Reynolds Family Spine Laboratory, Department of Neurosurgery, New Jersey Medical School, Rutgers, The State University of New Jersey, 205 South Orange Avenue, F-1204, Newark, NJ, 07103, USA.
J Neuroinflammation. 2020 Feb 25;17(1):73. doi: 10.1186/s12974-020-01748-x.
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.
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.
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.
TLR9 antagonism could create a favorable environment during SCI by supporting M2 macrophage polarization and chemotaxis via modulation of astrocyte-to-macrophage signals.
免疫系统细胞向中枢神经系统（CNS）的募集对损伤和疾病的结果有深远的影响。胶质细胞衍生的趋化因子，包括趋化因子，在这个过程中起着关键作用。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，浸润的巨噬细胞获得经典激活的 M1 或替代激活的 M2 表型。巨噬细胞向有害的 M1 表型与有益的 M2 表型的极化显著影响中枢神经系统的病理生理学。早期研究表明，Toll 样受体 9（TLR9）拮抗剂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，目前尚不清楚这些分子变化是否会影响星形胶质细胞诱导的趋化和巨噬细胞极化。本研究旨在解决这些问题。
通过 Transwell 共培养系统评估脊髓星形胶质细胞对小鼠腹腔巨噬细胞的趋化作用和极化作用。利用微阵列和 ELISA 定量分析纯星形胶质细胞培养物条件培养基（CM）中的趋化因子。免疫组织化学染色用于鉴定 M1 和 M2 特异性标记物，以确定巨噬细胞的表型。通过立体学方法在中胸段脊髓挫伤损伤（SCI）的小鼠中测定鞘内给予寡核苷酸 2088（TLR9 拮抗剂）后，在神经胶质瘢痕处 M2 巨噬细胞的百分比。使用双尾独立样本 t 检验和单因素方差分析（ANOVA），然后使用 Tukey 事后检验进行统计分析。p 值<0.05 被认为具有统计学意义。
TLR9 拮抗剂处理的星形胶质细胞通过释放趋化因子（C-C 基序）配体 1（CCL1）显著增加了腹腔巨噬细胞的趋化性。与 vehicle 处理的星形胶质细胞相比，vehicle 处理的星形胶质细胞将巨噬细胞极化到 M2 表型，而 TLR9 拮抗剂处理的星形胶质细胞促进了进一步的 M2 极化。星形胶质细胞对 TLR9 拮抗剂的反应中 CCL2 和 CCL9 的释放减少促进了 M2 表型的获得，这表明 CCL2 和 CCL9 是 M2 极化的负调节剂。与 vehicle 处理的损伤对照相比，在 SCI 后接受 TLR9 拮抗剂治疗的小鼠中，神经胶质瘢痕处的 M2 巨噬细胞百分比更高。
TLR9 拮抗作用可以通过调节星形胶质细胞-巨噬细胞信号，通过支持 M2 巨噬细胞极化和趋化作用，在 SCI 期间创造有利的环境。
