Lysosomes cell autonomously regulate myeloid cell states and immune responses

Myeloid cells maintain tissue homeostasis via the recognition, engulfment, and lysosomal clearance of dying cells and cellular debris, which is often accompanied by changes from homeostatic to reactive states. While a role for phagocytic receptors in gating these transitions has been described, less is known about if and how lysosomes can contribute to transcriptional and functional plasticity. To determine how lysosomal health impacts myeloid cell states, we evaluated microglia and macrophages deficient for progranulin (encoded by Grn), a lysosomal protein with pleiotropic functions whose loss is associated with several neurodegenerative diseases. Single-cell RNA-sequencing of the aged mouse brain identified a Grn knockout (KO)-specific microglial subpopulation marked by high GPNMB expression that displays hallmarks of lysosomal dysfunction, including lipofuscin accumulation. Epigenetic analysis of aged microglia revealed MITF/TFE transcription factors as key mediators of the transcriptional states associated with Grn deficiency. In addition to identifying a core myeloid cell transcriptional response to diverse lysosomal stressors, targeted perturbations of various lysosomal properties in vitro uncovered a cell autonomous, TREM2-independent, response to lysosomal deacidification (via v-ATPase or VPS34 loss of function) that overlaps with Grn KO microglia phenotypes, including the induction of a lysosomal gene program, increased proliferation, and secretion of pro-inflammatory cytokines. Compound loss-of-function approaches established GPNMB upregulation upon lysosomal stress is required for the compensatory response to enhance lysosomal function via promoting acidification. Finally, pharmacological endolysosomal reacidification through sodium/proton exchanger inhibition partially rescued Grn KO microglia phenotypes. Overall, these data establish a fundamental link between lysosomal health and myeloid cell epigenetic, transcriptional, and functional states observed in neurodegeneration models. Overall design: Two year old mice were anesthetized, transcardially perfused with PBS, and brains were removed for scRNA-seq or microglial H3K27ac analyses (see below). Hemibrains were separated and one half was frozen for nuclei isolation for H3K27ac ChIP-seq. Microglial nuclei sorted from two mice were pooled for each individual replicate, and analyses were performed with duplicates for each genotype.

髓系细胞（myeloid cells）可通过识别、吞噬并经溶酶体（lysosome）清除死亡细胞与细胞碎片来维持组织稳态，此过程往往伴随细胞从稳态向反应状态的转变。尽管已有研究表明吞噬受体（phagocytic receptors）可调控这类状态转变，但对于溶酶体是否以及如何参与调控转录与功能可塑性，目前尚不清楚。为明确溶酶体健康状态如何影响髓系细胞表型，我们对颗粒体蛋白（progranulin，由Grn编码）缺陷的小胶质细胞（microglia）与巨噬细胞（macrophages）开展了分析。颗粒体蛋白是具有多效性功能的溶酶体蛋白，其缺失与多种神经退行性疾病相关。对老龄小鼠大脑开展的单细胞RNA测序（single-cell RNA-sequencing，scRNA-seq）显示，存在一类Grn敲除（knockout，KO）特异性小胶质细胞亚群，该亚群以GPNMB高表达为特征，并表现出溶酶体功能障碍的典型标志，包括脂褐质（lipofuscin）沉积。对老龄小胶质细胞的表观遗传分析（epigenetic analysis）显示，MITF/TFE家族转录因子是介导Grn缺失相关转录状态的核心调控因子。除明确髓系细胞对多种溶酶体应激源（lysosomal stressors）的核心转录应答谱外，我们还通过体外靶向调控多种溶酶体特性，发现了一种不依赖TREM2（髓系细胞触发受体2，triggering receptor expressed on myeloid cells 2）的细胞自主性应答：该应答针对溶酶体去酸化过程（通过抑制v-ATPase（液泡型ATP酶，vacuolar-type H+-ATPase）或VPS34（液泡蛋白分选因子34，vacuolar protein sorting 34）的功能实现），其表型特征与Grn KO小胶质细胞高度重合，包括诱导溶酶体基因程序、增强细胞增殖以及促炎细胞因子（pro-inflammatory cytokines）分泌。通过复合功能缺失实验手段，我们证实溶酶体应激时GPNMB的上调表达，是通过促进酸化增强溶酶体功能的代偿性应答所必需的环节。最后，通过抑制钠/质子交换剂实现药物性内溶酶体再酸化，可部分挽救Grn KO小胶质细胞的异常表型。综上，本研究数据证实，在神经退行性疾病模型中，溶酶体健康状态与髓系细胞的表观遗传、转录及功能状态之间存在基础性关联。
实验整体设计：对24月龄（约2岁）老龄小鼠实施麻醉，经心腔灌注磷酸盐缓冲液（phosphate buffered saline, PBS）后取脑，分别用于单细胞RNA测序（scRNA-seq）或小胶质细胞H3K27ac分析（详见下文）。将大脑分为左右半球，其中一半脑组织冻存，用于后续提取细胞核以开展H3K27ac染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）分析。每一次生物学重复均将2只小鼠分选得到的小胶质细胞核混合，每种基因型均设置2次生物学重复用于分析。