Institute of Musculoskeletal Medicine, University Hospital Muenster, Albert-Schweitzer-Campus 1, Building D 3, 48149, Muenster, Germany.
Department of Trauma-, Hand- and Reconstructive Surgery, University Hospital Muenster, Albert-Schweitzer-Campus 1, Building W 1, 48149, Muenster, Germany.
Cell Death Dis. 2022 Mar 11;13(3):224. doi: 10.1038/s41419-022-04680-5.
Osteoarthritis (OA) is characterized by cartilage degradation that is induced by inflammation. Sterile inflammation can be caused by damage-associated molecular patterns that are released by chondrocytes and activate pattern recognition receptors. We evaluate the role of toll-like receptor-3-activating RNA in the pathogenesis of OA. Toll-like receptor 3 (TLR3) was detected by semiquantitative reverse transcriptase PCR, western blotting and microscopy. Rhodamine-labelled poly(I:C) was used to image uptake in chondrocytes and full-thickness cartilage. The production of IFNβ in chondrocytes after stimulation with poly(I:C) as well as in the synovial fluid of OA patients was measured using ELISA. Chondrocyte apoptosis was chemically induced using staurosporine. Immunohistochemistry was performed to examine TLR3 expression and apoptosis in human and murine OA cartilage. RNA in synovial fluid was quantified by RiboGreen assay. Destabilisation of the medial meniscus was performed in TLR3 and wildtype mice. OA was assessed after eight weeks using OARSI score. TLR3 expression was confirmed by western blot and RT-PCR. Poly(I:C) was internalised by chondrocytes as well as cartilage and caused an increase of IFNβ production in murine (11.46 ± 11.63 (wo) to 108.7 ± 25.53 pg/ml; N = 6) and human chondrocytes (1.88 ± 0.32 (wo) to 737.6 ± 130.5 pg/ml; N = 3; p < 0.001). OA cartilage showed significantly more TLR3-positive (KL0 = 0.22 ± 0.24; KL4 = 6.02 ± 6.75; N ≥ 15) and apoptotic chondrocytes (KL0 = 0.6 ± 1.02; KL4 = 9.78 ± 7.79; N ≥ 12) than healthy cartilage (p < 0.001). Staurosporine-induced chondrocyte apoptosis causes a dose-dependent RNA release (0 ng/ml = 1090 ± 39.1 ng/ml; 1000 ng/ml=2014 ± 160 ng/ml; N = 4; p < 0.001). Human OA synovial fluid contained increased concentrations of RNA (KL0-2 = 3408 ± 1129 ng/ml; KL4 = 4870 ± 1612ng/ml; N ≥ 7; p < 0.05) and IFNβ (KL0-2 = 41.95 ± 92.94 ng/ml; KL3 = 1181 ± 1865ng/ml; N ≥ 8; p < 0.05). TLR3 mice showed reduced cartilage degradation eight weeks after OA induction (OARSI WT = 5.5 ± 0.04; TLR3 = 3.75 ± 1.04; N ≥ 6) which was accompanied by gradually decreasing levels of TUNEL-positive cells (WT = 34.87 ± 24.10; TLR3 = 19.64 ± 7.89) resulting in decreased IFNβ expression (WT = 12.57 ± 5.43; TLR3 = 6.09 ± 2.07) in cartilage (p < 0.05). The release of RNA by apoptotic chondrocytes thus activating TLR3 signalling is one possible way of perpetuating inflammatory cartilage changes. The inhibition of TLR3 could be a possible therapeutic target for OA treatment.
骨关节炎（OA）的特征是软骨退化，由炎症引起。无菌性炎症可由软骨细胞释放的损伤相关分子模式引起，并激活模式识别受体。我们评估了 Toll 样受体 3 激活 RNA 在 OA 发病机制中的作用。通过半定量逆转录酶 PCR、western blot 和显微镜检测 Toll 样受体 3（TLR3）。用罗丹明标记的 poly（I：C）来观察软骨细胞和全层软骨中的摄取情况。用 ELISA 测量 poly（I：C）刺激后软骨细胞中 IFNβ的产生以及 OA 患者滑液中的 IFNβ产生。用 staurosporine 化学诱导软骨细胞凋亡。用免疫组化检测人及鼠 OA 软骨中的 TLR3 表达和凋亡。通过 RiboGreen 测定法对滑液中的 RNA 进行定量。在 TLR3 和野生型小鼠中进行内侧半月板的不稳定。八周后用 OARSI 评分评估 OA。通过 western blot 和 RT-PCR 确认 TLR3 表达。poly（I：C）被软骨细胞以及软骨摄取，并导致鼠（11.46±11.63（wo）至 108.7±25.53pg/ml；N=6）和人软骨细胞（1.88±0.32（wo）至 737.6±130.5pg/ml；N=3；p<0.001）中 IFNβ 产生增加。OA 软骨中 TLR3 阳性（KL0=0.22±0.24；KL4=6.02±6.75；N≥15）和凋亡软骨细胞（KL0=0.6±1.02；KL4=9.78±7.79；N≥12）明显多于健康软骨（p<0.001）。Staurosporine 诱导的软骨细胞凋亡导致 RNA 释放呈剂量依赖性（0ng/ml=1090±39.1ng/ml；1000ng/ml=2014±160ng/ml；N=4；p<0.001）。人 OA 滑液中含有增加的 RNA 浓度（KL0-2=3408±1129ng/ml；KL4=4870±1612ng/ml；N≥7；p<0.05）和 IFNβ（KL0-2=41.95±92.94ng/ml；KL3=1181±1865ng/ml；N≥8；p<0.05）。OA 诱导 8 周后，TLR3 小鼠的软骨降解减少（OARSI WT=5.5±0.04；TLR3=3.75±1.04；N≥6），这伴随着逐渐减少的 TUNEL 阳性细胞（WT=34.87±24.10；TLR3=19.64±7.89），导致软骨中 IFNβ 表达减少（WT=12.57±5.43；TLR3=6.09±2.07）（p<0.05）。凋亡软骨细胞释放的 RNA 通过激活 TLR3 信号从而激活 TLR3 信号是持续炎症性软骨变化的一种可能方式。TLR3 的抑制可能是 OA 治疗的一个潜在治疗靶点。
