Wu Yuqing, Li Cao, Riehle Andrea, Pollmeier Barbara, Gulbins Erich, Grassmé Heike
Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany.
Department of Surgery, University of Cincinnati, Cincinnati, Ohio, USA.
Cell Physiol Biochem. 2018;51(4):1815-1829. doi: 10.1159/000495683. Epub 2018 Nov 30.
BACKGROUND/AIMS: Mycobacteria-induced diseases, especially tuberculosis, cause more than 1 million deaths each year, which is higher than any other single bacterial pathogen. Neutral sphingomyelinase 2 (Nsm2) has been implied in many physiological processes and diseases, but the role of Nsm2 in pathogen-host interactions and mycobacterial infections has barely been studied.
We investigated the role of the Nsm2/ceramide system in systemic infection of mice and murine macrophages with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a model for mycobacterial infection. For in vitro assays we isolated bone marrow-derived macrophages from Wildtype mice or Nsm2-heterozygous and investigated the role of Nsm2 for macrophage migration/clustering as well as the involvement of p38 mitogen-activated protein kinases (p38K), c-Jun N-terminal kinase (JNK), β1-integrin and Rac1 activity by Western blot and microscopic studies. For in vivo assays we injected mice intravenously with BCG and analyzed infected tissues for the role of Nsm2-mediated activation of β1-integrin in granuloma formation and bacterial burden.
Our results reveal that BCG infection of macrophages results in rapid stimulation of Nsm2. Genetic and pharmacological studies demonstrate that Nsm2 stimulates a signaling cascade via p38K and JNK to an activation of surface β1-integrin and Rac1 that leads to the formation of granuloma-like macrophages clusters in vitro and granuloma in vivo. Heterozygosity of Nsm2 in macrophages or antibody-mediated neutralization of active b1-integrin reduced macrophage clusters in vitro and granuloma formation in vivo. Most importantly, Nsm2 heterozygosity or treatment with neutralizing antibodies against β1-integrin protected mice from systemic BCG infections and chronic infections of the liver and spleen.
The findings indicate that the Nsm2/ ceramide system plays an important role in systemic infection of mice with mycobacteria by regulating a signaling cascade via p38K, JNK, b1-integrin and Rac1.
背景/目的：分枝杆菌引起的疾病，尤其是结核病，每年导致超过100万人死亡，高于任何其他单一细菌病原体。中性鞘磷脂酶2（Nsm2）已被认为参与许多生理过程和疾病，但Nsm2在病原体与宿主相互作用及分枝杆菌感染中的作用几乎未被研究。
我们以牛分枝杆菌卡介苗（BCG）感染小鼠和鼠巨噬细胞作为分枝杆菌感染的模型，研究Nsm2/神经酰胺系统在其中的作用。在体外实验中，我们从野生型小鼠或Nsm2杂合子小鼠中分离出骨髓来源的巨噬细胞，通过蛋白质印迹法和显微镜研究，探讨Nsm2对巨噬细胞迁移/聚集的作用以及p38丝裂原活化蛋白激酶（p38K）、c-Jun氨基末端激酶（JNK）、β1整合素和Rac1活性的参与情况。在体内实验中，我们给小鼠静脉注射BCG，并分析感染组织中Nsm2介导的β1整合素激活在肉芽肿形成和细菌负荷中的作用。
我们的结果显示，巨噬细胞感染BCG会导致Nsm2迅速被激活。遗传学和药理学研究表明，Nsm2通过p38K和JNK刺激信号级联反应，激活表面β1整合素和Rac1，从而在体外导致形成肉芽肿样巨噬细胞簇，在体内导致形成肉芽肿。巨噬细胞中Nsm2的杂合性或抗体介导的活性β1整合素中和会减少体外巨噬细胞簇的形成和体内肉芽肿的形成。最重要的是，Nsm2杂合性或用抗β1整合素中和抗体治疗可保护小鼠免受全身性BCG感染以及肝脏和脾脏的慢性感染。
研究结果表明，Nsm2/神经酰胺系统通过调节经由p38K、JNK、β1整合素和Rac1的信号级联反应，在小鼠分枝杆菌全身性感染中起重要作用。
