Figure 1.
Trichomonasvirus positive T. vaginalis isolates (UR1, UH9) induce Type-1 Interferon and elevated proinflammatory responses as compared to virus-free (B7RC2) protozoa and purified lipophosphoglycan (UR1-LPG).
Endocervical (A–D), ectocervical (F–I) and vaginal (E, G) epithelial cells were exposed to trichomonad isolates, LPG or poly(I:C) for 24 h. Ectocervical immortalized cell monolayers (F,G) and the apical surface of primary polarized tissues grown on dual-compartment filters (H,I) were treated side-by-side. NF-κB-driven luciferase activity was assessed in cell lysates and soluble mediators were measured in monolayer supernatants or apical and basal compartments. Bars are means and S.E.M. from quadruplicate (A,C, F), triplicate (H,I) or duplicate cultures (D,E,G) representing one of at least three independent experiments. *p<0.05, **p<0.01 and ***p<0.001, different from medium (one-way ANOVA, Bonferroni).
Figure 2.
Trichomonasvirus (TVV)-positive protozoa upregulate TLR3 and trigger endosomal acidification-dependent inflammatory responses.
(A, B) TLR mRNA levels were measured by a multiplex nuclease protection assay in the endocervical epithelial cells (A) and in TLR-Null or TLR2/6+ or TLR4+ HEK293 cells (B). The endocervical cells were exposed to TVV-positive (UR1) and TVV-negative (B7RC2) trichomonad isolates, MALP-2, poly(I:C) or LPG for 24 h. **p<0.01, ***p<0.001 different from medium (two-way ANOVA, Bonferroni). (C) Levels of NF-κB-driven luciferase in cell lysates and IL-8 and IFNβ in culture supernatants were measured 12 h post stimulation in the presence or absence of endosomal acidification inhibitor bafilomycin A1 (BFA). (D) a multiplex immunoassay was applied to measure RANTES, IL-6 and MIP-3α in the same culture supernatants. +++p<0.001, treatment different from medium control ***p<0.001, bafilomycin A different from solvent (DMSO) control (two-way ANOVA, Bonferroni). Bars are means and S.E.M. of triplicate (for NF-κB) or duplicate (for cytokines) cultures representative of at least three independent experiments.
Figure 3.
TLR-3-dependent proinflammatory activation is a common trait for Trichomonasvirus(TVV)-infected T. vaginalis isolates.
(A–C) The endocervical NF-κB reporter cell line was challenged for 24 h with TVV positive or TVV negative trichomonads, poly(I:C), MALP-2 or LPG from trichomonas isolates UR1 and OC6 in the presence or absence of bafilomycin A (endosomal acidification inhibitor and known TLR3 antagonist) followed by luciferase, IL-8 and IFNβ measurements. (D) IL-8 was measured in supernatants from endocervical cells expressing dominant negative (dn)TRIF or control (mcs) plasmid and infected or stimulated under the same conditions. Bars are means and S.E.M. from duplicate cultures in two experiments. Fold change is computed over the average obtained for the medium solvent control. ***p<0.001, bafilomycin A1-treated or dnTRIF different from solvent or plasmid (mcs) control, respectively; +p<0.05 and +++p<0.001, infection/TLR ligand/LPG different from medium control (two-way ANOVA, Bonferroni).
Figure 4.
The cell-free culture phase of metronidazole treated TVV-positive trichomonads enhanced TLR3/TRIF-depedent proinflammatory responses.
(A–H) Endocervical cells expressing dominant negative (dn)TRIF or control mcs plasmid were exposed to cell free supernatants from trichomonads treated with 100 µM metronidazole for 24 h in the presence or absence of bafilomycin A1, followed by viability assessment via MTT assay (A) and cytokine measurement in the cell culture supernatants (B–H). Data are means and S.E.M. from duplicate cultures in one of three independent experiments. xp<0.05, xxxp<0.001, trichomonad supernatant different from medium (med.); +p<0.05, +++p<0.001, metronidazole different from ‘no drug’; *p<0.05, p<0.001***, bafilomycin A1 plus metronidazole different from metronidazole alone; dnTRIF different from plasmid control (two-way ANOVA, Bonferroni).
Figure 5.
The cell-free culture phase of metronidazole-treated TVV-positive trichomonads enhances TLR3-depedent IFNβ production.
Endocervical were exposed to cell-free supernatants from trichomonads treated with 100 µM metronidazole for 24 h in the presence or absence of bafilomycin A1. Data are means and S.E.M. from duplicate cultures in one of three independent experiments. xxxp<0.001, ‘no drug’ trichomonad supernatant different from medium (med.); +++p<0.001, metronidazole different from ‘no drug’; p<0.001***, bafilomycin A1 plus metronidazole different from metronidazole alone; (two-way ANOVA, Bonferroni).
Figure 6.
Metronidazole treatment of infection by TVV-positive trichomonads enhances TLR3/TRIF-depedent proinflammatory responses.
(A–H) Endocervical cells expressing dominant negative (dn)TRIF or control mcs plasmid were exposed to TVV-positive (OC6) or TVV-negative (B7RC2) trichomonads in the presence or absence of 100 µM metronidazole for 24 h followed by multiplex measurement of levels of all soluble mediators except IFNβ which was measured in a separate assay of the same cell culture supernatants. Data are means and S.E.M. from duplicate cultures in one of three experiments. Fold change is over medium control baseline in the absence of both drug and trichomonas. The difference between metronidazole-treated and no drug-treated OC6 is denoted by numeric p values (p<0.001); xxp<0.01and xxxp<0.001, OC6 infection different from respectful medium control; *p<0.05 and ***p<0.001, dnTRIF different from plasmid (mcs) control (two-way ANOVA, Bonferroni).
