Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health

Abstract

Cervicovaginal (CV) microbiota is associated with vaginal health and disease in non-pregnant women. Recent studies in pregnant women suggest that specific CV microbes are associated with preterm birth (PTB). While the associations between CV microbiota and adverse outcomes have been demonstrated, the mechanisms regulating the associations remain unclear. As the CV space contains an epithelial barrier, we postulate that CV microbiota can alter the epithelial barrier function. We investigated the biological, molecular, and epigenetic effects of Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis on the cervical epithelial barrier function and determined whether L. crispatus mitigates the effects of lipopolysaccharide (LPS) and G. vaginalis on the cervical epithelial barrier as a possible mechanism by which CV microbiota mitigates disease risk. Ectocervical and endocervical cells treated with L. crispatus, L. iners, and G. vaginalis bacteria-free supernatants alone or combined were used to measure cell permeability, adherens junction proteins, inflammatory mediators, and miRNAs. Ectocervical and endocervical permeability increased after L. iners and G. vaginalis exposure. Soluble epithelial cadherin increased after exposure to L. iners but not G. vaginalis or L. crispatus. A Luminex cytokine/chemokine panel revealed increased proinflammatory mediators in all three bacteria-free supernatants with L. iners and G. vaginalis having more diverse inflammatory effects. L. iners and G. vaginalis altered the expression of cervical-, microbial-, and inflammatory-associated miRNAs. L. crispatus mitigated the LPS or G. vaginalis-induced disruption of the cervical epithelial barrier and reversed the G. vaginalis-mediated increase in miRNA expression. G. vaginalis colonization of the CV space of a pregnant C57/B6 mouse resulted in 100% PTB. These findings demonstrate that L. iners and G. vaginalis alter the cervical epithelial barrier by regulating adherens junction proteins, cervical immune responses, and miRNA expressions. These results provide evidence that L. crispatus confers protection to the cervical epithelial barrier by mitigating LPS- or G. vaginalis-induced miRNAs associated with cervical remodeling, inflammation, and PTB. This study provides further evidence that the CV microbiota plays a role in cervical function by altering the cervical epithelial barrier and initiating PTB. Thus, targeting the CV microbiota and/or its effects on the cervical epithelium may be a potential therapeutic strategy to prevent PTB.

Differential Secretion of Inflammatory Cytokines by Human Trophoblasts in the Presence of <b><i>Escherichia coli</i></b>, <b><i>Lactobacillus crispatus</i></b>, and <b><i>Lactobacillus jensenii</i></b>

“…Previous work using HeLa cells showed that a vaginal strain of L. crispatus can induce NF-ķB activation and yet maintain low levels of several proinflammatory cytokines [63]. Another report, using human ectocervical and endocervical cell lines treated with soluble products produced by L. crispatus induced responses that may result in cervical epithelial barrier protection, including secretion of IL-10 -the same response we found in our experiments with trophoblasts, and that can be interpreted as being conducive to immunomodulation [64]. Recent evidence in non-pregnant women suggests that the endometrial cavity may have a characteristic microbiota with low biomass [65][66][67][68][69][70][71][72][73][74][75][76][77].…”

Cooperative Interactions between Trichomonas vaginalis and Associated Bacteria Enhance Paracellular Permeability of the Cervicovaginal Epithelium by Dysregulating Tight Junctions

“…Our study did not reveal the molecules involved in the physical disruption of the hEC barrier by these vaginal microorganisms, but the data possibly indicate that enhancement of phosphatase activity is among the major underlying mechanisms that lead to TJ dysregulation of the host cervicovaginal epithelium. Disruption of this barrier has been attributed to G. vaginalis (10,17,32), but the mechanisms are not yet understood and no phosphatases have been claimed to be involved. On the other hand, phosphatase activity has been found in various cellular compartments of T. vaginalis, including on its surface and in secreted fractions (50); those findings have been recently supported by proteomics data (51,52).…”

“…A link between these events and the composition of the vaginal microbiota has been claimed, and the CST-IV bacterium G. vaginalis, in particular, has been associated with this process (10,15,17,31). Protective lactobacilli, on the other hand, are known to promote cervical health by mitigating effects of proinflammatory molecules and counteracting G. vaginalisinduced disruption of the cervicovaginal barrier (32).…”

Interactions between the MicroRNAs and Microbiota in Cancer Development: Roles and Therapeutic Opportunities

“…Sierra et al evaluated the expression of certain miRNAs after contact to bacteria-free supernatants of Gardnella vaginalis (G. vaginalis), Lactobacillus crispatus (L. crispatus), and Lactobacillus iners (L. iners). Remarkably, the upregulation of miRNA-15a, miRNA-143, miRNA-145, miR-146, miR-223, and miR-148 by G. vaginalis bacteria-free supernatants and miR-146, miRNA-193b, and miR-223 by L. iners bacteria-free supernatants with no modification by L. crispatus reveals the existence of a bacterial species-specific modification in miRNA expression profiles [27].…”