脂滴(LD)和神经酰胺(Cer)的积累与非酒精性脂肪性肝病(NAFLD)有关,经常与2型糖尿病并存,免疫功能下降。慢性炎症和病毒感染中疾病严重程度的增加是肥胖相关免疫病理学的标志。中性鞘磷脂酶-2(NSM2)的上调已显示与组织中肥胖的病理有关。然而,鞘脂和特别是NSM2在调节免疫细胞对富含脂肪酸(FA)的环境的反应中的作用研究甚少。这里,我们使用抗坏血酸过氧化物酶APEX2催化的邻近依赖生物素标记方法鉴定了NSM2细胞内纳米环境中LD标记蛋白perilipin3(PLIN3)的存在.与此相符,超分辨率结构化照明显微镜(SIM)显示,在存在细胞外油酸(OA)浓度增加的情况下,LD细胞器中的NSM2和PLIN3共定位。此外,具有酶活性的NSM2与分离的LD的关联与这些脂质储存细胞器中Cer水平的增加有关。NSM2与LD结合不需要NSM2的酶活性,但对长链不饱和三酰甘油(TAG)物种的LD数量和细胞积累产生负面影响。同时,NSM2表达促进线粒体呼吸和脂肪酸氧化(FAO),以响应OA水平的增加,从而将细胞转移到高能状态。重要的是,内源性NSM2活性对于在富含FA的环境中原代人CD4+T细胞存活和增殖至关重要。最后,我们的研究显示了一种新的NSM2细胞内定位到LDs,以及酶活性NSM2在T细胞中FA浓度增加的代谢反应中的作用。
The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.