Microglial depletion and repopulation in brain slice culture normalizes sensitized proinflammatory signaling
Microglial depletion and repopulation in brain slice culture normalizes sensitized proinflammatory signaling
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DOI：
10.1186/s12974-019-1678-y
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发表时间：
2020-01-18
影响因子：
9.3
通讯作者：
Crews, Fulton T.
中科院分区：
文献类型：
--
作者：
Coleman, Leon G., Jr.;Zou, Jian;Crews, Fulton T.
背景：小胶质细胞是多种神经系统疾病的神经免疫病理的重要介质。小胶质细胞可以被Toll样受体(TLR)激活、乙醇、压力和其他侮辱持续激活或“启动”。因此，防止或逆转小胶质细胞启动的策略对于涉及逐渐增加小胶质细胞激活的情况可能是有益的。小胶质细胞耗竭和再生正在成为一种使慢性免疫激活正常化的潜在治疗方法。原代器官型海马片培养(OHSC)允许研究神经免疫激活以及小胶质细胞的耗竭和再生，而不涉及外周免疫激活。OHSC经历了功能成熟，并保持了与体内相似的细胞结构。方法在去除PLX3397后，用CSF1R拮抗剂PLX3397去除OHSC的小胶质细胞。在小胶质细胞耗竭和再生的环境中，评估了免疫、营养和突触基因对TLR2、3、4、7和9激动剂以及乙醇的反应变化。小胶质细胞的GI-DREADD抑制被用来证实所见的有耗竭的部分发现。同时还研究了小胶质细胞再生对慢性乙醇诱导的进行性促炎基因表达的抑制作用。结果PLX3397可使OHSC中的小胶质细胞减少90%。小胶质细胞的耗竭减弱了对几种TLR激动剂和乙醇的促炎反应，这与GI-DREADD抑制OHSC小胶质细胞的作用类似。去除PLX3397之后，小胶质细胞完全重新聚集。再生小胶质细胞的OHSC表现出抗炎细胞因子(例如IL-10)、小胶质细胞抑制信号(例如CX3CL1)和生长因子(例如BDNF)的基线表达增加。这与TLR4和TLR7激动剂对肿瘤坏死因子α和白介素1β的钝化诱导有关。此外，从体外4天(DIV)到16DIV的慢性循环酒精可以立即诱导2倍的肿瘤坏死因子α和IL-1β的诱导，到40DIV时，这些诱导作用会增长到年龄匹配的对照组切片的4倍。这种持续性炎症基因的表达可通过小胶质细胞的耗尽和慢性酒精后的再生而完全逆转。结论OHSC中的小胶质细胞介导了对TLR激动剂和乙醇的促炎反应。小胶质细胞的再生促进了抗炎、营养神经环境和促炎基因的正常表达。这支持了将小胶质细胞的再生作为逆转慢性神经免疫激活的一种策略的可能性。
Background Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or "primed" by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo. Methods OHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated. Results Microglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction ( 50%) of TNF alpha and IL-1 beta in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNF alpha and IL-1 beta that grew to 4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol. Conclusions Microglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.