布鲁氏菌性骨关节炎中Tfh/Tfr细胞失衡特征及关节损伤相关分子的变化OA

Objective The immuno-inflammatory mechanisms underlying acute and chronic Brucella osteoarthritis(BOA)remain unclear,and the dynamic changes in T follicular helper(Tfh)cells and follicular regulatory T(Tfr)cells in this disease,as well as their associations with immune-inflammatory responses,have not been systematically reported.This study aimed to characterize the stage-specific changes in Tfh and Tfr cells in peripheral blood and synovial tissue of BOA patients,and to analyze their relationships with related cytokines,transcription factors,and immunoglobulins,thereby providing the immunological evidence for pathogenesis and clinical management of the disease.Methods A cross-sectional study design was adopted in this research.A total of 13 patients with acute BOA and 21 patients with chronic BOA,who were diagnosed in the Department of Orthopedics at the First Affiliated Hospital of Shihezi University and its medical consortium hospitals from January 2024 to August 2025,were enrolled,and another 20 healthy individuals were subjected and served as the control group.Peripheral blood samples were collected from all participants,while synovial tissue specimens were obtained from 3 chronic BOA patients and 3 patients with non-infectious joint injury undergoing surgery for local tissue-level validation,which were not included in the overall clinical statistical analysis.Flow cytometry was performed to determine the proportions of Tfh cells(CD4⁺CXCR5⁺CD25⁻CD127⁺)and Tfr cells(CD4⁺CXCR5⁺CD25⁺CD127⁻)in peripheral blood,as well as the Tfh/Tfr ratio.Serum levels of IL-4,IL-21,IL-10,TGF-β,and immunoglobulins(IgA,IgG,IgE and IgM)were measured by ELISA.The mRNA expression of Tfh-related transcription factor BCL-6 and cytokine IL-21,as well as Tfr-related transcription factor FOXP3 and cytokine IL-10,was assessed in peripheral blood mononuclear cells(PBMCs)using qRT-PCR.Western blotting was used to detect the expression of IL-21,FOXP3,and joint matrix metabolism-related proteins MMP-13,type Ⅱ collagen(COLⅡ),and ADAMTS5)in synovial tissues.Results ① In the synovial tissues,the chronic BOA group showed significantly higher protein levels of FOXP3(P=0.020 1),MMP-13(P=0.004 8),and ADAMTS5(P=0.002 3),while lower levels of IL-21(P<0.000 1)and COLⅡ(P=0.009 2)compared with the control group.② Flow cytometric analysis of peripheral blood revealed that the proportion of Tfh cells in acute-phase patients was significantly higher than that in the control group(P=0.001 4)and the chronic-phase group(P<0.000 1);so was the Tfh/Tfr ratio when compared with that in the control group(P=0.020 4)and the chronic-phase group(P<0.000 1);whereas the proportion of Tfr cells was lower than that in the control group(P=0.015 5)and the chronic-phase group(P<0.000 1).③ qRT-PCR in PBMCs showed that the mRNA levels of the Tfh-related cytokine IL-21 and transcription factor BCL-6 were upregulated in acute-phase patients than the control group(P=0.000 8,P<0.000 1);those of the cytokine IL-10 and transcription factor FOXP3 were also enhanced in chronic-phase patients compared with the control group(both P<0.000 1).④ ELISA results showed that serum levels of IL-21,IL-4,and IgG were significantly elevated in acute-phase patients,all higher than those in the control group(all P<0.000 1);these 3 indicators showed a decreasing trend in the chronic phase compared with the acute group(P<0.05).In contrast,serum IL-10 level was significantly increased in the chronic phase,higher than that in the control group(P<0.000 1)and the acute-phase patients(P=0.034 3).Serum IgA and IgM levels in patients in both acute and chronic phases were significantly higher than those in the control group(P<0.05),but no significant difference was observed between the 2 phase groups.⑤ Spearman correlation analysis indicated that,in the acute phase,the proportion of Tfh cells was positively correlated with IL-21,IgA,and IgM(r=0.26,0.34,and 0.37,respectively),and negatively with Tfr cells(r=-0.54).IL-21 showed a strong positive correlation with IgG and IgM(r=0.75 and 0.58).In the chronic phase,the above correlations overall weakened,while correlations with Tfr-related indexes exhibited stronger associations compared with the other indicators.Conclusion This study preliminarily revealed the changes in Tfh/Tfr cell imbalance in BOA,with the acute phase characterized by Tfh cells and high expression of related factors,and the chronic phase by larger proportion of Tfr cells and high expression of related factors,accompanied by pathological joint damage.