TLR4信号通路在微量尼古丁刺激增强树突状细胞抗原交叉提呈中的作用研究

Efficient cross-presentation of dendritic cells (DCs) required mannose receptor (MR) mediating antigen uptake and endotoxin-induced, Toll-like receptor 4 (TLR4) and signaling molecule MyD88-dependent relocation of the transporter associated with antigen processing (TAP) to early endosomes. Our previous studies showed that nicotine stimulation （16.5ng/ml）could enhance DCs` cross-presentation and achieve anti-tumor effects, but the mechanisms of DCs` cross-presentation enhanced by nicotine treatment are still to be clarified and need further studies. In this project, the upregulations of α7 nAchR, MR and TLR4 with nicotine stimulation were firstly determined by Western blot and flow cytometry respectively; Then, using SiRNA transfection to silence TRL4 pathway related compotents, both the enhanced effects of relocation of MR, TAP, MHC and antigen in DCs`early endosomes by nicotine treatment and the relocations (MR, TAP, MHC and antigen) depending on TLR4 pathway activation were secondly explored by immunofluorescence; Thirdly, the activations of α7 nAchR-p38-PI3K-AKT-mTOR pathways by nicotine treatment were determined by Western blot; Forthly and importently, using kinase inhibitors or SiRNA to inhibit p38, AKT and mTOR kinases, the mechnisms of α7 nAchR, MR, TLR4 upregulation by nicotine treatment were explored by Western blot and Real time PCR respectively. The key point of our project is to explore the mechanisms of DCs` cross-presentation enhanced by nicotine treatment, which could provide fundamental theory support for clinical use of nicotine stimulated DCs to deal with tumor and viral infection.

MR(甘露糖受体)介导的抗原内吞和依赖TLR4通路活化的TAP于早期内体重定位是DCs交叉提呈抗原的关键。微量尼古丁（16.5ng/ml）虽可增强DCs交叉提呈抗原能力并具有抗肿瘤效应，但尼古丁增强DCs交叉提呈抗原的机制不清。本课题拟先以免疫印迹、FACS发现尼古丁对α7 nAchR 、MR、TLR4的调控作用；继以SiRNA沉默技术结合免疫荧光探讨尼古丁对DCs MR、TAP、MHC、抗原肽在早期内体共定位的影响及对TLR4通路TLR4、MyD88、TRIF的依赖程度；再以免疫印迹、Real time PCR结合使用激酶抑制剂或SiRNA分别沉默p38、AKT、mTOR，检测尼古丁激活α7 nAchR-p38-PI3K-AKT-mTOR通路情况及调控α7 nAchR 、MR、TLR4表达的机制，阐明尼古丁增强DCs交叉提呈抗原的机制，为应用尼古丁刺激DCs抗肿瘤免疫治疗提供理论依据。

DCs 交叉提呈依赖于甘露糖受体(MR)等分子所介导的抗原内体转位、内体对ATP酶p97、Sec61的招募。DCs是表达乙酰胆碱N型受体（nAchR）的APC。我们研究发现，尼古丁可调控DCs表面nAchR及CD80/86等表达、增强吞噬而促进DCs介导的交叉提呈；在LPS环境中，尼古丁对DCs交叉提呈的增强作用又转为免疫麻痹，但尼古丁对DCs的上述作用的机制不清。为此，本课题开展了如下工作：.1..先检测尼古丁对DCs表面MR、TLR4表达影响及MR、TLR4在尼古丁增强DCs交叉提呈中的作用。显示：尼古丁经PI3K-Akt-mTOR-p70S6通路增加人和小鼠DCs表面MR和TLR4分子表达；尼古丁对DCs交叉提呈的增强作用依赖于MR介导的抗原定位于内体和TLR4-MyD88-IRAK4介导的TAP内体转位。（此部分工作已发表）.2..探讨IFN-β逆转DCs交叉提呈致免疫麻痹的机制。显示： LPS不仅增强DCs抗原提呈能力、增加表面分子表达， 而且上调TGF-beta、TNF-alpha、 IL-6、IFN-beta表达； LPS诱生的 IFN-beta上调DCs表面共抑制分子B7-H1和GITRL表达；B7-H1与T细胞表面PD-1结合，抑制T细胞增殖；GITRL与GITR作用诱生Treg介导免疫麻痹。（此部分工作已发表）.3..观察泛素化过程在内体招募p97/Sec61的作用；发现泛素化调控p97/Sec61转位内体的机制。显示：抑制泛素化显著降低交叉提呈；LPS所致的TLR4、MyD88的内体转位可继而促进p97、Sec61内体转位，而泛素化抑制剂Thalidomide和PYR-41通过抑制NF-κB通路激活、抑制MyD88内体转位而显著逆转LPS对p97/Sec61的作用并影响交叉提呈和交叉致敏。（此部分工作待发表）.科学意义：本课题发现了尼古丁调控DCs表面模式识别受体的规律，初步阐明了尼古丁增强DCs交叉提呈抗原的分子机制；发现细菌性感染经IFN-β上调B7-H1（PD-L1）表达、诱导Treg致免疫麻痹的初步机制；明晰了交叉提呈中泛素化过程致ATP酶内体转位的机制。为开展尼古丁刺激DCs的免疫治疗、克服细菌感染致免疫麻痹及继发性感染提供理论依据及潜在分子靶点。