Introduction

Rheumatoid arthritis (RA) is a common systemic autoimmune disease characterized by chronic inflammation of synovial tissue, immune cell infiltration in joints, angiogenesis, and bone and cartilage damage [1]. The pathogenesis of RA is not completely understood. Current research suggests that the interaction between genetic factors and environmental factors contributes to the development of RA [2]. In addition, microbial infection and intestinal microbiota dysbiosis also participate in the occurrence and development of RA [3, 4].

In RA patients, there are large numbers of memory CD4+ T cells infiltrating the synovial tissue, which is associated with disease activity [5]. In RA patients, CD4+ T cells can recognize self-antigens, subsequently inducing an autoimmune response, and can infiltrate into the synovial tissue to cause inflammation [6, 7]. Therefore, correcting the functional abnormalities of CD4+ T cells may be an important approach for the treatment of RA.

Toll-like receptors (TLRs) sense the molecular signatures of microbial pathogens, and play a crucial role in initiating innate immune responses. TLRs signal via a common pathway that results in the expression of diverse inflammatory genes [8]. Previous studies have shown that TLRs are also expressed in various T cell subsets and can induce T cell effector or regulatory functions [9]. However, the role of TLRs in CD4+ T cells of RA patients and their signaling pathways in RA remain to be studied.

T cell metabolism is closely related to cell survival, activation, proliferation, and differentiation, and metabolic changes often cause changes in cell function [10, 11]. Glucose metabolism is crucial for cellular energy supply. Once glucose enters the cell, it follows three major metabolic pathways: glycolysis, oxidative phosphorylation, and pentose phosphate pathway [12]. Hexokinase 2 (HK2) is the first critical enzyme in glucose metabolism, regulating the common pathway of these three pathways [13]. Mitochondrial reactive oxygen species (ROS) are mainly generated during the electron transfer process of oxidative phosphorylation [14]. ROS are considered as risk factors and enhancers of autoimmune diseases, which are related to the pathogenesis of RA [15]. Whether CD4+ T cells in RA patients affect ROS by regulating glucose metabolism, thereby mediating cellular dysfunction, and the factors affecting the reprogramming of T cell glucose metabolism are still under investigation.

In our study, we aimed to explore the roles of TLR2 in regulating RA CD4+ T cell function and investigate the potential mechanism associated with cell glucose metabolism.

Materials and methods

Human blood samples

Peripheral blood samples were collected from rheumatoid arthritis (RA) patients at the Department of Rheumatology and Immunology of the Second Affiliated Hospital of Dalian Medical University in China. All RA patients included in this study met the revised criteria of the American College of Rheumatology (ACR) in 1987. These patients had no other autoimmune or systemic diseases. Peripheral blood samples from age and sex-matched healthy controls (HC) were collected from the Medical Examination Center of the Second Hospital of Dalian Medical University. The blood sampling procedure was approved by the ethics committee of Dalian Medical University (2018–061), and informed consent was obtained from all participants. The information of RA patients is listed in Supplemental Table 1. Meanwhile Information of healthy controls was also listed in Supplemental Table 1.

Cell isolation, purification, and activation

Peripheral blood mononuclear cells (PBMCs) were purified from peripheral blood using Ficoll-Paque plus (TBD science, China). CD4+ T were purified from PBMCs by CD4+ T Cell Isolation Kit (Biolegend, USA) according to the manufacturer's protocols. The purified CD4+ T cells were activated with anti-CD3 antibody (5 μg/mL, eBioscience, USA) and anti-CD28 antibody (2 μg/mL, eBioscience, USA) in RPMI-1640 containing 10% fetal bovine serum (FBS) and treated with or without Pam3CSK4 (100 ng/mL, InvivoGen, France). In several experiments, 2-DG (5 mM, Solarbio, China), rotenone (1 μg/mL, Sigma-Aldrich, USA), G6PDi-1 (10 mM, Shanghai yuanye Bio-Technology, China), Mitoquinone (200 nM, Biovision, USA), or 3PO (10 mM, Solarbio, China) were administrated to the cell culture.

Enzyme-linked immunosorbent assay (ELISA)

The concentration of soluble TLR2 in the serum was measured using the Human TLR2 DouSet ELISA Kit (RD system, USA) according to the instruction [16]. The plates were read at 450 nm by a microplate reader (Thermo, USA).

