Transcriptomics and biomarkers of fetal neuroinflammation

总结 这是对母基金HD 98389（2019-24）的行政补充提案，标题为“抗炎 减少不良妊娠结局的药物目标”。父母补助金的目标是开发阿那白滞素 （重组人IL-1受体拮抗剂）作为一种安全有效的治疗宫内 感染/炎症介导的早产和胎儿炎症。在父母补助金中，怀孕的恒河猴 在子宫内炎症（IUI）模型中给予猕猴阿那白滞素。我们现在将利用 家长补助金的资源，以扩大家长补助金的目的。胎儿神经炎症与 随着脑性瘫痪（CP）和自闭症谱系障碍（ASD）的发展， 公共卫生影响。作为父母资助活动的一部分，我们已经证明了抑制IL 1 使用阿那白滞素的信号传导显著降低了羊膜内注射E.杆菌 LPS。我们现在已经开发出从胎儿冷冻脑样品中提取完整细胞核的能力， 细胞测序与胞质RNA seq相比，核RNA seq具有不需要蛋白酶消化的优点 在37 ° C，从而消除人为的基因表达。此外，核RNA seq可以在存档中完成， 冷冻大脑样本，从而节省宝贵的动物使用和降低成本。在这份行政补充文件中， 授予，我们将测试的假设，抑制白细胞介素1信号传导的阿那白滞素将减少区域特异性 羊膜内脂多糖诱导的胎儿神经炎症。我们建议从2 大脑的区域-脑室周围的白色物质，和前额叶皮层-与大脑的活动有关的区域。 CP和ASD的发病机制。我们将比较三组中区域特异性单核mRNA序列， 约80%妊娠的恒河猴胎儿：1）对照-羊膜内注射（IA）盐水，2）IA LPS， 3)IA LPS +阿那白滞素。所有暴露均为16小时，我们先前证明了该时间点对于 建议的研究。由父母赠款资助的动物资源已经可用。因此，单细胞 转录组学研究将在获得资助的一年内得到极大的便利和可行性。这个胎儿的大脑 转录组学研究将揭示IUI引起的神经炎症的发病机制， 并为CP和ASD的生物标志物集确定线索。该研究还将进一步协助临床前开发 阿那白滞素作为IUI的候选治疗药物。

Summary This is an administrative supplement proposal to the parent grant HD98389 (2019-24) titled “Anti-inflammatory drug target to reduce adverse pregnancy outcomes”. The goal of the parent grant is to develop Anakinra (recombinant human IL1receptor antagonist) as a safe and effective therapeutic for intrauterine infection/inflammation mediated preterm labor and fetal inflammation. In the parent grant, pregnant Rhesus macaques will be given Anakinra in a model of intrauterine inflammation (IUI). We will now leverage the resources of the parent grant to expand the Aims of the parent grant. Fetal neuro-inflammation is associated with later development of cerebral palsy (CP) and autism spectrum disorders (ASD) with significant medical and public health implications. As part of the parent grant activities, we have demonstrated that inhibition of IL1 signaling using Anakinra significantly decreased fetal inflammation induced by intraamniotic injection of E. coli LPS. We have now developed capability of extracting intact nuclei from fetal frozen brain sample and to do single cell sequencing. Nuclear as compared to cytosolic RNA seq has the advantage of not needing protease digestion at 37oC thereby eliminating artifactual gene expression. Furthermore, nuclear RNA seq can be done in archived frozen brain sample thereby sparing precious animal use and reducing costs. In this administrative supplement grant, we will test the hypothesis that inhibition of IL1 signaling by Anakinra will decrease region specific fetal neuro inflammation induced by intraamniotic LPS. We propose doing single nuclear mRNA seq from 2 regions of the brain – the peri-ventricular white matter, and the prefrontal cortex – regions implicated in the pathogenesis of CP and ASD. We will compare region specific single nuclear mRNA seq in three groups of Rhesus macaque fetuses at about 80% gestation: 1) Controls – intraamniotic injection (IA) of saline, 2) IA LPS, 3) IA LPS + Anakinra. All exposures are for 16h, a time point that we previously demonstrated to be optimum for the proposed studies. The animal resources funded by the parent grant are already available. Thus, single cell transcriptomic studies will be greatly facilitated and feasible within the year of funding. This fetal brain transcriptomic study will reveal mechanistic insights in the pathogenesis of neuroinflammation resulting from IUI, and identify leads for biomarker sets for CP and ASD. The study will also further assist in pre-clinical development of Anakinra as a therapeutic candidate for IUI.