Center for Clinical and Translational Research, Virginia Commonwealth University , Richmond, Virginia.
Division of Pediatric Pulmonary Medicine, Children's Hospital of Richmond at Virginia Commonwealth University , Richmond, Virginia.
Am J Physiol Lung Cell Mol Physiol. 2018 Jan 1;314(1):L206-L214. doi: 10.1152/ajplung.00322.2017. Epub 2017 Oct 12.
Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high-mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long-chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long-chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by qRT-PCR and Western analyses, respectively. NE increased lung SPT long-chain subunit 2 (SPTLC2) protein levels but not SPTLC1 and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2 h before NE administration. Myriocin decreased BAL d and d ceramide, KC, and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.
鞘脂代谢改变与炎症增加有关；然而，炎症介质（包括中性粒细胞弹性蛋白酶 NE）对气道鞘脂动态平衡的影响尚不清楚。我们使用 NE 经口咽抽吸的一种经过充分特征描述的小鼠模型，研究了 NE 诱导的气道炎症与各种鞘脂类（包括神经酰胺）合成增加之间的潜在联系。使用反相高效液相色谱/电喷雾串联质谱分析来鉴定和定量支气管肺泡灌洗液 (BAL) 中的鞘脂。测定 BAL 总细胞和分类细胞计数、CXCL1/角质细胞化学引诱物 (KC) 蛋白水平和高迁移率族蛋白 1 (HMGB1) 蛋白水平。NE 暴露增加了 BAL 中的长链神经酰胺、总细胞和中性粒细胞计数，并上调了 KC 和 HMGB1。通过 qRT-PCR 和 Western 分析分别测定丝氨酸棕榈酰转移酶 (SPT) 长链亚基 1 和 2 的 mRNA 和蛋白水平，SPT 是负责从头合成神经酰胺的限速步骤的多聚酶。NE 增加了肺 SPT 长链亚基 2 (SPTLC2) 蛋白水平，但不增加 SPTLC1，对任一亚基的 mRNA 均无影响。为了评估从头合成神经酰胺是否是 NE 诱导炎症所必需的，在给予 NE 之前 2 小时通过腹膜内给予了 SPT 抑制剂——美拉诺坦或载体对照。美拉诺坦降低了 BAL 中由 NE 暴露引起的 d 和 d 神经酰胺、KC 和 HMGB1。这些结果支持了 NE 产生的神经酰胺和神经酰胺驱动的细胞因子信号转导的正反馈循环，这可能是慢性嗜中性粒细胞炎症为特征的肺部疾病的潜在干预靶点。