Flow cytometry analysis

Cell surface staining was performed using BD Biosciences or eBioscience reagents. The dead cells were excluded from the analysis by fixable viability dye (FVD) (eBioscience) before labeling the surface antibody [17]. For the detection of intracellular cytokines, a final concentration of 50 ng/ml PMA (Fcmacs Biotech, China), 1 μg/ml ionomycin (Fcmacs Biotech, China), and 10 μg/ml BFA (Fcmacs Biotech, China) were added to the cell culture system and incubated for 2 h [18]. After surface antibody staining, cells were fixed and permeabilized using the Cytofix/Cytoperm Intracellular Staining Kit (BD Biosciences, USA) and stained with intracellular antibodies. The flow antibodies are listed in Supplemental Table 2. All stained cells were analyzed on the Flow Cytometer (NovoCyte 2040R) and data were analyzed with NovoExpress software.

Intracellular total ROS and mitochondrial ROS detection

For intracellular total ROS and mitochondrial ROS detection, cells were incubated with DCFH-DA (1:1000, AAT Bioquest, USA) or MitoSOX (1:1000, eBioscience, USA) at 37 °C in the dark for 20 min. After washing with Hank's buffer, the cells were analyzed using a Flow Cytometer (NovoCyte 2040R, Agilent, USA).

Glucose uptake assay

Glucose uptake was measured by a fluorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) (Sigma-Aldrich, USA) [17]. Purified RA CD4+ T cells were treated with or without Pam3CSK4 for 24 h, followed by incubation in glucose-free RPMI 1640 medium for 30 min. After incubating with 2-NBDG (50 μM) for 20 min, the cells were cooled on ice and washed twice with ice-cold PBS. Subsequently, the cells were analyzed by flow cytometry immediately after staining with CD4 antibody.

Quantitative polymerase chain reaction (qPCR)

Total RNA was extracted from RA CD4+ T cells using RNA extraction reagent (Accurate, China) [19], and cDNA was transcribed using 5 × All-In-One RT MasterMix (Abm, Canada), both according to manufacturers' instructions. Real-time PCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate, China) on the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) [20]. The mRNA levels in each sample were normalized to the expression of the β-actin gene, and the relative gene expression was determined using the 2-ΔΔCT method. The specific primers used are listed in Supplemental Table 3.

Lactate assay

Purified RA CD4+ T cells were treated with or without Pam3CSK4 for 24 h. The concentrations of lactate in the cell culture supernatants were measured using the CheKine Lactate Assay Kit (Abbkine, USA) following the manufacturer's instructions [17].

Statistical analysis

Statistical analysis and graphical representation were performed using GraphPad Prism 9. Paired Student's t-test, unpaired Student's t-test, and one-way analysis of variance (ANOVA) were employed for statistical analysis. For statistics that did not pass the normal distribution, we used nonparametric tests to analyze. Data are presented as mean ± standard error of the mean (SEM). A significance level of P < 0.05 was considered statistically significant, indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Results

RA patients show elevated soluble TLR2 levels in the serum and up-regulated surface TLR2 expression on CD4+ T cells

Our previous serum protein microarray results demonstrated that RA patients had higher soluble TLR2 (sTLR2) levels in their serum compared to healthy individuals [21]. To validate this finding, we collected serum samples from 182 RA patients and 53 healthy individuals, and measured their sTLR2 levels using an ELISA assay. The results confirmed that RA patients had higher sTLR2 levels in their serum compared to healthy individuals (Fig. 1a). We further analyzed the relationship between sTLR2 levels and RA-related clinical indicators. The level of serum sTLR2 in RA patients showed positive correlations with C-reactive protein (CRP) and rheumatoid factor (RF), but no significant correlation was observed with the Disease Activity Score using 28 joint counts (DAS28), a simplified version of the Disease Activity Score (DAS) for RA (Fig. 1b). As sTLR2 is shed from the extracellular domain of membrane-bound TLR2 [22], we examined the expression of TLR2 on immune cells. The results demonstrated that CD4+ T cells and CD8+ T cells in RA patients had higher TLR2 expression levels compared to healthy individuals, while there was no significant difference in TLR2 expression levels on CD19+ B cells and CD14+ monocytes between the study groups (Fig. 1c). Given the important role of CD4+ T cells in systemic inflammatory responses in RA patients and the more pronounced up-regulation of TLR2 [5, 23], we focused on CD4+ T cells for further studies.

