA cells, we screened 10 DE-CRGs between RA samples and normal samples. Furthermore, to explore the impact of 10 DE-CRGs on RA, we examined the correlation between DE-CRGs and immune infiltration, immune factors, diagnostic efficacy, and predicted drugs. We assessed expression alterations in DE-CRGs across various RA cell clusters using single-cell RNA sequencing data. In summary, our findings provide valuable insights into characterizing copper homeostasis imbalance in RA and offer guidance for identifying potential therapeutic targets.

Cuproptosis has emerged as a novel mechanism of mitochondrial cell death triggered by disruptions in copper homeostasis. Increasing evidence from bioinformatics studies suggested that cuproptosis was involved in immune infiltration in numeric cancers, including clear cell renal cell carcinoma (36), head and neck squamous carcinomas (37), and lung adenocarcinoma (38). Cell death dysregulation plays a significant role in the initiation and development of RA, characterized by heightened chondrocyte apoptosis, diminished chondrocyte proliferation, and impaired extracellular matrix synthesis. However, the variation in copper content within RA tissue and specific RA cell clusters remains unclear. Given that cuproptosis stems from an imbalance in copper homeostasis driven by intracellular copper accumulation, we investigated this issue using nine DE-CRGs.

Our findings revealed a downregulation of GLS, PDHA1, and FDX1 expression, and an upregulation of NLRP3, DLST, ATP7B, ATP7A, NFE2L2, DBT, and SLC31A1 in whole RA tissues. SLC31A1 is involved in the transport of copper ions across cell membranes and plays a crucial role in maintaining cellular copper homeostasis. ATP7A is responsible for transporting copper ions across cell membranes and is involved in maintaining copper homeostasis within cells. Notably, both SLC31A1 and ATP7A are upregulated in response to elevated intracellular copper levels (39). DLST, an enzyme involved in the tricarboxylic acid (TCA) cycle, plays a crucial role in cellular metabolism by catalyzing the transfer of succinyl groups from succinyl-CoA to the enzyme lipoamide in the TCA cycle, facilitating the conversion of α-ketoglutarate to succinyl-CoA (40). However, the implications of these preliminary findings concerning copper reduction in whole RA tissue remained unclear. Therefore, we conducted further analysis on RA single-cell RNA sequencing (scRNA-seq) data to scrutinize the expression levels of the nine differentially expressed cuproptosis-related genes (DE-CRGs) across various cell clusters. Interestingly, we observed upregulated expression of all DE-CRGs, including DLST, SLC31A1, and GLS, in fibroblast and endothelial cell clusters. Conversely, minimal changes in the expression levels of the DE-CRGs were noted in other RA tissue cells, such as stem cells. GLS, an enzyme responsible for catalyzing the deamination of glutamine into glutamate, is a pivotal component of glutamine metabolism, serving as a fundamental energy source for proliferating cells (41, 42). Glutamine metabolism also plays a crucial role in various biological processes, including biosynthesis, antioxidant defense, and regulation of cell signaling (43). Moreover, glutamine induces a substantial carbon influx into the TCA cycle during cell proliferation. Therefore, the six identified differentially expressed cuproptosis-related genes (DE-CRGs) are intricately associated with protein lipoylation and energy metabolism processes within the mitochondria.

Subsequently, we scrutinized the biological functions, disease associations, and clinical correlations of the DE-CRGs, taking into account the tissue perspective. Our analyses, including GO and KEGG pathway analyses, unveiled the involvement of DE-CRGs in several critical pathways. Specifically, these pathways encompassed platinum drug resistance, the citrate cycle (TCA cycle), mineral absorption, central carbon metabolism in cancer, and carbon metabolism. The TCA cycle is crucial for cellular energy production and has been implicated in various inflammatory conditions. Alterations in mitochondrial function and energy metabolism are observed in RA, suggesting a potential link between TCA cycle dysregulation and RA pathogenesis (44). Mineral absorption, particularly of calcium and magnesium, plays a significant role in bone health. In RA patients, altered mineral metabolism can contribute to bone density loss and joint damage (45). Carbon metabolism, including the metabolism of glucose, fatty acids, and amino acids, is central to cellular function. In RA, dysregulated carbon metabolism contributes to inflammation and joint damage. Studies have identified metabolic changes in RA, such as increased glycolysis and altered fatty acid metabolism, which support the inflammatory processes characteristic of the disease (46, 47). Furthermore, our PPI analysis revealed that proteins sharing similar biological functions interacted closely with each other, potentially coordinating their roles in mediating essential cellular processes.

Our investigation into immune cell infiltration revealed significant alterations in various cell populations between the RA and normal groups. Specifically, B cells memory, plasma cells, T cells CD4 naive, CD4+ T cells memory activated, NK cells resting, monocytes, mast cells resting, and neutrophils exhibited elevated levels in the RA group compared to the normal group. Kishikawa et al. (48) demonstrated that the increased plasma nucleotide levels in RA patients. Lyu et al. (49) reported that CD305 reduction may mediate the excessive activation of memory CD4+ T cells and participate in the development of RA. Neutrophils play a crucial role in RA initiation and progression (50). After migrating into the articular cavity, they become activated, triggering inflammation (51). This leads to prolonged survival, excessive inflammatory activity, and increased oxidative stress, all of which contribute to RA pathogenesis (52). Conversely, T cells CD4 memory resting, T cells follicular helper, Tregs, NK cells activated, and eosinophils displayed decreased levels in the RA group. Sofi et al. (53) presented that eosinophilia occurred in several rheumatic diseases, including RA, and was associated with high disease activity and poor prognosis. Notably, we observed a negative correlation between activated and resting immune cells, while immune cells with similar functional states exhibited positive correlations. Plasma cells, arising from mature B lymphocytes, play a pivotal role in humoral immunity (54). Additionally, activated memory CD4+ T cells contribute to cellular immunity and collaborate with B cells (55). Neutrophils, as integral components of the innate immune system, act as frontline defenders and orchestrate subsequent adaptive immune responses (56). In summary, our findings suggest that RA triggers activation across humoral, cellular, and innate immune systems, which mutually reinforce each other. Furthermore, our analysis revealed a close association between differentially expressed cell regulatory genes (DE-CRGs) and resting immune cells, indicating their potential as novel targets for modulating excessive immune responses in RA.

