A total of 51 peripheral blood samples were collected from patients diagnosed with SLE, along with 28 peripheral blood samples from age-matched healthy individuals, following the receipt of informed consent. The SLEDAI-2K scale was used for disease activity score, ≥ 7 was classified as active SLE (20). Detailed patient information is listed in Supplementary Table 1 . This study obtained approval from the regional ethics committee and adhered to the principles outlined in the Declaration of Helsinki. Peripheral blood samples were collected using EDTA-containing anticoagulation blood vessels, centrifuged at 1500 rpm, centrifuged for 5 min, and the upper serum was stored at -80°C; meanwhile, the precipitated layer was used to isolated peripheral blood mononuclear cells (PBMC) or CD14+ monocytes.

PBMCs were isolated by density gradient centrifugation utilizing Ficoll-Paque™ (GE Healthcare, USA). Briefly, blood samples were diluted with sterile PBS and gently layered atop the Ficoll-Paque, then centrifuged at 1800 rpm for 20 minutes with the brake off at 20°C, then PBMC were collected from the interface. CD14+ monocytes were isolated using StraightFrom^® Whole Blood CD14 MicroBeads (Miltenyi Biotec, USA). Blood samples were incubated with CD14 magnetic beads for 15 minutes and then separated using a MACS column placed within a magnetic field. After two rinses with separation solution, CD14+ cells were collected with eluent buffer. The human monocytic cell line THP1 cells were obtained from the National Biomedical Experimental Cell Resource Bank (China). TCN2 heterozygous knockout THP1 cell (TCN2-KO cell) was purchased from UBIGENE (China). The knockout of TCN2 was achieved by CRISPR technology. Since the coding region sequence of exon 1 of TCN2 is too short, the gRNA targeting position was shifted to the most anterior region of exon 2. Exon 2 is well-organized among all coding transcripts, and targeting it affects all coding transcripts. Therefore, we deleted the fragment on exon 2 using double gRNA guidance. We confirmed the genotype of TCN2 KO cells through sanger sequencing. The results showed that of the two alleles, one gene was successfully knocked out and the other was a multiple of three genotypes ( Supplementary Figure 1 ). Cells were cultured in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C with 5% CO2. LPS (L4391) and S-adenosylmethionine (SAM, A7007) were purchased from Sigma (USA), N-acetylcysteine (NAC, HY-B0215), Hcy (homocysteine, HY-W010347) were purchased from MCE (China), and FA (IF0180) was purchased from Solarbio (China).

To establish normal, FA-deficient, or FA-supplemented conditions, THP-1 cells were cultured for 3 days in either normal RPMI-1640 medium (supplemented with 1 μg/ml FA) or FA-free RPMI-1640 medium (27016021, Thermo Fisher Scientific, USA) or by adding FA (with final concentration of 10 μg/ml FA) to normal RPMI-1640 medium (IF0180, Solarbio, China).

Cells were seeded into 96-well plates (5000 cells/well) and cultured for 0 to 5 days. To assess cell proliferation, 10 µl of CCK-8 reagent (Yeasen, China) was added to each well containing 100 µl of medium. The plates were then incubated at 37°C for 2 hours, after which the optical density (OD) was measured at 450 nm using a microplate reader (Thermo Scientific, USA). The BeyoClick™ EdU Cell Proliferation Kit (Beyotime, China) was utilized for EdU staining according to the manufacturer’s instructions. In brief, cells were incubated with a 10 μM EdU solution for 6 hours, then centrifugated at 300g for 5 minutes to collect the cells. Subsequently, the cells were fixed at room temperature in 4% paraformaldehyde for 15 minutes, washed with PBS three times, permeabilized with 0.3% Triton X-100 in PBS for 10 minutes, and incubated in Click Additive Solution in the dark for 30 minutes. Cell nuclei were stained with Hoechst for 10 minutes. For cell cycle analysis, cells (1×10⁶ cells/well) were incubated with DNA labeling solution (CYT-PIR-25, Cytognos, Spain) in the dark for 10 minutes at room temperature. Subsequently, cell cycle data were acquired using a flow cytometer (BD Calibur, USA) operating at a low flow rate.

THP-1 cells (1×10⁶ cells/well) were seeded in 12-well plates. After treatment, cells were exposed to 10 mM diacetyldichlorofluorescein (Beyotime, China) and 50 nM MitoSOX (MCE, China) for 30 minutes at room temperature, and washed twice with PBS. Cell surface markers were determined by direct staining with PerCP anti-human CD14 antibodies (301847, Biolegend) following the manufacturer’s protocol. ROS, mitoROS and CD14 levels were assessed using flow cytometry (Beckman, CytoFLEX, USA) and analyzed using FlowJo software (Version 10.8.1).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA), following the manufacturer’s instructions. Subsequently, the extracted RNA was reverse-transcribed into cDNA using Hifair III 1st strand cDNA Synthesis SuperMix (Yeasen, China). The relative expression levels of various target genes were quantified by q-PCR employing Hieff UNICONT^® Universal Blue qPCR SYBR Master Mix (Yeasen, China). For normalization, gene expression was referenced to ACTB. The primer sequences for qPCR are listed in Supplementary Table 2 .

