【作者】 陈鸿鹄；

【导师】 严杰； 潘建平；

【作者基本信息】 浙江大学 ， 病原生物学， 2013， 博士

【摘要】 背景和立题依据：由病原微生物感染引起的传染病是人类健康和生命的主要威胁之一。目前发现,90%以上的人类传染病经由黏膜途径感染。然而,黏膜抗感染免疫机制至今不明。黏膜抗感染免疫不同于系统免疫,与系统免疫系统相比,黏膜处于一个长期带菌的环境,接触不同来源和性质的抗原,这就要求黏膜免疫系统对有害抗原和无害抗原具有区别能力,并反馈为不同的免疫反应。对于食物,正常菌群等无害抗原,肠道黏膜免疫系统表现为免疫耐受,避免发生超敏反应和自身免疫性疾病,而与无害抗原相反,肠道内的病原体等有害抗原可以被肠道黏膜免疫系统识别,并引起免疫应答。然而,黏膜免疫的机制至今不明。肠道黏膜免疫系统是机体免疫系统中最大也是最为复杂的部分,也最具有代表性。大量研究数据揭示,肠道黏膜免疫系统的免疫耐受机制主要有调节性T细胞(regulatory T cells,Treg)介导,而诱导性Treg的产生与肠道黏膜微环境密不可分,这种特殊联系来源于肠道黏膜免疫系统的几种细胞或细胞亚群分泌的维甲酸。上述资料提示,肠道具有分泌维甲酸能力的细胞或细胞亚群可能在肠道免疫耐受机制中发挥了重要作用。研究内容及意义：本文通过研究肠道派氏结中不同树突状细胞亚群维甲酸合成限速酶——醛脱氢酶活性来筛选诱导肠道免疫耐受的免疫细胞,研究过程中,发现了一群高表达醛脱氢酶活性的未知细胞,鉴定结果是嗜酸性粒细胞,并定量检测这群肠道嗜酸性粒细胞维甲酸的分泌量,肠道嗜酸性粒细胞Treg诱导相关的细胞因子—-TGF-β和IL-2分泌量,以及肠道嗜酸性粒细胞与初始型T细胞共培养后,T细胞的分化方向。为了检测非黏膜来源的嗜酸性粒细胞是否具有诱导初始型T细胞向Treg分化的能力,分离纯化外周血嗜酸性粒细胞,鉴定外周血嗜酸性粒细胞的表型,外周血嗜酸性粒细胞的维甲酸分泌量,以及外周血嗜酸性粒细胞与初始型T细胞共培养后,Treg的表达情况。以期待揭示嗜酸性粒细胞在肠道黏膜免疫中的功能和机制。研究方法：本文采用美天尼磁珠分选和流式细胞分选来收集和纯化野生型小鼠肠道黏膜中的树突状细胞以及肠黏膜和外周血中嗜酸性粒细胞；流式细胞仪检测醛脱氢酶活性检测试剂标记的细胞的醛脱氢酶活性；流式细胞检测细胞表型鉴定和瑞氏染色形态学鉴定高表达醛脱氢酶活性细胞的细胞类型；液相色谱-质谱联用定量检测肠道黏膜以及外周血来源的嗜酸性粒细胞的维甲酸分泌量；ELISA检测肠道黏膜以及外周血来源的嗜酸性粒细胞单独培养上清中TGF-β和IL-2的浓度；纯化的肠道黏膜或外周血来源的嗜酸性粒细胞与纯化的CD4+CD62L+OT-II CD4+初始型T细胞共培养数天后,流式细胞仪检测CD4+细胞中CD25+Foxp3+Treg细胞的比例,ELISA检测培养上清中IL-4, IL-17, IFN-γ, TGF-β and IL-2的浓度。实验结果：实验结果表明,肠道派氏结中,存在一群高表达醛脱氢酶的细胞,经表型和形态学鉴定确认为嗜酸性粒细胞,经液相色谱质谱联用定量检测肠道嗜酸性粒细胞培养上清,发现这群肠道嗜酸性粒细胞分泌高浓度维甲酸,采用ELISA检测上清,证明这群肠道嗜酸性粒细胞同时也分泌TGF-p。将肠道嗜酸性粒细胞与初始型T细胞共培养后,流式细胞检测发现,6.52%CD4+T细胞表达CD25+Foxp3+,说明初始型T细胞向Treg方向分化。为了验证非黏膜来源的嗜酸性粒细胞是否具备诱导Treg的功能,分离和纯化小鼠外周血嗜酸性粒细胞,采用流式鉴定细胞表型,结果显示,肠道嗜酸性粒细胞与外周血嗜酸性粒细胞表型有差异,肠道嗜酸性粒细胞表达CD80和CDllc,而外周血嗜酸性粒细胞不表达。同时,外周血来源的嗜酸性粒细胞不能分泌维甲酸。外周血嗜酸性粒细胞与初始型T细胞共培养后,并不诱导CD4+CD25+Foxp3+T细胞的产生。结论：肠道黏膜来源嗜酸性粒细胞高表达醛脱氢酶活性,分泌高浓度维甲酸和TGF-β。肠道黏膜来源嗜酸性粒细胞诱导初始型T细胞向Treg方向分化,抑制初始型T细胞向Th1/Th17方向分化。非黏膜来源的嗜酸性粒细胞——外周血嗜酸性粒细胞表型与肠道嗜酸性粒细胞不同,不能分泌维甲酸,也不能诱导初始型T细胞向Treg方向分化。更多还原

【Abstract】 Background:Infectious diseases caused by pathogenic microorganism are one of the main threat to human health and life. It is well known that the mucosa is the largest immune organ in the body, and it is generally believed that almost all infectious diseases are initiated at mucosal surface. However, little is known about mucosal immunity against pathogens infection.Within the immune system, a series of anatomically distinct compartments can be distinguished, each of which is specially adapted to generate a response to pathogens present in a particular set of body tissues. The previous part of the chapter illustrated the general principles underlying the initiation of an adaptive immune response in the compartment comprising the peripheral lymph nodes and spleen. This is the compartment that responds to antigens that have entered the tissues or spread into the blood. A second compartment of the adaptive immune system of equal size to this, and located near the surfaces where most pathogens invade, is the mucosal immune system.The gastrointestinal tract is most classic component of the body’s mucosal immune system. In fact, the intestine possesses the largest mass of lymphoid tissue in the human body. The intestinal immune system must constantly maintains immunological tolerance to harmless food antigens and commensal bacteria yet recognizes harmful pathogens and responses to eliminate them. The mechanisms that maintain this balance of intestinal immune homeostasis are poorly understood. Gut-associated