Fig. 1
figure 1

RA patients show elevated soluble TLR2 levels in the serum and up-regulated surface TLR2 expression on CD4+ T cells. a Detection of sTLR2 levels in serum from RA patients (n = 182) and healthy controls (HC) (n = 53) using an ELISA kit method. b Statistical analysis of the correlation between sTLR2 levels in serum from RA patients and CRP, RF, and DAS28. (c) Flow cytometry detection of TLR2 expression on CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD14.+ monocytes. Data are presented as mean ± SEM. ns: no significant difference, *P < 0.05, **P < 0.01

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Activation level of RA CD4+TLR2+ T cells is higher than that of CD4+TLR2− T cells

The abnormal activation of CD4+ T cells plays a crucial role in the pathogenesis and persistent chronic inflammation of RA [23]. Therefore, we detected the cell surface activation molecules CD40L, CD25, CD69, PD-1, and ICOS in RA patient CD4+T cells using flow cytometry. As shown in Fig. 2a, we divided the RA CD4+ T cells into TLR2+ and TLR2− groups. The results demonstrated that the expression proportions of the five activation molecules and their mean fluorescence intensities (MFIs) were significantly higher in RA patient CD4+TLR2+ T cells compared to CD4+TLR2− T cells (Fig. 2b). These findings suggest that the expression of TLR2 on the surface of CD4+ T cells may promote their abnormal activation.

Fig. 2
figure 2

Activation level of RA CD4+TLR2+ T cells is higher than that of CD4+TLR2− T cells. a CD4+ T cells in RA patients' PBMC were divided into TLR2+ and TLR2− populations by flow cytometry. b The expression and MFI of cell surface activation molecules CD40L, CD25, CD69, PD-1, and ICOS in CD4+TLR2+ and CD4+TLR2.− T cells. Data are presented as mean ± SEM. **P < 0.01,****P < 0.0001

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TLR2 stimulation promotes CD4+ T cells activation and TNF-α secretion in RA patients

To determine the functional significance of TLR2 expression on RA CD4+ T cell activation and cytokine secretion, purified CD4+ T cells (Fig. 3a) of RA patients were cultured for 72 h in the presence or absence of Pam3CSK4, a ligand for TLR1/2 complex, and then the activation levels and cytokine secretion were detected by flow cytometry. The results showed that after Pam3CSK4 stimulation, the expression of CD25, CD40L, CD69, and PD-1 was up-regulated in CD4+ T cells, while ICOS expression did not show significant changes (Fig. 3b). In addition, TLR2 activation promoted the production of TNF-α (Fig. 3c). These findings suggest that TLR2 stimulation promotes CD4+ T cells activation and TNF-α secretion in RA patients, which may be one of the factors contributing to the development and maintenance of chronic inflammation in RA patients.

Fig. 3
figure 3

TLR2 stimulation promotes CD4+ T cells activation and TNF-α secretion in RA patients. a The purity of MACS selected CD4+ T cells was detected by flow cytometry. The purified CD4.+ T cells were stimulated with TLR2 agonist Pam3CSK4 or not, and the expression of surface activation molecules (b), as well as the production levels of cytokines (c), were detected by flow cytometry. Data are presented as mean ± SEM. ns: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001

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TLR2 activation induces cell hyperactivation and TNF-α secretion in CD4+T cells of RA patients by promoting ROS production

Given that ROS can act as a second messenger to activate NF-κB and amplify the inflammatory response, as well as being a downstream product of NF-κB [24, 25], we investigated the relationship between TLR2 and ROS in RA CD4+ T cells. We found that after TLR2 signal activation the production of both total ROS and mitochondrial ROS (mitoROS) in RA CD4+ T cells increased (Fig. 4a). Since the increase in total ROS is mainly due to an increase in mitochondrial ROS, we next used Mitoquinone to inhibit mitochondrial ROS generation. We observed that Pam3CSK4 treatment could up-regulate the expression of CD25 and CD40L and promote TNF-α secretion, while Mitoquinone treatment could down-regulate the expression of CD25 and CD40L and reduce TNF-α secretion compared to the control group (Fig. 4b. More importantly, Pam3CSK4 treatment could not promote CD25 and CD40L expression and TNF-α secretion after mitochondrial ROS generation was inhibited (Fig. 4b). These results indicate that TLR2 activation can induce the abnormal activation of RA patient CD4+ T cells and the pro-inflammatory factor TNF-α secretion by promoting mitochondrial ROS production.

