Caspase-1调控单核—巨噬细胞分化的效应及机制

发布时间：2018-04-13 20:20

本文选题：单核-巨噬细胞分化 + caspase-1　；参考：《南京大学》2012年博士论文

【摘要】：巨噬细胞是单核吞噬细胞系统中的重要成员,在天然免疫和获得性免疫中都发挥着重要作用。体内巨噬细胞的来源包括单核细胞起源的巨噬细胞、原位增殖而生成的巨噬细胞以及卵黄囊巨噬细胞来源的巨噬细胞,其中单核细胞起源的巨噬细胞是组织巨噬细胞的主要来源。在机体稳态或发生炎症的时候,单核细胞都能在微环境中一些因子的调控下向巨噬细胞分化。单核-巨噬细胞分化过程的紊乱与多种疾病的发生发展密切相关,如自身免疫疾病、自身炎症性疾病、代谢性疾病以及肿瘤等。因此,对于单核-巨噬细胞分化过程相关调控及分子机制的研究一直以来都是免疫学领域的热点。Caspase-1是caspases家族中与炎症密切相关的一个成员,对于巨噬细胞的功能具有关键的调控作用。Caspase-1缺失的巨噬细胞释放炎症因子IL-1p和IL-18的能力显著下降;同时caspase-1还能调控巨噬细胞由M2型到M1型的极化。此外,caspase-1也能参与细胞分化程序的控制。Caspase-1能够通过调控IL-1p的加工、释放而控制脂肪细胞和Th17细胞的分化。但是,对于caspase-1在单核-巨噬细胞分化中的功能我们还知之甚少。基于caspase-1广泛的生物学功能及其在单核细胞、巨噬细胞的核心地位,caspase-1在单核-巨噬细胞的分化过程中也具有重要的调控功能。在本研究中,我们通过构建体外诱导人单核细胞白血病细胞株(THP-1、U937)或人外周血单核细胞(PBMCs)向巨噬细胞分化的模型,深入探讨了 caspase-1对于单核-巨噬细胞分化过程的调控效应以及所涉及的分子机理,主要研究内容和结果如下:1.我们运用PMA成功诱导THP-1和U937细胞向巨噬细胞分化,并通过分析此过程中细胞形态、表面标志分子(markers)及吞噬功能的变化情况而将单核-巨噬细胞的分化过程分为早期和晚期两个阶段。2.我们发现caspase-1在THP-1和U937细胞中呈组成型的高表达;然而随着分化的进行其活性不断升高,并伴随有IL-1β和IL-18的释放。Caspase-1活性升高的现象在M-CSF或GM-CSF诱导PBMCs向巨噬细胞分化的模型中也得到了证实。并且通过分析炎症小体各组分基因表达结果显示caspase-1的活化与炎症小体的组装相关。3. Caspase-1特异性抑制剂WEHD能够剂量依赖性地抑制PMA诱导的巨噬细胞makers (CD11b、CD14)的表达,并且这种抑制作用主要发生在分化的晚期;同时WEHD还能明显抑制巨噬细胞的吞噬功能。在PBMCs的分化中,WEHD也具有明显的抑分化效果。4.干扰caspase-1的表达能抑制caspase-1的激活,并能显著抑制CD1 1b和CD14的表达以及细胞的吞噬活性。我们发现急性单核细胞白血病(AML-M5b)病人骨髓单核细胞中caspase-1的基因水平和蛋白水平均发生显著下调,这与AML-M5b中细胞分化程序的阻滞和单核细胞的积累密切相关。5.基于以上的生物学效应,我们探讨了 caspase-1促进单核-巨噬细胞分化的分子机制:(1) 在分化早期PPARγ的表达逐渐升高;而在晚期PPARγ的表达则明显下降。Caspase-1的特异性抑制剂或shRNA能够逆转分化晚期PPARy的下调,但是其mRNA水平却没有明显改变。在HEK293T细胞中重组NLRP3炎症小体能有效激活caspase-1,并能下调PPARγ的蛋白表达,但caspase-1不能直接切割PPARγ。(2) 在分化早期PPARγ的表达上调,转录活性也升高;PPARγ能够调控细胞周期相关蛋白cyclinD1和p21的表达,从而抑制细胞周期并促进单核-巨噬细胞的分化进程。在分化晚期,PPARy配体troglitazone能够通过抑制caspase-1的活性而逆转PPARγ下调的现象,并诱导NF-κB转录活性的抑制,从而抑制单核-巨噬细胞的分化进程。(3) 我们还对caspase-1的经典底物在单核-巨噬细胞分化中的作用进行了分析。运用washing away、补加细胞因子或补加细胞因子对应中和抗体的方法,结果表明单核-巨噬细胞的分化不依赖于IL-1β和IL-18的释放。IL-1α和IL-33在分化过程中不释放,也不具有调控单核-巨噬细胞分化的作用。6.通过构建原位乳腺癌种植瘤模型,我们发现caspase-1抑制剂YVAD能够诱导肿瘤微环境中髓源性抑制细胞(MDSCs)的积累并能促进肿瘤的生长,但不影响TAMs的浸润。Caspase-1的活化能够在体内调控MDSCs的分化。我们的实验首次阐明了 caspase-1在单核-巨噬细胞分化中的重要调控功能;caspase-1能够通过下调晚期分化中PPARγ的表达而促进巨噬细胞分化的进程。并详细论述了 PPARγ在巨噬细胞分化的早期阶段和晚期阶段发挥的不同功能。本研究还揭示了 caspase-1的下调与AML-M5b之间的联系,以及caspase-1调控肿瘤微环境中MDSCs分化的效应。这些发现表明caspase-1可能成为治疗白血病等肿瘤的有效分子靶点。
[Abstract]:Macrophage is an important member of the mononuclear phagocytic cells in the system, in innate and acquired immunity plays an important role. The source of macrophages in vivo including monocyte macrophage origin, generated in situ proliferation of macrophages and macrophages in the yolk sac of macrophage, monocyte macrophage origin which is the main source of tissue macrophages. When body homeostasis or inflammation, mononuclear cells in the microenvironment can control some factors to macrophage differentiation. Closely related to the occurrence and development of mononuclear macrophage differentiation disorder with various diseases, such as autoimmune diseases, autoinflammatory disease, metabolic diseases and cancer. Therefore for the study, mononuclear macrophage differentiation regulation and molecular mechanism of.Ca has been a hotspot in the field of Immunology Spase-1 is a closely related with inflammation of the members of the caspases family, with lack of regulation of.Caspase-1 key for the function of macrophages macrophages to release inflammatory factors IL-1p and IL-18 decreased significantly; at the same time, caspase-1 can also regulate macrophage polarization from type M2 to type M1. In addition,.Caspase-1 caspase-1 can also participate in cell differentiation program through processing the regulation of IL-1p differentiation and control release of fat cells and Th17 cells. However, the function of caspase-1 in monocyte macrophage differentiation are unknown. Based on extensive and biological function of caspase-1 in mononuclear cells, the core position of macrophages, also has an important regulatory function of caspase-1 in monocytes macrophage differentiation. In this study, we constructed an in vitro human monocytic leukemia cell line (T HP-1, U937) or human peripheral blood mononuclear cells (PBMCs) to macrophage differentiation model, discussed the caspase-1 effect for the regulation of monocyte macrophage differentiation and molecular mechanism involved, the main research contents and results are as