lymphatic tissue (GALT) maintain the symbiosis between commensal bacteria and the gut for the fine balance of intestinal immune homeostasis. When the GALT receives signals from the intestinal flora or food antigens, it must limit the magnitude of effector responses and allow the establishment of immunological tolerance. Regulatory T cells (Treg) play an indispensable role in maintaining self tolerance.Although a role for Treg in the maintenance of immune tolerance has been demonstrated in both humans and mice, the origin of these cells is still not completely understood.Tregs play an indispensable role in maintaining self tolerance, aside from Tregs arise in the thymus, peripheral conversion of Tregs occurs primarily in the GALT, suggested that the GALT microenviroment is particularly well suited for peripheral conversion of Treg. It has been proved that retinoic acid promote de novo generation of Foxp3+Treg cells via retinoic acid (RA)The vitamin A metabolite RA is a lipophilic molecule that controls the activity of a constellation of genes via binding to nuclear receptors. Vitamin A is derived from the diet, and the liver constitutes a large reservoir of vitamin A in the form of retinyl esters. Retinyl esters are hydrolyzed to retinol and released into the blood. Once retinol enters cells expressing appropriate enzymes, it is converted successively into retinal and RA. The first step of the conversion is catalyzed by alcohol dehydrogenases and by microsomal retinol dehydrogenases that are expressed by most cells, including dendritic cells (DCs). The second step consists of the oxidation of retinal into RA and is catalyzed by3aldehyde dehydrogenases (ALDHs), known as RALDH1,2, and3and encoded by the Aldhlal,-2, and-3genes, respectively. RALDH expression is limited to certain cell types and, despite the widespread availability of retinol, only cells expressing one of the RALDHs can oxidize retinaldehyde to RA.Recently, DCs that are located in GALT and express Aldhla2have gained considerable attention because of their ability to produce RA. On migration to mesenteric lymph nodes (MLNs), this exclusive property allows them to promote the expression of the gut-tropic α4β7integrin and CCR9chemokine receptor on antigen-responsive T cells and in turn confer them gut-seeking properties. Importantly, RA production by GALT-associated DCs is also involved in the generation of induced Foxp3+Treg(iTreg). Treg can be distinguished from "naturally occurring" Foxp3+Treg (iTreg) on the basis of their development. Whereas nTreg develop in the thymus, iTreg develop de novo in secondary lymphoid organs from conventional, naive CD4+T cells. This conversion that is triggered by DCs requires submitogenic dose of antigen and low costimulation, high levels of transforming growth factor-β (TGF-β) and is greatly enhanced by the presence of RA. The exact mechanism through which DC-produced RA impacts on the generation of iTreg is still a matter of debate. For instance, RA has been proposed to enhance the TGF-β-dependent differentiation of naive CD4+T cells into Foxp3+iTreg by blocking their differentiation into proinflammatory T cells. Alternatively, RA may indirectly affects iTreg generation by preventing memory CD4+T cells from producing cytokines (interleukin-4[IL-4], IL-21, and