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

Compound heterozygosity for lossâ ofâ function GARS variants results in a multisystem developmental syndrome that includes severe growth retardation

Aminoacylâ tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycylâ tRNA synthetase (GARS) is a bifunctional ARS that charges tRNAGly in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcotâ Marieâ Tooth disease but have not been convincingly implicated in recessive phenotypes. Here, we describe a patient from the NIH Undiagnosed Diseases Program with a multisystem, developmental phenotype. Wholeâ exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a lossâ ofâ function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARSâ related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138288/1/humu23287-sup-0001-text.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138288/2/humu23287.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138288/3/humu23287_am.pd

Ribavirin improves the IFN-γ response of natural killer cells to IFN-based therapy of hepatitis C virus infection

Ribavirin (RBV) is an important component of interferon (IFN)-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBV's mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by signal transducer and activator of transcription (STAT)1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56dim NK cells with cytotoxic effector functions and the frequency of CD56bright NK cells with the capacity to produce IFN-γ decreased (P=0.049 and P=0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P<0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-α. This was associated with an increase in IFN-γ production but not cytotoxicity of NK cells during subsequent IFN-α-based therapy. The frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders. Conclusion: RBV enhances the pSTAT4 and IFN-γ response of NK cells to IFN-α-stimulation

<i>Trichomonasvirus</i> (TVV)-positive protozoa upregulate TLR3 and trigger endosomal acidification-dependent inflammatory responses.

<p>(<b>A, B</b>) TLR mRNA levels were measured by a multiplex nuclease protection assay in the endocervical epithelial cells (<b>A</b>) and in TLR-Null or TLR2/6+ or TLR4+ HEK293 cells (<b>B</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). (<b>C</b>) 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). (<b>D</b>) 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.</p

Metronidazole treatment of infection by TVV-positive trichomonads enhances 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 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 <i>p</i> 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 (<i>mcs</i>) control (two-way ANOVA, Bonferroni).</p

Model of <i>T. vaginalis</i> virulence and <i>Trichomonasvirus</i> (TVV) interaction with the human host.

<p>The protozoan parasite adherent to the human epithelial cells signals via its major surface lipophosphoglycan (LPG) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048418#pone.0048418-Fichorova2" target="_blank">[22]</a> 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048418#pone.0048418-Singh1" target="_blank">[23]</a>. 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.</p

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

Purified <i>Trichomonasvirus</i> (TVV) virions provoke TLR3- and TRIF-dependent inflammatory responses.

<p>(<b>A</b>) Transmission electron micrograph of TVV1 virions isolated from UH9 trichomonads (bar = 100 nm). (<b>C</b>) IL-8 and (<b>D</b>) IFNβ levels in supernatants from endocervical epithelial cells transfected with <i>mcs</i> or dnTRF and exposed to B7RC2, UH9, TVV1 and poly(I:C) for 24 h with MTT viability assay (<b>B</b>) performed on the same cells at the time of supernatant collection. (<b>E</b>) NF-κB activation and (<b>F</b>) 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. (<b>G</b>) NF-κB activation, (<b>H</b>) IL-8, (<b>I</b>) RANTES and (<b>J</b>) 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 (<i>mcs</i>) (two-way ANOVA, Bonferroni).</p

<i>Trichomonasvirus</i> positive <i>T. vaginalis</i> isolates (UR1, UH9) induce Type-1 Interferon and elevated proinflammatory responses as compared to virus-free (B7RC2) protozoa and purified lipophosphoglycan (UR1-LPG).

<p>Endocervical (<b>A–D</b>), ectocervical (<b>F–I</b>) and vaginal (<b>E, G</b>) epithelial cells were exposed to trichomonad isolates, LPG or poly(I:C) for 24 h. Ectocervical immortalized cell monolayers (<b>F,G</b>) and the apical surface of primary polarized tissues grown on dual-compartment filters (<b>H,I</b>) 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 (<b>A,C, F</b>)<b>,</b> triplicate (<b>H,I</b>) or duplicate cultures (<b>D,E,G</b>) 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).</p

Effect of ShortCut RNAse III on TVV dsRNA signaling.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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).</p