Human Three-Dimensional Endometrial Epithelial Cell Model To Study Host Interactions with Vaginal Bacteria and Neisseria gonorrhoeae

Abstract

Colonization of the endometrium by pathogenic bacteria ascending from the lower female reproductive tract (FRT) is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions in vitro, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and the rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue, including cell differentiation, the presence of junctional complexes/desmosomes and microvilli, and the production of membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with poly(I·C) and flagellin but not with synthetic lipoprotein (fibroblast-stimulating lipoprotein 1 [FSL-1]) or lipopolysaccharide (LPS) significantly induced proinflammatory mediators in a dose-dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria: Lactobacillus crispatus, Gardnerella vaginalis, and Neisseria gonorrhoeae. All vaginal microbiota and N. gonorrhoeae efficiently colonized the 3-D surface, localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic N. gonorrhoeae and not infection with the other bacteria tested significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells. The latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of the 3-D EEC model for the study of the pathogenesis of N. gonorrhoeae using a well-characterized ΔpilT mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT.

Interaction of Gardnerella vaginalis and Vaginolysin with the Apical versus Basolateral Face of a Three-Dimensional Model of Vaginal Epithelium

“…These discrepancies could be due to a lack of cell polarization or stratification and a lack of bacterial growth in the monolayer system. The results from a three-dimensional tissue model of G. vaginalis infection are in agreement with this study in that no significant increases in the secretion of TNF-␣, IL-6, or IL-8 were detected (60). These data partly reflect in vivo cytokine profiles in BV-positive women.…”

“…Moreover, cervicovaginal colonization by G. vaginalis in pregnant mice resulted in increased levels of IL-6 in cervicovaginal fluid as well as increased IL-1␤, IL-8, and IL-10 gene expression in cervical tissue (59). In a human three-dimensional endometrial epithelial cell model, however, G. vaginalis did not elicit any significant changes in the secretion of IL-6, IL-8, or tumor necrosis factor alpha (TNF-␣) compared to a mock-infected control (60). Similarly, when G. vaginalis was added to organotypic cultures of human vaginal epithelium containing dendritic cells, there was no stimulation of H␤D-2, IL-8, or IL-1 (␣ or ␤) expression, albeit bacteria were heat inactivated prior to inoculation of tissues (61).…”

The relationship between sex hormones, the vaginal microbiome and immunity in HIV-1 susceptibility in women

“…Similar to the VMB, the endometrial microbiota may be able to modulate inflammation. In vitro co-cultures of endometrial epithelial cells with pathogenic bacteria ( Neisseria gonorrhoeae ) induced proinflammatory mediators ( Christodoulides et al, 2000 ), whereas other bacteria typically found in the reproductive microbiotas ( Lactobacillus crispatus , Gardnerella vaginalis ) did not ( Łaniewski et al, 2017 ). This suggests that the endometrial microbiota may be able to modulate endometrial inflammation in the host.…”

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age

“…It was the first technology used to develop 3-D models for infection studies with bacterial (Salmonella) and viral (rhinovirus) pathogens (11,51). A range of RWV-derived 3-D models have been developed using cell lines, stem cells, and/or primary cells, including small and large intestine (11,80,141,143,145,146,152,(168)(169)(170)(171)(172)(173)(174)(175)(176)(177), lung (144,147,(178)(179)(180)(181)(182), liver (148,153,174,183,184), bladder (8,(185)(186)(187), reproductive tissue (149)(150)(151)(188)(189)(190), heart (191)(192)(193), prostate (142,186,194), pancreas (195,<...>…”