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 significa...