follows: 1.. We use the PMA THP-1 and U937 cells to induce macrophage differentiation, and through the analysis of cell morphology in this process, the surface marker (markers) and the changes of phagocytosis and differentiation of mononuclear macrophages were divided into early and late two stages of.2. we found that caspase-1 was highly expressed constitutively in THP-1 and U937 cells; however with the differentiation of its activity gradually increased and with the increase of IL-1 beta and IL-18 release of.Caspase-1 activity induced by PBMCs in M-CSF or GM-CSF phenomenon to macrophage differentiation model has also been confirmed. And through the analysis of inflammation Body components of gene expression showed that caspase-1 inflammasome activation and assembly of.3. Caspase-1 specific inhibitor WEHD dose dependently inhibited PMA induced macrophage makers (CD11b, CD14) expression, and this inhibition occurs mainly in the differentiation of the advanced stage; while WEHD can obviously inhibit the phagocytosis of macrophages. The differentiation of PBMCs, WEHD also has the effect of.4. interference inhibiting the expression of differentiation caspase-1 can obviously inhibit the activity of Caspase-1, and the expression of CD1 and CD14 can significantly inhibit 1b and phagocytic activity. We found that acute monocytic leukemia (AML-M5b) patients with caspase-1 bone marrow mononuclear cells in gene and protein levels there were significantly reduced, and the arrest of cell differentiation in the AML-M5b program and the accumulation of monocytes is closely related to the biological effects of.5. on the basis of the above, I We discussed the molecular mechanism of Caspase-1 promoting monocyte macrophage differentiation: (1) the expression of PPAR increased gradually during the early differentiation of gamma; expression in advanced PPAR gamma decreased the specific inhibitor of.Caspase-1 or shRNA can down regulate the differentiation of reversal of advanced PPARy, but the mRNA level did not changed obviously in HEK293T cells. Recombinant NLRP3 inflammasome can effectively activate caspase-1, and down regulate the expression of PPAR gamma protein, but caspase-1 cannot cut PPAR gamma directly. (2) raised in the early differentiation of PPAR gamma expression and transcriptional activity also increased; PPAR expression can regulate cell cycle related proteins cyclinD1 and p21, and inhibit the cell cycle and promote monocyte macrophage differentiation process. In the late differentiation stage, PPARy ligand troglitazone can inhibit caspase-1 activity and reverse PPAR gamma cut, and induce NF- kappa B transcription activity Inhibition inhibits monocyte macrophage differentiation process. (3) we are also on the classic caspase-1 substrate on monocyte macrophage differentiation were analyzed. Using washing away method, adding additional cytokines or cytokine corresponding neutralizing antibodies, the results show that the differentiation of mononuclear macrophage not depending on the IL-1 beta and IL-18 release of.IL-1 alpha and IL-33 was not released in the process of differentiation,.6. also has no regulation of monocyte macrophage differentiation by constructing tumor model of breast cancer in situ cultivation, we found that Caspase-1 inhibitor YVAD can induce tumor myeloid derived suppressor cells in the microenvironment (MDSCs) accumulation and to promote tumor growth, but does not affect the differentiation of TAMs activation in vivo invasion of.Caspase-1 can regulate MDSCs. Our experiments for the first time to clarify the important caspase-1 on monocyte macrophage differentiation in control Function; caspase-1 can promote macrophage differentiation process through the expression of differentiation in the late stage of PPAR gamma. And discusses the different functions of PPAR gamma in macrophage differentiation in the early stage and late stage play. This study also reveals that the downregulation of caspase-1 and AML-M5b between the contact and the regulation effect of caspase-1 in tumor microenvironment MDSCs differentiation. These findings suggest that caspase-1 may be an effective molecular target for the treatment of leukemia and other tumors.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【参考文献】