BMDCs stimulated with EphrinB2 upregulate <i>EphB2</i> mRNA.

<p>Naïve BMDCs were plated onto plate-bound Ephrin-B1-Fc fusion protein (A, B, C) or plate bound Ephrin-B2-Fc fusion protein (D, E, F). The transcription of <i>EphB2</i> with and without the addition of recombinant interferon-γ (IFN-γ) was monitored by qPCR over 24 hours of culture (A, D). The efficiency of the Eph receptor ligation was monitored by assessing tyrosine phosphorylation on DCs by western blot (B, E) and the phosphorylation quantified using densitometry (C, F). All graphs represent the median value of pooled data from 3 independent DC preparations ±SD.</p

Stimulation of BMDCs from EphB2+/+ and EphB2-/- mice with TLR receptor agonists results in similar levels of secreted cytokine.

<p>BMDCs from EphB2+/+ and EphB2-/- mice were incubated with TLR ligands LPS and CpG1668 with and without the addition of recombinant interferon (IFN)-γ for 20 hours. Interleukin (IL)-12p40 (A), IL-12p70 (B), IL-10 (C) and tumor necrosis factor (TNF)-α (D) were measured in the culture supernatant using Luminex. The graphs are representative of 3 individual experiments and bars represent the mean ±SD of 2 replicate wells plated.</p

EphB2 expression on BMDCs can be modulated by ligation with Toll-like receptor ligands.

<p>(A) BMDC were incubated with a Toll Like receptor (TLR)-4 agonist (lipopolysaccharide (LPS) 1μg/ml) and (B) a TLR-9 agonist (CpG1668 1μM) with or without recombinant mouse interferon (IFN)-γ at a concentration of 20ng/ml. <i>EphB2</i> mRNA was quantified by qPCR at different time points post-stimulation. The modulation of transcription for known co-stimulatory molecules CD80 and CD86 as well as the cytokine interleukin (IL)-12p40 in response to TLR stimulation in the same experiments are shown for comparison. (C) The change in EphB2 protein expression at 22 hours post-incubation with LPS and IFN-γ is shown and the mean fluorescence quantified for different conditions. All graphs represent the median value of pooled data across 3 independent BMDC preparations ±SD and data analyzed using One-way ANOVA– Kruskal Wallis test and Dunn’s multiple comparisons post-test. *P<0.05. MFI = Mean Fluorescence Intensity.</p

EphB2 co-localizes with MHC-II on BMDCs.

<p>Two examples are shown: (A) Representative BMDCs stimulated with 1μg/ml lipopolysaccharide (LPS) and 20ng/ml recombinant mouse interferon (IFN)-γ for 22 hours; (B) shows unstimulated cells. EphB2 is shown in red (Northernlights557), MHC-II is shown in green (FITC) and DAPI is shown to demarcate the nuclei of the cells. Magnification 100x; Scale bar, 20μm.</p

Splenic CD11c^hi and bone marrow-derived DCs express EphB2.

<p>(A) Eph receptor expression on the surface of naïve splenic CD11c^hi DCs was detected by incubation with Ephrin-B2-Fc chimeric protein and binding was detected using a biotinylated anti-Fc antibody and streptavidin-APC (SA-APC) using flow cytometry. (B) The expression of EphB2 on BMDCs incubated with anti-EphB2 antibody or isotype control (IgG2a) detected by immunofluorescence. Magnification 40x; Scale bar, 10μm.</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 minimally activates human DCs.

<p><b>(A)</b> moDCs were left uninfected (“Mock”) or infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 (n = 6–8 donors). Cells were collected at 48hpi and labeled for ZIKV E protein and indicated DC activation markers. Cells were categorized as being viral E protein- or viral E protein+ and activation marker surface expression quantitated by flow cytometry. Values are represented as median fluorescence intensity (MFI) for each individual donor with uninfected and ZIKV infected samples from the same donor connected with a line. Statistical significance (p< 0.05) was determined using a Friedman test with comparisons made to donor-paired, uninfected cells. <b>(B)</b> moDCs infected with PR-2015 at MOI of 1 were stratified into “low” (n = 3 donors) and “high” (n = 5 donors) infection on the basis of viral E protein staining. MFIs are shown as the mean +/- SD. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006164#ppat.1006164.s003" target="_blank">S3 Fig</a>.</p

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

Innate immune signaling restricts ZIKV viral replication within human DCs.

<p><b>(A)</b> moDCs were infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 (n = 4 donors). After viral attachment and entry at 1hpi, cells were treated with RIG-I agonist (10ng/1e5 cells), human IFNβ (100 IU/mL), or left untreated. <b>(B)</b> Supernatants were collected at 48hpi and assessed for infectious virus release by FFA. Values for each individual donor are shown with the mean +/- SD. Statistical significance (p< 0.05) was determined using a Friedman test with comparisons made to donor-paired, untreated, ZIKV-infected cells. The assay limit of detection is indicated with a dashed line.</p