Immune Gene Regulation in Human Trophoblast Cells

描述(申请人提供)：成功的怀孕需要抗原性不同的胎儿避免母亲的免疫系统攻击。胎盘滋养层细胞(TbCs)是唯一与母体血液直接接触的胚泡来源细胞，在调节母体对妊娠的免疫反应中发挥着重要作用。干扰素-3(IFN-3)是一种促炎细胞因子，在多种细胞过程中发挥重要作用，包括诱导细胞凋亡、抑制细胞增殖和启动适应性免疫反应。重要的是，在正常妊娠期间，干扰素-3存在于子宫中。来自所有胎龄的人脐带血对干扰素-3介导的细胞凋亡和多态MHC抗原表达的激活都具有抵抗力。这表明人类的脐带血细胞已经进化出逃避干扰素-3的有害影响的机制。对干扰素-3的反应是通过JAK-STAT-1途径激活转录介导的。重要的是，多种病原体通过扰乱JAK-STAT通路对干扰素-3的反应来逃避宿主免疫反应。细胞对干扰素-3反应的幅度和持续时间由正向作用的JAK和STATs的活性与负向作用的蛋白酪氨酸磷酸酶(PTPs)和细胞因子信号转导抑制物(SOCs)之间的平衡决定。调节干扰素-3信号的PTP在结构上是活跃的，但在细胞暴露于干扰素-3后会暂时失活。PTPs的失活导致JAK的最大激活和细胞因子信号的传播。我们先前证明，通过PTP介导的JAK的抑制，人的初代足月胎肾细胞和TBC来源的绒毛膜癌(CC)细胞中的干扰素-3反应被抑制。我们的动力学研究表明，即使在干扰素-3治疗的早期时间点，JAK在TBCs中也受到抑制。这些结果表明，在暴露于干扰素-3的TBCs中，PTP没有被灭活。我们最近还证明了SOCS-1和SOCS-3在TBCs中有结构性表达。因此，在特定的目标I中，我们将研究1)在干扰素-3处理的TBCs中，PTPs是否与其他类型的细胞有不同的调控，以及2)SOCS-1和SOCS-3在调控TBCs中的干扰素-3反应中所起的作用。我们的集体研究表明，抑制JAK是TBCs抑制干扰素-3反应的主要机制，但它们没有解决JAK或STAT-1异常激活对滋养细胞对干扰素-3反应的后果。最近的研究发现，原因不明的妊娠丢失与JAK-2的结构性活性(CA)突变之间存在关联。此外，STAT-1在肿瘤的免疫监测中起着至关重要的作用。因此，在特定的目标II中，我们将研究JAK和STAT-1的CA突变对TBC干扰素-3应答的影响。目前尚不清楚脐带血中干扰素-3信号的抑制是发生在整个妊娠过程中，还是仅限于妊娠的特定三个月。因此，在特定目标III中，我们将检查妊娠1、2和3个月的脐带血中的干扰素-3反应。我们的研究将为胎儿如何逃避对干扰素-3的有害反应，以及脐带血中异常的干扰素-3信号如何导致妊娠并发症提供见解。公共卫生相关性：最近的研究表明，怀孕是一种受控的炎症状态，胎盘中干扰素-3水平的变化在胎盘病理与生殖成功之间起着关键作用。我们拟议的研究将通过全面分析脐带血细胞调节干扰素-3反应的分子机制，阐明脐带血细胞如何补偿正常妊娠期间和感染后子宫中促炎症干扰素-3的存在(S)。因此，我们的研究将为了解胚胎如何逃避对干扰素-3的潜在有害反应，以及脐带血细胞中异常的细胞因子信号如何导致妊娠并发症提供见解。这项工作对肿瘤免疫也有重要意义，因为肿瘤细胞对干扰素-3的反应性丧失在肿瘤免疫逃避中起着至关重要的作用。

DESCRIPTION (provided by applicant): Successful pregnancy requires that the antigenically disparate fetus avoid attack by the mother's immune system. Placental trophoblast cells (TBCs) are the only blastocyst-derived cells in direct contact with maternal blood, and they play essential roles in modulating maternal immune responses to the conceptus. Interferon-3 (IFN-3) is a pro-inflammatory cytokine that plays critical roles in diverse cellular processes, including induction of apoptosis, inhibition of cell proliferation and initiation of adaptive immune responses. Importantly, IFN-3 is present in the uterus during normal pregnancy. Human TBCs from all gestational ages examined are resistant to both IFN-3-mediated apoptosis, and activation of polymorphic MHC antigen expression. This suggests that human TBCs have evolved mechanisms to evade the harmful effects of IFN-3. Responses to IFN-3 are mediated by activation of transcription through the JAK-STAT-1 pathway. Importantly, multiple pathogens evade host immune responses by disrupting the JAK-STAT pathway in response to IFN-3. The magnitude and duration of cellular responses to IFN-3 are dictated by the equilibrium between the activities of the positively acting JAKs and STATs, versus negatively acting protein tyrosine phosphatases (PTPs) and suppressors of cytokine signaling (SOCS). The PTPs that regulate IFN-3 signaling are constitutively active, but are transiently inactivated following exposure of cells to IFN-3. Inactivation of the PTPs results in maximal activation of the JAKs and propagation of the cytokine signal. We previously demonstrated that IFN-3 responses are inhibited in human primary term TBCs and TBC-derived choriocarcinoma (CC) cells by PTP-mediated repression of the JAKs. Our kinetic studies demonstrate that JAKs are inhibited in TBCs even at early time points of IFN-3 treatment. These results suggest that the PTPs are not inactivated in TBCs exposed to IFN-3. We also recently demonstrated that SOCS-1 and SOCS-3 are constitutively expressed in TBCs. Thus, in Specific Aim I, we will investigate 1) whether PTPs are differentially regulated in IFN-3-treated TBCs versus other cell types, and 2) the roles of SOCS-1 and SOCS-3 in regulating IFN-3 responses in TBCs. Our collective studies suggest that inhibition of the JAKs is the primary mechanism by which TBCs suppress IFN-3 responses, but they do not address the consequences of aberrant JAK or STAT-1 activation on trophoblastic responses to IFN-3. Recent studies identified an association between unexplained pregnancy loss and a constitutively active (CA) mutant of JAK-2. Moreover, STAT-1 plays an essential role in immunosurveillance of tumors. Thus, in Specific Aim II, we will investigate the effects of CA mutants of the JAKs and STAT-1 on TBC IFN-3 responses. It is currently unclear whether inhibition of IFN-3 signaling in TBCs occurs throughout gestation, or is restricted to specific trimesters of pregnancy. Thus, in Specific Aim III we will examine IFN-3 responses in 1st, 2nd and 3rd trimester TBCs. Our studies will provide insights into how the conceptus evades deleterious responses to IFN-3, and how aberrant IFN-3 signaling in TBCs may lead to complications of pregnancy. PUBLIC HEALTH RELEVANCE: Recent studies suggest that pregnancy is a state of controlled inflammation, and that alterations in placental levels of IFN-3 play a critical role in placental pathology versus reproductive success. Our proposed studies will shed light on how TBCs compensate for the presence of pro-inflammatory IFN-3 in the uterus during normal pregnancy and following infection by providing a comprehensive analysis of the molecular mechanism(s) by which TBCs regulate responses to IFN-3. Thus, our studies will provide insights into how the conceptus evades potentially deleterious responses to IFN-3, and how aberrant cytokine signaling in TBCs may lead to pregnancy complications. This work also has important implications for tumor immunity, for loss of responsiveness to IFN-3 by tumor cells plays an essential role in tumor immunoevasion.