Approximately 20 µg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). Following a 1-hour block in 5% bovine serum albumin (Yeasen, China), the membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hour. β-actin was employed as a loading control. The bound antibodies were visualized using the enhanced chemiluminescence (ECL) western blotting detection system (Merck Millipore, USA). The information for primary antibodies used in the experiment is listed in Supplementary Table 3 .

After treatment, the cells were washed with pre-cooled PBS, then fixed with 4% paraformaldehyde for 10 minutes, and dropped on a slide to dry. Pap Pen circled cell location, PBS washed cells three times. The cells were further permeabilized with 0.3% Triton X-100 in PBS for 20 minutes, blocked with 3% BSA for 30 minutes at room temperature, then incubated with anti-pp65 (1:500) antibody at 4°C overnight, incubated with Alexa Fluor 488 secondary antibody (1:1000, Abcam) for 1 hour at room temperature. Nuclei were stained with DAPI. Finally, images were captured using a Leica X10 Confocal (Mannheim, German).

THP-1 cells (6 × 10⁶ cells/well) were collected. Cells were frozen in liquid nitrogen and were thawed in 37°C water bath. Repeated the freeze-thaw cycle 3 times and stored at -80°C before use. UHPLC-MRM-MS/MS was performed by Shanghai Biotree Biotech Limited Company (China). The abundance of metabolites was measured and compared to internal standard controls according to protocols.

After treatment for 48 hours, the cell culture supernatant was collected to measure the release of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1/CCL2) and CXCL10. A simple plex assay, a multiplex biomarker immunoassay panel cartridge (ProteinSimple, Bio-Techne, SPCKE-PS-009400), was used to measure the content of these cytokines in the serum samples according to the manufacturer’s instructions. Briefly, the samples were removed from the freezer, thawed, diluted with sample buffer diluent at a 1:1 ratio, pipetted, mixed well, centrifuged to remove visible particles, added to the samples, and analyzed with the Ella platform (ProteinSimple, Bio-Techne).

The LPS, vitamin B12 level, and glutathione/oxidized glutathione (GSH/GSSG) content were measured using the LPS (E09945h, CUSABIO), vitamin B12 ELISA kit (E07903h, CUSABIO) and GSH and GSSG (S0053, Beyotime), following the manufacturer’s instructions. After treatment, the cell culture supernatant and cells were separated by centrifugation at 1000g for 5 minutes. The cells were washed once with cold PBS and diluted to a concentration of 100 million/ml, after which the cells and supernatant were placed at -20°C overnight and detected the vitamin B12 and GSH/GSSG radio according to manufacturer’s protocol.

Microarray datasets (GSE72509 and GSE112087) and the SLE patient monocyte RNA-seq dataset (GSE164457) were collected from the Gene Expression Omnibus database. The large-scale bulk transcriptome study of 27 immune cell types for SLE was obtained from the National Bioscience Database Center Human Database (Dataset ID: JGAS000486) (21). Microarray datasets were integrated and subjected to differential expression analysis using the limma package. Correlation analysis of TCN2 expression with clinical symptoms, clinical test results, and TLR4 expression, as well as ISGs score, were performed using the ggstatsplot and BayesFactor packages in the R environment.

Experimental results are expressed as mean ± SD and were subjected to unpaired Student’s t-test, one-way ANOVA and (or) two-way ANOVA, followed by Tukey’s or Dunnett’s multiple comparisons posttest. All analyses of experimental data were executed using GraphPad Prism 9 software (San Diego, USA). Statistical significance was established at p < 0.05.

We identified elevated TCN2 expression in individuals with SLE through the analysis of public available microarray datasets (GSE72509 and GSE112087) and our RNA-seq analysis ( Figures 1A, B ) (22), while vitamin B12 transporter TCN1 of the same family was not ( Supplementary Figure 2 ) (16). Subsequently, qPCR results confirmed the upregulation of TCN2 in PBMC from SLE patients compared to healthy control (HC, Supplementary Figure 3A ). To pinpoint the cells responsible for TCN2 expression changes in PBMC, we conducted a search using a large-scale bulk transcriptome study of 27 immune cell types for SLE (21). The results indicated a significant elevation of TCN2 expression in non-transforming memory B cells from inactive SLE patients compared to healthy individuals. More importantly, TCN2 expression in all types of monocytes was notably increased in patients with high disease activity compared to those with inactive SLE ( Figure 1C ), implying the potential involvement of TCN2 in SLE progression through the regulation of monocyte function.

