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

基本信息

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
项目类别：
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

项目摘要

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着不同，挑战现有的，还原主义的体外模型系统的实用性，以解决体内问题的复合物。怀孕期间 de骨基质细胞（DSC）和细胞增多质细胞（CTB）形成螺旋菌，这是螺旋体，妊娠（胎儿）膜和绒毛膜内的免疫细胞偏向于耐受性表型。但是，细菌感染会引起感染（绒毛膜炎），这可能导致早产（PTB）和其他不利结果。新证据表明DSC和CTB会积极参与免疫监视并塑造先天免疫调查以感染。我们有证据表明 DSC和CTB可以以不同的方式调节巨噬细胞对细菌感染的反应当所有三个细胞都是共培养的响应时，也很明显，强调了对新模型的需求杂细胞组织免疫生物学系统。在此提案中，我们使用创新的器官芯片杂细胞组织模型，以测试中心假设，即microRNA（miRNA）含有细胞外蔬菜（EV）介导NFκB依赖性Mφ免疫反应的旁分泌调节对细菌 DSC和CTB在胎儿中感染。 AIM 1将定义CTB和/或DSC的程度调节Mφ对感染的反应，检验了具有Mφ的CTB和DSC TRI培养的特定假设促进对细菌感染的独特而特异的Mφ炎症反应。我们将培养CTB， DSC，Mφ和评估细胞因子的产生，主要免疫途径激活以及记者测定法促炎转录因子NFκB，并将其与单栽培和2向的免疫特征进行比较共同文化。 AIM 2将确定脉络膜衍生的EV货物对Mφ激活的影响细菌感染，检验EV miRNA抑制Mφ细胞因子产生的特定假设。 Subaim 2a 将确定电动汽车参与Mφ免疫调节。我们将从未经治疗或感染中净化电动汽车 CTB和/或DSC培养通过细胞因子释放和NFκB的激活。 Subaim 2b将比较电动汽车的转录组与细胞产生它们。我们将执行1）CTB，2）CTB衍生的EV，3）DSC和4）DSC-的miRNA分析了。派生的EV并确定特定的miRNA序列是否在EV中有选择地包装。我们将使用基因沉默方法来确定EV中发现的miRNA可能抑制MφNFκB 激活（例如miR146a，miR155）和细胞因子激活。该项目将定义精度免疫使用新型的微流体，在组织水平的人妊娠膜内进行调节有机系统。我们研究的发现可以确定可行的预防目标或怀孕期间的插入细菌感染的治疗，对母子健康构成了重大威胁。