Chen Xueting, Du Rong, Wang Pan, Qiu WenLin, Chen Lu, Wan Jian, Qiu Hui, Xiong Li, Nandakumar Kutty Selva, Holmdahl Rikard, Geng Hui
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, China.
Department of Rheumatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Front Immunol. 2025 Apr 9;16:1563426. doi: 10.3389/fimmu.2025.1563426. eCollection 2025.
Carbamylated proteins and dysregulated neutrophils are implicated in rheumatoid arthritis (RA) pathogenesis. Herein, we characterized the neutrophils present in RA synovial fluid (SF) using proteomic techniques and evaluated their contribution to protein carbamylation.
RA-SF neutrophil proteomic profile and SF proteome signature were investigated using high-resolution mass spectrometry. Carbamylated proteins and the degree of protein carbamylation were evaluated by mass spectrometric analysis. ELISA and chemiluminescence kits were used to examine myeloperoxidase (MPO) activity, and hydrogen peroxide (HO) generation.
SF neutrophils exhibited a shift in proteomic cargo with up-regulated proteins involved in defense responses, neutrophil degranulation, and reactive oxygen species metabolic processes, while proteins down-regulated were associated with megakaryocyte differentiation, leukocyte migration, and integrin-mediated signaling pathway. Elevated levels of neutrophil-derived proteins were detected in RA-SF. In addition, we specifically identified many carbamylated proteins and observed an increased frequency of protein carbamylation in RA-SF samples. Functionally, neutrophils from RA-SF showed a significantly increased level of MPO release and HHO generation. Moreover, MPO activity was higher in RA-SF than in autologous blood samples, which correlated well with the degree of protein carbamylation in RA-SF.
Synovial neutrophils were found to be activated and increased releasing protein cargo, including MPO and ROS, into the synovial fluid. Presence of many carbamylated proteins in RA-SF and an increased MPO activity showed a strong correlation to the degree of protein carbamylation, suggesting neutrophil-derived MPO in promoting generation of aberrantly carbamylated proteins.
氨甲酰化蛋白和失调的中性粒细胞与类风湿性关节炎（RA）的发病机制有关。在此，我们使用蛋白质组学技术对RA滑膜液（SF）中的中性粒细胞进行了表征，并评估了它们对蛋白质氨甲酰化的作用。
使用高分辨率质谱研究RA-SF中性粒细胞蛋白质组图谱和SF蛋白质组特征。通过质谱分析评估氨甲酰化蛋白和蛋白质氨甲酰化程度。使用ELISA和化学发光试剂盒检测髓过氧化物酶（MPO）活性和过氧化氢（HO）生成。
SF中性粒细胞的蛋白质组货物发生了变化，参与防御反应、中性粒细胞脱颗粒和活性氧代谢过程的蛋白质上调，而下调的蛋白质与巨核细胞分化、白细胞迁移和整合素介导的信号通路有关。在RA-SF中检测到中性粒细胞衍生蛋白水平升高。此外，我们特异性鉴定了许多氨甲酰化蛋白，并观察到RA-SF样本中蛋白质氨甲酰化频率增加。在功能上，来自RA-SF的中性粒细胞显示MPO释放水平和HHO生成显著增加。此外，RA-SF中的MPO活性高于自体血样本，这与RA-SF中蛋白质氨甲酰化程度密切相关。
发现滑膜中性粒细胞被激活并增加向滑膜液中释放蛋白质货物，包括MPO和ROS。RA-SF中许多氨甲酰化蛋白的存在以及MPO活性增加与蛋白质氨甲酰化程度密切相关，表明中性粒细胞衍生的MPO促进异常氨甲酰化蛋白的生成。
