天然免疫分子TLR4在造血干细胞移植术后GVHD中的作用及其机制研究

发布时间：2018-06-02 09:53

本文选题：Toll样受体4 + 脂多糖　；参考：《浙江大学》2011年博士论文

【摘要】：造血干细胞移植(hematopoietic stem cell transplantation,HSCT)是当今治疗恶性血液肿瘤的重要手段,移植物抗宿主病(graft-versus-host disease,GVHD)等移植并发症严重影响移植效果和临床的广泛应用。本项目从临床和动物实验模型入手,对造血干细胞移植供受者TLR4(Toll-like receptors, TLRs)基因单核苷酸多态性(Single Nucleotide Polymorphisms,SNP)与移植术后GVHD发病率和严重程度以及术后感染的关系进行了研究。同时也建立了TLR4基因缺陷的小鼠骨髓移植模型,以期研究TLR4分子表达对GVHD发病率和移植生存期的影响,观察TLR4基因缺陷对DC(dendritic cells,DCs)成熟、抗原递呈功能和诱导T细胞异基因免疫反应能力的影响,阐明TLR4在GVHD中的分子机制。本研究将为恶性血液肿瘤移植术后GVHD的预后因素和防治的研究提供新的思路,为造血干细胞移植治疗恶性血液病提供新的理论依据,具有重要的研究意义。关于TLR4对GVHD的影响,国际上有少量报道涉及,而且结论尚有争议。有学者认为TLR4可能减少GVHD的发病风险,而另外学者认为TLR4会增加或不影响GVHD的发生。为明确TLR4在异基因移植后GVHD发生中扮演的角色,我们采用PCR扩增直接测序法和聚合酶链反应-限制性片段多态性分析技术(PolymeraseChain Reaction-Restriction Fragment Length Polymorphism,PCR-RFLP)去对208例在我移植中心行异基因造血干细胞移植术的病人及相配对的亲缘或非亲缘供者进行TLR4基因Asp299Gly和Thr399Ile两个单核苷酸多态性变异位点的基因型测序。Ncol内切酶分析Asp299Gly突变,Hinfl内切酶分析Thr339Ile突变,我们研究发现：在所有被检测的造血干细胞移植供受者人群样本中均没有检测到TLR4的Asp299Gly和Thr399Ile基因多态性存在。我们得出结论：TLR4基因的多态性在不同的地区和人种中分布是不同的,在非洲、欧洲人种中分布相对较高,最高突变报道出现在伊朗西部人群中,而在其他亚洲人种中分布罕见。我们的实验证明了在中国尤其是浙江及周边地区移植人群中TLR4基因Asp299Gly和Thr399Ile的基因多态性非常罕见。由于这个结果使我们无法用统计学方法阐明TLR4基因和GVHD之间的联系,因此,我们试图通过TLR4基因敲除小鼠GVHD模型来证实这个假设：供者或受者的TLR4基因突变可能影响到GVHD的发生。我们通过小鼠移植模型探讨了TLR4基因在骨髓移植后GVHD的发生中扮演的角色。GVHD的发生有3个因素,其中关键因素是供者淋巴细胞识别供者或受者的抗原递呈细胞(antigen -presenting cell, APC)而活化,转移到靶组织器官对受者的组织器官发生攻击,引起组织破坏。树突状细胞作为一类具有最强抗原提呈功能的细胞群体,在识别和递呈抗原启动免疫应答、诱导移植排斥中起重要的作用。人们一直认为,DC是抗原提呈能力最强的APC,是唯一能够激活初始型T细胞的APC,具有激活移植排斥反应的作用。我们利用小鼠同种异基因骨髓移植模型观察TLR4基因缺陷小鼠DC诱导同种移植免疫耐受效果,并分析了相关的免疫机制。通过研究TLR4基因敲除小鼠DC在体内外对供者T细胞成熟、分化和功能的影响以及致耐受作用的不同来了解TLR4在GVHD中所扮演的角色。 BALB/c、C57BL/6小鼠是纯系的同种小鼠,这两种小鼠的主要和次要组织相容性抗原(MHC-Ⅰ、Ⅱ)均不相同,可进行同种间移植排斥反应的免疫学研究。TLR4-/-是TLR4基因敲除小鼠其背景与C57BL/6小鼠(TLR4+/+)相同。研究表明,异基因骨髓移植后供者和受者来源的APC均可出现在受者的二级淋巴组织中,供者的T细胞受体(T cell receptor, TCR)可以识别受者APC(直接递呈)或供者APC(间接递呈)呈递的异基因抗原。我们将实验设计为TLR4+/+和TLR4-/-小鼠分别做供者或受者与BALB/c小鼠之间相互进行骨髓移植,了解TLR4-/(?)与TLR4+/+的DC在参与直接递呈和间接递呈识别过程中,两者相比受鼠发生GVHD的不同。通过观察我们发现TLR4-/-小鼠无论作供鼠还是受鼠其移植后的嵌合体与TLR4+/+组相比发生GVHD程度均较轻、体重下降较慢、临床GVHD评分较低、肝脏及小肠等器官、组织的受损程度较小,均预示TLR4-/-小鼠可以诱导机体产生针对特异性抗原的耐受,导致机体同种异体器官移植的免疫耐受。此外我们还观察到TLR4-/-小鼠做供鼠时移植后受鼠BALB/c发生GVHD时间(中位时间16.1天)较TLR4-/-小鼠做受鼠时其嵌合体发生典型GVHD(中位时间12.3天)的时间也要退后一些,解剖发现受鼠肝脏、小肠表面出血点程度更轻；我们认为这可能是移植后受体淋巴组织中供体或受体来源的DC所占比例不同所造成的,当TLR4-/-作为供鼠时,其提供的DC在移植后2周为嵌合小鼠淋巴组织中主要DC,因其递呈异基因抗原使T细胞激活的作用较弱,故诱导发生GVHD的程度要轻。