Trichomonas vaginalis lipophosphoglycan triggers a selective upregulation of cytokines by human female reproductive tract epithelial cells

Abstract

Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3alpha, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1beta release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.

FIG. 1.

A. Inhibition of T. vaginalis adhesion with T. vaginalis LPG. Approximately 8 × 10^{5 32}S-labeled parasites were added to confluent HVECs (5 × 10⁴) containing different concentrations of LPG in a total volume of 0.6 ml and incubated for 30 min at 37°C (5% CO₂). B. Inhibition of T. vaginalis adhesion to HVECs with various densities of unlabeled parasites. Unlabeled parasites were coincubated with 8 × 10^{5 32}S-labeled parasites and HVECs in 1.2 ml for 30 min at 37°C (5% CO₂). Binding was determined as described in the text.

FIG. 2.

Effects of T. vaginalis LPG on cell viability and IL-8 production. A. LPG doses of <60 μg/ml were nontoxic to the ectocervical (Ect1/E6E7) epithelial cells. Similar results were obtained for the vaginal and endocervical epithelial cell lines. B. T. vaginalis but not T. foetus LPG stimulates IL-8 production in ectocervical (Ect1/E6E7) epithelial cells. C. IL-8 production adjusted per viable cell counts in endocervical (End1/E6E7) and vaginal (Vk2/E6E7) epithelial cells. Results represent means and standard errors of the means from triplicate cultures in one of two independent experiments with each cell line. *, P < 0.05; **, P < 0.01.

FIG. 3.

Multiplex cytokine analysis of ectocervical (Ect1/E6E7) cultures stimulated with T. vaginalis LPG for 24 h. Cytokine concentrations were measured by a Meso-Scale 2400 imager. LPG induced IL-6, IL-8, and MIP-3α but not IL-10, IL-1β, and TNF-α release. Results represent means and standard errors of the means from triplicate cultures.

FIG. 4.

Inhibition of LPG signaling with dnMyD88. A. MIP-3α concentrations measured by enzyme-linked immunosorbent assay in supernatants from Vk2/E6E7 cells collected 24 h after stimulation with T. vaginalis LPG and IL-1β. B. IL-8 measured in the same culture supernatants. C. NF-κB activation (presented as firefly/Renilla luciferase ratio) in Ect1/E6E7 cells transfected with control plasmid pDeNy-mcs or with dnMyD88 and stimulated for 4 h with IL-1β (20 ng/ml) or 15 μg/ml of LPG. The bars represent means and standard errors of the means from triplicate cultures. **, P < 0.01, different from nontreated (medium) control; n.s., no significant difference from medium control.