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

妊娠膜内细胞间通讯对细菌感染的反应

• 批准号: 10392749
• 负责人: Alison Joan Eastman
• 金额: $ 2.94万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-18 至 2021-10-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392749
• 关键词: Address; Allogenic; Antibody Formation; Bacterial Infections; Biological Assay; Biological Models; Biology; Cell Communication; Cell Culture Techniques; Cell model; Cells; Childbirth; Chorion; Coculture Techniques; Complex; Cytokine Activation; Data; Disease; Fetal Development; Fetal Membranes; Fetus; Gene Silencing; Goals; Health; Host Defense; Human; Immune; Immune response; Immunity; Immunobiology; Immunologic Surveillance; Individual; Infection; Inflammation; Inflammatory Response; Innate Immune Response; Intervention; Macrophage Activation; Maternal and Child Health; Mediating; Membrane; MicroRNAs; Microfluidics; Modeling; Molecular; Outcome; Paracrine Communication; Pathogenesis; Pathway interactions; Phagocytes; Phenotype; Placenta; Play; Pregnancy; Pregnancy Complications; Pregnancy Outcome; Premature Birth; Premature Labor; Prevention; Production; Protein Activation Pathway; Protein Kinase; Regulation; Reporter; Research; Resolution; Role; Rupture; Shapes; Signal Transduction; Stromal Cells; System; Techniques; Testing; Tissue Model; Tissues; adverse outcome; antibody conjugate; cell type; cytokine; cytotrophoblast; exosome; experimental study; extracellular; extracellular vesicles; immune activation; immunoregulation; improved; in vitro Model; in vivo; innovation; insight; intraamniotic infection; macrophage; miRNA expression profiling; microbial; neonatal infection; new therapeutic target; novel; organ on a chip; paracrine; preterm premature rupture of membranes; prevent; response; stillbirth; transcription factor; transcriptome; transcriptome sequencing; vesicular release

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.

组织对感染的反应可能与单个细胞类型的反应显著不同，这挑战了免疫组织化学。 利用现有的简化体外模型系统来解决复杂的体内问题。在怀孕期间， 蜕膜基质细胞（DSC）和细胞滋养层细胞（CTB）形成绒毛膜蜕膜，绒毛膜蜕膜的外层。 妊娠（胎儿）膜，绒毛膜蜕膜内的免疫细胞倾向于耐受性 表型然而，细菌感染引起炎症（绒毛膜炎），这可能导致 早产（PTB）和其他不良后果。新的证据表明，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 DSCs和CTB可以通过不同的方式调节巨噬细胞（M φ）对细菌感染的反应 当所有三种细胞共培养时，反应也是不同的，强调了对新模型的需要。 异细胞组织免疫生物学系统。在这个提案中，我们使用创新的器官芯片， 异质细胞组织模型，以测试一个中心假设，即含有microRNA（miRNA）的细胞外 囊泡（EV）介导NF κ B依赖性M φ对细菌免疫反应的旁分泌调节 感染胎儿膜内的DSC和CTB。目标1将界定CTB和/或DSC 调节M φ对感染的反应，测试CTB和DSC与M φ三培养的特定假设 促进对细菌感染的一组独特和特异的M φ炎症反应。我们会培养CTB， DSC和M φ，并评估细胞因子的产生、主要免疫途径的激活和报告基因测定。 促炎性转录因子NF κ B，并将其与单培养和双向培养的免疫概况进行比较。 共培养。目的2将确定绒毛膜蜕膜来源的EV货物对M φ激活的影响， 细菌感染，测试EV miRNA抑制M φ细胞因子产生的特定假设。Subaim 2a 将决定EV参与M φ免疫调节。我们将从未经治疗或感染的EV中纯化 CTB和/或DSC培养以刺激M φ，选择性阻断CTB或DSC EV释放并评估M φ活化 通过细胞因子释放和NF κ B活化。Subaim 2b将比较EV与细胞的转录组 生产它们的人。我们将对1）CTB、2）CTB衍生的EV、3）DSC和4）DSC-1进行miRNA分析。 并确定特定的miRNA序列是否被选择性地包装在EV内。我们将 使用基因沉默方法来确定EV中发现的哪些miRNA可能抑制M φ NF κ B 激活（例如，miR146a、miR155）和细胞因子活化。这个项目将定义精确的免疫 在组织水平上使用新型微流体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治疗的可行目标， 治疗妊娠期宫内细菌感染，严重威胁母婴健康。