在进一步的体内外实验中我们比较了TLR4-/-小鼠DC的抗原提呈能力、与同种异基因T细胞的混合淋巴细胞反应(mixed lymphocyte reaction,MLR)、抑制T辅助细胞1型(T help cell type 1,Thl)亚群的分化以及体外诱导同种抗原特异性T细胞低反应性等来解释其发生移植免疫耐受的免疫机制。近来的研究认为在T细胞反应的早期,当童贞T细胞识别APCs提呈的抗原时,APCs是否能够表达足够的CD86、CD80、CD40等共刺激分子决定了T细胞是否被完全活化,产生免疫应答,或是未被活化导致凋亡或无能。体外实验我们发现TLR4-/-可以保护非成熟型DC不被外源性脂多糖(lipopolysaccharide,LPS)刺激所激活。我们的FACS数据表明,给予外源性LPS(1μg/ml)刺激24h, TLR4+/+小鼠Day-5-DC表达高水平CD80、CD86、CD40和MHC-Ⅱ类分子,显示出成熟型DC的表型特征,白介素12(Interleukin 12,IL-12)分泌水平明显升高；而TLR4-/-小鼠Day-5-DC则维持在非成熟型状态,DC的表型和IL-12分泌在LPS刺激前后几乎无改变,对T细胞为低刺激活性。 IL-12是由DC分泌的重要的免疫调节因子,可以促进干扰素-γ(Interferongamma, IFN-γ)的分泌,引起CD4+T细胞增殖并向Th1细胞分化,IL-12的缺乏则T细胞增殖受抑。TLR4-/-小鼠在LPS刺激后DC的非成熟状态明显抑制了其抗原提呈能力,减弱DC与同种异基因T细胞的混合淋巴细胞反应,引起T细胞增殖的作用较弱,从而减轻GVHD的发生。在体内实验我们对骨髓移植后21天小鼠进行研究,供鼠是TLR4-/-组小鼠脾细胞来源的DC其表面共刺激分子CD80、CD86的表达与供鼠TLR4+/+组相比也明显降低。TLR4基因的缺失可使DC维持非成熟型状态,具备对T细胞的低刺激活性。一般认为CD4+T细胞Th1亚群向Th2亚群漂移,可以改变移植物局部的免疫反应,抑制细胞介导的排斥反应。IL-2和IFN-γ是Th1亚群的特征细胞因子,而IL-10和IL-4是Th2亚群来源的细胞因子；IL-17为第三个T细胞亚群,在预防胞外病原体中扮演重要角色,其缺乏可导致炎症的进展和严重的自身免疫疾病。为了探讨TLR4-/-小鼠的DC与异基因CD4+T细胞在混合淋巴细胞反应中对T细胞分化的影响,我们测定并分析了同种MLR反应体外培养体系上清中Th1和Th2来源的细胞因子,发现TLR4-/-小鼠来源非成熟型DC与新鲜分离BALB/c小鼠的CD4+T细胞作MLR后,代表Th1亚群的细胞因子IFN-γ、IL-2水平明显降低(p0.01),有意思的是代表Th2亚群的细胞因子IL-10和IL-4水平也表现为下降(p0.05)。结果说明TLR4-/-小鼠DC可抑制Th1亚群的增生反应,但并未诱导Th1亚群向Th2亚群偏移。此外TLR4+/+小鼠MLR上清中IL-17的水平与TLR4-/-组相比显著升高,说明IL-17对GVHD的进展起促进作用。我们在MLR实验中通过流式和羧基荧光素二醋酸盐琥珀酰亚胺酯(carbox fluorescenceindiacetate succinimidyl ester, CFSE)标记检测发现TLR4-/- DC对异基因T细胞的活化作用减弱,与TLR4+/+组相比T细胞增殖明显减少；本结果说明TLR4基因的缺失使DC在体外同种MLR反应中能够维持处于非成熟型状态,不能充分活化T细胞,从而诱导T细胞对同种抗原的低反应性。血清中Th1亚群分泌的细胞因子与严重的GVHD和死亡率相关。为了解小鼠移植后体内来自Th1亚群和Th2亚群细胞因子水平的变化；推测TLR4-/- DC在体内对T细胞分化的影响；我们收集了移植后7天嵌合体小鼠的血清,供鼠为TLR4-/-的移植后受鼠血清中代表Th1亚群的IL-2水平明显较低；而令人惊讶的是其血清中IFN-γ的水平正好相反,移植后第7天受鼠血清中IFN-γ水平与TLR4+/+小鼠组的比较明显升高,考虑可能是除T细胞以外的其他细胞如自然杀伤细胞(Natural killer cells, NK cells)、APC分泌的较多量IFN-γ所至。这结果也与其他一些学者观点符合：IFN-γ对GVHD起到保护作用,IFN-γ对活化T细胞起负调控作用,抑制细胞分化,促进细胞死亡,保护受体器官不受损坏。研究发现Th2相关的细胞因子可以下调细胞介导的免疫反应,拮抗Th1亚群的细胞因子效应,从而减轻GVHD的发生。我们检测发现代表Th2亚群的细胞因子IL-10水平在供鼠为TLR4-/-的嵌合小鼠血清中表现为明显升高,提示可以抑制小鼠移植后GVHD的发生。此外移植后第7天最大量成熟的DC迁移到淋巴组织,参与到递呈抗原,活化T细胞的过程中,这一定程度解释了实验中发现移植后嵌合体小鼠血清中细胞因子检测为何在第7天水平最高这一现象。影响GVHD的因素很多,在体内这些因素相互关联形成复杂的网络最终影响到GVHD的发生和严重程度,LPS/TLR4的信号传导通路也是其中影响因素之一。我们从这个方面入手了解天然免疫分子TLR4在GVHD中所起的作用,最后得出结论TLR4基因在T细胞对APCs的刺激产生明显的活化和增殖反应中起着重要的桥梁作用,是同种反应性T淋巴细胞启动介导同种免疫应答的关键因素；TLR4基因的缺失可诱导机体产生针对特异性抗原的耐受,导致机体同种异体器官移植的免疫耐受从而显著减少受体GVHD的发生。针对TLR4基因研发新的靶向治疗药物也许可以为今后预防和减轻异基因造血干细胞移植后GVHD的发生提供了一个新的治疗思路。
