Role of host sphingolipids against fungal infections

摘要 本项目的目标是研究鞘磷脂合成酶(Sms1和Sms2)的作用和机制 参与控制由致病真菌新生隐球菌(CN)引起的感染。 我们的实验室率先研究了真菌鞘脂在调节感染性疾病中的作用。 疾病。除了研究真菌鞘脂外，我们最近还发现某些宿主鞘脂发挥作用 在控制对人类真菌病原体CN的免疫反应中起关键作用。一种寄主鞘脂 被证明调节免疫反应的是由鞘磷脂合成酶(SMS)产生的鞘磷脂(SM)，由 SMS1和SMS2基因1，2SMS将胆碱磷酸部分从磷脂酰胆碱(PC)转移到神经酰胺， 产生SM和二酰甘油(DAG)(图1)。3，4这些脂质与许多细胞功能有关，包括 促炎反应的激活，5提示免疫细胞中SMS活性的调节可能 在控制感染方面发挥关键作用。事实上，我们之前已经表明，由短信产生的DAG 可能通过依赖蛋白激酶D(PKD)介导吞噬细胞对CN的体外杀伤 机制，SMS-DAG-PKD信号通路通过吞噬细胞介导抗菌肽的分泌 细胞，特别是防御素(§进度报告，新的图9和图6，7)。 非常有趣的是，我们目前的研究还揭示了SM在富含胆固醇膜的调节中的关键作用 巨噬细胞中的木筏。我们发现，巨噬细胞外膜SM的耗竭显著减少 吞噬作用(图3和图5)，并使FCG受体(FcgR)从点缀的、簇生的变为弥漫的和 均匀分布(图7)。当胆固醇被耗尽时，这种现象得到了验证(图7)， 证实了FcgR与脂筏的联系。这些枯竭的结果是显著的减少 抗体介导的CN吞噬作用(图3、4和5)。 重要的是，在缺乏FcgR的小鼠肺泡巨噬细胞中也观察到了FcgR的移位 Sms1(Sms1-/-)或Sms2(Sms2-/-)，也显示抗体介导的吞噬功能减少(新图12)。 综上所述，这些结果表明，丹参通过调节巨噬细胞对CN细胞的内化 产生SM，它在质膜上稳定富含胆固醇的脂筏(新图8)，用于锚定 FcgR。 Sms1(Sms1-/-)或Sms2(Sms2-/-)的缺失使动物对CN感染非常敏感 (图10)。吸入后，CN细胞在肺内迅速复制，并迅速扩散到Sms1-/-的大脑 或sms2-/-小鼠(新图11)。初步的流式细胞术显示Sms2-/-的免疫细胞成分不同 与WT肺(新图15)相比，sms2-/-小鼠不能形成有效的肺肉芽肿(新图13)。 这些结果表明，在肺环境中，SMS对CN-巨噬细胞界面的调节可能具有 在调节宿主整体免疫力和疾病转归方面发挥关键作用。 基于这些研究，我们假设，通过SM和DAG的产生，SMS调节 吞噬/杀死CN，刺激针对CN的有效宿主免疫反应。因此，我们提出如下建议 目标： 1)确定丹参对CN吞噬及吞噬杀伤作用的调节机制 2)建立丹参调节宿主整体免疫的作用和机制(S)。 CN. 通过研究Sms在CN-吞噬细胞相互作用和感染过程中的作用，我们将能够识别 新的宿主保护机制对开发新的免疫疗法以更好地 控制这种危及生命的疾病。

ABSTRACT The goal of this project is to study the role and mechanisms by which sphingomyelin synthases (Sms1 and Sms2) are involved in controlling the infection caused by the pathogenic fungus Cryptococcus neoformans (Cn). Our laboratory has pioneered studies dealing with the role of fungal sphingolipids in the regulation of infectious diseases. In addition to studying fungal sphingolipids, we have recently discovered that certain host sphingolipids play a key role in controlling the immune response against the human fungal pathogen Cn. One of the host sphingolipid shown to regulate immune responses is sphingomyelin (SM) produced by sphingomyelin synthase (SMS), encoded by the SMS1 and SMS2 genes.1,2 SMS transfers a choline phosphate moiety from phosphatidylcholine (PC) to ceramide, producing SM and diacylglycerol (DAG) (Fig. 1).3,4 These lipids have been implicated in many cellular functions including the activation of pro-inflammatory responses,5 suggesting that the regulation of SMS activity in immune cells may assume a critical role in controlling infections. In fact, we have shown previously that the DAG produced by SMS mediates the in vitro extracellular killing of Cn by phagocytic cells possibly through a protein kinase D (PKD) dependent mechanism, and the SMS-DAG-PKD signaling pathway mediates the secretion of antimicrobial peptides by phagocytic cells, particularly defensins (§ Progress Report, new Fig. 9, and6,7). Very intriguingly, our current studies also revealed a key role for SM in the regulation of cholesterol-rich membrane rafts in macrophages. We found that depletion of SM in the outer membrane of macrophages dramatically decreases phagocytosis (Figs. 3 and 5) and displaces the Fcg receptor (FcgR) from a punctuated, clustered to a diffused and homogeneous distribution (Fig. 7). This phenomenon was validated when cholesterol was depleted (Fig. 7), corroborating the association of the FcgR with lipid rafts. The resulting effect of these depletions is a significant decrease of antibody-mediated phagocytosis of Cn (Figs. 3, 4 and 5). Importantly, the displacement of the FcgR was also observed in alveolar macrophages isolated from mice lacking Sms1 (sms1-/-) or Sms2 (sms2-/-), which also showed a decrease of antibody-mediated phagocytosis (new Fig. 12). Taken together, these results suggest that SMS regulates the internalization of Cn cells by macrophages through the production of SM, which, at the plasma membrane, stabilizes cholesterol-rich lipid rafts (new Fig. 8) for anchoring the FcgR. Deletion of Sms1 (sms1-/-) or Sms2 (sms2-/-) renders the animals significantly hypersusceptible to Cn infection (Fig. 10). Upon inhalation, Cn cells are rapidly replicating in the lung and quickly disseminating to the brain of sms1-/- or sms2-/- mice (new Fig. 11). Preliminary flow cytometry shows a different immuno cellular composition in sms2-/- compared to WT lungs (new Fig. 15), and the sms2-/- mice cannot form an efficient lung granuloma (new Fig. 13). These results suggest that the regulation of SMS at the Cn-macrophage interface in the lung environment may have a key role in the regulation of the overall host immunity and the outcome of the disease. Based on these studies, we hypothesize that, through the production of SM and DAG, SMS regulates the phagocytosis/killing of Cn, stimulating an effective host immune response against Cn. Thus, we propose the following aims: 1) To determine the mechanism by which SMS regulates phagocytosis and killing of Cn by phagocytic cells; and 2) To establish the role and mechanism(s) by which SMS regulates the overall host immunity against Cn. By studying the role of SMS during Cn-phagocytes interaction and during the infection we will be able to identify new mechanisms of host protection with important insights into the development of new immunotherapies to better control this life-threatening disease.