interferon-y), which inhibit the differentiation of iTreg. RA production by GALT-associated DCs has been proposed to maintain the balance between effector and Treg in the gastrointestinal tract and to constitute a major mechanism underlying oral tolerance. The production of RA by gut DCs is restricted to mDCs expressing the integrin αE chain CD103and requires the presence of both granulocyte-macrophage colony stimulating factor (GM-CSF) and RA in the lamina propria (LP).Considering that, under physiologic conditions, ALDH expression constitutes the only parameter that limits RA production, we used a flowcytometry-based assay to measure ALDH activity at the single-cell level and performed a comprehensive analysis of the RA-producing cell populations present in Peyer’s patches under steady-state conditions.Methods:Isolation of cells with microbeads on LS MACS columns and FACS sorting. To identified the cells with high ALDH activity, we employed flowcytometry-based assay to measure phenotype and May-Grunwald-Giemsa staining to observe morphology. LC (liquid chromatography)/MS/MS assay was used to detect the RA secreting by intestinal eosinophil(Eos), the intestinal cells with high ALDH activity. ELISA assay was performed to test the secretion of TGF-P and IL-2by intestinal Eos. For in vitro stimulation, purified peripheral blood Eos or intestinal Eos were cultured together with unlabeled naive CD4+CD62L+OT-II CD4+T cells, Foxp3expression in CD4+T cells was evaluated using the Foxp3staining set. ELISA assay was performed to test the concentration of IL-4, IL-17, IFN-γ, TGF-β and IL-2in the co-culture supernatant.Results:There is no significant difference among the ALDH activity of three distinct DC in Peyer’s patches. However, a group of cells(CD11c+CD11b+CD8-) in intestinal expressed high level of ALDH activity. CD11c+CD11b+CD8-intestinal cells had moderate expression of Siglec-F+CCR3+, which indicated a Eos character. The image show that the CD11c+CD11b+CD8-intestinal cells are Eos with uniquely shaped nuclei and eosinophilic granules. The RA and TGF-β secreting ability of intestinal eosinophils were demonstrated by culture supernatant quantification. After coculture with naive CD4+CD62L+OT-II CD4+T cells in the presence of antigen and TGF-β, approximately6.5%of cells expressed the marker Foxp3and CD25. After coculture with naive CD4+CD62L+OT-II CD4+T cells in the presence of antigen and TGF-β, approximately6.5%of cells expressed the marker Foxp3and CD25. Culture with intestinal eosinophils lead to strong reduction of IFN-y and IL-17, and correlated with increased TGF-β production. The expression of phenotype is different between intestinal and peripheral blood Eos, which is no expression of CD11c and CD80. Peripheral blood Eos have low level of ALDH activity and no RA secreting. After coculture with naive T cells in the presence of antigen and TGF-β, almost no cells expressed the marker Foxp3and CD25.Conclusion:Intestinal Eos inducing the differentiation of naive T cell into regulatory T cell, and inhibiting the differentiation of Th1and Th17. The mechanism is cause via RA and TGF-β. The expression of phenotype is different between intestinal and peripheral blood Eos. Peripheral blood Eos cannot induce the differentiation of naive T cell into regulatory T cell.更多还原