Inhibition of PGE2 secretion by <i>Viscum album</i> in A549 cells.

<p>Cells were treated with IL-1β (10 ng/ml) and increasing concentrations of VA Qu Spez for 18 hours. PGE2 was analyzed in culture supernatants by EIA. Results are mean±SEM from 4 independent experiments (*<i>p</i><0.05 versus control cells, **<i>p</i><0.05 versus cells treated with IL-1β, analyzed by paired Student-t-test).</p

Effect of <i>Viscum album</i> on COX-1 expression in A549 cells.

<p><b>A and B.</b> A549 cells were treated with increasing concentrations of VA Qu Spez in presence of IL-1β (10 ng/ml) for 18 hours. Protein expression of COX-1 was analyzed by western blot. A representative western-blot depicting the effect of VA on COX-1 expression under IL-1β-stimulated culture conditions (A). Relative expression (mean ± SEM) of COX-1 protein from three independent experiments as quantified by densitometry (ratio of density of COX-1: that of β-actin). ns, non-significant (B). <b>C.</b> Cells were cultured either in medium alone or treated with increasing concentrations of VA Qu Spez without IL-1β for 18 hours. 20 µg of total cellular protein was separated by 10% SDS-PAGE followed by western blotting to analyze the expression of COX-1. Results are representative of two experiments.</p

Host immune response to pathogens and predisposition to infections due to autoimmunity.

<p>Antigens from invading pathogens are recognized and presented by innate immune cells (A) such as macrophages and dendritic cells to CD4+ and CD8+ T cells (CTL) (B). CD8+ T cells recognize endogenous antigens presented by MHC class I molecules and exert cytotoxic functions upon activation. CD4+ T cells recognize antigens presented in the context of MHC class II molecules, and under the influence of innate cells and cytokine milieu, CD4+ T cells can be polarized into different subsets such as Th1, Th2, Th17, and regulatory T cells (Tregs) that secrete distinct cytokines. CD4+ T cells provide help to B cells to produce antigen-specific antibodies (C). However, due to autoimmunity, neutralizing autoantibodies can be produced against any of these key components of the immune system critical for mounting anti-microbial responses and might either predispose to an increased risk of bacterial, viral, and opportunistic fungal infections or exacerbate the ongoing infectious diseases. Indeed, in patients with infections, the occurrence of neutralizing autoantibodies against several key cytokines such as IFN-γ, IL-6, GM-CSF, IL-17, and IL-22 (highlighted in red boxes) that interfere with the host immune response to pathogens have been demonstrated. In addition, autoantibodies are also reported against type I IFNs and IL-12 that might play role in predisposition to infections (highlighted in blue boxes). CTLA-4, cytotoxic T lymphocyte antigen-4; CTL, cytotoxic T lymphocyte; FasL, Fas ligand; GM-CSF, granulocyte/macrophage–colony stimulating factor.</p

Inhibition of cytokine-induced COX-2 protein expression by <i>Viscum album</i> in A549 cells.

<p>A549 cells were either cultured in medium alone or treated with IL-1β (10 ng/ml) with or without increasing concentration of VA Qu Spez for 18 hours. Using 20 µg of total cellular protein, expression of COX-2 was analyzed by western blotting. <b>A.</b> A representative western-blot depicting the effect of VA on expression of IL-1β-induced COX-2. <b>B.</b> Relative expression (mean ± SEM) of COX-2 protein from four independent experiments as quantified by densitometry (ratio of density of COX-2: that of β-actin). *<i>P</i><0.05 versus control cells and ** <i>p</i><0.05 versus IL-1β-stimulated cells as analyzed by paired Student-t-test. <b>C.</b> Kinetics of COX-2 protein expression upon treatment of A549 cells with various doses of VA. The cells were either cultured in medium alone or stimulated with IL-1β with our without VA for 12, 18, 24 and 36 hours. Expression of COX-2 protein was analyzed by western blotting and relative expression of COX-2 protein was quantified by densitometry (ratio of density of COX-2: that of β-actin). Results are representative of two experiments.</p

Selective inhibition of COX-2 expression by <i>Viscum album</i>.

<p>Quantitative comparison between expression of COX-1 and COX-2 proteins as revealed by western blot. Protein expression was quantified by densitometry and expressed as percentage protein expression normalized to that in cells stimulated with IL-1β. Results (mean ± SEM) obtained from 3 independent experiments are presented. * <i>P</i><0.05 versus control cells for COX-2 and ** <i>p</i><0.05 versus IL-1β-stimulated cells for COX-2 as analyzed by paired Student-t-test. The values for COX-1 are non-significant.</p

Effect of VA Q Spez on IL-1β-induced COX-2 transcription.

<p>Total cellular RNA was isolated from A549 cells that were cultured either in medium alone or stimulated with IL-1β (10 ng/ml) with or without various concentrations of VA for 18 hours. mRNA expression of COX-2 was analyzed by semi quantitative RT-PCR using COX-2-specific primers. Amplification of a house keeping gene GAPDH is used as an internal control. (A) PCR products were separated on 1% agarose gel and a representative gel picture is shown. (B) Relative expression (mean ± SEM) of COX-2 mRNA from six independent experiments as quantified by densitometry (ratio of density of COX-2: that of GAPDH). * P<0.05 analyzed by paired Student-t-test, ns refers to non-significant, compared to IL-1β stimulated cells.</p

Oligonucleotide primers used in the study.

<p>Oligonucleotide primers used in the study.</p

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells

<div><p>Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target.</p></div

ZIKV infection induces an antiviral state within human DCs.

<p>moDCs were infected with ZIKV PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 (n = 6–8 donors). Cells were collected at indicated hours post-infection and antiviral gene expression was determined by qRT-PCR. Gene expression was normalized to <i>GAPDH</i> transcript levels in each respective sample and represented as the averaged log₂ normalized fold increase above donor and time-point matched uninfected cells. The averaged log₁₀ normalized levels of infectious virus (FFU/mL) at each time point is depicted beneath the gene expression heat map. <b>(A)</b> RLR gene expression. <b>(B)</b> Antiviral effector gene expression. <b>(C)</b> moDCs were left untreated (“Mock”), treated with RIG-I agonist (10ng/1e5 cells), or infected with ZIKV PR-2015 (MOIs of 1 and 10) or MR-1947 (MOI 1). After 18hrs of agonist treatment or at 48hpi with ZIKV, whole-cell lysates were collected for western blot analysis of host antiviral effector protein expression. Western blots are shown for a single donor and are representative of data obtained from two donors. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006164#ppat.1006164.s005" target="_blank">S5 Fig</a>.</p

Differential infection of human DCs by evolutionarily distinct ZIKV strains.

<p>moDCs were infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 and assessed for viral replication at the indicated hours post-infection. <b>(A)</b> Infectious virus release into the supernatant was determined by FFA. Shown as the mean +/- SEM from 6–9 donors. <b>(B)</b> Infectious virus release for 6 of the individual donors summarized in panel A. <b>(C)</b> Percent infected cells assessed by ZIKV E protein staining and flow cytometry. Shown as the mean +/- SEM from 6–9 donors. <b>(D)</b> Percent infected cells in 6 of the individual donors summarized in panel C. <b>(E)</b> Cell viability of infected moDCs assessed by Ghost Red 780 (Tonbo) viability staining and flow cytometry. Shown as the mean +/- SEM from 6–9 donors. Statistical significance (p< 0.05) was determined using a two-way ANOVA with comparisons made to mock-infected cells. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006164#ppat.1006164.s007" target="_blank">S1 Table</a>.</p