BACKGROUND The recruitment of immune system cells into the central nervous system (CNS) has a profound effect on the outcomes of injury and disease. Glia-derived chemoattractants, including chemokines, play a pivotal role in this process. In addition, cytokines and chemokines influence the phenotype of infiltrating immune cells. Depending on the stimuli present in the local milieu, infiltrating macrophages acquire the classically activated M1 or alternatively activated M2 phenotypes. The polarization of macrophages into detrimental M1 versus beneficial M2 phenotypes significantly influences CNS pathophysiology. Earlier studies indicated that a toll-like receptor 9 (TLR9) antagonist modulates astrocyte-derived cytokine and chemokine release. However, it is not known whether these molecular changes affect astrocyte-induced chemotaxis and polarization of macrophages. The present studies were undertaken to address these issues. METHODS The chemotaxis and polarization of mouse peritoneal macrophages by spinal cord astrocytes were evaluated in a Transwell co-culture system. Arrays and ELISA were utilized to quantify chemokines in the conditioned medium (CM) of pure astrocyte cultures. Immunostaining for M1- and M2-specific markers characterized the macrophage phenotype. The percentage of M2 macrophages at the glial scar was determined by stereological approaches in mice sustaining a mid-thoracic spinal cord contusion injury (SCI) and intrathecally treated with oligodeoxynucleotide 2088 (ODN 2088), the TLR9 antagonist. Statistical analyses used two-tailed independent-sample t-test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p value < 0.05 was considered to be statistically significant. RESULTS ODN 2088-treated astrocytes significantly increased the chemotaxis of peritoneal macrophages via release of chemokine (C-C motif) ligand 1 (CCL1). Vehicle-treated astrocytes polarized macrophages into the M2 phenotype and ODN 2088-treated astrocytes promoted further M2 polarization. Reduced CCL2 and CCL9 release by astrocytes in response to ODN 2088 facilitated the acquisition of the M2 phenotype, suggesting that CCL2 and CCL9 are negative regulators of M2 polarization. The percentage of M2 macrophages at the glial scar was higher in mice sustaining a SCI and receiving ODN 2088 treatment as compared to vehicle-treated injured controls. CONCLUSIONS TLR9 antagonism could create a favorable environment during SCI by supporting M2 macrophage polarization and chemotaxis via modulation of astrocyte-to-macrophage signals.

中文翻译：

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星形胶质细胞 TLR9 拮抗作用通过调节星形胶质细胞衍生信号促进巨噬细胞的趋化性和替代激活：对脊髓损伤的影响。


背景技术免疫系统细胞募集至中枢神经系统（CNS）对损伤和疾病的结果具有深远的影响。神经胶质细胞衍生的化学引诱剂，包括趋化因子，在此过程中发挥着关键作用。此外，细胞因子和趋化因子影响浸润免疫细胞的表型。根据局部环境中存在的刺激，浸润巨噬细胞获得经典激活的 M1 或替代激活的 M2 表型。巨噬细胞极化为有害的 M1 与有益的 M2 表型显着影响 CNS 病理生理学。早期研究表明，Toll 样受体 9 (TLR9​​) 拮抗剂可调节星形胶质细胞衍生的细胞因子和趋化因子的释放。然而，尚不清楚这些分子变化是否影响星形胶质细胞诱导的巨噬细胞的趋化性和极化。目前的研究就是为了解决这些问题而进行的。方法 在 Transwell 共培养系统中评估脊髓星形胶质细胞对小鼠腹膜巨噬细胞的趋化性和极化作用。利用阵列和 ELISA 来定量纯星形胶质细胞培养物的条件培养基 (CM) 中的趋化因子。 M1 和 M2 特异性标记物的免疫染色表征了巨噬细胞表型。在遭受中胸脊髓挫伤 (SCI) 并用 TLR9 拮抗剂寡脱氧核苷酸 2088 (ODN 2088) 鞘内治疗的小鼠中，通过体视学方法测定神经胶质疤痕处 M2 巨噬细胞的百分比。统计分析使用双尾独立样本 t 检验和单向方差分析 (ANOVA)，然后进行 Tukey 事后检验。 p 值< 0.05 被认为具有统计显着性。 结果 ODN 2088 处理的星形胶质细胞通过释放趋化因子（CC 基序）配体 1 (CCL1) 显着增加腹膜巨噬细胞的趋化性。载体处理的星形胶质细胞将巨噬细胞极化为 M2 表型，ODN 2088 处理的星形胶质细胞促进进一步的 M2 极化。星形胶质细胞响应 ODN 2088 而减少 CCL2 和 CCL9 释放，促进了 M2 表型的获得，表明 CCL2 和 CCL9 是 M2 极化的负调节因子。与媒介物治疗的受伤对照相比，遭受 SCI 并接受 ODN 2088 治疗的小鼠中，神经胶质疤痕处的 M2 巨噬细胞百分比更高。结论 TLR9 拮抗作用可以通过调节星形胶质细胞至巨噬细胞信号来支持 M2 巨噬细胞极化和趋化性，从而在 SCI 期间创造有利的环境。