背景:热带桃花心木\'Neem\'(Azadirachtaindica)的成熟叶子的水溶性成分,被鉴定为糖蛋白,因此被命名为“印鉴叶糖蛋白”(NLGP)。这种无毒的叶片成分使癌性小鼠肿瘤(黑色素瘤,癌,肉瘤)在不同的实验情况下通过增强主要的抗肿瘤免疫属性而反复出现。这种抗肿瘤免疫调节,辅助细胞毒性T细胞(Tc)为基础的肿瘤细胞湮灭。这项研究的重点是识别和表征启动这种全身免疫调节的信号网关。在寻找这个网关时,探索了抗原呈递细胞(APC),激活并诱导Tc细胞的细胞毒性推力。
方法:在APC上发现了六种糖蛋白结合C型凝集素,即,MBR,Dectin-1,Dectin-2,DC-SIGN,在来自C57BL/6J小鼠的骨髓来源的树突状细胞上筛选DEC205和DNGR-1。荧光显微镜,RT-PCR,流式细胞术和ELISA显示Dectin-1为NLGP结合受体,然后通过RNAi进行验证。在检测NLGP中的β-葡聚糖后,在计算机上探索了它们与Dectin-1的相互作用。通过免疫共沉淀研究了第二信使和转录因子在下游信号中的作用,西方印迹,和染色质免疫沉淀。通过处理DC的共焦显微照片观察到FITC偶联的NLGP的细胞内化。
结果:考虑到NLGP驱动的细胞因子IL-10和IL-12p35的受体中和转录速率的阻碍程度,发现树突细胞上的Dectin-1受体通过配体的外周β-葡聚糖链结合NLGP。产生的信号磷酸化PKCδ,形成CARD9,Bcl10和MALT1的三分子复合物,进而激活转录调节的经典NFκB途径。因此,NFκB-异二聚体p65:p50增强Il12a转录,p50:p50同二聚体抑制Il10转录,导致基于细胞因子的系统偏向1型免疫环境。Further,NLGP被树突状细胞吞噬,可能通过Dectin-1的内吞活性。
结论:NLGP与小鼠树突状细胞上的Dectin-1受体结合,随后是细胞内信号,导致NFκB介导的细胞因子转录的对比调节,启动促炎免疫极化,通过包括Tc细胞在内的应答免疫细胞进一步扩增,除了它们增强的细胞毒性。这些对NLGP在细胞和分子水平上启动哺乳动物全身免疫调节的见解,可能有助于揭示其作为一种新型免疫调节剂对抗人类癌症的作用方式,在临床试验之后。
BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany \'Neem\' (Azadirachta indica), was identified as glycoprotein, thus being named as \'Neem Leaf Glycoprotein\' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells.
METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of β-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs.
RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand\'s peripheral β-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1.
CONCLUSIONS: NLGP\'s binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.