Toll样受体对子宫NK细胞的调控特征及其与母胎耐受的关系
发布时间：2018-09-17 14:11
【摘要】： 长久以来胎儿被认为是一种自体移植物，但令人困惑的是妊娠的建立和维持无法用经典的移植免疫耐受或肿瘤免疫逃逸理论来解释。由于母体激素的作用，会有很多可溶性免疫介质和免疫细胞聚集在哺乳动物的生殖道内，它们不仅没有引起强烈的免疫排斥反应，反而可以维持和促进正常的月经周期、胚胎植入和胎儿发育。通过对人和啮齿类动物的生殖免疫研究，发现过去被视为对肿瘤组织具有杀伤能力的NK细胞和整个妊娠过程有着极为密切的关系，子宫NK细胞还可以从不同方面影响母胎耐受。哺乳动物胚泡在着床开始后，为了进一步为入侵子宫内膜基质做准备，子宫内膜蜕膜化。此时，大量的子宫NK细胞在一系列粘附分子、趋化因子和细胞因子的作用下从外周淋巴组织被招募到母胎界面处发挥其免疫调节功能。 在妊娠这一特殊的生理阶段，机体任何的细胞免疫反应都会受到雌激素、孕激素等的调控，子宫NK细胞或其前体细胞向蜕膜的迁移也是一样。我们首先通过细胞转输实验分析了妊娠早期的C5781／6J小鼠的子宫NK细胞迁移特征。给怀孕第6.5天的小鼠输入CFDA-SE标记的正常脾淋巴细胞，24小时后发现NK1.1~+细胞比CD3~+细胞细胞更具有迁移优势，流式检测表明：相对于肝脏(0.96±0.14％)、脾脏(0.15±0.08％)，蜕膜组织有1.13±0.18％的CFDA-SE~+NK1.1~+细胞驻留。这说明至少在蜕膜反应阶段，母胎界面是NK细胞归巢的主要和重要部位。但是Toll样受体(TLR)信号的激活可以打破上述NK细胞的稳态平衡：在进行细胞转输的同时给受体鼠腹腔注射聚肌胞苷酸(polyⅠ：C，TLR3配体)后，发现转输的CFSE-DA~+NK1.1~+细胞大量的在肝脏聚集，而不是子宫或脾脏。这也表明妊娠期间外周淋巴细胞向子宫募集是有前提条件的，外界dsRNA刺激可以从时间和空间角度影响NK细胞向母胎界面迁移。 本研究还证实炎性刺激可以导致子宫内膜TLR介导的免疫应答。TLR3信号可以调节机体抗病毒反应，促进细胞因子分泌。由于许多生殖道病毒包涵或表达dsRNA，因此我们利用小鼠流产模型进行polyⅠ：C干预，并以子宫NK细胞为中心，着眼于妊娠早期，分析母胎界面TLR3的表达特征以及TLR3激动剂干预诱发的子宫局部免疫反应对妊娠结果的影响，阐述子宫NK细胞的调节性作用及局部微环境的天然免疫学特点。在妊娠第6.5天，对CBA×DBA／2小鼠模型分别腹腔注射polyⅠ：C和PBS，于第7.5、8.5、10.5、12.5天处死动物进行相应研究。结果表明在妊娠中期，CBA×DBA／2小鼠胚胎吸收率为11.0±3.0％，polyⅠ：C处理使其显著增加到40.2±1.7％，并伴随有明显的子宫螺旋动脉重铸抑制等病理变化，如管腔变小、管壁变厚，量化分析发现其血管外、内横截面积比值显著增加。同时研究还发现，尽管polyⅠ：C处理24小时后(妊娠第7.5天)表观检查未能发现CBA×DBA／2小鼠有胚胎吸收的明显征兆，与对照组相比母胎界面IFN-γ含量也没有明显变化，但其蜕膜区TLR3表达明显上调，且局部组织TNF-α含量增加2.7倍。上述结果说明子宫蜕膜TNF-α分泌是一个重要的炎性信号，它可能抑制子宫螺旋动脉重铸并最终影响CBA×DBA／2小鼠的妊娠结局。 另外，24小时后检测发现polyⅠ：C处理明显上调了蜕膜NK(DX5~+CD3~-)细胞的胞内因子TNF-α和IFN-γ的阳性率。出乎意料的是小鼠注射polyⅠ：C后，流式检测(DX5~+CD3~+)和细胞组化(DBA~+)分析结果都证实母胎界面子宫NK细胞数目显著减少。此外，局部NKT(DX5~+CD3~-)、T(DX5~+CD3~-)细胞以及树突状细胞(CD11c~+)均不受polyⅠ：C的影响。实际上，母胎界面存在的每一种免疫细胞都对妊娠过程是有益的。由于DX5~+细胞下调，使得母胎界面DC／NK比例发生改变，从而影响了包括细胞因子在内的局部免疫反馈调节过程。由于DC-NK的相互作用不要求细胞的直接接触，，因而尽管蜕膜DC和NK细胞的空间分布没有完全重合，二者的相互影响也不容忽视。 综上所述，本研究证明淋巴细胞在小鼠妊娠早期(蜕膜化阶段)向母胎界面募集时，外周NK细胞比T细胞有更大的迁移优势。PolyⅠ：C作用不仅可以影响NK细胞的归巢和组织特异性分布，还可以激活蜕膜TLR3信号。子宫NK细胞的高度活化表现为细胞因子分泌能力的增强，继而促进母胎界面的炎症反应，加剧在妊娠过程中的病理性变化如螺旋动脉重铸抑制，导致胚胎吸收的增加。由此可见子宫NK细胞和局部TLR信号对小鼠妊娠具有重要调节功能，本动物实验的结果可以外推到临床研究和实践中，即通过关注子宫NK细胞与其它免疫细胞的相互作用来治疗生殖免疫疾病、预测妊娠结果。