Figure 7.
Purified Trichomonasvirus (TVV) virions provoke TLR3- and TRIF-dependent inflammatory responses.
(A) Transmission electron micrograph of TVV1 virions isolated from UH9 trichomonads (bar = 100 nm). (C) IL-8 and (D) IFNβ levels in supernatants from endocervical epithelial cells transfected with mcs or dnTRF and exposed to B7RC2, UH9, TVV1 and poly(I:C) for 24 h with MTT viability assay (B) performed on the same cells at the time of supernatant collection. (E) NF-κB activation and (F) IL-8 expressed by endocervical epithelial cells in response to trichomonads (B7RC2 and UH9), purified virions (TVV1 from UH9), poly(I:C) or MALP-2 in the presence or absence of TLR3 inhibitor BFA. (G) NF-κB activation, (H) IL-8, (I) RANTES and (J) IFNβ production in response to virions (TVV1 from UH9), virion extraction buffer control at equivalent concentration, poly(I:C) and MALP-12 in the presence or absence of 100 µM metronidazole. Bars represent means and S.E.M. of duplicate (b,c,d) or triplicate (e,f-h) cultures in two of four independent experiments. +++p<0.01 and +++p<0.001, stimulated/infected different from medium control; *<0.05, ***p<0.001 bafilomycin A1 different from solvent (DMSO) control or dnTRIF different from plasmid control (mcs) (two-way ANOVA, Bonferroni).
Figure 8.
Effect of ShortCut RNAse III on TVV dsRNA signaling.
(A) A dose of 2 U/ml efficiently digested purified TVV1 dsRNA: 1 = No Rnase control, 2 = Company buffer provided by manufacturer with the RNase kit, 3 = KSFM culture medium, 4 = modified Diamond medium. (B) Comparison of enzymatic effects on intact TVV1 (lanes 1–4) versus pre-heated TVV1 (lanes 5–8): 1, 5 = no RNase, 2, 6 = 200 U/mL, 3,7 = 20 U/mL, 4,8 = 2 U/mL of RNase III, all efficiently digesting the dsRNA but only if released from pre-heated virions. (C) IFNβ measurement in endocervical epithelial cells exposed to medium (med.), supernatants (sup.) from B7RC2 and UH9 TV isolates treated with metronidazole or DMSO control, intact virions (UH9 TVV) and poly(I:C). Bars represent means and S.E.M. of biological triplicates. ***p<0.001, RNase different from medium control (two-way ANOVA, Bonferroni).
Figure 9.
IRF3 phosphorylation in response to T. vaginalis infection.
(A) 1 h stimulation of endocervical epithelial cells: 1 = medium control; 2 = UH9 virions; 3 = B7RC2 live TV, 4 = UR1 live TV, 5 = UH9 live TV; 6 = poly(I:C). (B) 3 h stimulation of endocervical epithelial cells: 1 = medium control, 2 = poly(I:C), 3 = live UR1 TV, 4 = UH9 live TV. (C) Stimulation of vaginal epithelial cells for 1 h (lane 1) and 5 h (lanes 2–6): 1 = poly(I:C) control, 2 = −medium control, 3 = B7RC2 live TV, 4 = B7RC2 24 h supernatant, 5 = poly(I:C), 6 = UH9 live TV, 7 = UH9 24 h supernatant. (D) Endocervical cells were infected with B7RC2, UR1 and UH9 for 24 h, supernatants from infected cells and medium control were then transferred to fresh endocervical epithelial cells for another 5 h: 1 = medium control, 2 = epithelial cell conditioned medium, 3 = epithelial supernatants post B7RC2 infection; 4 = epithelial supernatants post UR1 infection; 5 = epithelial supernatants post UH9 infection. The protein ladders indicate from top to bottom 130.743 kD, 87.91 kD and 42.466 kD. The upper band in each panel represents IRF3 and the lower band represents β-Actin.
Figure 10.
Model of T. vaginalis virulence and Trichomonasvirus (TVV) interaction with the human host.
The protozoan parasite adherent to the human epithelial cells signals via its major surface lipophosphoglycan (LPG) [22] and one or more still unknown host receptors triggering NF-κB and other previously identified proinflammatory signal transduction pathways (ERK1/2, MEK1/2, AP-1, p38 and JNK) [23]. Protozoan proteins and nucleic acids may also signal to the human host although the latter has not been shown. This study demonstrates for the first time that genomic dsRNA and purified virions from TVV-inhabited trichomonads trigger NF-κB activation, proinflammatory mediators and interferon Type 1 via endosomal TLR3/TRIF-dependent pathways upstream from Interferon regulatory factor (IRF) 3. We also demonstrate that anti-protozoan antibiotic treatment (metronidazole) exaggerates the proinflammatory responses e.g. NF-κB, IL-8, and prototype virus stress inducible genes e.g. IFNβ, RANTES, IL-6, MIP-3α and ICAM-1. The mode of TVV entry or TVV dsRNA transfer to the human host and the role of cytosolic host receptors and kinases (e.g. Nod-like receptors, NLR, RIG-I-like receptors, RLR, and interferon-induced dsRNA-dependent protein kinase, PKR) remain to be elucidated.