Placental Treg Activation and Pregnancy Outcome in the FIV-infected Cat Model

描述（由申请人提供）：艾滋病大流行的一个重要组成部分是儿科感染。每年有数十万儿童感染艾滋病毒。至少90%的儿童感染是母婴传播（MTCT）的结果。流产率的增加也与艾滋病毒感染有关。FIV感染的猫是一种很好的MTCT小动物模型。在实验性感染FIV-B-2542的猫中，胎盘和胎儿感染率非常高，并且在妊娠早期和晚期经常发生生殖失败。胎盘免疫参数的初步分析表明，炎症伴随着生殖失败。胎盘调节性T细胞（TCRs）在维持妊娠中至关重要，因为它们的免疫抑制功能允许母体对半同种异体胎儿的免疫耐受。甲状腺素的消耗与生殖失败有关。FIV优先在受感染的猫的T细胞的这种表型中复制，从而在慢性感染期间引起Treg耗尽和随后的超免疫激活。这些信息支持以下假设：1）由于FIV感染这些细胞，发生胎盘T细胞的群体动力学和功能改变。2)感染的这种免疫病理学效应导致无法抑制母胎界面的炎症，并导致病毒性MTCT和/或妊娠受损。本项目将解决两个目标：1）确定感染和对照猫在妊娠早期和晚期的Treg群体动态和活化; 2）检查接近Treg的胎盘微环境，以确定免疫调节剂的表达是否受到Treg活化状态的影响。将使用妊娠早期和晚期的冷冻或福尔马林固定胎盘组织。将通过用CD 4、CD 25和FoxP 3抗体进行免疫荧光标记，在胎盘切片中鉴定Treg细胞。共聚焦显微镜将用于量化这些细胞。将进行免疫组织化学以标记用于激光捕获显微切割（LCM）的TcB。将进行LCM，以显微解剖胫骨，沿着来自邻近微环境的组织。将从显微切割的组织中纯化RNA。使用真实的时间逆转录酶（RT）-PCR定量靶基因和B-肌动蛋白表达。将从Treg RNA定量Treg活化标志物（FoxP 3、CTLA 4、TGF-b、B7、CD 25）、受体（CD 134和CXCR 4）和细胞因子（IL-2和IL-10）。还将定量这些细胞中的FIV RNA负荷。将在相邻微环境中评价的免疫调节剂将包括吲哚胺2，3-双加氧酶（IDO），一种由抗原呈递细胞（APC）和滋养层产生的免疫抑制酶，由APC产生的细胞因子INF-c、TNF-α、IL-1b、IL-2、IL-6和IL-12，细胞毒性T细胞和NK细胞。这项研究将揭示胎盘Treg功能在FIV的MTCT和猫模型中的生殖失败中的作用，从而拓宽我们对HIV感染妇女的胎儿感染和生殖结果的理解。公共卫生相关性：儿童艾滋病毒感染主要是母婴传播的结果，感染艾滋病毒的妇女流产率较高。已知胎盘中的调节性T细胞（免疫抑制性T细胞）通过允许母体对发育中的胎儿的耐受来支持成功的妊娠，然而HIV可以扰乱调节性T细胞功能。该项目的目的是评估艾滋病毒感染对调节性T细胞功能的影响，使用感染猫免疫缺陷病毒（FIV）的猫来模拟母胎感染和妊娠受损。

DESCRIPTION (provided by applicant): An important component of the AIDS pandemic is pediatric infection. Each year, hundreds of thousands of children become infected with HIV. At least 90% of pediatric infections are a result of mother-to-child transmission (MTCT). Increased rates of miscarriage also are associated with HIV infection. The FIV-infected cat is an excellent small-animal model of MTCT. Very high rates of placental and fetal infection occur in cats experimentally infected with FIV-B-2542, and reproductive failure is frequent at early and late-term pregnancy. Preliminary analyses of placental immune parameters indicate that inflammation accompanies reproductive failure. Placental regulatory T cells (Tregs) are centrally-important in the maintenance of pregnancy because their immunosuppressive function allows maternal immune tolerance to the semi-allogeneic fetus. Depletion of Tregs is associated with reproductive failure. FIV preferentially replicates in this phenotype of T cells in the infected cat, thereby causing Treg depletion during chronic infection and consequent hyperimmune activation. This information supports the following hypotheses: 1) Altered population dynamics and function of placental Tregs occurs as a result of FIV infection of these cells. 2) This immunopathological effect of infection results in the failure to suppress inflammation at the maternal-fetal interface and contributes to viral MTCT and/or compromised pregnancy. Two aims will be addressed in this project: 1) Determine Treg population dynamics and activation at early and late pregnancy in infected and control cats; 2) Examine the placental microenvironment proximate to Tregs to determine whether expression of immunomodulators is affected by the Treg activation state. Frozen or formalin-fixed placental tissues from early and late gestation will be used. Treg cells will be identified in placental sections by immunofluorescent labeling with antibodies to CD4, CD25, and FoxP3. Confocal microscopy will be used to quantify these cells. Immunohistochemistry will be done to label Tregs for laser capture microdissection (LCM). LCM will be performed to microdissect Tregs, along with tissue from the proximate microenvironment. RNA will be purified from microdissected tissues. Target gene and b- actin expression will be quantified using real time reverse-transcriptase (RT)-PCR. Treg activation markers (FoxP3, CTLA4, TGF-b, B7, CD25), receptors (CD134 and CXCR4) and cytokines (IL-2 and IL-10) will be quantified from the Treg RNA. FIV RNA load in these cells also will be quantified. The immunomodulators that will be evaluated in the adjacent microenvironment will include indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme produced by antigen presenting cells (APCs) and trophoblasts, cytokines INF-c, TNF-", IL-1b, IL-2, IL-6, and IL-12, produced by APCs, cytotoxic T cells, and NK cells. This study should reveal the role of placental Treg function in MTCT of FIV and reproductive failure in the feline model, thus broadening our understanding of fetal infection and reproductive outcome in HIV-infected women. PUBLIC HEALTH RELEVANCE: Pediatric HIV infections are largely a result of maternal-fetal transmission, and miscarriages occur at an elevated rate in HIV-infected women. Regulatory T cells (immunosuppressive T cells) in the placenta are known to support successful pregnancy by allowing maternal tolerance of the developing fetus, yet HIV can perturb regulatory T cell function. The purpose of this project is to evaluate the effect of HIV infection on the function of regulatory T cells, using the feline immunodeficiency virus (FIV)-infected cat to model maternal-fetal infection and compromised pregnancy.