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童感染。每年都有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）的结果。流产率的增加也与艾滋病毒感染有关。感染fiv的猫是一个很好的MTCT小动物模型。在实验感染FIV-B-2542的猫中，胎盘和胎儿感染率非常高，在妊娠早期和晚期，生殖失败是常见的。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）在维持妊娠中起着至关重要的作用，因为它们的免疫抑制功能允许母体对半异体胎儿产生免疫耐受。Tregs的耗竭与生殖失败有关。FIV在感染猫的这种表型T细胞中优先复制，从而导致慢性感染期间Treg耗竭和随之而来的超免疫激活。这些信息支持以下假设：1)胎盘treg细胞的种群动态和功能的改变是FIV感染这些细胞的结果。2)这种感染的免疫病理作用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。该项目将解决两个目标：1)确定感染猫和对照猫妊娠早期和晚期Treg种群动态和激活；2)检测Treg附近的胎盘微环境，确定免疫调节剂的表达是否受到Treg激活状态的影响。冷冻或福尔马林固定胎盘组织从早期和晚期妊娠将被使用。通过免疫荧光标记CD4、CD25和FoxP3抗体，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。免疫组织化学标记treg用于激光捕获显微解剖（LCM）。LCM将用于微解剖treg，以及来自邻近微环境的组织。RNA将从显微解剖组织中纯化。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激活标记（FoxP3, CTLA4, TGF-b, B7, CD25），受体（CD134和CXCR4）和细胞因子（IL-2和IL-10）将从Treg RNA中定量。这些细胞中的FIV RNA负载也将被量化。将在邻近微环境中评估的免疫调节剂包括吲哚胺2,3-双加氧酶（IDO），一种由抗原呈递细胞（APCs）和滋养层细胞产生的免疫抑制酶，细胞因子INF-c， TNF-", IL-1b, IL-2， IL-6和IL-12，由APCs，细胞毒性T细胞和NK细胞产生。本研究旨在揭示胎盘Treg功能在FIV MTCT和猫模型生殖失败中的作用，从而拓宽我们对hiv感染妇女胎儿感染和生殖结局的认识。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。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性来支持成功妊娠，但HIV可以扰乱调节性T细胞的功能。本项目的目的是评估HIV感染对调节性T细胞功能的影响，使用猫免疫缺陷病毒（FIV）感染的猫来模拟母胎感染和受损妊娠。

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.