Dectin-1内在化表达介导氧依赖性方式杀伤白色念珠菌

Intracellular expression of Dectin-1 mediates the killing of Candida albicans by human neutrophils in a manner dependent on ROS production

目的探讨Dectin-1介导人嗜中性粒细胞以氧依赖性方式杀伤调理吞噬后白色念珠菌孢子的机制。方法在白色念珠菌孢子作用前后，流式细胞术和激光共聚焦显微镜检测人嗜中性粒细胞中Dectin．1的表达率及表达部位；并通过Dectin-1阻断试验分析嗜中性粒细胞中Dectin-1的表达与胞内活性氧簇（reactiveoxygenspecies，ROS）的产生及对白色念珠菌孢子杀伤的相关性。结果在白色念珠菌孢子刺激30或60min后，细胞内Dectin-1的表达率明显上调（0min，8．32％；30min，16．82％；60rain，23．88％。与0min相比，P均〈0．01），且Dectin-1与ROS在胞内的表达均被募集到吞噬的白色念珠菌孢子表面。Dectin-1的阻断分别与ROS峰值的抑制（R2=0．306，P〈0．01）及白色念珠菌杀伤率的降低（R2=0．251，P〈0．01）呈剂量依存关系。结论Dectin-1可通过内在化的表达模式介导人嗜中性粒细胞以ROS依赖性方式杀伤调理吞噬的白色念珠菌孢子。

Objective To investigate the mechanism by which Dectin-1 mediates the killing of Candida albicans （C. albicans） by human neutrophils in a manner dependent on the production of reactive oxygen species （ROS）. Methods After stimulation with FITC-C. albicans at a multiplicity of infection （MOI） of 10 for 30 or 60 rain, PE-anfi-human Dectin-1 monoclonal antibody （2.5μg/10^6 ceils） was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry. For Dectin-1 inhibition test and ROS assay, human neutrophils （2×10^6/ml） were respectively pre-incubated with different concentrations of blocking antibody （0.5, 1, 2.5 and 5 μg/ml） for 60 min at 4℃, and then with 25 μmol/L 2＇ ,7＇-Dichlo- rofluorescein diacetate for another 20 min at room temperature. Afterwards, under stimulation with live C. al- bicans at a MOI of 10, the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min. In addition, localization of Dectin-1 and ROS in human neutrophils was observed by confocal/two-photon laser scanning microscopy after stimulation with live C. albicans. For the detection of candicidal activity, after pre-treatment with different concentrations of Dectin-1 blocking antibody as mentioned above, neutrophils were stimulated with live C. albicans （ MOI = 1 ） for 60 min, serial dilutions of cell lysate were plated onto yeast agar, and CFU were enumerated after incuba- tion at 37℃ for 48 h. The candicidal activity was represented as [ 1-（ CFU_blocking group/CFU_buffer ）] ×100%. Results Under stimulation with FITC-C. albicans at a MOI of 10 for 30 and 60 rain, positive percentage of intracellular Dectin-l-expressing neutrophils increased significantly when compared with initial level （0 rain, 8.32% ; 30 min, 16.82% ; 60 min, 23.88% ） （ versus 0 rain, P〈0. 01 ）. However, positive percentage of cell-surface Dectin-l-expressing neutrophils remained almost unchanged after stimulation for 30 and 60 min （versus 0 rain, P〉0.05）. Upon blockage of Dectin-1, the stimulated ROS generation （R2= 0.306, P〈 0.01 ） and candicidal activity （R2 =0.251, P〈0.01 ） of nentrophils were partly and irreversibly inhibited in a dose-dependent manner when compared with control group. In addition, the intracellular Dectin-1 is re- cruited and co-distributed with ROS on the surface of phagocytized C. albicans as observed by confocal mi- croscopy. Conclusion Taken together, these results demonstrated that an internalized expression pattern ofhuman Dectin-1 might contribute to the ROS-dependent killing of serum-opsonzied C. albicans which was phagocytized by human neutrophils.