Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae

The Villain Team-Up or how Trichomonas vaginalis and bacterial vaginosis alter innate immunity in concert

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

Trichomonas vaginalis Lipophosphoglycan Triggers a Selective Upregulation of Cytokines by Human Female Reproductive Tract Epithelial Cells

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 3α, 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-1β release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88

M1 Macrophage Polarization Is Dependent on TRPC1-Mediated Calcium Entry

Summary: Macrophage plasticity is essential for innate immunity, but in-depth signaling mechanism(s) regulating their functional phenotypes are ill-defined. Here we report that interferon (IFN) γ priming of naive macrophages induces store-mediated Ca2+ entry and inhibition of Ca2+ entry impairs polarization to M1 inflammatory phenotype. In vitro and in vivo functional analyses revealed ORAI1 to be a primary contributor to basal Ca2+ influx in macrophages, whereas IFNγ-induced Ca2+ influx was mediated by TRPC1. Deficiency of TRPC1 displayed abrogated IFNγ-induced M1 inflammatory mediators in macrophages. In a preclinical model of peritonitis by Klebsiella pneumoniae infection, macrophages showed increased Ca2+ influx, which was TRPC1 dependent. Macrophages from infected TRPC1−/− mice showed inhibited expression of M1-associated signature molecules. Furthermore, in human patients with systemic inflammatory response syndrome, the level of TRPC1 expression in circulating macrophages directly correlated with M1 inflammatory mediators. Overall, TRPC1-mediated Ca2+ influx is essential for the induction/shaping of macrophage polarization to M1 inflammatory phenotype. : Biological Sciences; Immune Response; Immunology Subject Areas: Biological Sciences, Immune Response, Immunolog

The cell-free culture phase of metronidazole treated TVV-positive trichomonads enhanced TLR3/TRIF-depedent proinflammatory responses.

<p>(<b>A–H</b>) Endocervical cells expressing dominant negative (dn)TRIF or control <i>mcs</i> 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 (<b>A</b>) and cytokine measurement in the cell culture supernatants (<b>B–H</b>). 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).</p

TLR-3-dependent proinflammatory activation is a common trait for <i>Trichomonasvirus(TVV)</i>-infected <i>T. vaginalis</i> isolates.

<p>(<b>A–C</b>) 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. (<b>D</b>) IL-8 was measured in supernatants from endocervical cells expressing dominant negative (dn)TRIF or control (<i>mcs</i>) 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 (<i>mcs</i>) control, respectively; ⁺p<0.05 and ⁺⁺⁺p<0.001, infection/TLR ligand/LPG different from medium control (two-way ANOVA, Bonferroni).</p