LAPF和CD11b负向调控天然免疫应答及其机制研究

发布时间：2018-07-29 13:07

【摘要】：天然免疫应答是机体抵抗病原微生物入侵的第一道防线,但是,过度活化的天然免疫反应会导致组织器官的损伤,可导致自身免疫病的产生甚至造成病人死亡,所以,天然免疫应答反应需要及时活化以有效清除入侵的病原体,同时,其应答程度与持续时间也应控制在适度范围内以维持机体平衡,可见,天然免疫应答反应的负向调控对于机体维持免疫自稳至关重要。近年来,发现与研究能够负向调控天然免疫的细胞和分子逐步成为天然免疫研究领域的前沿热点。本课题围绕着两个负向调控分子的作用与相关机制,分两部分开展了负向调控天然免疫应答及其机制研究,包括"LAPF抑制TLR触发巨噬细胞产生TNF-α及其机制研究”和"CD11b-Src信号通路促进M2型巨噬细胞产生及其机制研究”,以期通过我们的研究,加深对天然免疫调节和免疫自稳的认识和理解。第一部分LAPF抑制TLR触发巨噬细胞产生TNF-a及其机制研究包含PH和FYVE结构域的溶酶体相关凋亡诱导蛋白LAPF(又叫PLEKHF1)是我们实验室从人树突状细胞cDNA文库中通过随机大规模测序发现的一个新的溶酶体相关蛋白并于2005年报道其参与TNF-a诱导的肿瘤细胞凋亡。然而有关LAPF在天然免疫中的功能还没有研究。在本课题中,我们制备了LAPF条件缺失的小鼠( lapfloxp/loxplyz2Cre/+),发现LAPF条件缺失小鼠相比于杂合子对照小鼠在内毒素休克模型中炎性细胞因子分泌增加,其中TNF-α显著升高,同时肺脏和脾脏中TNF-α的mRNA水平也显著升高,提示LAPF可能负向调控天然免疫反应与炎症发生。通过体外细胞实验,发现LAPF缺失的巨噬细胞在TLR配体刺激下分泌TNF-α升高并且TAK-、 IKKa/β和p65的磷酸化活化水平显著提高,表明LAPF能够抑制NF-κB信号通路的活化。利用Ip-质谱分析结合体外IP验证,我们发现LAPF可以与TLR信号关键转运蛋白TIRAP和MyD88结合,为进一步研究LAPF如何调控NF-κB信号通路、影响炎性细胞因子释放奠定了基础。第二部分CDllb-Src信号通路促进M2型巨噬细胞产生及其机制研究整合素信号对于天然免疫应答的调控一直是天然免疫的前沿热点问题。整合素对于天然免疫细胞的分化、发育、迁移、粘附和活化均有十分重要的作用。CDllb是整合素Mac-1的a链,CDllb的抗体常常用来筛选出巨噬细胞或者其它髓系来源的单核吞噬细胞。巨噬细胞根据表型和行使功能的不同又可以分为M1型(经典性活化的)巨噬细胞和M2型(替代性活化的)巨噬细胞,分别释放出促炎性细胞因子和抑炎性细胞因子,M1和M2型巨噬细胞的平衡对于天然免疫的调节和自稳至关重要。我们实验室以往发现CDllb能够通过Src-Syk泛素化降解MyD88和TRIF,从而抑制天然免疫过程中TNF-a的产生。但是,CD11b-Src在巨噬细胞M1和M2的表型和功能中的作用并不清楚,值得进一步研究。我们发现在体外诱导肠炎模型中,CD1lb缺陷的小鼠以及利用Src抑制剂Dasatinib腹腔注射处理的小鼠,结肠部位的炎症反应均更为严重,小鼠TNF-a产量增加但IL-10的产量减少,同时,iNOS的表达增强但Arg-1的表达减弱。TNF-a和iNOS是M1型巨噬细胞的标志性分子,IL-10和Arg1是M2型巨噬细胞的标志性分子,这个现象提示了CD11b-Src信号可能促使巨噬细胞趋向于M2型巨噬细胞。为此,我们直接检测了用Dasatinib处理并且诱导肠炎的小鼠结肠部位的巨噬细胞类型,发现M1型巨噬细胞比例增加、M2型巨噬细胞比例减少。随后我们开展了CD11b-Src使巨噬细胞更趋向于M2型巨噬细胞相关机制研究,发现1)骨髓来源的巨噬细胞中CD11b-Src可以抑制IFN-γ诱导的M1型巨噬细胞标志分子iNOS的表达,CD11b-Src还能够促进IL-4诱导的M2型巨噬细胞标志分子Arg-1和YM-1的表达；2)CDllb通过促进Src-Akt信号通路活化,促进了TLR诱导的STAT6活化和IL-10释放。进一步的机制研究发现,Src能够增强IL-4信号通路的Akt和STAT6活化,这两者均被报道能够促进M2型巨噬细胞表型分子的表达。但是,只有过表达STAT6能够逆转Src抑制剂对Argl的下调作用,提示STAT6在此过程的重要作用。Src能够和STAT6相互作用,形成复合体,也进一步提示Src能够参与活化STAT6.在TLR信号刺激下,Src则通过促进P13K的调节亚基p85的泛素化降解,促进PI3K-Akt信号通路活化,进而通过负向调控GSK促进AP-1的活化入核,最终促进IL-10的产生。综上,CD11b-Src通过促进STAT6和Akt活化而使巨噬细胞更趋向于M2型巨噬细胞。可见,CD11b-Src具有重要的天然免疫负向调控与控制炎症的作用,深入研究CD11b-Src的机制将有助于深入认识炎症的发生发展过程和抗炎药物的研发。
[Abstract]:The natural immune response is the first line of defense for the organism to resist the invasion of pathogenic microbes. However, the excessive activation of natural immune response can lead to the injury of the tissues and organs, which can lead to the production of autoimmune diseases and even cause the death of the patients. Therefore, the natural immune response should be activated in time to effectively remove the invading pathogens. The degree and duration of answer should also be controlled in a moderate range to maintain the balance of the body. It can be seen that the negative regulation of natural immune response is crucial to the maintenance of immune self stability. In recent years, the discovery and study of cells and molecules that can negatively regulate natural immunity gradually become the forefront of the field of natural immune research. Around the role of two negative regulators and related mechanisms, two parts of the negative regulation of natural immune response and its mechanism are carried out, including "LAPF inhibition of TLR triggering macrophages to produce TNF- alpha and its mechanism research" and "CD11b-Src signaling pathway to promote the production and mechanism of M2 macrophages", with a view to through our research To deepen the understanding and understanding of natural immune regulation and immune self stability. Part 1 LAPF inhibits TLR triggering macrophages to produce TNF-a and its mechanism, which includes PH and FYVE domain lysosome related apoptosis induced protein LAPF (also called PLEKHF1) which is by random large-scale sequencing from human dendritic cell cDNA library. A new lysosome related protein is now reported to be involved in the apoptosis of tumor cells induced by TNF-a in 2005. However, the function of LAPF in natural immunity has not been studied. In this subject, we prepared the LAPF deficient mice (lapfloxp/loxplyz2Cre/+), and found that the LAPF condition deficient mice were compared to the heterozygote control mice. The secretion of inflammatory cytokines increased in the endotoxin shock model, in which TNF- alpha was increased significantly, while the mRNA level of TNF- a in the lungs and spleen also increased significantly, suggesting that LAPF may