背景:类风湿性关节炎(RA)是一种自身免疫性疾病,其特征是关节的炎症反应和组织损伤。临床长期用药常伴有不良反应。体外光分离疗法(ECP)是一种副作用少的免疫调节疗法,通过诱导免疫耐受,为RA提供了一个潜在的和安全的治疗替代方案。本研究旨在利用胶原诱导的关节炎(CIA)模型研究ECP对RA的治疗作用。以及探讨其在体内的免疫调节作用。此外,我们特别关注单核细胞在ECP过程中的重要作用.
方法:通过向DBA/1J小鼠施用两次注射牛II型胶原来建立类风湿性关节炎的小鼠模型。ECP,ECP-MD(在ECP期间单个核细胞被耗尽),MTX,和PBS处理应用于CIA小鼠。在治疗过程中,密切监测CIA小鼠的临床评分和体重变化.经过六次治疗,捕获来自活小鼠的后爪的显微CT图像。收集小鼠的踝关节和爪子并处理用于组织学评价。收集脾脏样本以测量Th17/Treg细胞比率,收集血清样品以评估细胞因子和抗II型胶原IgG水平。流式细胞术检测体外ECP前后的单核细胞和树突状细胞群。
结果:ECP治疗可显着减弱CIA的进展,减轻CIA小鼠临床症状的严重程度,有效抑制滑膜增生,炎症,和软骨损伤。体内CD3+CD4+CD25+FoxP3+Tregs的百分比增加,CD3+CD4+IL17A+Th17细胞减少。此外,ECP降低血清促炎细胞因子IL-6水平(53.47±7.074pg/mLvs5.142±1.779pg/mL,P<0.05)和IL-17A(3.077±0.401pg/mLvs0.238±0.082pg/mL,P<0.0001)与PBS相比。有趣的是,ECP过程中单核细胞的消耗未导致CIA小鼠的临床症状或组织学评分得到任何改善.此外,Th17/Treg细胞比例的失衡变得更加明显,伴随着促炎细胞因子IL-6和IL-17A的分泌增加。体外,与未经ECP处理的细胞相比,CD11b+细胞比例显著降低(P<0.01),ECP处理24h后CD11c+细胞比例显著升高(P<0.001)。此外,MHCⅡ的表达(P<0.0001),CD80(P<0.01),ECP处理24h后CD11c+细胞中CD86下调(P<0.001)。
结论:我们的研究表明,ECP在CIA小鼠中表现出与常规疗法相当的治疗效果,ECP对RA的保护机制涉及Th17/Treg细胞比例,这导致IL-6和IL-17A减少。值得注意的是,来自CIA小鼠的单核细胞是ECP治疗疗效不可或缺的部分,体外ECP处理后,单核细胞比例降低,耐受性树突状细胞比例增加。
Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory reactions and tissue damage in the joints. Long-term drug use in clinical practice is often accompanied by adverse reactions. Extracorporeal photopheresis (ECP) is an immunomodulatory therapy with few side effects, offering a potential and safe therapeutic alternative for RA through the induction of immune tolerance. This study aimed to investigate the therapeutic effects of ECP on RA using a collagen-induced arthritis (CIA) murine model, as well as to explore its immunomodulatory effects in vivo. Additionally, particular attention was given to the significant role of monocytes during the ECP process.
A murine model of rheumatoid arthritis was established by administering two injections of bovine type II collagen to DBA/1J mice. ECP, ECP-MD (mononuclear cells were depleted during the ECP), MTX, and PBS treatment were applied to the CIA mice. During the treatment process, clinical scores and body weight changes of CIA mice were closely monitored. After six treatment sessions, micro-CT images of the hind paws from live mice were captured. Ankle joints and paws of the mice were collected and processed for histological evaluation. Spleen samples were collected to measure the Th17/Treg cells ratio, and serum samples were collected to assess cytokine and anti-type II collagen IgG levels. Monocytes and dendritic cells populations before and after ECP in vitro were detected by flow cytometry.
ECP therapy significantly attenuated the progression of CIA, alleviated the severity of clinical symptoms in CIA mice and effectively suppressed synovial hyperplasia, inflammation, and cartilage damage. There was an expansion in the percentage of CD3 + CD4 + CD25 + FoxP3 + Tregs and a decrease in CD3 + CD4 + IL17A + Th17 cells in vivo. Furthermore, ECP reduced the serum levels of pro-inflammatory cytokines IL-6 (53.47 ± 7.074 pg/mL vs 5.142 ± 1.779 pg/mL, P < 0.05) and IL-17A (3.077 ± 0.401 pg/mL vs 0.238 ± 0.082 pg/mlL, P < 0.0001) compared with PBS. Interestingly, the depletion of monocytes during the ECP process did not lead to any improvement in clinical symptoms or histological scores in CIA mice. Moreover, the imbalance in the Th17/Treg cells ratio became even more pronounced, accompanied by an augmented secretion of pro-inflammatory cytokines IL-6 and IL-17A. In vitro, compared with cells without ECP treatment, the proportion of CD11b + cells were significantly reduced (P < 0.01), the proportion of CD11c + cells were significantly elevated (P < 0.001) 24 h after ECP treatment. Additionally, the expression of MHC II (P < 0.0001), CD80 (P < 0.01), and CD86 (P < 0.001) was downregulated in CD11c + cells 24 h after ECP treatment.
Our study demonstrates that ECP exhibits a therapeutic effect comparable to conventional therapy in CIA mice, and the protective mechanisms of ECP against RA involve Th17/Treg cells ratio, which result in decreased IL-6 and IL-17A. Notably, monocytes derived from CIA mice are an indispensable part to the efficacy of ECP treatment, and the proportion of monocytes decreased and the proportion of tolerogenic dendritic cells increased after ECP treatment in vitro.