Fig. 4
figure 4

TLR2 activation induces cell hyperactivation and TNF-α secretion in CD4+T cells of RA patients by promoting ROS production. Isolation of PBMCs from RA patients and selection of CD4+ T cells by MACS. Flow cytometry detection of intracellular total ROS and mitochondrial ROS levels (a), CD25 and CD40L expression, and TNF-α secretion (b). MitoQ, mitochondrial ROS clearance agent Mitoquinone. Data are presented as mean ± SEM. ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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TLR2 regulates ROS production and the function of RA CD4+ T cells via HK2

ROS production is closely related to cellular metabolism, and thus we further investigated the changes in glucose and lipid metabolism in RA CD4+ T cells after TLR2 signal activation. We found that intracellular 2-NBDG fluorescence intensity was enhanced in CD4+ T cells treated with Pam3CSK4, indicating increased glucose uptake ability (Fig. 5a). We also detected the content of lactate, the end product of glycolysis, in the culture supernatant using a lactate detection kit. The result showed that the lactate level did not increase, indicating that Pam3CSK4 had no significant impact on the glycolysis (Fig. 5b). Moreover, the expression of HK2 was up-regulated, while the expression levels of phosphatidylinositol-dependent protein kinase-1 (PDK1) and lactate dehydrogenase A (LDHA) did not show significant changes (Fig. 5c). More importantly, the expression of phosphofructokinase-2 / fructose-2,6-bisphosphatase 3 (PFKFB3) was significantly reduced after Pam3CSK4 stimulation, and the ratio of glucose-6-phosphate dehydrogenase (G6PD) to PFKFB3 (G6PD/PFKFB3) was significantly higher compared to the control group (Fig. 5c). However, TLR2 activation has little impact on lipid metabolism in RA CD4+ T cells (Fig. S1). These results indicate that TLR2 activation reprograms glucose metabolism towards the pentose phosphate pathway in RA CD4+ T cells.

Fig. 5
figure 5

TLR2 regulates ROS production and the function of RA CD4+ T cells via HK2. a Flow cytometry analysis of 2-NBDG uptake level in CD4+ T cells from RA patients. b Lactate content in the culture supernatant was detected by a lactate detection kit. c The mRNA expression of glycolysis-related enzymes was measured by qPCR. d-e Isolation of PBMCs from RA patients and selection of CD4+ T cells by MACS. Flow cytometry detection of intracellular total ROS and mitochondrial ROS levels (d), CD25 and CD40L expression, and TNF-α secretion (e). Data are presented as mean ± SEM. ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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Furthermore, we employed 2-DG, the HK2 inhibitor, to investigate whether TLR2 regulates glucose metabolism to affect ROS levels and thereby disturb the function of CD4+ T cells in RA patients. The results showed that after the addition of 2-DG, intracellular total ROS and mitochondrial ROS production in RA CD4+ T cells were reduced (Fig. 5d), and the expression of CD25 and CD40L as well as the secretion of TNF-α were reduced ((Fig. 5e), indicating that blocking HK2 significantly impaired RA CD4+ T cell function, potentially by reducing intracellular mitochondrial ROS production. Moreover, compared to inhibiting HK2 alone, stimulation of the cells with Pam3CSK4 after inhibiting HK2 did not significantly change cell function (Fig. 5e), suggesting that TLR2 promotes glucose metabolism through HK2 to affect mitochondrial ROS production in RA CD4+ T cells and thereby disturb their function.

TLR2 activation mediates cell hyperactivation and TNF-α secretion of CD4+ T cells in RA patients via oxidative phosphorylation and pentose phosphate pathway

In the three pathways of glucose metabolism, since TLR2 had no significant impact on glycolysis in CD4+ T cells, we examined the changes in cell function after inhibiting oxidative phosphorylation with rotenone and inhibiting the pentose phosphate pathway with G6PDi, respectively. The results showed that inhibition of oxidative phosphorylation led to a decrease in the expression of CD25 and CD40L, and stimulation with Pam3CSK4 could not restore the original level (Fig. 6a). In contrast, inhibition of the pentose phosphate pathway resulted in a decreased trend in CD40L expression, but stimulation with TLR2 ligand could partially restore it to a certain level (Fig. 6b). These findings suggest that TLR2 activation mainly occurs through the oxidative phosphorylation pathway, with a partial contribution from the pentose phosphate pathway, and mediates the abnormal function of CD4+ T cells.