To clarify changes in TCN2 expression in monocytes of SLE patients, we isolated monocytes from both SLE patients and HC. We confirmed that TCN2 was significantly upregulated in SLE patients, particularly in those with active SLE ( Figure 1D , Supplementary Figure 3B ). We then investigated the correlation between monocyte TCN2 expression and disease progression as well as tissue damage. The expression of TCN2 in monocytes of SLE patients showed a positive correlation with SLEDAI-2K score, and a negative association with complement C3 and C4 levels ( Figure 1E ). Patients were divided into high and low groups based on TCN2 expression, and the high group exhibited higher SLEDAI-2K scores and lower complement C3 and C4 levels ( Figure 1F ). In addition, TCN2 expression was found to be associated with certain symptoms such as alopecia, elevated urinary proteins (>0.5 g/24 hours), arthritis, skin rash, light sensitivity, and various autoantibodies including anti-dsDNA, SM, RNP, histone, and nucleosome antibody ( Figure 1G ). Patients exhibiting these symptoms or autoantibodies displayed higher TCN2 expression in monocytes ( Figure 1H , Supplementary Figure 3C ). Taken together, monocytes from patients with SLE exhibit elevated expression of TCN2, which is associated with disease activity and involvement of the skin, kidneys, and joints.

To investigate the reasons for the upregulation of TCN2 expression, CD14+ monocytes were isolated from HC and treated with serum from patients with active SLE. Increased TCN2 expression was observed after treatment with serum from SLE patients ( Figure 2A ).

LPS is a classical TLR4 agonist that has been reported increased in SLE patients (23, 24). Monocytes are one of the major cells responsible for TLR4-mediated inflammatory responses (3, 25). Elevated LPS may promote TCN2 expression in monocytes. We analyzed the correlation between TCN2 and TLR4 in the transcriptome of SLE patients and found a positive association between TCN2 and TLR4 levels ( Figure 2B ). Additionally, LPS level was elevated in the serum of SLE patients and was positively correlated with TCN2 expression in monocytes (P=8.34e-03, R² = 0.47), as determined by ELISA and correlation analysis ( Figure 2C ). Furthermore, the mRNA and protein level of TCN2 increased in monocytes after direct treatment with LPS ( Figures 2D, E ). These results confirmed that increased LPS induces monocytes to upregulate TCN2 expression in SLE patients.

To explore the role of TCN2 in vitro, heterozygous knockout TCN2 THP1 cell was constructed ( Figure 3A ). The qPCR and western blot analysis confirmed successful downregulation of TCN2 in THP1 cells ( Figures 3B, C ).

The CCK-8 assay indicated that TCN2-KO cells exhibited slower proliferation compared to wild-type (TCN2-WT) cells ( Figure 3D ). The heterozygous knockout of TCN2 resulted in reduced DNA replication ( Figure 3E ) and G2/M phase arrest ( Figure 3F ). This suggests that cells carry out DNA replication and repair during the mitosis phase. Furthermore, western blot analysis indicated that TCN2-KO cells exhibited reduced expression of the checkpoint protein CDK1/2 and increased expression of the suppressor protein p21 compared to TCN2-WT cells ( Figure 3G ).

We further evaluated the impact of TCN2 heterozygous knockout on cytokines and proinflammatory factors related to disease progression. As depicted in Figure 4A , TNFA and IL6 mRNA increases were not induced in TCN2-KO cells after LPS treatment. Although LPS induced the upregulation of CCL2 and CXCL10 expression in TCN2-KO cells, their expression levels were significantly lower than those in LPS-induced TCN2-WT cells. More importantly, Ella results confirmed that TCN2-KO reduced the release of CCL2, CXCL10, IL6, and TNFα induced by LPS. The levels of these cytokines after LPS stimulation were not significantly different from those before treatment ( Figure 4B ).

Furthermore, TCN2 heterozygous knockout significantly reduced the phosphorylation of nuclear factor kappa B (NF-κB) p65, as well as the nuclear translocation of pp65 under LPS stimuli ( Figures 4C, D ). These results suggested that TCN2 heterozygous knockout alleviates LPS-induced inflammatory response.

In most cells of the human body, vitamin B12 binds to TCN2 and mediates endocytic uptake through CD320 (26). We subsequently assessed vitamin B12 levels and the transfer factor CD320 in TCN2 KO cells. As illustrated in Figures 5A and B , heterozygous TCN2 knockout resulted in a decreased cellular vitamin B12 and CD320 expression, indicating that vitamin B12 absorption was inhibited. In addition, MTR and MTRR were downregulation in TCN2-KO cells, implying an inhibition in conversion of homocysteine to methionine ( Figure 5C ). We then assessed changes in catalytic enzyme and metabolite levels in one-carbon metabolism. As expected, the qPCR analysis identified a significant downregulation of enzymes involved in the folate cycle and methionine cycle after TCN2 dysfunction ( Supplementary Figure 4 ). Consistently, metabolites from the one-carbon pathway were lower in TCN2-KO cells than in TCN2-WT cells ( Figures 5D, E ), suggesting impaired folate one-carbon metabolism ( Figure 5F ).