negatively regulate the natural immune response and inflammation. Through in vitro cell experiments, it was found that the LAPF deficient macrophages secreted the increase of TNF- alpha under the TLR ligand stimulation. And TAK-, the activation level of phosphorylation of IKKa/ beta and p65 significantly increased, indicating that LAPF could inhibit the activation of NF- kappa B signaling pathway. Using Ip- mass spectrometry analysis and in vitro IP validation, we found that LAPF can be combined with TIRAP and MyD88 with TLR signal key transporter protein, which can further study the regulation of inflammatory cells and affect inflammatory cells. Factor release lays the foundation. Second CDllb-Src signaling pathway promotes the production and mechanism of M2 type macrophages. The regulation of integrin signal for natural immune response has been a hot issue in the frontier of natural immunity. Integrin has a very important role in the differentiation, development, migration, adhesion and activation of natural immune cells. CDllb is the a chain of integrin Mac-1, and CDllb antibodies are often used to screen out macrophages or other myeloid mononuclear phagocytes. Macrophages can be divided into M1 type (classic activated) macrophages and M2 type macrophages based on the phenotype and function of the macrophages, which release proinflammatory cytokines, respectively. The balance between M1 and M2 macrophages is essential for the regulation and self stability of natural immunity. In our laboratory, we have found that CDllb can degrade MyD88 and TRIF through Src-Syk ubiquitination, thus inhibiting the production of TNF-a in natural immune processes. However, CD11b-Src is in the phenotype and function of M1 and M2 in macrophages. The effect was not clear and worthy of further study. In the model of in vitro induced enteritis, we found that the CD1lb deficient mice and the mice treated with the Src inhibitor Dasatinib intraperitoneally were more severe in the colon, and the production of TNF-a in the mice was increased but the production of IL-10 decreased, and the expression of iNOS was enhanced but the expression of Arg-1 was enhanced. Weakened.TNF-a and iNOS are marker molecules of M1 type macrophages, IL-10 and Arg1 are marker molecules of M2 macrophages. This phenomenon suggests that CD11b-Src signals may induce macrophages to tend to M2 type macrophages. To this end, we directly detected the macrophages in the colon of mice that were treated with Dasatinib and induced enteritis. The proportion of M1 type macrophages increased and the proportion of M2 type macrophages decreased. Then we carried out CD11b-Src to make macrophages more inclined to M2 macrophage related mechanisms, and found that CD11b-Src in bone marrow derived macrophages can inhibit the expression of the M1 type macrophage marker molecule iNOS, which is induced by IFN- gamma, and CD11b-Src also can be used. Promote the expression of IL-4 induced M2 macrophage markers Arg-1 and YM-1; 2) CDllb promotes TLR induced STAT6 activation and IL-10 release by promoting the activation of Src-Akt signaling pathway. Further mechanism studies found that Src enhances Akt and activation of IL-4 signaling pathways, both of which are reported to promote macrophages. The expression of phenotypic molecules. However, only overexpression of STAT6 can reverse the downregulation of Src inhibitors to Argl, suggesting the important role of STAT6 in this process,.Src can interact with STAT6 to form a complex, and further hints that Src can participate in activated STAT6. in the TLR signal stimulation, Src by promoting P13K to regulate the ubiquitin of subunit p85. Degradation, promoting activation of PI3K-Akt signaling pathway, and further promoting the activation of AP-1 through negative regulation of GSK, eventually promotes the production of IL-10. In conclusion, CD11b-Src makes macrophages more likely to be M2 type macrophages by promoting STAT6 and Akt activation. Thus, CD11b-Src has an important role in natural immune negative regulation and control of inflammation. Studying the mechanism of CD11b-Src will help us to further understand the occurrence and development of inflammation and the research and development of anti-inflammatory drugs.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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