Fig. 6
figure 6

TLR2 activation mediates cell hyperactivation and TNF-α secretion of CD4+ T cells in RA patients via oxidative phosphorylation and pentose phosphate pathway. The purified CD4.+ T cells were stimulated with TLR2 agonist Pam3CSK4 or not, with or without rotenone (a) or G6PDi (b), the expression of CD25 and CD40L, and TNF-α secretion were detected by flow cytometry. Data are presented as mean ± SEM. ns: no significant difference, *P < 0.05, **P < 0.01

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Discussion

The pathogenesis of RA is not fully understood, but some research suggests that the onset of RA is divided into two stages: first, genetic susceptibility leads to the production of self-reactive T cells and B cells; second, a triggering event is required, such as microbial infection or tissue injury, which provides antigens to activate antigen-presenting cells and thereby activate the self-reactive lymphocytes, leading to the interruption of immune tolerance and the development of RA [26]. In the innate immune response, TLR2 recognizes pathogen-associated molecular patterns and induces immune cells to produce pro-inflammatory cytokines, playing a crucial role in the occurrence and development of RA [27, 28]. It is likely that TLR2 is an important molecule playing a role in the triggering stage of RA onset [29]. Our research has found that the expression of TLR2 on CD4+ T cells in RA patients is increased, and activating TLR2 leads to cell activation and TNF-α production by affecting glucose metabolism and inducing ROS generation.

Our previous study found that the serum level of sTLR2 was higher in RA patients than in healthy individuals [21]. In this study, we found that serum sTLR2 levels in RA patients positively correlated with the clinical indicators CRP and RF. sTLR2 is formed by the extracellular domain of membrane-bound TLR2, and its content may be related to the expression of membrane TLR2 [22]. Although the role of TLR2 on innate immune cells is well known, the effect of TLR2 on T cells is still not completely clear. Our study found that the expression of TLR2 on CD4+ T cells in RA patients was higher than that in healthy individuals. More importantly, in RA patients, the activation level of CD4+ T cells expressing TLR2 was significantly higher than those not expressing TLR2. Furthermore, stimulation with TLR2 agonists resulted in an increased activation level of CD4+ T cells and more TNF-α secretion. This suggests that TLR2 on CD4+ T cells may be an important triggering and enhancing factor for RA. TLR2 is the pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) in microbial species [30]. TLR2 has the capability to recognize the broadest range of PAMPs, which is particularly significant for the recognition of mycobacteria and gram-positive bacteria [31]. In addition, TLR2 can recognize damage-associated molecular patterns (DAMPs), such as oxidized phospholipids. The activation of these immune receptors leads to an increase in the expression of cytokines and chemokines, stimulating the recruitment of inflammatory cells to the tissue, which can result in chronic pathological damage. [32,33,34]. Lipid-associated membrane proteins (LAMPs) are potent inducers of the host innate immune system, can be recognized by TLR2, thereby promoting disease progression [35, 36]. Dysregulated TLR-mediated responses are characteristic of RA patients and contribute to the establishment of a chronic inflammatory state. Following the detection of conserved changes in PAMPs and DAMPs generated after cellular injury, an innate immune response is elicited. Consequently, new therapeutic and preventive strategies targeting TLRs for the treatment of RA are continuously emerging [37, 38].

Activation of TLR2 signaling can recruit myeloid differentiation factor 88 (MyD88), which is then activated through a series of intracellular signaling to activate nuclear factor-kappa B (NF-κB) and thereby induce inflammatory responses [39]. Reactive oxygen species (ROS) can act as a second messenger to activate NF-κB, amplifying inflammatory responses, or be regulated as a downstream product of NF-κB [33]. Studies have shown that TLR2 signaling activation can promote the production of ROS by peritoneal macrophages in mice [34]. We found that after TLR2 activation, the production levels of total ROS and mitochondrial ROS in RA CD4+ T cells were increased, and the increase in total ROS might be mainly due to the increase in mitochondrial ROS. Activating TLR2 did not promote the activation and pro-inflammatory functions of RA CD4+ T cells, after inhibiting mitochondrial ROS production, indicating that TLR2 might be regulating RA CD4+ T cell function in a mitochondrial ROS-dependent manner. Researches have shown that the role of ROS in RA is multifaceted. On one hand, ROS are key molecules in the signaling of T cell activation, proliferation, and differentiation, maintaining the normal physiological functions of immune cells [33]. A decrease in ROS production can lead to more severe autoimmune reactions and exacerbate chronic arthritis [40]. On the other hand, limiting macrophage ROS production can improve the symptoms of type II collagen-induced arthritis in NOX2-deficient mice [41]. Therefore, we cannot simply use antioxidants to improve RA symptoms but should target different cells and take different intervention strategies.

