DataSheet1_Pathological Neuroinflammatory Conversion of Reactive Astrocytes Is Induced by Microglia and Involves Chromatin Remodeling.docx

Following brain injury or in neurodegenerative diseases, astrocytes become reactive and may suffer pathological remodeling, features of which are the loss of their homeostatic functions and a pro-inflammatory gain of function that facilitates neurodegeneration. Pharmacological intervention to modulate this astroglial response and neuroinflammation is an interesting new therapeutic research strategy, but it still requires a deeper understanding of the underlying cellular and molecular mechanisms of the phenomenon. Based on the known microglial–astroglial interaction, the prominent role of the nuclear factor kappa B (NF-κB) pathway in mediating astroglial pathological pro-inflammatory gain of function, and its ability to recruit chromatin-remodeling enzymes, we first explored the microglial role in the initiation of astroglial pro-inflammatory conversion and then monitored the progression of epigenetic changes in the astrocytic chromatin. Different configurations of primary glial culture were used to modulate microglia–astrocyte crosstalk while inducing pro-inflammatory gain of function by lipopolysaccharide (LPS) exposure. In vivo, brain ischemia by cortical devascularization (pial disruption) was performed to verify the presence of epigenetic marks in reactive astrocytes. Our results showed that 1) microglia is required to initiate the pathological conversion of astrocytes by triggering the NF-κB signaling pathway; 2) this interaction is mediated by soluble factors and induces stable astroglial phenotypic changes; 3) the pathological conversion promotes chromatin remodeling with stable increase in H3K9K14ac, temporary increase in H3K27ac, and temporary reduction in heterochromatin mark H3K9me3; and 4) in vivo reactive astrocytes show increased H3K27ac mark in the neuroinflammatory milieu from the ischemic penumbra. Our findings indicate that astroglial pathological pro-inflammatory gain of function is associated with profound changes in the configuration of astrocytic chromatin, which in turn are initiated by microglia-derived cues. These results open a new avenue in the study of potential pharmacological interventions that modify the initiation and stabilization of astroglial pathological remodeling, which would be useful in acute and chronic CNS injury. Epigenetic changes represent a plausible pharmacological target to interfere with the stabilization of the pathological astroglial phenotype.当发生脑损伤或罹患神经退行性疾病时，星形胶质细胞会发生反应性活化，并可能出现病理性重塑，其特征为稳态功能丧失以及促炎功能获得，后者会加剧神经退行性病变的进展。针对此类星形胶质细胞反应与神经炎症进行调节的药物干预手段，是极具潜力的新型治疗研究方向，但目前仍需深入阐明该现象背后的细胞与分子机制。基于已探明的小胶质细胞-星形胶质细胞互作（microglial–astroglial interaction）、核因子κB（nuclear factor kappa B, NF-κB）通路在介导星形胶质细胞病理性促炎功能获得中的核心作用，以及该通路招募染色质重塑酶（chromatin-remodeling enzymes）的能力，本研究首先探究了小胶质细胞在启动星形胶质细胞促炎转化中的作用，随后监测了星形胶质细胞染色质的表观遗传变化进程。本研究采用多种配置的原代胶质细胞培养（primary glial culture）体系，调节小胶质细胞-星形胶质细胞的串扰，并通过脂多糖（lipopolysaccharide, LPS）暴露诱导星形胶质细胞发生促炎功能获得。在体内实验中，本研究通过皮层血管切断（cortical devascularization，软膜破坏（pial disruption））构建脑缺血模型，以验证反应性星形胶质细胞中的表观遗传标记（epigenetic marks）特征。本研究结果显示：1）小胶质细胞可通过激活核因子κB信号通路，启动星形胶质细胞的病理性转化；2）该互作由可溶性因子介导，并可诱导星形胶质细胞表型发生稳定改变；3）病理性转化会促进染色质重塑（chromatin remodeling），具体表现为H3K9K14乙酰化（H3K9K14ac）持续升高、H3K27乙酰化（H3K27ac）暂时升高，以及异染色质标记H3K9三甲基化（H3K9me3）暂时降低；4）在体实验中，缺血半暗带（ischemic penumbra）神经炎症微环境内的反应性星形胶质细胞，其H3K27ac标记水平显著升高。本研究结果表明，星形胶质细胞的病理性促炎功能获得与星形胶质细胞染色质构型的显著改变密切相关，而此类染色质改变由小胶质细胞分泌的信号分子启动。上述研究结果为探索靶向调控星形胶质细胞病理性重塑的启动与稳定过程的潜在药物干预手段开辟了新方向，该策略或可用于急性与慢性中枢神经系统（CNS）损伤的治疗。表观遗传变化可作为潜在的药物靶点，用于干预病理性星形胶质细胞表型的稳定过程。