[Abstract]:Hematopoietic stem cell transplantation (HSCT) is an important means for the treatment of malignant hematological tumors. Graft versus host disease (graft-versus-host disease, GVHD) and other transplantation complications seriously affect the effect of transplantation and extensive clinical application. This project starts with the clinical and animal model of hematopoiesis. The single nucleotide polymorphisms of TLR4 (Toll-like receptors, TLRs) gene (Single Nucleotide Polymorphisms, SNP) and the relationship between the morbidity and severity of GVHD and the postoperative infection after transplantation were studied. The mouse bone marrow transplantation model of the TLR4 gene defect was also established in order to study the TLR4 molecule expression for GVHD hair. The influence of the rate of disease and the survival time of the transplant, to observe the effect of TLR4 gene defect on the maturation of DC (dendritic cells, DCs), the antigen presenting function and the ability to induce the allogeneic immune response of T cells, and to elucidate the molecular mechanism of TLR4 in GVHD. This study will provide a new idea for the study of the prognostic factors and the prevention and treatment of GVHD after malignant hematological tumor transplantation. It provides a new theoretical basis for hematopoietic stem cell transplantation in the treatment of hematological malignancies, and has important research significance.
There are a few international reports about the impact of TLR4 on GVHD, and there are controversial conclusions. Some scholars believe that TLR4 may reduce the risk of GVHD, while other scholars believe that TLR4 will increase or do not affect the occurrence of GVHD. In order to clarify the role of TLR4 in the occurrence of GVHD after allogeneic transplantation, we use PCR direct sequencing and aggregation. PolymeraseChain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was used to perform the TLR4 gene Asp299Gly and Thr399Ile two single nucleotide sequences in 208 patients with allogeneic hematopoietic stem cell transplantation at the center of my