[Abstract]:The fetus has long been thought of as an autograft, but the establishment and maintenance of pregnancy cannot be explained by classical transplant tolerance or tumor immune escape theories. Through the study of reproductive immunity in humans and rodents, it was found that NK cells, which were previously considered to be cytotoxic to tumor tissues, were closely related to the whole pregnancy process, and uterine NK cells were also found. Mammalian blastocysts are recruited from peripheral lymphoid tissue to the maternal-fetal interface by a series of adhesion molecules, chemokines, and cytokines to prepare for invasion of endometrial matrix. Its immunomodulatory function.
In this particular physiological stage of pregnancy, any cellular immune response is regulated by estrogen, progesterone, and so on. The migration of uterine NK cells or their precursors to decidua is also the same. We first analyzed the migration characteristics of uterine NK cells in C5781/6J mice in early pregnancy by cell transfer experiments. The normal splenic lymphocytes labeled with CFDA-SE were transfused into the spleen of day 1 mice. NK1.1~+ cells had more migration advantages than CD3~+ cells 24 hours later. Flow cytometry showed that 1.13 (+ 0.18%) CFDA-SE ~+ NK1.1~+ cells resided in the decidua tissue than in the liver (0.96 (+ 0.14%) and the spleen (0.15 (+ 0.08%). The maternal-fetal interface is the main and important site of NK cell homing. However, the activation of Toll-like receptor (TLR) signal can break the homeostasis of NK cells mentioned above. After intraperitoneal injection of Polyinosine (poly I: C, TLR3 ligand) into recipient mice, it was found that a large number of C FSE-DA~+NK1.1~+ cells accumulated in the liver, while Toll-like receptor (TLR) signal was transported. This also indicates that peripheral lymphocyte recruitment to the uterus during pregnancy is preconditioned. External dsRNA stimulation can influence the migration of NK cells to the maternal-fetal interface in time and space.
