Effects of MRSA and FTY720 S-phosphonate on H3K9 acetylation in human lung endothelial cells

Methicillin-resistant Staph. Aureus (MRSA) is a common cause of severe pneumonia and sepsis that can lead to Acute Respiratory Distress Syndrome (ARDS). MRSA causes lung endothelial cell (EC) dysfunction, a critical step in the pathogenesis and progression of lung injury. Our previous studies have demonstrated that FTY720 S-phosphonate (Tysiponate, Tys), an analog of sphingosine-1-phosphate, ameliorates MRSA-induced lung EC activation and barrier disruption (PMID: 35015568). To advance our mechanistic understanding of MRSA and Tys effects on lung EC, we investigated associated epigenetic changes. Specifically, we studied histone lysine acetylation, which is a central epigenetic alteration that has been linked to gene transcription and functional regulation of endothelial responses to inflammatory stimuli. We therefore determined the effects of MRSA exposure in the presence or absence of Tys on lung EC acetylation at the 9th lysine residue of the histone H3 protein (H3K9ac), which is an important chromatin modification associated with active promoters and gene activation. ChIP-seq analysis was employed to perform an unbiased genome-wide profiling of H3K9ac epigenetic patterns in human lung EC. This analysis identified multiple genes that are differentially targeted by acetylation when EC are exposed to MRSA±Tys. Overall design: Chromatin immunoprecipitation sequencing (Chip-Seq) for histone modification H3K9 acetylation in human pulmonary artery endothelial cells treated with heat-killed methicillin resistant Staph Aureus (HK-MRSA), Tysiponate (FTY720 S-phosphonate; S1P analogue),or HK-MRSA +Tys for 30 min. Cells treated only with vehicle were used as control.

耐甲氧西林金黄色葡萄球菌（Methicillin-resistant Staph. Aureus, MRSA）是引发重症肺炎与脓毒症的常见病原菌，此类感染可进展为急性呼吸窘迫综合征（Acute Respiratory Distress Syndrome, ARDS）。MRSA可诱导肺内皮细胞（lung endothelial cell, EC）功能障碍，这是肺损伤发病机制与病程进展中的关键环节。本团队既往研究证实，1-磷酸鞘氨醇（sphingosine-1-phosphate）的类似物FTY720 S-磷酸酯（Tysiponate，简称Tys）可缓解MRSA诱导的肺内皮细胞活化及屏障破坏（PMID: 35015568）。为进一步解析MRSA与Tys对肺内皮细胞的作用机制，本研究针对相关表观遗传改变展开了探究。具体而言，我们聚焦组蛋白赖氨酸乙酰化——这一类核心表观遗传修饰，其与基因转录及内皮细胞对炎症刺激的应答功能调控密切相关。据此，我们检测了在Tys存在与否的条件下，MRSA暴露对肺内皮细胞组蛋白H3蛋白第9位赖氨酸残基乙酰化（H3K9ac）的影响；H3K9ac是与活跃启动子及基因激活相关的重要染色质修饰。本研究采用染色质免疫共沉淀测序（Chromatin immunoprecipitation sequencing, ChIP-seq）技术，对人肺内皮细胞的H3K9ac表观遗传图谱开展了无偏倚的全基因组分析。该分析鉴定出多个当肺内皮细胞暴露于MRSA±Tys时，乙酰化修饰存在差异的靶基因。

实验整体设计：以人肺动脉内皮细胞为研究对象，分别采用热灭活耐甲氧西林金黄色葡萄球菌（heat-killed methicillin resistant Staph Aureus, HK-MRSA）、Tysiponate（FTY720 S-磷酸酯；1-磷酸鞘氨醇类似物）、HK-MRSA联合Tys进行处理，处理时长均为30分钟；以仅经溶剂处理的细胞作为空白对照。