The microRNA Cargo of Human Vaginal Extracellular Vesicles Differentiates Parasitic and Pathobiont Infections from Colonization by Homeostatic Bacteria.

The disturbed vaginal microbiome defined as bacterial vaginosis (BV) and the parasitic infection by Trichomonas vaginalis (TV), the most common non-viral sexually transmitted pathogen, have well-established adverse effects on reproductive outcomes and susceptibility to infection and cancer. Molecular mechanisms underlying these associations and the failure of antibiotic therapy to mitigate adverse consequences are not fully elucidated. In an in vitro human vaginal colonization model, we tested the hypothesis that responses to TV and/or BV-bacteria will disrupt the micro(mi)RNA cargo of extracellular vesicles (EV) with the potential to modify pathways associated with reproductive function, cancer, and infection. miRNAs were quantified by HTG EdgeSeq. MiRNA differential expression (DE) was established in response to TV, the BV signature pathobiont Prevotella bivia and a homeostatic Lactobacillus crispatus with adjusted p < 0.05 using R. Validated gene targets, pathways, protein-protein interaction networks, and hub genes were identified by miRWalk, STRING, Cytoscape, and CytoHubba. In contrast to L. crispatus, TV and the BV pathobiont dysregulated a massive number of EV-miRNAs, over 50% shared by both pathogens. Corresponding target pathways, protein interaction clusters and top hub genes were related to cancer, infectious disease, circadian rhythm, steroid hormone signaling, pregnancy, and reproductive tissue terms. These data support the emerging concept that bacteria and parasitic eukaryotes disturbing the human vaginal microbiome may impact reproductive health through EV-miRNA dysregulation.

Aberrant cervical innate immunity predicts onset of dysbiosis and sexually transmitted infections in women of reproductive age.

Sexually transmitted infections (STIs) and vaginal dysbiosis (disturbed resident microbiota presenting with abnormal Nugent score or candidiasis) have been associated with mucosal inflammation and risk of HIV-1 infection, cancer and poor reproductive outcomes. To date, the temporal relationships between aberrant cervical innate immunity and the clinical onset of microbial disturbance have not been studied in a large population of reproductive age women. We examined data from a longitudinal cohort of 934 Ugandan and Zimbabwean women contributing 3,274 HIV-negative visits who had complete laboratory, clinical and demographic data. Among those, 207 women later acquired HIV, and 584 women were intermittently diagnosed with C. trachomatis (CT), N. gonorrhoeae (NG), genital herpes (HSV-2), T. vaginalis (TV), candidiasis, and abnormal intermediate (4-6) or high (7-10) Nugent score, i.e. bacterial vaginosis (BV). Immune biomarker concentrations in cervical swabs were analyzed by generalized linear and mixed effect models adjusting for site, age, hormonal contraceptive use (HC), pregnancy, breastfeeding, genital practices, unprotected sex and overlapping infections. High likelihood ratios (1.5-4.9) denoted the values of cervical immune biomarkers to predict onset of abnormal Nugent score and candidiasis at the next visits. When controlling for covariates, higher levels of ß-defensin-2 were antecedent to BV, CT and HSV-2, lower anti-inflammatory ratio IL-1RA:IL-1ß-to intermediate Nugent scores and candida, lower levels of the serine protease inhibitor SLPI-to candida, lower levels of the adhesion molecule ICAM-1 -to TV, and lower levels of the oxidative stress mitigator and endothelial activation marker VEGF-to NG. Changes in innate immunity following onset of dysbiosis and infections were dependent on HC use when controlling for all other covariates. In conclusion, imminent female genital tract dysbiosis or infection can be predicted by distinct patterns of innate immunity. Future research should characterize biotic and abiotic determinants of this pre-existing innate immunity state.

Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity.

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.