The villain team-up or how Trichomonas vaginalis and bacterial vaginosis alter innate immunity in concert

Abstract

Objectives: Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia).

Methods: Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells.

Results: TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines.

Conclusions: These molecular host-parasite-endosymbiont-bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.

Keywords: BACTERIAL VAGINOSIS; IMMUNOLOGY; TRICHOMONAS; VAGINAL MICROBIOLOGY; WOMEN.

Figure 1

Effect of Trichomonas vaginalis (TV) on bacterial colonisation in an in vitro cervicovaginal colonisation model. (A) Colony-forming units (CFU) associated with endocervical epithelial cells 48 h postcolonisation and 24 h after exposure to live TV or purified TV virulence factors. Bars represent means±SEM of duplicate measurements representing consistent results from five independent experiments. ***p<0.001, **p<0.01, *p<0.05, live TV, lipophosphoglycan, ceramide-phosphoinositol-glycan core and Trichomonas vaginalis virus virions different from medium control, two-way ANOVA, Bonferroni post-test. Similar results were obtained with vaginal and ectocervical cells (data not shown). (B) Electron micrographs from the vaginal colonisation model. Arrows in the higher magnification image (upper panel) indicate adherent or internalised Atopobium vaginae in vaginal epithelial cells. (C–H) CFU numbers assessed after 4 h antibiotic treatment of epithelia-free bacterial suspensions (C–E) or vaginal epithelial monolayers (F–H) precolonised with bacteria for 24 h. Data are means±SEM of duplicate cultures representing one of three experiments. med, antibiotic-free medium, p/s, combined penicillin–streptomycin; metro, metronidazole, Vk, vaginal epithelial cells. *p<0.05, **p<0.01, ***p<0.001, antibiotics different from no antibiotics, +p<0.05, ++p<0.01, +++p<0.001, supernatant different from lysate. (I) Reduction of planktonic or epithelia-associated CFU after 24 h infection with TV. Data are means and SEM from duplicate cultures of vaginal (Vk), ectocervical (Ect) and endocervical (End) epithelial cells representing two independent experiments. *p<0.05, **p<0.01, ***p<0.001, TV—infected compared with non-infected control; ++, p<0.01, +++p<0.001, planktonic compared with epithelial-associated CFU reduction (two-way ANOVA, Bonferroni post-test).

Figure 2

Effect of Trichomonas vaginalis (TV) and bacteria on chemokine levels in an in vitro cervicovaginal epithelial model. (A–C) Levels of IL-8 (A), RANTES (B) and secretory leucocyte protease inhibitor (C) measured in epithelial-cell supernatants collected 48 h after bacterial colonisation as indicated (1–7, bacteria identified below panel C) and 24 h after exposure to TV isolate UR1 (TV), lipophosphoglycan (LPG), ceramide-phosphoinositol-glycan core (CPI-GC), Trichomonas vaginalis virus (TVV) virions or MALP-2. Data are means±SEM from duplicate cultures in one of five independent experiments. ***p<0.001, **p<0.01, *p<0.05, TV, LPG, CPI-GC and TVV different from medium control within each group, two-way ANOVA, Bonferroni post-test; +++p<0.001, ++ p<0.01, +p<0.05, medium+bacteria or medium+MALP-2 different from ‘no bacteria’ medium control, one-way ANOVA, Dunnett post-test. (D–G) Levels of IL-8 (D), RANTES (E), MIP3a (F) and IP-10 (G) assessed simultaneously by Meso Scale Discovery multiplex in epithelial-cell supernatants collected 48 h after colonisation with Lacidophilus acidophilus and Prevotella bivia and 24 h after exposure to TV (1–6, conditions identified below panel G). ***p<0.001, **p<0.01, different from medium control, +++p<0.001, ++p<0.01, P bivia different from P bivia+ TV, one-way ANOVA, Bonferroni post-test.

Figure 3

Schematic presentation of chemokine response and suppressed secretory leucocyte protease inhibitor in response to Trichomonas vaginalis (TV) virulence factors and combined bacterial vaginosis (BV)-TV infection. Arrows show magnitude and direction of change. Arrows in parentheses indicate responses dependent on type of BV bacteria.