Transcobalamin 2 orchestrates monocyte proliferation and TLR4-driven inflammation in systemic lupus erythematosus via folate one-carbon metabolism

BACKGROUND: SLE is a complex autoimmune disease with deleterious effects on various organs. Accumulating evidence has shown abnormal vitamin B12 and one-carbon flux contribute to immune dysfunction. Transcobalamin II (TCN2) belongs to the vitamin B12-binding protein family responsible for the cellular uptake of vitamin B12. The role of TCN2 in SLE is still unclear.
METHODS: We collected clinical information and blood from 51 patients with SLE and 28 healthy controls. RNA sequencing analysis, qPCR and western blot confirmed the alteration of TCN2 in disease monocytes. The correlation between TCN2 expression and clinical features and serological abnormalities was analyzed. TCN2 heterozygous knockout THP1 cells were used to explore the effects of TCN2 dysfunction on monocytes. CCK-8 assay and EdU staining were used to detect cell proliferation. ELISA was conducted to assess vitamin B12, glutathione and cytokines changes. UHPLC-MRM-MS/MS was used to detect changes in the intermediates of the one-carbon cycle. Flow cytometry is used to detect cell cycle, ROS, mitoROS and CD14 changes.
RESULTS: Elevated TCN2 in monocytes was correlated positively with disease progression and specific tissue injuries. Using CD14+ monocytes and TCN2 genetically modified THP1 cell lines, we found that the TCN2 was induced by LPS in serum from SLE patients. TCN2 heterozygous knockout inhibited cellular vitamin B12 uptake and one-carbon metabolism, leading to cell proliferation arrest and decreased Toll-like receptor 4 (TLR4)-mediated CCL2 release. Methionine cycle metabolites, s-adenosylmethionine and homocysteine, rescued these effects, whereas folate treatment proved to be ineffective. Folate deficiency also failed to replicate the impact of TCN2 downregulation on THP1 inflammatory response.
CONCLUSION: Our study elucidated the unique involvement of TCN2-driven one-carbon flux on SLE-associated monocyte behavior. Increased TCN2 may promote disease progression and tissue damage by enhancing one-carbon flux, fostering monocyte proliferation and exacerbating TLR4 mediated inflammatory responses. The inhibition of TCN2 may be a promising therapeutic approach to ameliorate SLE.

研究背景：系统性红斑狼疮（Systemic Lupus Erythematosus, SLE）是一种累及多器官的复杂自身免疫性疾病。日益积累的研究证据表明，维生素B12水平异常与一碳代谢流紊乱均可导致免疫功能紊乱。转钴胺素II（Transcobalamin II, TCN2）属于维生素B12结合蛋白家族，负责介导维生素B12的细胞摄取，但其在SLE中的作用仍未明确。
研究方法：本研究纳入51例SLE患者与28例健康对照者，收集其临床资料与血液样本。通过RNA测序、实时荧光定量PCR（quantitative real-time PCR, qPCR）与蛋白质免疫印迹（Western Blot）实验，证实了疾病状态下单核细胞中TCN2的表达异常。分析了TCN2表达水平与临床特征及血清学异常指标的相关性。采用TCN2杂合敲除的THP1细胞系，探究TCN2功能异常对单核细胞的影响。采用CCK-8实验与5-乙炔基-2'-脱氧尿苷（5-Ethynyl-2'-deoxyuridine, EdU）染色检测细胞增殖情况。采用酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）检测维生素B12、谷胱甘肽与细胞因子的水平变化。采用超高效液相色谱-多反应监测质谱联用技术（Ultra High Performance Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry, UHPLC-MRM-MS/MS）检测一碳循环中间代谢物的水平变化。采用流式细胞术检测细胞周期、活性氧（Reactive Oxygen Species, ROS）、线粒体活性氧（mitoROS）以及CD14的表达变化。
研究结果：单核细胞中TCN2的表达上调与疾病进展及特异性组织损伤呈正相关。借助CD14阳性单核细胞与TCN2基因修饰的THP1细胞系，本研究发现SLE患者血清中的脂多糖（Lipopolysaccharide, LPS）可诱导TCN2的表达。TCN2杂合敲除可抑制细胞对维生素B12的摄取与一碳代谢，进而导致细胞增殖停滞，并下调Toll样受体4（Toll-like Receptor 4, TLR4）介导的CCL2释放。甲硫氨酸循环代谢物S-腺苷甲硫氨酸（S-adenosylmethionine）与同型半胱氨酸（homocysteine）可逆转上述效应，而叶酸干预则无此效果。叶酸缺乏同样无法模拟TCN2下调对THP1细胞炎症应答的影响。
研究结论：本研究阐明了TCN2介导的一碳代谢流在SLE相关单核细胞功能异常中的独特作用。TCN2表达上调可通过增强一碳代谢流、促进单核细胞增殖以及加剧TLR4介导的炎症应答，进而推动疾病进展与组织损伤。抑制TCN2或可成为改善SLE的潜在治疗策略。