TLR Mediated Trophoblast Injury and Poor Pregnancy Outcomes in S. japonica

描述(申请人提供)：人类血吸虫病感染2亿人，包括4000万育龄妇女，是该寄生虫流行地区发病率和死亡率的一个重要原因。吡喹酮(PZQ)治疗是许多血吸虫感染者的标准治疗方案，但孕妇被系统地排除在这一治疗方案之外。尽管孕期感染血吸虫的啮齿动物模型清楚地显示了不良的出生结果，但在人类中仍然很少有这种现象的研究。我们的初步研究数据表明，人类感染血吸虫可导致滋养层细胞产生和凋亡增加，以及出生体重下降。这一应用在评价血吸虫释放的可溶性虫卵抗原(SEA)对人滋养层细胞的影响方面是新颖的。我们假设SEA将激活人类合体滋养层(STB)表面的Toll样受体(TLR)-2和-4，从而导致该细胞层诱导促炎级联反应。我们将利用现有的最相关的模型系统，从生活在日本血吸虫病流行地区的菲律宾妇女身上分离出足月的原代滋养层细胞。滋养层细胞将在分化后进行检查，因为STB是与母体血液直接接触的细胞，因此最有可能在体内暴露在SEA中。特定目标1将从健康的、未感染的妇女中分离出足月细胞滋养层细胞。体外合胞化后，细胞暴露于SEA和TLR激活，通过凋亡率、细胞内肿瘤坏死因子水平、细胞因子产生、MAPK和NF？B激活来评估。完成具体目标1将使我们能够报告SEA对人类STB的具体影响。特定目标2将从妊娠早期接受PZQ治疗的感染妇女中分离出足月细胞滋养层细胞。这些样本是正在进行的一项由U01资助的随机对照试验的一部分，该试验评估了PZQ治疗血吸虫病妇女的前三个月的疗效。STB将被培养，TLR的激活被评估为特定的目标1。尽管PZQ治疗，组织虫卵负荷不会立即减少，因此特定目标2的完成将确定剩余的血吸虫卵以及PZQ治疗对STB的影响。特定目标3将利用小鼠模型来确定SEA激活滋养层细胞上的特异性TLR(S)。根据其他生理系统的数据，我们假设TLR2和-4都将被激活。这将通过缺乏TLR2或-4的遗传模型中的滋养细胞对血吸虫感染的反应来检验。来自特定目标3的发现将促进该领域的机械性理解，允许更好地照顾患有血吸虫病的孕妇。据我们所知，这项应用将是第一次评估STB对血吸虫感染期间释放到循环中的抗原的特异性反应。这项工作至关重要，因为它将允许对怀孕期间受血吸虫病影响的妇女及其后代进行更全面的护理。 公共卫生相关性：在任何给定时间，全球约有4000万育龄妇女感染血吸虫。在啮齿动物模型中的多项研究以及在人类中的一些报告表明，血吸虫感染会导致不良的妊娠结局。这种现象的机制尚不清楚，将在这一应用中进行研究，以加强对患有血吸虫病的孕妇及其后代的治疗和健康。

DESCRIPTION (provided by applicant): Human schistosomiasis infects 200 million individuals, including 40 million women of child bearing age, and is a significant cause of morbidity and mortality in areas where the parasite is endemic. Treatment with praziquantel (PZQ) is the standard of care for many of those suffering from schistosome infections, however pregnant women have been systematically excluded from this treatment option. Although rodent models of schistosome infection during pregnancy clearly demonstrate adverse birth outcomes, there remains little examination of such a phenomenon in humans. Data from our pilot study suggests schistosome infection in humans can result in increased trophoblast TNF-a; production and apoptosis, as well as lower birth weights. This application is novel in its evaluation of the effect of schistosome-released soluble egg antigen (SEA) on human trophoblasts. We hypothesize that SEA will activate toll-like receptors (TLR)-2 and -4 on the surface of the human syncytiotrophoblast (STB), leading to induction of a pro-inflammatory cascade by this cell layer. We will utilize the most relevant model system available, primary trophoblasts isolated at term from women living in a region of The Philippines in which S. japonicum is endemic. Trophoblasts will be examined after differentiation, as the STB is the cell in direct contact with maternal blood, and thus most likely to be exposed to SEA in vivo. Specific Aim 1 will isolate term cytotrophoblasts from healthy, uninfected women. After in vitro syncytialization the cells will be exposed to SEA and TLR activation assessed through apoptosis rates, intracellular TNF-a; levels, cytokine production, MAPK and NF?B activation. Completion of Specific Aim 1 will allow us to report on specific effects of SEA on STBs in humans. Specific Aim 2 will isolate term cytotrophoblasts from infected women PZQ treatment given during the first trimester. These samples are being collected as part of an ongoing, U01 funded, randomized controlled trial evaluating PZQ treatment in the first trimester in women with schistosomiasis. STB will be cultured and TLR activation assessed as in Specific Aim 1. Despite PZQ treatment, the tissue egg burden does not immediately decrease, thus completion of Specific Aim 2 will identify effects of remaining schistosome eggs as well as PZQ treatment on the STB. Specific Aim 3 will utilize a murine model to determine the specific TLR(s) activated by SEA on trophoblast cells. Based on data in other physiological systems, we hypothesize that both TLR2 and -4 will be activated. This will be examined by trophoblast response to schistosome infection in genetic models deficient in either TLR2 or -4. Findings from Specific Aim 3 will advance mechanistic understanding in the field, allowing for better care of pregnant women with schistosomiasis. To our knowledge, this application will be the first to evaluate STBs for a specific response to antigens released into circulation during schistosome infection. This work is crucial in that it will allow for more comprehensive care of women and their offspring affected by schistosomiasis during pregnancy. PUBLIC HEALTH RELEVANCE: Approximately 40 million women of childbearing age suffer from schistosome infection globally at any given time. Multiple studies in rodent models, as well as a few reports in humans, suggest that schistosome infection results in poor pregnancy outcomes. The mechanisms of this phenomenon are unclear, and will be investigated in this application in order to enhance the treatment and health of pregnant women with schistosomiasis, as well as that of their affected offspring.