In the murine K/BxN serum transfer rheumatoid arthritis (RA) model, tactile allodynia persists after resolution of inflammation in male and partially in female wild type (WT) mice, which is absent in Toll-like receptor (TLR)4 deficient animals. We assessed the role of TLR4 on allodynia, bone remodeling and afferent sprouting in this model of arthritis. K/BxN sera were injected into male and female mice with conditional or stable TLR4 deletion and controls. Paw swelling was scored and allodynia assessed by von Frey filaments. At day 28, synovial neural fibers were visualized with confocal microscopy and bone density assayed with microCT. Microglial activity and TLR4 dimerization in spinal cords were examined by immunofluorescence and flow cytometry. In the synovium, K/BxN injected WT male and female mice showed robust increases in calcitonin gene related-peptide (CGRP+), tyrosine hydroxylase (TH)+ and GAP43+ nerve fibers. Trabecular bone density by microCT was significantly decreased in K/BxN WT female but not in WT male mice. The number of osteoclasts increased in both sexes of WT mice, but not in Tlr4-/- K/BxN mice. We used conditional strains with Cre drivers for monocytes/osteoclasts (lysozyme M), microglia (Tmem119 and Cx3CR1), astrocytes (GFAP) and sensory neurons (advillin) for Tlr4f/f disruption. All strains developed similar arthritis scores after K/BxN serum injection with the exception being the Tlr4Tmem119 mice which showed a reduction. Both sexes of Tlr4Lyz2, Tlr4Tmem119 and Tlr4Cx3cr1 mice displayed a partial reversal of the chronic pain phenotype but not in Tlr4Avil, and Tlr4Gfap mice. WT K/BxN male mice showed increases in spinal Iba1, but not GFAP, compared to Tlr4-/- male mice. To determine whether spinal TLR4 was indeed activated in the K/BxN mice, flow cytometry of lumbar spinal cords of WT K/BxN male mice was performed and revealed that TLR4 in microglia cells (CD11b+ /TMEM119+) demonstrated dimerization (e.g. activation) and a characteristic increase in lipid rafts. These results demonstrated a complex chronic allodynia phenotype associated with TLR4 in microglia and monocytic cell lineages, and a parallel spinal TLR4 activation. However, TLR4 is dispensable for the development of peripheral nerve sprouting in this model.

中文翻译：

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TLR4 激活和信号转导在血清转移性关节炎中骨重塑和传入发芽中的作用


在小鼠 K/BxN 血清转移类风湿性关节炎 （RA） 模型中，雄性野生型 （WT） 小鼠炎症消退后触觉异常性疼痛持续存在，雌性野生型 （WT） 小鼠部分存在，这在 Toll 样受体 （TLR）4 缺陷动物中不存在。我们评估了 TLR4 对该关节炎模型中异常性疼痛、骨重塑和传入发芽的作用。将 K/BxN 血清注射到具有条件或稳定 TLR4 缺失的雄性和雌性小鼠和对照中。对爪肿胀进行评分，并通过 von Frey 细丝评估异常性疼痛。第 28 天，用共聚焦显微镜观察滑膜神经纤维，用 microCT 测定骨密度。通过免疫荧光和流式细胞术检查脊髓中的小胶质细胞活性和 TLR4 二聚化。在滑膜中，注射 K/BxN 的 WT 雄性和雌性小鼠表现出降钙素基因相关肽 （CGRP+） 、酪氨酸羟化酶 （TH）+ 和 GAP43+ 神经纤维的强劲增加。microCT 显示 K/BxN WT 雌性小鼠的小梁骨密度显著降低，而 WT 雄性小鼠则未显著降低。WT 小鼠两性破骨细胞的数量均增加，但 Tlr4 - / - K/BxN 小鼠的破骨细胞数量没有增加。我们使用具有 Cre 驱动程序的条件菌株来破坏单核细胞/破骨细胞 （溶菌酶 M）、小胶质细胞 （Tmem119 和 Cx3CR1） 、星形胶质细胞 （GFAP） 和感觉神经元 （advillin） 来破坏 Tlr4f/f。所有菌株在注射 K/BxN 血清后均出现相似的关节炎评分，但 Tlr4Tmem119 小鼠除外，其显示降低。Tlr4Lyz2 、 Tlr4Tmem119 和 Tlr4Cx3cr1 小鼠的两性均表现出慢性疼痛表型的部分逆转，但在 Tlr4Avil 和 Tlr4Gfap 小鼠中则没有。与 Tlr4-/-雄性小鼠相比，WT K/BxN 雄性小鼠的脊髓 Iba1 增加，但 GFAP 没有增加。 为了确定脊髓 TLR4 是否确实在 K/BxN 小鼠中被激活，对 WT K/BxN 雄性小鼠的腰脊髓进行了流式细胞术，结果显示小胶质细胞 （CD11b+ /TMEM119+） 中的 TLR4 表现出二聚化（例如 激活）和脂筏的特征性增加。这些结果表明，在小胶质细胞和单核细胞谱系中，与 TLR4 相关的复杂慢性异常性疼痛表型，以及平行的脊髓 TLR4 激活。然而，在该模型中，TLR4 对于周围神经发芽的发育是必不可少的。