TLR3 signal can regulate the body's antiviral response and promote the secretion of cytokines. Because many reproductive tract viruses contain or express dsRNA, we used the mouse abortion model poly I:C intervention, and the uterine NK cells as the center, focusing on pregnancy. In early pregnancy, the expression of TLR3 at maternal-fetal interface and the effect of TLR3 agonist on the outcome of pregnancy were analyzed. The regulatory effect of uterine NK cells and the natural immunological characteristics of local microenvironment were elaborated. 7.5, 8.5, 10.5 and 12.5 days after the treatment, the animals were sacrificed. The results showed that in the second trimester of pregnancy, the embryo absorption rate of CBA *DBA/2 mice was 11.0 +3.0%. Poly I: C treatment significantly increased the embryo absorption rate to 40.2 +1.7%. It was accompanied by obvious pathological changes, such as the reduction of lumen and the thickening of tube wall. At the same time, the study also found that although the apparent examination of poly I:C treatment 24 hours after pregnancy (7.5 days) did not show obvious signs of embryonic absorption in CBA *DBA/2 mice, and compared with the control group, there was no significant change in IFN-gamma content at the maternal-fetal interface, but the expression of TLR3 in the decidual region was significantly up-regulated, and local. These results suggest that the secretion of TNF-a in decidua is an important inflammatory signal, which may inhibit the recasting of uterine spiral artery and ultimately affect the pregnancy outcome of CBA *DBA/2 mice.
In addition, 24 hours later, poly I:C treatment significantly increased the positive rates of TNF-alpha and IFN-gamma in decidual NK (DX5~+CD3~-) cells. To our surprise, flow cytometry (DX5~+CD3~+) and cytohistochemistry (DBA~+) analysis showed a significant decrease in the number of uterine NK cells at the maternal-fetal interface. NKT (DX5~+CD3~-), T (DX5~+CD3~-) cells and dendritic cells (CD11c~+) were not affected by poly I: C. In fact, every immune cell at the maternal-fetal interface was beneficial to the pregnancy process. Because of the downregulation of DX5~+ cells, the DC/NK ratio at the maternal-fetal interface was changed, which affected local factors including cytokines. Because DC-NK interactions do not require direct cell contact, the spatial distribution of DC and NK cells in decidua does not coincide completely, and the interaction between DC and NK cells should not be neglected.
In conclusion, this study demonstrates that peripheral NK cells have a greater migration advantage than T cells when lymphocytes are recruited at the maternal-fetal interface in early pregnancy (decidualization stage). Poly I:C not only affects the homing and tissue-specific distribution of NK cells, but also activates TLR3 signal in decidua. The highly activated uterine NK cells are manifested by high activation of TLR3 signal. The enhancement of cytokine secretion and the subsequent inflammatory response at the maternal-fetal interface can aggravate the pathological changes during pregnancy, such as the inhibition of spiral artery recasting, leading to the increase of embryo absorption. In bed research and practice, we treat reproductive immune diseases and predict pregnancy outcomes by focusing on the interaction between uterine NK cells and other immune cells.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2246177
[Abstract]:The fetus has long been thought of as an autograft, but the establishment and maintenance of pregnancy cannot be explained by classical transplant tolerance or tumor immune escape theories. Through the study of reproductive immunity in humans and rodents, it was found that NK cells, which were previously considered to be cytotoxic to tumor tissues, were closely related to the whole pregnancy process, and uterine NK cells were also found. Mammalian blastocysts are recruited from peripheral lymphoid tissue to the maternal-fetal interface by a series of adhesion molecules, chemokines, and cytokines to prepare for invasion of endometrial matrix. Its immunomodulatory function.
