Department of Immunology, Institute of Tuberculosis Control, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, China; Department of Laboratory Medicine, Guangzhou First Municipal People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou 510500, China.
Department of Immunology, Institute of Tuberculosis Control, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, China; Shenzhen Key Laboratory of Reproductive Immunology for Peri-implantation, Fertility Center, Shenzhen Zhongshan Urology Hospital, Shenzhen 518045, China.
J Infect. 2015 Apr;70(4):415-26. doi: 10.1016/j.jinf.2014.09.013. Epub 2014 Oct 13.
To explore the role of myeloid-related protein 8/14 in mycobacterial infection.
The mRNA and protein expression levels of MRP8 or MRP14 were measured by real-time PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry.
MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROS-dependent manner.
The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases.
探讨髓系细胞表达的触发受体-1（MRP8/14）在分枝杆菌感染中的作用。
采用实时 PCR 和流式细胞术分别检测 MRP8 或 MRP14 的 mRNA 和蛋白表达水平。通过过表达或 RNA 干扰实验检测 MRP8/14 的作用。采用流式细胞术和集落形成单位实验分别检测细胞内牛分枝杆菌（BCG）的吞噬作用和存活情况。通过 Western blot 和免疫荧光检测由 MRP8/14 介导的自噬。通过共聚焦显微镜检测 BCG 吞噬体与自噬体或溶酶体的共定位。通过流式细胞术检测 ROS 产生。
分枝杆菌刺激后，人单核细胞 THP1 细胞和原代巨噬细胞中 MRP8/14 的表达上调。沉默 MRP8/14 抑制了细菌杀伤，但对 BCG 的吞噬作用没有影响。重要的是，沉默 MRP8/14 减少了 THP1 衍生巨噬细胞中的自噬和 BCG 吞噬体成熟，从而增加了 BCG 的存活。此外，我们证明 MRP8/14 通过促进 ROS 生成来促进自噬。
本研究揭示了 MRP8/14 在通过促进 ROS 生成介导细胞内 BCG 的自噬消除中的新作用，这可能为结核病和其他细胞内细菌感染性疾病提供有希望的治疗靶点。