GSH is an essential downstream metabolite of the methionine cycle which functions as an antioxidant (27). We evaluated the alterations in GSH and ROS after TCN2 knockout. We found a recognizable decrease in GSH and GSH/GSSG levels, coupled with an increased ROS and mitoROS, but no significant increase in inflammatory factors expression (CCL2, CXCL10, IL-6, and TNFA) ( Figures 5G–I ), suggesting that a slight increase in ROS does not trigger the release of inflammatory factors in TCN2-KO cells. Furthermore, the addition of NAC, a precursor to GSH, resulted in a significantly reduced ROS, mitoROS, and expression of CCL2 and TNFA in TCN2-KO cells ( Figures 5H, I ). LPS can induce massive ROS production (28). Importantly, GSH levels, GSH/GSSG ratios, and ROS remained stable after LPS treatment in TCN2-KO cells ( Figures 5J, K ), suggesting that TCN2 knockdown prevented reactive ROS elevation and GSH depletion. CD14 acts as a co-receptor for LPS and regulates cellular TLR4 signaling. In line with this, TCN2-KO cells did not exhibit a noticeable increase in CD14 expression under LPS stimuli, whereas TCN2-WT cells showed a significant increase ( Figure 5L ).

Folate one-carbon metabolism includes the folate cycle and the methionine cycle. To investigate whether TCN2 regulates monocyte function by affecting folate one-carbon metabolism, we treated TCN2-KO cells with the support of the folate cycle (FA) or the methionine cycle (SAM) ( Figure 6A ). FA enters cells through its transporters (29). We first examined whether TCN2 KO affects FA transport. After TCN2 inhibition, the folate cycle is inhibited, which reduces the cell’s demand for FA and downregulates the folate transporter (FORLR2 and SLC19A1). However, they increased to normal levels after FA supplementation ( Supplementary Figure 5 ), suggesting that folate absorption was less affected by TCN2 knockout.

Subsequently, we assessed cell proliferation and found SAM treatment reduced the G2/M phase rate and promoted cell proliferation in TCN2-KO cells. In contrast, FA treatment did not significantly affect the proliferation of TCN2-KO cells ( Figures 6B–D ). These findings suggested that TCN2 knockout suppresses cell proliferation by inhibiting the methionine cycle. Furthermore, we detected the release of inflammatory factors induced by LPS. Figure 6E showed an increase in CCL2 secretion induced by LPS after SAM supplementation, while CXCL10 expression was further inhibited. IL6 and TNF levels remained unchanged. The release of CCL2 is dependent on ROS (30, 31). As expected, TCN2-KO cells with SAM pretreatment showed a significant increase in LPS-induced ROS and GSH consumption, coupled with upregulation of CD14 ( Figures 6F–G ). Consistently, Hcy inhibited TCN2-mediated suppression of ROS and mitochondrial ROS, while increasing CD14 ( Supplementary Figure 6 ). Furthermore, the changes in inflammatory factors after FA supplementation were not significantly different from those in the control group. These findings suggested that TCN2 enhances LPS-induced CCL2 release by regulating the methionine cycle rather than the folate cycle ( Figure 6H ).

Folate has been implicated in the inflammatory response (32). We investigated whether folate deficiency could mimic the effects of TCN2 on monocyte cells by depleting THP1 cells of folate using FA-free medium ( Figure 7A ).

As anticipated, folate depletion resulted in decreased cellular expression of vitamin B12 and TCN2 ( Figures 7B, C ). In line with previous research (9), FA depletion caused cell growth retardation, while FA supplementation did not significantly affect cell proliferation ( Figures 7D, E , Supplementary Figures 7A, B ). However, folate deficiency induced a stronger inflammatory response, as evidenced by elevated CD14 expression and increased release of CCL2 and TNFα in cells with low folate levels compared to those with normal levels. Additionally, pretreatment with FA did not result in a reduced response to LPS stimulation ( Figures 7F, G , Supplementary Figure 7C ). Moreover, folate-deficient cells exhibited a significant increase in ROS and mitoROS compared to cells with folate supplements and the normal folate group under LPS stimuli ( Figures 7H, I ). These findings suggest that the cellular response under FA deficiency differs from the impact of TCN2 inhibition, emphasizing the intricate role of TCN2 in modulating cellular responses.