Organ-on-chip traffic dynamics of bacterial vaginosis-associated bacteria in the genital tract, gestational tissues, and induction of compartmentalized inflammatory response.

• Grant number: 24/07342-9
• Support Opportunities: Regular Research Grants
• Start date: September 01, 2024
• End date: August 31, 2026
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Márcia Guimarães da Silva
• Grantee: Márcia Guimarães da Silva
• Host Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated researchers: Jossimara Polettini ; Ramkumar Menon
• Associated scholarship(s): 24/17377-4 - Vaginal Cervical Decidual Fetal-Maternal Interface Organ-on-Chip (VCD-FMi-OOC) Model for Analyzing the Ascension of the Pathological Core of Bacterial Vaginosis to the Maternal-Fetal Interface during Metronidazole Treatment, BP.PD

Abstract

Introduction: Genomic studies have revealed the potential for variations in the vaginal microbiota during gestation, leading to the development of classical dysbioses. The association between bacterial vaginosis and preterm birth lies in the capability of various bacterial species to ascend to the amniotic cavity and elicit an inflammatory response at the maternal-fetal interface. Although spontaneous preterm birth has a multifactorial etiology and amniotic cavity infection is well understood, there exists complexity within this polybacterial scenario, as well as in the response elicited by these interactions. Objective: The primary aim of this study is to characterize the dynamics of bacterial species traffic belonging to the pathological core of bacterial vaginosis along the female genital tract and in gestational tissues, analyzing compartmental inflammatory responses in an organ-on-chip (OOC) model. Secondly, utilizing exosomes isolated from vaginal cell cultures previously stimulated by a polymicrobial scenario, this study seeks to characterize and quantify the dynamics of exosome traffic along the female genital tract and the fetal-maternal interface, analyzing compartmentalized inflammatory profiles in an OOC model. Methodology: OOC devices will be established, integrating the regions of the vagina, cervix, decidua (VCD-OOC), and chorioamniotic membranes (FMi-OOC) as a model for studying polymicrobial infection of the amniotic cavity. To ensure morphological and physiological integrity parameters of the cells in the devices VCD-OOC and FMi-OOC, the effects of infection on cellular layers will be analyzed by microscopy, observing cell migratory capacity, as well as apoptosis and necrosis analyses by fluorescence. Cellular senescence will be verified by ²-galactosidase colorimetric assay. Vaginal epithelial cells will be stimulated with the main bacterial species identified in the pathological core of bacterial vaginosis. Bacterial stimulus-free samples will be used as negative controls, and lipopolysaccharide will be used as a positive control. Bacterial counting in each compartment will be performed by flow cytometry. To evaluate cytokine production regulation, supernatants obtained from various cellular layers will be collected and stored at -80°C. Supernatants from cultures will be assessed using Luminex technology employing a panel for IL-1², IL-6, sIL-6R, sgp130, mIL-6R, gp130, IL-8, IL-10, IL-13, TNF-±, GM-CSF, TLR-2, and PTX3. Real-time PCR quantification will be conducted to assess HBD1, HBD2, HBD3, TLR-4, TLR-6, and TLR-8. Exosomes derived from vaginal epithelial cells cultured in a polymicrobial scenario will be isolated and labeled with fluorescence (DiO), added to the outer chamber of the VCD-OOC, and their traffic between chambers will be evaluated by microscopy. (AU)