Cell-to-cell communication within gestational membranes in response to bacterial infection

组织对感染的反应可能与单个细胞类型的反应显着不同，这对 利用现有的还原论体外模型系统来解决复杂的体内问题。怀孕期间， 蜕膜基质细胞（DSC）和细胞滋养层细胞（CTB）形成绒毛膜蜕膜，即蜕膜的外层。 妊娠（胎儿）膜和绒毛蜕膜内的免疫细胞偏向于耐受性 表型。然而，细菌感染会引起炎症（绒毛膜羊膜炎），从而导致 早产（PTB）和其他不良后果。新证据表明 DSC 和 CTB 积极 参与免疫监视并塑造对感染的先天免疫反应。我们有证据表明 DSC 和 CTB 各自可以以不同的方式调节巨噬细胞 (Mφ) 对细菌感染的反应 当所有三种细胞共培养时，反应也不同，强调了新模型的需要 异细胞组织免疫生物学系统。在这个提案中，我们使用创新的片上器官 异细胞组织模型来测试一个中心假设，即含有 microRNA (miRNA) 的细胞外 囊泡 (EV) 介导 NFκB 依赖性 Mφ 细菌免疫反应的旁分泌调节 胎膜内 DSC 和 CTB 的感染。目标 1 将定义 CTB 和/或 DSC 的程度 调节 Mφ 对感染的反应，检验 CTB 和 DSC 与 Mφ 进行三培养的具体假设 促进对细菌感染的一组独特且特定的 Mφ 炎症反应。我们将文化CTB， DSC 和 Mφ 并评估细胞因子的产生、主要免疫途径的激活以及报告基因检测 促炎转录因子 NFκB 并将其与单一培养物和 2-way 的免疫特征进行比较 共培养。目标 2 将确定脉络膜蜕膜衍生的 EV 货物对 Mφ 激活的影响 细菌感染，检验 EV miRNA 抑制 Mφ 细胞因子产生的具体假设。苏拜姆2a 将决定 EVs 参与 Mφ 免疫调节。我们将净化未经处理或感染的电动汽车 CTB 和/或 DSC 培养物刺激 Mφ，选择性阻断 CTB 或 DSC EV 释放并评估 Mφ 激活 通过细胞因子释放和 NFκB 激活。 Subaim 2b 将比较 EV 与细胞的转录组 产生它们的。我们将对 1) CTB、2) CTB 衍生的 EV、3) DSC 和 4) DSC 进行 miRNA 分析- 衍生 EV 并确定特定 miRNA 序列是否选择性包装在 EV 内。我们会 使用基因沉默方法来确定 EV 中发现的哪些 miRNA 可能抑制 Mφ NFκB 激活（例如 miR146a、miR155）和细胞因子激活。该项目将定义精确的免疫 使用新型微流体在组织水平上在人类妊娠膜内进行调节 器官型系统。我们的研究结果可以确定预防或预防的可行目标 妊娠期宫内细菌感染的治疗，对母婴健康构成重大威胁。

The response of tissues to infection can significantly differ from that of individual cell types, challenging the utility of existing, reductionist in vitro model systems to solve complex in vivo problems. During pregnancy, decidual stromal cells (DSC) and cytotrophoblasts (CTB) form the choriodecidua, the outer layer of the gestational (fetal) membrane, and immune cells within the choriodecidua are skewed towards a tolerogenic phenotype. However, bacterial infection provokes inflammation (chorioamnionitis), which can result in preterm birth (PTB) and other adverse outcomes. New evidence suggests that DSCs and CTBs actively participate in immune surveillance and shape innate immune responses to infection. We have evidence that DSCs and CTBs can each regulate the response of macrophages (Mφ) to bacterial infection in different ways and when all three cells are cocultured responses are also distinct, underscoring the need for new model systems of heterocellular tissue immunobiology. In this proposal we use innovative organ-on-chip heterocellular tissue models to test a central hypothesis that microRNA (miRNA)-containing extracellular vesicles (EVs) mediate the paracrine regulation of NFκB-dependent Mφ immune responses to bacterial infection by DSCs and CTBs within fetal membranes. Aim 1 will define the extent to which CTBs and/or DSCs modulate Mφ responses to infection, testing the specific hypothesis that CTB and DSC tri-culture with Mφ promote a unique and specific set of Mφ inflammatory responses to bacterial infection. We will culture CTB, DSC, and Mφ and assess cytokine production, major immune pathway activation, and reporter assays for the proinflammatory transcription factor NFκB and compare this to immune profiles of monoculture and 2-way co-culture. Aim 2 will determine the impact of choriodecidually-derived EV cargo on Mφ activation during bacterial infection, testing the specific hypothesis that EV miRNAs inhibit Mφ cytokine production. Subaim 2a will determine involvement of EVs in Mφ immune modulation. We will purify EVs from untreated or infected CTB and/or DSC culture to stimulate Mφ, selectively block CTB or DSC EV release and assess Mφ activation by cytokine release and activation of NFκB. Subaim 2b will compare the transcriptome of EVs with the cells that produce them. We will perform miRNA profiling of 1) CTB, 2) CTB-derived EVs, 3) DSC, and 4) DSC- derived EVs and determine whether specific miRNA sequences are selectively packaged within EVs. We will use gene silencing approaches to determine which miRNAs found in EVs might be inhibiting Mφ NFκB activation (e.g., miR146a, miR155) and cytokine activation. This project will define the precise immune regulation taking place within human gestational membranes at the tissue level using a novel, microfluidic organotypic system. Findings from our research could identify actionable targets for the prevention or treatment of intrauterine bacterial infection during pregnancy, a significant threat to maternal-child health.