脓毒症中IL-3与Treg细胞功能变化的内在联系与机制研究

发布时间：2018-04-24 22:04

本文选题：调节性T细胞 + 白细胞介素-3　；参考：《天津医科大学》2017年博士论文

【摘要】：近来研究发现白介素-3(IL-3)是参与脓毒症免疫紊乱进展,放大炎症效应的重要驱动因子,阻断IL-3的活性能够明显改善脓毒症预后,临床研究亦显示IL-3水平升高可作为脓毒症患者预后的重要预测因素。而,调节性T细胞(Treg)对于维持自身免疫稳态非常关键,在感染免疫中,是影响机体有效细菌清除与避免过度炎症反应病理损害的重要调节因素,其在脓毒症病生理过程中的核心作用已得到共识。但,在脓毒症免疫炎症反应过程中,IL-3与Treg之间是否存在内在的功能联系等,目前尚未见研究报道。对两者间内在联系与作用机制的深入研究将无疑有助于进一步阐释脓毒症的发病机制,并可能提示新的潜在治疗靶点与治疗策略。为此,本课题首先证实在Treg细胞膜表达有IL-3受体,显示Treg细胞可能受到IL-3的功能调控;进而,我们发现Treg细胞自身亦能够生成IL-3;并且,通过改变Treg细胞内IL-3的生成,我们分析了Treg细胞功能的变化,结果显示下调IL-3表达,Treg细胞中Foxp3和CTLA-4表达上升,IL-10与TGF-β生成增加,对效应T细胞的增殖抑制活性明显增强,而上调IL-3表达,Treg细胞功能呈现相反变化;因此,我们的研究结果表明:作为一种自分泌机制,IL-3对Treg细胞功能活性的发挥具有重要的负反馈调节效应。在此基础上,为了探讨脓毒症中IL-3与Treg细胞间的功能联系,我们使用体外LPS刺激模拟脓毒症细胞模型,首先分析了外源添加与抗体阻断IL-3对LPS活化Treg细胞功能活性的影响,结果显示,IL-3作用会抑制Treg细胞中Foxp3和CTLA-4表达、IL-10与TGF-β生成和对效应T细胞的增殖效应,而,应用IL-3Ab能够阻断上述效应;进一步,我们应用慢病毒载体转染,上调或下调Treg细胞内IL-3表达,证实上调Treg细胞内IL-3的水平会明显抑制Treg细胞Foxp3和CTLA-4表达、IL-10与TGF-β生成和对效应T细胞的增殖效应,反之亦然;因此,以上研究结果显示,脓毒症病生理过程中,IL-3可能作为一种重要的负反馈调节机制,负向调控LPS对Treg细胞的活化效应,这可能是机体保持免疫平衡,避免免疫紊乱的一种保护性机制。进而,通过采用盲肠结扎穿孔脓毒症(CLP)模型,应用IL-3Ab,我们首先证实,阻断IL-3的效应,能够有效降低TNF等炎症因子水平,减轻肝、肾、肺等脏器损害,改善脓毒症预后;同时,我们发现阻断IL-3,也能够增加Treg细胞中Foxp3和CTLA-4的表达,促进IL-10与TGF-β的生成,并使其对效应T细胞的增殖抑制活性明显增强;因此,我们的研究结果在证实IL-3Ab的有效性和阻断炎症反应效应的同时,揭示了IL-3参与脓毒症病生理过程的新机制,即,IL-3在放大天然免疫反应的同时,也会下调Treg细胞的免疫抑制活性,参与脓毒症免疫紊乱的发生发展,这一研究发现不仅阐释了IL-3Ab治疗效应的作用机制,也为我们提示了一个新的免疫调控治疗脓毒症的策略途径。综上所述,本研究首先阐释了IL-3与Treg细胞间内在联系的生物学基础,即Treg细胞表达IL-3R,且自身能生成IL-3,并证实自分泌的IL-3对Treg细胞具有负反馈调节效应;在此基础上,通过体内外脓毒症模型,证实脓毒症病生理过程中,IL-3不仅可通过促进中性粒细胞等的生成放大炎症反应,同时,也会下调Treg细胞的活性,共同参与脓毒症免疫紊乱的发生发展。本项目研究揭示了脓毒症中IL-3与Treg之间的内在联系和作用机制,不仅有助于深化对脓毒症病生理机制的认识,也为探索有效防控脓毒症提供潜在靶点和治疗策略的新启示。
[Abstract]:Recent studies have found that interleukins -3 (IL-3) is an important driving factor involved in the progression of sepsis immune disorder and amplifying the inflammatory effect. Blocking IL-3 activity can significantly improve the prognosis of sepsis. Clinical studies also show that the elevated level of IL-3 can be an important pretest factor for the prognosis of patients with sepsis. And regulatory T cells (Treg) can maintain themselves. Immune homeostasis is very important. In infection immunity, it is an important regulatory factor affecting the pathological damage of effective bacteria clearance and avoidance of excessive inflammation. Its core role in the physiological process of sepsis has been recognized. However, there is an inherent functional relationship between IL-3 and Treg during the immune inflammatory response of sepsis. An in-depth study of the intrinsic links and mechanisms of action between the two will undoubtedly help further explain the pathogenesis of sepsis, and may suggest new potential therapeutic targets and therapeutic strategies. For this reason, this topic first confirms that the expression of IL-3 receptors in the Treg cell membrane, indicating that Treg cells may be subject to the work of IL-3 In addition, we found that Treg cells could also produce IL-3, and by changing the formation of IL-3 in Treg cells, we analyzed the changes in the function of Treg cells. The results showed that the expression of IL-3, the expression of Foxp3 and CTLA-4 in Treg cells increased, IL-10 and TGF- beta was increased, and the proliferation inhibition activity of the effect T cells was significantly enhanced. In addition, the Treg cell function is opposite to IL-3 expression; therefore, our results show that as a kind of autocrine mechanism, IL-3 plays an important negative feedback regulation