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

描述(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，数十万儿童感染艾滋病毒。至少90%的儿科感染是母婴传播(MTCT)造成的。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。在实验中感染FIV-B-2542的猫，胎盘和胎儿感染率很高，在怀孕早期和晚期经常发生生殖失败。对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(Tregs)在维持妊娠中起重要作用，因为它们的免疫抑制功能允许母体对半异基因胎儿的免疫耐受。Tregs的枯竭与生殖失败有关。在感染的猫中，FIV优先在这种表型的T细胞中复制，从而在慢性感染期间导致Treg耗尽，并随之而来的高免疫激活。这一信息支持以下假设：1)胎盘Tregs的种群动态和功能发生了改变，这是FIV感染这些细胞的结果。2)感染的这种免疫病理效应导致未能抑制母胎界面的炎症，并导致病毒性母婴传播和/或不良妊娠。该项目将解决两个目标：1)确定感染和对照猫妊娠早期和晚期的Treg种群动态和激活；2)检测Treg附近的胎盘微环境，以确定免疫调节剂的表达是否受到Treg激活状态的影响。将使用来自妊娠早期和晚期的冷冻或福尔马林固定的胎盘组织。胎盘切片中的Treg细胞将通过抗CD4、CD25和FoxP3抗体的免疫荧光标记来鉴定。共聚焦显微镜将被用于对这些细胞进行量化。将进行免疫组织化学标记Tregs以进行激光捕获显微切割(LCM)。LCM将与邻近微环境中的组织一起进行Tregs的显微解剖。RNA将从显微解剖的组织中提纯。目的基因和b-肌动蛋白的表达将使用实时逆转录酶(RT)-PCR进行定量。Treg的激活标志物(FoxP3、CTLA4、TGF-b、B7、CD25)、受体(CD134和CXCR4)以及细胞因子(IL-2和IL-10)将从Treg RNA中定量。这些细胞中的FIV RNA载量也将被量化。将在邻近微环境中评估的免疫调节剂将包括吲哚胺2，3-双加氧酶(IDO)，一种由抗原提呈细胞(APC)和滋养层细胞产生的免疫抑制酶，以及由APC、细胞毒性T细胞和NK细胞产生的细胞因子INF-c、TNF-“、IL-1b、IL-2、IL-6和IL-12。这项研究将揭示胎盘Treg功能在FIV的MTCT和猫模型中生殖失败中的作用，从而拓宽我们对HIV感染妇女胎儿感染和生殖结局的理解。公共卫生相关性：儿童艾滋病毒感染在很大程度上是母婴传播的结果，艾滋病毒感染妇女的流产率较高。众所周知，胎盘中的调节性T细胞(免疫抑制T细胞)通过允许母体对发育中的胎儿的耐受来支持成功怀孕，但HIV可以扰乱调节性T细胞的功能。本项目的目的是利用猫免疫缺陷病毒(FIV)感染的猫建立母婴感染和不良妊娠的模型，以评估HIV感染对调节性T细胞功能的影响。

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.