ROS production is closely related to cellular metabolism, with the mitochondrial respiratory chain involved in glucose oxidation being the main source of mitochondrial ROS [42]. Lipid metabolism abnormalities may also induce ROS production [43]. Therefore, cellular metabolism is a potential therapeutic target for treating RA [44]. Our results found that activating TLR2 upregulates the expression of key glycolytic enzyme HK2 and inhibits the expression of PFKFB3, leading to glucose metabolism diverting towards the pentose phosphate pathway. Previous studies have reported that glucose metabolism in CD4+ T cells of RA patients shunts towards the pentose phosphate pathway [45,46,47], and our results suggest that TLR2 activation exacerbates this phenomenon. Additionally, TLR2 activation does not seem to affect lipid metabolism in CD4+ T cells. After inhibiting HK2 with 2-DG, the production of mitochondrial ROS in CD4+ T cells is suppressed, along with the suppression of cell activation and TNF-α production, indicating that TLR2 activation can promote RA CD4+ T cell glucose metabolism through upregulating HK2, leading to increased ROS production and enhanced cell functionality. Similar findings were also observed in CD8+ T cells [48], where TLR2 can promote CD8+ T cell glucose metabolism, inducing CD8+ T cell activation and proliferation. Inhibition of HK2 can suppress CD8+ T cell activation and IFN-γ secretion [42]. However, it remains unclear whether their glucose metabolism pathway diverts towards the pentose phosphate pathway and whether ROS are involved in the regulation of CD8+ T cell function by glycolysis.

Glucose is metabolized through three pathways, which are glycolysis producing lactic acid, aerobic oxidation with oxidative phosphorylation, and the pentose phosphate pathway. It is reported that lactate level in RA synovial fluid is increased [49], however, our study did not show any significant changes in lactate production after Pam3CSK4 stimulation, indicating that TLR2 activation does not affect CD4+ T cell function by glycolysis. We used inhibitors to block the other two pathways and found that TLR2 activation mainly regulates RA CD4+ T cell activation through oxidative phosphorylation, with a smaller contribution from the pentose phosphate pathway. In contrast to T cell activation, oxidative phosphorylation or the pentose phosphate pathway inhibition did not affect TNF-α secretion. There may be two possible reasons for this: first, there is a compensation mechanism between the two pathways, resulting in no impact on TNF-α secretion when only one pathway is inhibited; second, as a pro-inflammatory factor, TNF-α production mainly depends on the activation of transcription factors such as NF-κB and activating protein-1 (AP-1), and metabolism cannot affect it. Additionally, inhibition of HK2 leads to a decrease in TNF-α secretion, possibly because inhibition of HK2 results in reduced ROS production, which can act as an upstream factor for transcription factors such as NF-κB and AP-1. The reduced ROS level inhibits the activation of these transcription factors, leading to a decrease in TNF-α production.

The article elucidated from the perspective of glucose metabolism the specific mechanisms by which TLR2 participates in the progression of RA. TLR2 activated CD4+T cells and promoted the secretion of the TNF-α cytokine through ROS mediation, thereby advancing the course of RA. But this study lacked corresponding animal experiments. Treatments targeting TLR2 were not tested in vivo in collagen-induced arthritis (CIA) models. Besides, The TLR2 agonist Pam3CSK4 is derived from PAMPs, and we did not validate its effects on other pattern molecules derived from DAMPs and LAMPS. In the future, targeted drugs against TLR receptors could be developed for the treatment of autoimmune diseases like RA, offering new avenues for consideration in the therapy of RA.

In summary, as shown in Fig. S2, our study demonstrates that TLR2 signaling can activate RA CD4+ T cells and promote the production of TNF-α by reprogramming glucose metabolism and increasing ROS. This provides new strategies for regulating cellular metabolism to intervene in the development and progression of RA, as well as reducing infections by pathogenic microorganisms to prevent or slow down the progression of RA.