Kirkley Kelly S, Popichak Katriana A, Afzali Maryam F, Legare Marie E, Tjalkens Ronald B
Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 1680 Campus Delivery, Fort Collins, CO, 80523-1680, USA.
Center for Environmental Medicine, Colorado State University, Fort Collins, CO, USA.
J Neuroinflammation. 2017 May 5;14(1):99. doi: 10.1186/s12974-017-0871-0.
As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood.
Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes.
Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1β, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-κB in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes.
These results provide evidence that NF-κB signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.
作为中枢神经系统中的主要免疫反应细胞，小胶质细胞不断监测微环境，并对压力、感染和损伤迅速做出反应，使其成为神经炎症反应的重要调节因子。在帕金森病、阿尔茨海默病、多发性硬化症和人类免疫缺陷病毒引起的痴呆等疾病中，小胶质细胞的激活先于星形胶质细胞增生和明显的神经元丢失。尽管小胶质细胞增生与锰（Mn）神经毒性有关，但小胶质细胞和神经胶质细胞间相互作用在锰诱导的神经退行性变中的作用仍知之甚少。
实验采用无柱磁分离法免疫纯化小鼠小胶质细胞和星形胶质细胞。通过基因表达分析、锰摄取测量、蛋白质产生及形态变化研究锰对小胶质细胞的影响。此外，利用基因表达分析确定经锰处理的小胶质细胞对暴露于锰的星形胶质细胞炎症反应的影响。
对免疫纯化的小胶质细胞和星形胶质细胞进行免疫荧光和流式细胞术分析表明，培养物的纯度分别为97%和90%。小胶质细胞中的锰处理导致促炎基因表达呈剂量依赖性增加，转变为混合的M1/M2表型，并出现去分支形态。暴露于锰的小胶质细胞的条件培养基（MCM）显著增强了星形胶质细胞中Tnf、Il-1β、Il-6、Ccl2和Ccl5的mRNA表达，与在共培养的小胶质细胞存在下暴露于锰的情况相同。MCM中细胞因子和趋化因子（包括IL-6、TNF、CCL2和CCL5）的水平升高。使用Bay 11-7082对小胶质细胞中的NF-κB进行药理学抑制可完全阻断小胶质细胞诱导的星形胶质细胞激活，而在原代小胶质细胞中对Tnf进行siRNA敲低仅部分抑制星形胶质细胞中的神经炎症反应。
这些结果提供了证据，表明小胶质细胞中的NF-κB信号通过调节放大星形胶质细胞激活的细胞因子和趋化因子，在锰毒性的炎症反应中起重要作用。