In this particular physiological stage of pregnancy, any cellular immune response is regulated by estrogen, progesterone, and so on. The migration of uterine NK cells or their precursors to decidua is also the same. We first analyzed the migration characteristics of uterine NK cells in C5781/6J mice in early pregnancy by cell transfer experiments. The normal splenic lymphocytes labeled with CFDA-SE were transfused into the spleen of day 1 mice. NK1.1~+ cells had more migration advantages than CD3~+ cells 24 hours later. Flow cytometry showed that 1.13 (+ 0.18%) CFDA-SE ~+ NK1.1~+ cells resided in the decidua tissue than in the liver (0.96 (+ 0.14%) and the spleen (0.15 (+ 0.08%). The maternal-fetal interface is the main and important site of NK cell homing. However, the activation of Toll-like receptor (TLR) signal can break the homeostasis of NK cells mentioned above. After intraperitoneal injection of Polyinosine (poly I: C, TLR3 ligand) into recipient mice, it was found that a large number of C FSE-DA~+NK1.1~+ cells accumulated in the liver, while Toll-like receptor (TLR) signal was transported. This also indicates that peripheral lymphocyte recruitment to the uterus during pregnancy is preconditioned. External dsRNA stimulation can influence the migration of NK cells to the maternal-fetal interface in time and space.
TLR3 signal can regulate the body's antiviral response and promote the secretion of cytokines. Because many reproductive tract viruses contain or express dsRNA, we used the mouse abortion model poly I:C intervention, and the uterine NK cells as the center, focusing on pregnancy. In early pregnancy, the expression of TLR3 at maternal-fetal interface and the effect of TLR3 agonist on the outcome of pregnancy were analyzed. The regulatory effect of uterine NK cells and the natural immunological characteristics of local microenvironment were elaborated. 7.5, 8.5, 10.5 and 12.5 days after the treatment, the animals were sacrificed. The results showed that in the second trimester of pregnancy, the embryo absorption rate of CBA *DBA/2 mice was 11.0 +3.0%. Poly I: C treatment significantly increased the embryo absorption rate to 40.2 +1.7%. It was accompanied by obvious pathological changes, such as the reduction of lumen and the thickening of tube wall. At the same time, the study also found that although the apparent examination of poly I:C treatment 24 hours after pregnancy (7.5 days) did not show obvious signs of embryonic absorption in CBA *DBA/2 mice, and compared with the control group, there was no significant change in IFN-gamma content at the maternal-fetal interface, but the expression of TLR3 in the decidual region was significantly up-regulated, and local. These results suggest that the secretion of TNF-a in decidua is an important inflammatory signal, which may inhibit the recasting of uterine spiral artery and ultimately affect the pregnancy outcome of CBA *DBA/2 mice.
In addition, 24 hours later, poly I:C treatment significantly increased the positive rates of TNF-alpha and IFN-gamma in decidual NK (DX5~+CD3~-) cells. To our surprise, flow cytometry (DX5~+CD3~+) and cytohistochemistry (DBA~+) analysis showed a significant decrease in the number of uterine NK cells at the maternal-fetal interface. NKT (DX5~+CD3~-), T (DX5~+CD3~-) cells and dendritic cells (CD11c~+) were not affected by poly I: C. In fact, every immune cell at the maternal-fetal interface was beneficial to the pregnancy process. Because of the downregulation of DX5~+ cells, the DC/NK ratio at the maternal-fetal interface was changed, which affected local factors including cytokines. Because DC-NK interactions do not require direct cell contact, the spatial distribution of DC and NK cells in decidua does not coincide completely, and the interaction between DC and NK cells should not be neglected.
In conclusion, this study demonstrates that peripheral NK cells have a greater migration advantage than T cells when lymphocytes are recruited at the maternal-fetal interface in early pregnancy (decidualization stage). Poly I:C not only affects the homing and tissue-specific distribution of NK cells, but also activates TLR3 signal in decidua. The highly activated uterine NK cells are manifested by high activation of TLR3 signal. The enhancement of cytokine secretion and the subsequent inflammatory response at the maternal-fetal interface can aggravate the pathological changes during pregnancy, such as the inhibition of spiral artery recasting, leading to the increase of embryo absorption. In bed research and practice, we treat reproductive immune diseases and predict pregnancy outcomes by focusing on the interaction between uterine NK cells and other immune cells.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 张建红;C57Bl/6J小鼠妊娠早-中期子宫自然杀伤细胞研究[J];生殖医学杂志;2005年01期
本文编号：2246177
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