Microglia innate immune memory is retained after enforced repopulation [ATAC-seq]

Microglia are crucial for CNS homeostasis and involved in a wide range of neurodegenerative and neuroinflammatory diseases. Systemic inflammation and infections can contribute to neurodegeneration later in life by affecting microglia. Like other innate immune cells, microglia can develop innate immune memory in response to a challenge, altering their response to future stimuli. Innate immune memory can ameliorate or worsen CNS pathology, but its persistence is unclear. Recently, several methods have been developed to stimulate microglia turnover. Here, we investigated whether colony-stimulating factor 1 (CSF1R)-dependent microglia depletion followed by repopulation reversed microglial tolerance to systemic inflammation in mice. Repopulated microglia displayed a reduced expression of homeostatic genes and genes related to mitochondrial respiration and TCA cycle metabolism and an increased expression of immune effector and activation genes. Nonetheless, the blunted inflammatory gene expression induced by the LPS-preconditioning was retained after a depletion-repopulation cycle. Our study highlights the persistence of epigenetic changes underlying microglial tolerance and indicates potential implications of microglia depletion and repopulation therapies on microglia functions. We pre-conditioned mice with intraperitoneal LPS (or PBS), transiently depleted microglia with BLZ945, and, after repopulation, assessed microglia gene expression by bulk RNA-seq and chromatin accessibility by bulk ATAC-seq after a secondary LPS (or PBS) challenge. 10-week-old male mice were challenged with 1 mg/kg intraperitoneal (i.p.) LPS from Escherichia coli O111:B4. For unchallenged (PP, PBP) and naïve (PL, PBL) mice, sham injections consisted of the same volume/body mass of Dulbecco’s phosphate-buffered saline (DPBS). A week after the first challenge, microglia depletion was induced by colony-stimulating factor 1 receptor (CSF1R) inhibition for five days. Microglia depletion was established with daily administration via oral gavage of the CSF1R inhibitor (CSF1Ri) BLZ945 (or Sotuletinib) at a dosage of 169 mg/kg per day. Control (non-depleted) animals were treated with the same volume/body mass of vehicle containing 20% (w/v) 2-hydroxypropyl-beta-cyclodextrin (HPβCD). Five weeks after the initial challenge, mice were similarly re-challenged with 1 mg/kg LPS or PBS (i.p.) and housed in warming cabinets for three hours before brain tissue collection. Microglia were isolated via FACS, and their chromatin accessibility was assessed by bulk ATAC-seq.

小胶质细胞（Microglia）对中枢神经系统（Central Nervous System, CNS）稳态至关重要，且参与多种神经退行性与神经炎症性疾病的发生发展。全身性炎症与感染可通过影响小胶质细胞，进而在晚年促成神经退行性病变。与其他固有免疫细胞类似，小胶质细胞可在受到刺激后形成固有免疫记忆，改变其对后续刺激的应答反应。固有免疫记忆可改善或加重中枢神经系统病理损伤，但其持续时长尚不明确。近期已有多种方法被开发用于诱导小胶质细胞更新。本研究旨在探究：以集落刺激因子1受体（colony-stimulating factor 1 receptor, CSF1R）依赖的方式清除小胶质细胞并随后实现其增殖重建，是否能够逆转小鼠体内小胶质细胞对全身性炎症的耐受状态。经增殖重建的小胶质细胞，其稳态相关基因、线粒体呼吸与三羧酸循环代谢相关基因的表达水平降低，而免疫效应分子与活化相关基因的表达水平升高。尽管如此，经脂多糖（Lipopolysaccharide, LPS）预刺激所诱导的炎症基因表达受抑状态，在小胶质细胞清除-增殖重建循环后仍得以保留。本研究证实了小胶质细胞耐受背后表观遗传改变的持久性，并提示小胶质细胞清除与增殖重建疗法可能对小胶质细胞功能产生潜在影响。我们首先通过腹腔注射脂多糖（或磷酸盐缓冲液）对小鼠进行预刺激，随后使用BLZ945暂时性清除小胶质细胞；待小胶质细胞增殖重建完成后，再次给予脂多糖（或磷酸盐缓冲液）刺激，通过批量RNA测序（bulk RNA-seq）检测小胶质细胞基因表达水平，通过批量转座酶可及性测序（bulk ATAC-seq）检测染色质开放状态。本研究使用10周龄的雄性小鼠，以1 mg/kg剂量的大肠杆菌O111:B4来源的脂多糖进行腹腔内（intraperitoneal, i.p.）注射刺激。对于未接受二次刺激（PP、PBP组）与未预刺激的小鼠（PL、PBL组），假手术组均给予同等体积/体重的杜氏磷酸缓冲盐溶液（Dulbecco’s phosphate-buffered saline, DPBS）。首次刺激一周后，通过抑制集落刺激因子1受体（CSF1R）持续五天以诱导小胶质细胞清除。每日通过灌胃给予集落刺激因子1受体抑制剂（CSF1Ri）BLZ945（或Sotuletinib），剂量为169 mg/kg/天，以实现小胶质细胞清除。对照组（未清除小胶质细胞）小鼠给予同等体积/体重的溶剂，该溶剂含20%（w/v）羟丙基-β-环糊精（2-hydroxypropyl-beta-cyclodextrin, HPβCD）。首次刺激五周后，我们再次以1 mg/kg剂量的脂多糖或磷酸盐缓冲液对小鼠进行腹腔注射，并将小鼠置于加温饲养箱中饲养三小时后收集脑组织。通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离小胶质细胞，并通过批量转座酶可及性测序检测其染色质开放状态。