We observed correlations between the 10 DE-CRGs and numeric immune factors. Particularly within the chemokine-related genes, our analysis revealed that SLC31A1 exhibited a negative correlation with CCL21, CCL24, and CCL27, while it displayed positive correlations with CCL14, CCL16, CXCL12, and CXCL14. Chemokines constitute a large protein family characterized by low molecular weight structures, and they play pivotal roles in regulating immune cell residence, migration, and inflammatory responses. The CC chemokine family primarily facilitates the recruitment of lymphocytes, whereas the CXC chemokine family is closely associated with neutrophils and monocytes (57). Additionally, CXCL12 and CXCL14 have been shown to augment macrophage function (58, 59). Therefore, our findings suggest that SLC31A1 is more inclined toward recruiting neutrophils and macrophages rather than lymphocytes.

Among the immunoinhibitory-related genes, we observed significant correlations. Specifically, SLC31A1 exhibited notably negative correlations with CXCR4, ICOSLG, TNFRSF13B, and TNFSF9, while it displayed positive correlations with C10orf54, CD276, CD40, CD48, IL6R, MICB, PVR, TNFSF14, TNFSF15, and TNFSF4. Similarly, PDHA1 demonstrated remarkable negative correlations with CD276, CD48, CD40, CD80, IL6R, MICB, PVR, RAET1E, TNFRSF17, TNFRSF18, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF13B, TNFSF14, TNFSF15, and TNFSF4, while being positively correlated with CXCR4, ICOSLG, TNFRSF13B, and TNFSF9. The protein encoded by the CD276 gene belongs to the immunoglobulin superfamily and is believed to play a role in regulating T-cell-mediated immune responses (60). CD40, also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), is a cell surface receptor protein found on antigen-presenting cells such as B cells, dendritic cells, and macrophages. It plays a crucial role in the immune system by providing co-stimulatory signals necessary for the activation of B cells and other immune cells (61). CD48 exhibits anti-inflammatory properties in Staphylococcus aureus Enterotoxin B-induced eosinophilic inflammation (62), while CXCR4 has been associated with the anti-inflammatory properties of mesenchymal stromal cells (63). Additionally, ICOS/ICOSL upregulation has been linked to inflammatory responses and endothelial dysfunction in type 2 diabetes mellitus (64). Our findings suggest that SLC31A1 and PDHA1 may be associated with anti-inflammatory effects based on these correlations.

In assessing the predictive potential of DE-CRGs in RA, our findings revealed ATP7B to exhibit the most robust diagnostic effect, followed by PDHA1 and DLST. The ATP7B gene encodes a protein called ATPase copper-transporting beta (ATP7B), which is primarily involved in transporting copper ions across cell membranes. This protein is crucial for maintaining copper homeostasis within the body by facilitating the incorporation of copper into ceruloplasmin and its subsequent secretion into the bloodstream (65). Despite its known role in copper transport, the precise impact of ATP7B on RA pathogenesis remains unclear. However, considering our results alongside those of previous investigations, PDHA1 and DLST emerge as candidates capable of disrupting intracellular copper homeostasis and mitochondrial function in the context of RA. Consequently, these findings may offer insights into novel etiological mechanisms and therapeutic targets for RA.

In the realm of predictive drugs for RA treatment, focusing on DE-CRGs, our analysis pinpointed several key targets, notably ATP7A, ATP7B, SLC31A1, GLS, and NFE2L2. The drugs identified for targeting ATP7A, ATP7B, and SLC31A1 primarily comprised anticancer agents, including thalidomide, progesterone, docetaxel, and various platinum-based chemotherapies such as oxaliplatin, carboplatin, daunorubicin, and cisplatin. Additionally, a subset of compounds, such as methotrexate, glutamine (Gln), and dexamethasone, were highlighted for their potential to mitigate the catabolism of Gln catalyzed by GLS by bolstering substrate levels. Of particular interest were drugs predicted to modulate the NFE2L2 gene, including Lagascatriol, Andalusol, Irofulven, NK-252, RTA-408, and Sulforaphane, which are commonly employed to alleviate inflammation associated with RA in clinical practice. These findings offer insights into novel candidates for systemic antimicrobial therapy. Furthermore, our analysis revealed upregulated expression of ATP7B and NFE2L2 in both RA tissues and cell clusters, suggesting that attenuating ATP7B decomposition may represent a promising avenue for future research into RA treatment.

This research has sparked numerous inquiries that warrant further exploration. A heightened emphasis on copper intake from daily essentials and diets could yield intriguing insights, enhancing our comprehension of the acquired induction and prevention of RA. Substantial efforts will be required to elucidate alterations in the transcription of DE-CRGs and subsequent post-translational modifications of coded proteins associated with mitochondrial dysfunction. Moreover, future studies could evaluate the potential therapeutic benefits of drugs targeting GLS, ATP7B, and ATP7A in the context of RA. Although the study has several limitations, particularly the lack of experimental validation, we plan to address these through follow-up research. This will include in vitro assays to explore the functional roles of ATP7A, ATP7B, and GLS in RA pathogenesis, as well as in vivo models to evaluate their therapeutic potential.