Role of host sphingolipids against fungal infections

宿主鞘脂对抗真菌感染的作用

• 批准号: 10427149
• 负责人: Maurizio Del Poeta
• 金额: --
• 依托单位: NORTHPORT VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-10-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427149
• 关键词: Alveolar Macrophages; Animals; Antibodies; Brain; Cell membrane; Cell physiology; Cells; Cellular Immunity; Ceramides; Cholesterol; Communicable Diseases; Cryptococcosis; Cryptococcus neoformans; Cryptococcus neoformans infection; Cyclic AMP-Dependent Protein Kinases; Defensins; Development; Diffuse; Diglycerides; Disease; Disease Outcome; Environment; Exposure to; Flow Cytometry; Functional disorder; Genes; Goals; Granuloma; Host resistance; Human; Immune; Immune response; Immunity; Immunocompetent; Immunotherapy; In Vitro; Individual; Infection; Infection Control; Inflammatory Response; Inhalation; Laboratories; Lecithin; Life; Lipids; Lung; Mediating; Membrane; Membrane Microdomains; Military Personnel; Mus; Mycoses; Nitric Oxide; Pathway interactions; Peptides; Phagocytes; Phagocytosis; Phosphorylcholine; Play; Predisposition; Production; Progress Reports; Reactive Oxygen Species; Regulation; Role; Signal Pathway; Site; Sphingolipids; Sphingomyelins; Testing; antimicrobial; antimicrobial peptide; base; extracellular; insight; macrophage; novel therapeutic intervention; pathogenic fungus; protein kinase D; pulmonary granuloma; receptor; sphingomyelin synthase; Alveolar Macrophages; Animals; Antibodies; Brain; Cell membrane; Cell physiology; Cells; Cellular Immunity; Ceramides; Cholesterol; Communicable Diseases; Cryptococcosis; Cryptococcus neoformans; Cryptococcus neoformans infection; Cyclic AMP-Dependent Protein Kinases; Defensins; Development; Diffuse; Diglycerides; Disease; Disease Outcome; Environment; Exposure to; Flow Cytometry; Functional disorder; Genes; Goals; Granuloma; Host resistance; Human; Immune; Immune response; Immunity; Immunocompetent; Immunotherapy; In Vitro; Individual; Infection; Infection Control; Inflammatory Response; Inhalation; Laboratories; Lecithin; Life; Lipids; Lung; Mediating; Membrane; Membrane Microdomains; Military Personnel; Mus; Mycoses; Nitric Oxide; Pathway interactions; Peptides; Phagocytes; Phagocytosis; Phosphorylcholine; Play; Predisposition; Production; Progress Reports; Reactive Oxygen Species; Regulation; Role; Signal Pathway; Site; Sphingolipids; Sphingomyelins; Testing; antimicrobial; antimicrobial peptide; base; extracellular; insight; macrophage; novel therapeutic intervention; pathogenic fungus; protein kinase D; pulmonary granuloma; receptor; sphingomyelin synthase

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.

抽象的 该项目的目的是研究鞘磷脂合酶的作用和机制（SMS1和SMS2） 参与控制由致病真菌新甲虫（CN）引起的感染。 我们的实验室进行了开创性的研究，涉及真菌鞘脂在传染性调节中的作用 疾病。除了研究真菌鞘脂，我们最近发现某些宿主鞘脂玩具 控制针对人类真菌病原体CN的免疫反应的关键作用。主机鞘脂 显示用于调节免疫反应的是由鞘磷脂合酶（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和二酰基甘油（DAG）（图1）.3,4这些脂质在包括 促炎反应的激活，5表明免疫细胞中SMS活性的调节可能 在控制感染中扮演关键作用。实际上，我们先前已经证明了SMS产生的DAG 通过蛋白激酶D（PKD）依赖于吞噬细胞对CN的体外细胞外杀死的体外细胞外杀死 机理，SMS-DAG-PKD信号通路介导吞噬细胞的抗菌胡椒的分泌 细胞，特别是防御素（§进度报告，新图9和6,7）。 非常有趣的是，我们目前的研究还揭示了SM在调节富含胆固醇的膜中的关键作用 巨噬细胞中的筏。我们发现，巨噬细胞外膜中SM的耗竭急剧下降 吞噬作用（图3和5），并将FCG受体（FCGR）从点状，聚集到扩散和 均匀分布（图7）。当胆固醇耗尽时，该现象得到了验证（图7）， 证实了FCGR与脂质筏的关联。这些耗竭的结果是显着减少 CN抗体介导的吞噬作用（图3、4和5）。 重要的是，在缺乏的小鼠中分离出的肺泡巨噬细胞中还观察到了FCGR的位移 SMS1（SMS1 - / - ）或SMS2（SMS2 - / - ），也显示出抗体介导的吞噬作用的减少（新图12）。 综上所述，这些结果表明，SMS通过巨噬细胞通过 SM的产生，该SM在质膜上稳定了富含胆固醇的脂质筏（新图8），用于锚定 FCGR。 删除SMS1（SMS1 - / - ）或SMS2（SMS2 - / - ）使动物显着过度启发CN感染 （图10）。吸入后，CN细胞正在肺部迅速复制，并迅速传播到SMS1 - / - 的大脑 或SMS2 - / - 小鼠（新图11）。初步流式细胞术显示SMS2 - / - 中不同的免疫细胞组成 与WT肺（新图15）相比，SMS2 - / - 小鼠无法形成有效的肺肉芽肿（新图13）。 这些结果表明，在肺部环境中CN巨噬细胞界面处的SMS调节可能具有 在整体宿主免疫和疾病结果调节中的关键作用。 基于这些研究，我们假设通过生产SM和DAG，SMS调节了 CN的吞噬作用/杀死，刺激针对CN的有效宿主免疫反应。那，我们提出以下内容 目标： 1）确定SMS调节吞噬作用和通过吞噬细胞杀死的机制 细胞； 2）建立SMS调节整体宿主免疫的作用和机制 CN。 通过研究SMS在CN-Phagocytes相互作用中的作用以及在感染期间，我们将能够识别 宿主保护的新机制，具有对新免疫疗法发展的重要见解，以改善 控制这种威胁生命的疾病。