transplant and the matched or unrelated donors in my transplant center. Asp299Gly mutation and Hinfl endonuclease analysis of Thr339Ile mutations were analyzed by genotype sequencing.Ncol endonucleases of polymorphic polymorphisms. We found that there was no Asp299Gly and Thr399Ile polymorphism in TLR4 in all samples of the donor recipients who were detected. We concluded: TLR4 gene The polymorphism distribution is different in different regions and ethnicity, in Africa, the European ethnicity is relatively high, the highest mutation report appears in the western Iran population, and is rare in other Asian people. Our experiment proved that the TLR4 gene Asp299Gly and Thr39 in the transplant population in China, especially in Zhejiang and the surrounding areas. The genetic polymorphism of 9Ile is very rare. Because this result makes it impossible to statistically clarify the connection between TLR4 and GVHD, we attempt to confirm this hypothesis by the GVHD model of the TLR4 knockout mice. The mutation of the donor or recipient's TLR4 gene may sound to the occurrence of GVHD.
We explored the role of the TLR4 gene in the occurrence of GVHD after bone marrow transplantation by 3 factors, the key factor is the activation of the donor lymphocyte recognition donor or recipient's antigen presenting cell (antigen -presenting cell, APC) and transferred to the target tissue and organs to the tissues and organs of the recipient. As a class of cell populations with the strongest antigen presentation function, dendritic cells play an important role in identifying and presenting antigens to initiate immune responses and induce transplant rejection. It has been believed that DC is the most powerful APC for antigen presentation, and is the only APC capable of activating the initial T cells. The effect of graft rejection.
We used the mouse allogeneic allogeneic bone marrow transplantation model to observe the effect of DC induced allograft tolerance in TLR4 gene deficient mice, and analyzed the related immune mechanisms. By studying the effect of DC on the maturation, differentiation and function of donor T cells in vivo and outside the donor TLR4 gene knockout mice and the difference in tolerance, the TLR4 in GVHD was understood. The role played in it.
