IRF1通过启动BRD4-EP300活性介导气道上皮固有记忆

摘要(中文译文)

气道上皮的哨兵细胞会反复暴露于RNA病毒感染。在此过程中，病原体相关分子模式（PAMPs）的产生激活先天免疫应答（IIR），诱导抗病毒防御、激活适应性免疫并刺激上皮修复。反复暴露是否会产生先天“记忆”——即在后续暴露时产生增强的炎症和损伤/修复反应——目前尚不清楚。我们通过反复激活Toll样受体（TLR3）通路（即“训练”），在肿瘤蛋白（TP63）+/角蛋白（KRT）5+人小气道上皮基底细胞中建立了一个可重复的先天记忆模型。通过CRISPR/Cas9敲低、染色质免疫沉淀、共免疫沉淀以及使用BRD4染色质重塑复合物（CRC）选择性抑制剂，研究了IRF1依赖性靶基因重塑的机制。我们观察到“训练”增加了内在IIR基因网络的基础表达，在后续暴露时减少呼吸道合胞病毒（RSV）的复制。此外，经过训练的人小气道上皮细胞（hSAECs）进入上皮-间充质可塑性（EMP）程序，其特征为细胞形态改变、细胞质波形蛋白和核心间充质调控因子表达增加。在机制上，我们发现干扰素调节因子1（IRF1）是先天记忆所必需的，因为IRF1缺陷细胞无法激活基础内在IIR。在TLR3激活过程中，IRF1被整合到含溴结构域蛋白4（BRD4）染色质重塑复合物（CRC）中，该复合物含有组蛋白H3乙酰转移酶EP300/p300。在急性TLR3激活中，BRD4在内在IIR启动子上的结合增加，但这种结合不受训练影响。相反，我们发现训练诱导EP300结合并增加这些区域的H3K27乙酰化（H3K27ac）。小分子抑制剂实验表明，BRD4是建立先天记忆和H3K27ac积累所必需的。最后，我们发现训练增强了BRD4-EP300相互作用，且记忆诱导的H3K27ac形成依赖于EP300。我们得出结论：hSAECs通过IRF1与BRD4-EP300 CRC偶联来激活内在IIR和细胞状态转换基因，从而获得先天记忆。我们的数据表明，先天训练是动态BRD4 CRC相互作用通过增强EP300募集来启动转录反应性的结果。

Abstract

Sentinel cells of the airway epithelium are repetitively exposed to RNA virus infections. Here, the production of pathogen-associated molecular patterns (PAMPs) activates the innate immune response (IIR), inducing antiviral defenses, activating adaptive immunity and stimulating epithelial repair. The question whether repeated exposures produce innate "memory", where heightened inflammatory and injury/repair responses are produced upon subsequent exposure, is unknown. We establish a reproducible model of innate memory in tumor protein (TP63)+/keratin (KRT)5+ human small airway basal epithelial cells by repetitive activation of the Toll like receptor (TLR3) pathway (a.k.a. "training"). Mechanisms of IRF1-dependent target gene remodeling are investigated by CRISPR/Cas9 knockdown, chromatin immunoprecipitation, co-immunoprecipitation and through use of selective inhibitors of the BRD4 chromatin remodeling complex (CRC). We observe "training" increases basal expression of a network of intrinsic IIR genes, reducing Respiratory Syncytial Virus (RSV) replication upon subsequent exposure. In addition, trained hSAECs enter an epithelial mesenchymal plasticity (EMP) program, characterized by cell shape changes, increased expression of cytoplasmic vimentin and core mesenchymal regulators. Mechanistically, we find that Interferon Regulatory Factor 1 (IRF1) is required for innate memory because IRF1-deficient cells fail to activate basal intrinsic IIR. In TLR3 activation, IRF1 is incorporated into the bromodomain containing protein 4 (BRD4) chromatin remodeling complex (CRC), a complex containing the histone H3 acetyltransferase, EP300/p300. In acute TLR3 activation, the binding of BRD4 is increased on intrinsic IIR promoters, yet this binding is unaffected by training. Instead, we find training induces EP300 binding and increases H3K27 acetylation (H3K27ac) at these regions. Small molecule inhibitors show that BRD4 is required for establishing innate memory and H3K27ac accumulation. Finally, we find that training enhances BRD4-EP300 interactions and that memory-induced H3K27ac formation is EP300-dependent. We conclude that hSAECs acquire innate memory through IRF1 coupling with the BRD4-EP300 CRC to activate intrinsic IIR and cell-state transition genes. Our data suggest that innate training is the consequence of dynamic BRD4 CRC interactions through enhanced EP300 recruitment that primes transcriptional responsiveness.

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