Abstract：
OBJECTIVE: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation.
METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors.
RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts.
CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.
METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors.
RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts.
CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.
摘要：
目的：单核细胞衍生的树突状细胞（DC）是诱导炎症的关键参与者，类风湿关节炎（RA）的自身反应性T细胞活化和耐受性丧失，但是它们激活的确切机制仍然难以捉摸。这里,我们假设RA滑液中释放的细胞外microRNAs可能代表了一种新的，生理刺激通过表达TLR8的DC刺激触发不需要的免疫应答。
方法:用富含GU的miRNA在RA组织中上调并在滑液中释放的混合物(Ex-miRNA)刺激人单核细胞来源的DC。通过Westernblot根据NF-κB激活评估DC的激活，通过ELISA，细胞因子产生T细胞增殖和极化受同种异体混合淋巴细胞反响。根据抗酒石酸酸性磷酸酶的产生和牙本质切片中吸收凹坑的形成来评估DC向破骨细胞的分化。使用DC诱导的关节炎的鼠模型评估体内关节炎症的诱导。通过特异性抑制剂评估TLR7/8参与。
结果：Ex-miRNA激活DC分泌TNFα，诱导关节炎症，启动早期自身免疫反应并增强DC向侵袭性破骨细胞的分化。
结论：这项工作证明了在RA关节中过表达的细胞外miRNAs库可以通过刺激人DC表达的TLR8作为炎症的生理激活剂，进而发挥关节功能。在这种情况下，药物抑制TLR8可能为减少RA的炎症和破骨细胞介导的骨破坏提供新的治疗选择.
方法:用富含GU的miRNA在RA组织中上调并在滑液中释放的混合物(Ex-miRNA)刺激人单核细胞来源的DC。通过Westernblot根据NF-κB激活评估DC的激活，通过ELISA，细胞因子产生T细胞增殖和极化受同种异体混合淋巴细胞反响。根据抗酒石酸酸性磷酸酶的产生和牙本质切片中吸收凹坑的形成来评估DC向破骨细胞的分化。使用DC诱导的关节炎的鼠模型评估体内关节炎症的诱导。通过特异性抑制剂评估TLR7/8参与。
结果：Ex-miRNA激活DC分泌TNFα，诱导关节炎症，启动早期自身免疫反应并增强DC向侵袭性破骨细胞的分化。
结论：这项工作证明了在RA关节中过表达的细胞外miRNAs库可以通过刺激人DC表达的TLR8作为炎症的生理激活剂，进而发挥关节功能。在这种情况下，药物抑制TLR8可能为减少RA的炎症和破骨细胞介导的骨破坏提供新的治疗选择.
