Temporal regulation of the inflammatory response in in vitro models of polybacterial infection of immortalized amniotic epithelial cells with different bacterial species associated with preterm birth, in the presence of Lactobacillus spp.

• Grant number: 21/13773-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2022
• End date: December 31, 2022
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Márcia Guimarães da Silva
• Grantee: Vitória Carvalho Troitiño
• Host Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Although the etiology of preterm birth is multifactorial, several microorganisms have been associated with its pathogenesis, since the bacterial species most frequently included in the amniotic fluid of patients with intra-amniotic infection are bacteria associated with bacterial vaginosis. Even though the infection of the amniotic cavity is the main cause of spontaneous preterm birth, there is a complexity in this infectious scenario because it has several bacterial communities involved, different interactions between species, different responses to antibiotics, and different clinical results. Objective: To evaluate the temporal regulation of the inflammatory response in vitro models of polybacterial interaction in amnion epithelial cells with different associations of bacterial species related to preterm birth, in the presence of Lactobacillus spp. Methodology: Immortalized amnion epithelial cells will be stimulated with different species and bacterial loads, a scenario frequently found in the lower genital tract of pregnant women who have preterm births. Cell culture without stimulation will be used as a negative control and, lipopolysaccharide from Escherichia coli asa positive control. To evaluate the regulation of expression and production of different inflammatory markers, culture supernatants collected at different times will be used (0h, 6h,12h, 18h, and 24h after stimulation). The supernatants will be collected and used for protein quantification by Enzyme Immunoassay (ELISA) for Interleukin (IL)-1², IL-6, IL-8, IL-10, IL-13, Granulocyte and Macrophage Colony Stimulating Factor (GM-CSF), Tumor Necrosis Factor (TNF) -±, Human-² Defensin (HBD)1, HBD2, HBD3 and Pentraxin 3 (PTX3). The ultrastructural characterization of immortalized amnion epithelial cells, after stimulation with different combinations of bacterial species, will be performed by confocal microscopy.(AU)