BALB/c, C57BL/6 mice are homozygous homologous mice. The main and secondary histocompatibility antigens (MHC- I, II) of these two mice are all different. The immunological study of the allograft rejection reaction is the same as the C57BL/6 mouse (TLR4+/+) in the TLR4 gene knockout mice. The study showed that the donor and the donor after allogeneic bone marrow transplantation were the same. The recipient's APC may appear in the recipient's two stage lymphoid tissue, and the donor's T cell receptor (T cell receptor, TCR) can identify the recipient APC (direct presentation) or the donor APC (indirect presentation) presentation of the allogeneic antigen. We designed TLR4+/+ and TLR4-/- mice to act as donors or recipients and BALB/c mice, respectively. Bone marrow transplantation (BMT) was used to understand the difference between the TLR4-/ (?) and the TLR4+/+ DC in the direct presentation and indirect presentation, compared with the GVHD in the rats. Through observation, we found that the chimerism of the TLR4-/- mice, no matter the donor or the recipient, was lighter than the TLR4+/+ group, and the loss of weight was slower, and the clinical GVHD evaluation was less than that of the TLR4+/+ group. The liver and small intestine and other organs were less damaged, which indicated that the TLR4-/- mice could induce the tolerance of the body to specific antigen, and lead to the immune tolerance of the allogenic organ transplantation. In addition, we also observed that the time of GVHD (16.1 days in the median time) of the rat BALB/c after transplantation was also observed in TLR4-/- mice. The time of the typical GVHD (12.3 days of the median time) of the chimeras at the time of the TLR4-/- mice should also be reduced. The anatomy found that the liver of the rat and the bleeding point on the surface of the small intestine are lighter; we think this may be the result of the different proportion of DC in the donor or receptor source in the recipient lymphoid tissue, when TLR4-/- is used as the donor. In the 2 week after transplantation, the DC is the main DC in the chimeric lymphoid tissue, and the activation of T cells is weak because of its presentation of the allogenic antigen, so the degree of induction of GVHD is light.
In the further experiments, we compared the antigen presenting ability of DC in TLR4-/- mice, the mixed lymphocyte reaction (mixed lymphocyte reaction, MLR) with the allogeneic T cells, the inhibition of the differentiation of the T auxiliary cell 1 (T help cell type 1) subgroup and the induction of the specific antigen specific cells in vitro. The recent study suggests that when the virginal T cells identify the antigen presented by APCs at the early stage of the T cell response, whether APCs can express enough CD86, CD80, CD40 and other co stimulators determines whether T cells are fully activated, produce an immune response, or are not activated to lead to apoptosis or inactivation. In vitro experiments, we found that TLR4-/- can protect immature DC from exogenous LPS (lipopolysaccharide, LPS) stimulation. Our FACS data show that exogenous LPS (1 mu g/ml) stimulates 24h, TLR4+/+ mice Day-5-DC expresses high level CD80, CD86, CD86, and class II molecules, showing the phenotypic characteristics of mature type. The secretion level of prime 12 (Interleukin 12, IL-12) increased significantly, while Day-5-DC in TLR4-/- mice was maintained in the non mature state. The phenotype of DC and the secretion of IL-12 were almost unchanged before and after LPS stimulation, and the activity of T cells was low.
IL-12 is an important immunomodulatory factor secreted by DC, which can promote the secretion of interferon - gamma (Interferongamma, IFN- gamma), cause CD4+T cells to proliferate and differentiate into Th1 cells. The proliferation of IL-12 in the proliferation of T cells inhibits the antigen presenting ability of the.TLR4-/- mice in DC after LPS stimulation, and reduces the DC and isobasis. Because of the mixed lymphocyte reaction of T cells, the effect of T cell proliferation is weak, thus reducing the occurrence of GVHD. In vivo experiment, we studied the mice 21 days after bone marrow transplantation, and the rat was the DC surface CO stimulator of DC in the TLR4-/- group, and the expression of CD86 was significantly lower than that of the donor TLR4+/+ group. The absence of DC can maintain a non mature state and have a low activation of T cells.
