Siglec-1 介导组织巨噬细胞炎症反应参与类风湿关节炎发病的机制研究

Siglec-1 (CD169, Sn) is constitutively expressed on subpopulations of tissue-resident macrophages (Mφ). In a variety of inflammatory and autoimmune diseases, Sn is highly expressed and involved in the process of inflammation and T cell activation. rheumatoid arthritis (RA) is an chronic systemic autoimmune disease characterized by arthrosynovitis and immuno-inflammation mediated by monocyte/macrophage is particularly important in the initiation and progression of the disease. But the role of Sn in RA has not been elucidated. Our preliminary study found that Sn was highly expressed on peripheral blood monocyte surface in RA patients and its expression was positively correlated with disease severity and inflammatory activity. We also found that type Ⅱ collagen can up-regulate the expression of Siglec-1, enhance IL-1β, IFN-γ and TNF-α secretion and active the NF-κb pathway. Whereas siRNA of Siglec-1 can decrease these cytokines secretion and signaling pathway activation. Therefore, we speculate that Sn may participate in the pathogenesis of RA through synovial macrophage inflammation, pro-inflammatory cytokines secretion and T cell activation. Therefore, we intend to perform our experiments by three levels which including peripheral blood analysis, cell culture and animal experiments, with either Siglec-1 inhibition or elevation, to explore the role of Siglec-1 in RA and to investigate its possible mechanism. We hope our study will shed new light on the prevention and treatment of rheumatoid arthritis.

Siglec-1（CD169，Sn）表达于特定组织巨噬细胞（Mφ）胞膜，在多种炎症和自身免疫性疾病中表达增加，参与介导炎症反应和T 细胞活化。类风湿关节炎（RA）是一种以关节滑膜炎为特征的慢性全身性自身免疫性疾病，单核巨噬细胞在免疫炎症的发生发展中起关键作用，但Sn在RA中的作用尚未见报道。我们前期研究发现，RA患者外周血单核细胞膜Sn表达增加，并与疾病严重程度和活动度呈正相关，Ⅱ型胶原能刺激其表达上调，分泌IL-1β、IFN-γ、TNF-α等炎症因子增加，NF-κb信号通路活化，而RNA干扰巨噬细胞Sn表达后上述作用减弱。因此，我们推测：Sn可能通过参与Mφ活化并介导滑膜炎症反应，分泌促炎性细胞因子，激活T 淋巴细胞等过程参与RA发病。为此，本研究拟从外周血表型分析，细胞培养和动物实验三个层次，Sn表达抑制和增强两方面对其在RA发病中的作用及机制进行研究，为RA的预防和治疗提供新的思路。

Siglec-1（Sn）表达于特定组织巨噬细胞（Mφ）胞膜，在多种炎症和自身免疫性疾病中介导了炎症反应和T细胞活化。类风湿关节炎（RA）是一种以关节滑膜炎为特征的慢性全身性自身免疫性疾病，单核巨噬细胞在RA发病中起关键作用，但siglec-1在RA发病中作用研究甚少。本项目通过外周血表型分析，细胞培养和动物实验三个层次，siglec-1表达抑制和增强两方面对其在RA中的作用及机制进行了较为详细的研究，结果发现：（1）Siglec-1蛋白和mRNA在RA患者外周血表达升高，明显高于骨关节炎组和健康对照组。并且RA患者Siglec-1的表达与疾病活动度评分DAS28以及炎症程度指标hs-CRP之间均呈正相关（r=0.89, P<0.001；r=0.48, P<0.001）。（2）成功利用组织块贴壁法培养滑膜成纤维细胞并鉴定表型。（3）Ⅱ型胶原、IFN-γ和TNF-α均可以浓度依赖性刺激骨髓巨噬细胞、腹腔巨噬细胞和RAW264.7细胞表达Siglec-1。（4）通过单核细胞和淋巴细胞共培养，加入抗Siglec-1抗体或si-RNA靶向干扰Siglec-1表达能降低RA患者巨噬细胞刺激CD4+、CD8+ T淋巴细胞增殖能力和分泌炎性细胞因子IFN-γ和TNF-α。（5）通过关节腔注射+尾静脉注射联合注射Lv-shSiglec-1慢病毒载体对Siglec-1靶向抑制后，能明显减轻小鼠关节腔炎症情况，减少炎症细胞浸润，减轻关节滑膜破坏程度。免疫组化染色发现小鼠关节腔T、B、巨噬细胞、成纤维细胞等浸润减少。（6）高通量细胞因子检测发现Lv-shSiglec-1小鼠血浆IL-1a、IL-6、Eotaxin、CXCL1、CXCL9表达明显降低，与Lv-shNC组或生理盐水组相比，差异具有统计学意义(P<0.05)。关节引流淋巴结T细胞增殖和分泌IL-17a、CXCL1、MCP-1、CXCL9、MCP-3炎性细胞因子和趋化因子能力减弱。综上，对Siglec-1在类风湿关节炎中的发病机制研究将有助于为临床治疗RA提供新的思路。