effect on the function of Treg cells. On this basis, in order to explore the functional relationship between IL-3 and Treg cells in sepsis, we use an in vitro LPS stimulation model. In the Pseudomonas cell model, the effects of exogenous and antibody blocking IL-3 on the functional activity of LPS activated Treg cells were first analyzed. The results showed that the effect of IL-3 could inhibit the expression of Foxp3 and CTLA-4 in Treg cells, the production of IL-10 and TGF- beta and the effect on the proliferation of effect T cells, while IL-3Ab could be used to block the above effects. Further, we applied it. Lentivirus vector transfection, up or down regulation of the expression of IL-3 in Treg cells, confirmed that the level of IL-3 in Treg cells can obviously inhibit the expression of Foxp3 and CTLA-4 in Treg cells, IL-10 and TGF- beta generation and the effect on the proliferation of effect T cells, and vice versa; therefore, the above results show that IL-3 may be a heavy weight in the physiological process of sepsis. The negative feedback regulation mechanism, which negatively regulates the activation effect of LPS on Treg cells, may be a protective mechanism for the body to maintain the immune balance and avoid immune disorders. Then, by using the cecum ligation of the perforated sepsis (CLP) model, the application of IL-3Ab, first of all, to block the effect of IL-3, can effectively reduce the TNF and other inflammatory factors. Level, reduce the liver, kidney, lung and other visceral damage, improve the prognosis of sepsis. At the same time, we found that blocking IL-3, also can increase the expression of Foxp3 and CTLA-4 in Treg cells, promote the formation of IL-10 and TGF- beta, and make it significantly increase the inhibitory activity of the effect T cells; therefore, our research results confirm the effectiveness and blocking of IL-3Ab. At the same time, the effect of inflammatory reaction reveals a new mechanism of IL-3's participation in the physiological process of sepsis. That is, IL-3 also reduces the immunosuppressive activity of Treg cells and participates in the development of the immune disorder of sepsis, while amplifying the natural immune response. This study not only explained the mechanism of the effect of IL-3Ab, but also provided us with the mechanism of the therapeutic effect of the sepsis. A new strategy for the treatment of sepsis with a new immunoregulation is shown. To sum up, this study first explains the biological basis of the intrinsic link between IL-3 and Treg cells, that is, the expression of IL-3R by Treg cells and the self generation of IL-3, and that the autocrine IL-3 has a negative feedback regulation effect on Treg cells; on this basis, it passes through the abscess of the body and the body. In the physiological process of sepsis, it is proved that IL-3 can not only enlarge the inflammatory response by promoting the production of neutrophils, but also reduce the activity of Treg cells, and participate in the development of the immune disorder of sepsis. This project reveals the internal relationship and mechanism of IL-3 and Treg in sepsis, not only It helps deepen our understanding of the physiological mechanism of sepsis and provides new insights into the potential targets and therapeutic strategies for effective prevention and control of sepsis.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R459.7

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