Objective

Joint injury-induced perturbations to the endocannabinoid system (ECS), a regulator of both inflammation and nociception, remain largely uncharacterized. We employed a mouse model of ACL rupture to assess alterations to nociception, inflammation, and the ECS while using in vitro models to determine whether CB2 agonism can mitigate inflammatory signaling in macrophages and fibroblast-like synoviocytes (FLS).

Design

Mice underwent noninvasive ACL rupture (ACLR) via tibial compression-based loading. Nociception was measured longitudinally using mechanical allodynia and knee hyperalgesia testing. Synovitis was assessed using histological scoring and histomorphometry. Gene and protein markers of inflammation were characterized in whole joints and synovium. Immunohistochemistry assessed injury-induced alterations to CB1+, CB2+, and F4/80+ cells in synovium. To assess whether CB2 agonism can inhibit pro-inflammatory macrophage polarization, murine bone marrow-derived macrophages (mBMDM) were stimulated with IL-1β or conditioned medium from IL-1β-treated FLS and treated with vehicle (DMSO), the CB2 agonist HU308, or cannabidiol (CBD). Macrophage polarization was assessed as the ratio of M1-associated (IL1b, MMP1b, and IL6) to M2-associated (IL10, IL4, and CD206) gene expression. Human FLS (hFLS) isolated from synovial tissue of OA patients were treated with vehicle (DMSO) or HU308 following TNF-α or IL-1β stimulation to assess inhibition of catabolic/inflammatory gene expression.

Results

ACLR induces synovitis, progressively-worsening PTOA severity, and an immediate and sustained increase in both mechanical allodynia and knee hyperalgesia, which persist beyond the resolution of molecular inflammation. Enrichment of CB2, but not CB1, was observed in ACLR synovium at 3d, 14d, and 28d, and CB2 was found to be associated with F4/80 (+) cells, which are increased in number in ACLR synovium at all time points. The CB2 agonist HU308 strongly inhibited mBMDM M1-type polarization following stimulation with either IL-1β or conditioned medium from IL-1β-treated mFLS, which was characterized by reductions in Il1b, Mmp1b, and Il6 and increases in Cd206 gene expression. Cannabidiol similarly inhibited IL-1β-induced mBMDM M1 polarization via a reduction in Il1b and an increase in Cd206 and Il4 gene expression. Lastly, in OA hFLS, HU308 treatment inhibited IL-1β-induced CCL2, MMP1, MMP3, and IL6 expression and further inhibited TNF-α-induced CCL2, MMP1, and GMCSF expression, demonstrating human OA-relevant anti-inflammatory effects by targeting CB2.

Conclusions

Joint injury perturbs the intra-articular ECS, characterized by an increase in synovial F4/80(+) cells, which express CB2, but not CB1. Targeting CB2 in murine macrophages and human FLS induced potent anti-inflammatory and anti-catabolic effects, which indicates that the CB2 receptor plays a key role in regulating inflammatory signaling in the two primary effector cells in the synovium. The intraarticular ECS is therefore a potential therapeutic target for blocking pathological inflammation in future disease-modifying PTOA treatments.

中文翻译：

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关节损伤后，2 型大麻素受体在滑膜中上调，并介导滑膜成纤维细胞和巨噬细胞的抗炎作用

客观的

关节损伤引起的对内源性大麻素系统 (ECS) 的扰动（炎症和伤害感受的调节剂）在很大程度上仍未被表征。我们采用 ACL 破裂的小鼠模型来评估伤害感受、炎症和 ECS 的改变，同时使用体外模型来确定 CB2 激动是否可以减轻巨噬细胞和成纤维细胞样滑膜细胞 (FLS) 中的炎症信号。

设计

小鼠通过基于胫骨压缩的负荷进行无创 ACL 破裂 (ACLR)。使用机械异常性疼痛和膝痛觉过敏测试纵向测量伤害感受。使用组织学评分和组织形态学评估滑膜炎。炎症的基因和蛋白质标记物在整个关节和滑膜中都有特征。免疫组织化学评估了损伤诱导的滑膜中 CB1+、CB2+ 和 F4/80+ 细胞的改变。为了评估 CB2 激动是否可以抑制促炎性巨噬细胞极化，用 IL-1β 或来自经 IL-1β 处理的 FLS 的条件培养基刺激小鼠骨髓来源的巨噬细胞 (mBMDM)，并用载体 (DMSO)、CB2 激动剂 HU308 进行处理, 或大麻二酚 (CBD)。巨噬细胞极化被评估为 M1 相关（IL1b，MMP1b和IL6）对 M2 相关（IL10、IL4和CD206）基因表达的影响。从 OA 患者的滑膜组织分离的人 FLS (hFLS) 在 TNF-α 或 IL-1β 刺激后用载体 (DMSO) 或 HU308 处理，以评估分解代谢/炎症基因表达的抑制。

结果

ACLR 诱发滑膜炎、逐渐恶化的 PTOA 严重程度，以及机械异常性疼痛和膝痛觉过敏的立即和持续增加，其持续超过分子炎症的消退。在第 3 天、第 14 天和第 28 天在 ACLR 滑膜中观察到 CB2 而不是 CB1 的富集，并且发现 CB2 与 F4/80 (+) 细胞相关，这些细胞在所有时间点都在 ACLR 滑膜中增加。CB2 激动剂 HU308 在用 IL-1β 或来自 IL-1β 处理的 mFLS 的条件培养基刺激后强烈抑制 mBMDM M1 型极化，其特征是Il1b、Mmp1b和Il6减少以及Cd206基因表达增加。大麻二酚通过减少Il1b以及Cd206和Il4基因表达的增加。最后，在 OA hFLS 中，HU308 治疗抑制了 IL-1β 诱导的CCL2、MMP1、MMP3和IL6表达，并进一步抑制了 TNF-α 诱导的CCL2、MMP1和GMCSF表达，证明了人类 OA 相关的抗炎作用通过靶向CB2。

结论

关节损伤扰乱了关节内 ECS，其特点是滑膜 F4/80(+) 细胞增加，表达 CB2，但不表达 CB1。靶向小鼠巨噬细胞和人类 FLS 中的 CB2 可诱导有效的抗炎和抗分解代谢作用，这表明 CB2 受体在调节滑膜中两个主要效应细胞的炎症信号传导中起着关键作用。因此，关节内 ECS 是未来疾病缓解 PTOA 治疗中阻断病理性炎症的潜在治疗靶点。