【作者】 张伟；

【导师】 汪洌； 王建莉；

【作者基本信息】 浙江大学 ， 免疫学， 2012， 博士

【摘要】 巨噬细胞是固有免疫反应重要的效应细胞,在特异性免疫发挥作用前对机体防御病原微生物中起着重要的作用,同时也是一类主要的抗原递呈细胞,是连接固有免疫和适应性免疫的枢纽,在特异性免疫应答的诱导与调节中起着关键的作用。最近的研究主要把巨噬细胞分为两类,一类称为经典激活的巨噬细胞,即M1；另一类称为选择激活的巨噬细胞,即M2。骨髓细胞可以在GM-CSF和M-CSF的诱导条件条件下,分别形成两种类似于M1和M2的表型,在这里我们称之为GM-BMM和M-BMM。 GM-BMM可以产生大量的炎症因子,可以介导对病原菌的抵抗；而M-BMM可以分泌抑炎因子,有助于帮助机体进行组织修复和重建。M-BMM在用LPS刺激时,会有一种高表达IL-10低表达IL-12的状态,但是这种状态是特别是IL-12的调控是如何形成的,一直未有研究。KLF家族是一类锌指蛋白转录因子家族,在其C末端或者接近C末端包含由3个C2H2锌指结构组成的高度保守的DNA结合区域。通过调控基因的转录,KLFs能够在多种生理或者病理过程中发挥功能。Klf10属于Kriippel类转录因子家族,其最初被描述为转化生长因子p诱导的早期基因-(TIEGl,transforming growth factor-beta inducible early gene1)。我们的研究发现K1f10在M-CSF诱导的骨髓来源的小鼠原代巨噬细胞中能够调节IL-12p40的表达,IL-12p40作为IL-12p70和IL-23的共同亚基,在细胞因子的调节网络中起着重要的作用。K1f10基因敲除小鼠中IL-12p40的表达明显上调。而我们通过流式细胞术分析发现K1f10对巨噬细胞表面的一些特定分子标志并没有明显影响,暗示了K1f10并没有影响到巨噬细胞的分化。此外,我们发现Klf家族中与K1f10高度同源的另外一个成员Klf11也能协同K1f10一起来调控IL-12p40的表达。深入的机制的研究发现K1f10能够通过结合到IL-12p40启动子的CACCC位点来抑制其转录。同时我们发现在K1f10敲除小鼠中,CCAAT/增强子结合蛋白-α(C/EBP-a)和干扰素调节因子8(IRF8)都受到了明显影响,这两个基因都被报道过能够调控IL-12p40的表达,因此K1f10可能通过调节C/EBP-a和IRF8来间接调控IL-12p40的合成。接下来我们发现在一个用LPS诱导的小鼠内毒素休克模型中,K1f10敲除小鼠也有着上调的IL-12p40的表达。综上,我们在巨噬细胞中找到了一种新的调控IL-12p40的转录因子K1f10,进一步完善了IL-12p40的调控网络,并对K1f10在固有免疫及炎症反应中的作用的进一步研究提供一些新的启示。更多还原

【Abstract】 Macrophages are considered to be important effector cells that play critical roles in innate immune system and also important antigen presenting cells which contribute to adaptive immunity. Macrophages are now classified as M1(classic activated macrophages) and M2(alternative activated macrophages).Bone marrow derived macrophage (BMM) can be respectively treated with GM-CSF or M-CSF and induced to GM-BMM or M-BMM which resembles a M1or M2profile. GM-BMMs make large amounts of proinflammatory cytokines and mediate resistance to pathogens while M-BMMs produce anti-inflammatory cytokines that contribute to tissue repairing and remodeling. M-BMMs stimulated with LPS are in an anti-inflammatory state with an IL-12lowIL-10high phenotype, but how this is regulated still remains unclear.K1f10belongs to the family of Kruppel-like transcription factors and is initially described as transforming growth factor-beta (TGF-beta) inducible early gene1(TIEG1). Here we report our studies that K1f10can regulate the production of IL-12p40, a common subunit shared by IL-12p70and IL-23in M-BMMs. We found IL-12p40was dramatically up-regulated in lipopolysaccharide (LPS) stimulated M-BMMs derived from K1f10deficient mouse. Phenotype analysis of cell surface markers revealed that there were no significant differences in M-BMMs between K1f10deficient mouse and wildtype mouse, which indicated that K1f10may not be involved in the differentiation of macrophages. Klfll, another member of KLF family which share high conserved consensus sequences with K1f10, could also participate in the regulation of IL-12p40.Mechanistic studies demonstrated that K1f10can bind to the CACCC element of the IL-12p40promoter and inhibit its transcription. The expression of CCAAT/enhancer-binding protein-a(C/EBP-a) and interferon regulatory factor8(IRF8) were also affected, which may be involved in the regulation of IL-12p40. In addition, the regulation of IL-12p40by K1f10was also found in a LPS mediated murine endotoxin shock model, which indicated a critical role of K1f10in inflammation induced by bacterial infections. Taken together, we identified K1f10as a transcription factor in M-BMMs that were able to regulate the expression of IL-12p40. Furthermore, it may play an important role in host innate and adaptive immune responses.更多还原