It is generally believed that the Th1 subgroup of CD4+T cells drifts to the Th2 subgroup, which can change the local immune response to the graft, inhibit the cell mediated rejection of.IL-2 and IFN- gamma as the characteristic cytokine of the Th1 subgroup, while IL-10 and IL-4 are the cytokines of the Th2 subgroup, and IL-17 is the third T subsets that play an important role in the prevention of extracellular pathogens. Lack of color can lead to inflammation and severe autoimmune diseases.
In order to investigate the effect of DC and CD4+T cells in TLR4-/- mice on the differentiation of T cells in mixed lymphocyte reaction, we measured and analyzed the cytokines of Th1 and Th2 in the supernatant of the culture system of the same MLR reaction in the culture system, and found that the non mature DC from TLR4-/- mice and CD4+T cells of the newly separated BALB/c mice were substituted for MLR. The cytokine IFN- gamma and IL-2 level of the subgroup of table Th1 were significantly decreased (P0.01). It was interesting that the level of cytokine IL-10 and IL-4 representing Th2 subgroup was also decreased (P0.05). The results showed that DC in the TLR4-/- mice could inhibit the proliferation reaction of Th1 subgroup, but did not induce the Th1 subgroup to shift to the subgroup. The level of IL-17 was significantly higher than that in the TLR4-/- group, indicating that the progress of GVHD was promoted. In the MLR experiment, we found that the activation of TLR4-/- DC on the allogeneic T cells was weakened by the flow and carboxyl fluorescein two acetate succinimide (carbox fluorescenceindiacetate succinimidyl ester, CFSE) markers. The proliferation of T cells was significantly reduced, and the results showed that the deletion of the TLR4 gene could keep the DC in the non mature state in the MLR reaction in vitro, and not fully activate the T cells, thus inducing the low responsiveness of the T cells to the same antigen.
The cytokines secreted by the Th1 subgroup in the serum are associated with severe GVHD and mortality. To understand the changes in the level of Th1 subsets and Th2 subsets in the mice after transplantation, and to speculate on the effect of TLR4-/- DC on the differentiation of T cells in the body; we collected the serum of the chimeric mice 7 days after transplantation, and the mice were transplanted to TLR4-/- after transplantation. The IL-2 level of the Th1 subgroup in the rat serum was significantly lower, but it was surprising that the level of IFN- gamma in the serum was just the opposite. The level of IFN- gamma in the rat serum was significantly higher than that in the TLR4+/+ mice seventh days after the transplantation, considering that other cells except the T cells such as the natural killer cells (Natural killer cells, NK cells). APC secretes more IFN- gamma. This result also conforms to some other scholars' Views: IFN- gamma plays a protective role on GVHD, IFN- gamma plays a negative regulatory role in activating T cells, inhibits cell differentiation, promotes cell death, and protects receptor organs from damage. The study found that the cytokine of Th2 phase can reduce cell mediated immune responses. We should antagonize the cytokine effect of the Th1 subgroup and reduce the occurrence of GVHD. We found that the level of cytokine IL-10 representing the Th2 subgroup was significantly elevated in the mouse sera of the chimeric mice fed as TLR4-/-, suggesting that the occurrence of GVHD in mice after transplantation was inhibited. In addition, the most mature DC migrated to the lymphoid group at seventh days after transplantation. In the process of participating in the delivery of antigen and activating T cells, this explains to some extent the phenomenon that the detection of cytokines in the serum of chimeric mice after transplantation is the highest at the seventh day level.
There are many factors affecting the GVHD, which are related to the formation of complex networks in the body and ultimately affect the occurrence and severity of GVHD. The signal transduction pathway of LPS/TLR4 is also one of the influencing factors. We begin with this aspect to understand the role of the natural immune molecule TLR4 in GVHD, and finally conclude that the TLR4 gene is in T. Cells play an important role in the activation and proliferation of APCs stimulation. It is the key factor for the homologous immune response to the activation of the homologous T lymphocyte, and the deletion of TLR4 gene can induce the specific specificity of the organism.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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