Cytokines influence the level of IFN-α production by enriched pDC in a concentration dependent manner.

<p>IFN-α responses of CD172a⁺-sorted cells stimulated with CpG (10 µg/ml; grey line) or FMDV (MOI of 5 TCID₅₀/cell; black line) for 24 h in presence of cytokines at indicated concentration. After stimulation, supernatants were collected and IFN-α was measured by ELISA. The marker represents the mean value of 2 independent experiments with each condition performed in triplicate cultures. The error bar represents the standard deviation.</p

Boxplots showing the statistical analysis for the influence of cytokines on IFN-α production induced by CpG or FMDV in enriched pDC.

<p>Cells were cultured in presence of GM-CSF (100 U/ml), Flt3-L (100 U/ml), IFN-β (100 U/ml), IFN-γ (10 ng/ml), IL-4 (100 U/ml) or IL-10 (10 ng/ml), and either directly stimulated for 24 h (grey bars, A, C) or pre-incubated for 16 h before stimulation with CpG or FMDV for another 24 h (white bars, B, D). IFN-α in the supernatants was measured by ELISA. Values are shown as box plots representing at least 3 independent experiments with each condition performed in triplicate cultures. The black line represents the median value and the red line the mean value. The error bars represent the standard deviation. The asterisk indicates statistical significance between non-treated and treated cells (Mann-Withney Rank Sum test, P<0.05).</p

CpG but not FMDV deplete pDC population by inducing apoptosis.

<p>CD172a⁺ sorted cells were untreated (A) or stimulated with 10 µg/ml of CpG for 16 h or FMDV for 24 h (B, C, D). A. Three-color FCM of CD4, CD172a and AnnexinV, with a CD4/CD172a dot plot defining the pDC gate (CD4^highCD172a^low) and the monocyte/cDC gate (CD4⁻CD172a⁺) after culture in medium. On the left side plot, putative live cells and live and dead cells were defined according to the FSC/SSC profile, excluding lymphocytes and cell debris. On the right side plots, the AnnexinV expression of the gated pDC (blue) and monocytes/cDC (red) with percentages are shown. B. Apoptosis of pDC and monocytes/cDC after stimulation with CpG, Mock antigen and FMDV. On the left side plot, pDC and monocytes/cDC with percentages were gated on live/dead cells as described in A. On the right side plot, the AnnexinV expression of the gated cell populations with percentages is shown after CpG or FMDV stimulation. C, D. Absolute number of pDC and monocytes/cDC on gated live cells after culture of enriched pDC with CpG, FMDV or only medium. Each marker represents results from one individual experiment and the bar represents the mean value. The asterisk indicates statistical significance between treated and non-treated cells (t-test, P<0.05).</p

Synergistic effects of IFNs with other cytokines on IFN-α by enriched pDC.

<p>IFN-α responses of CD172a⁺-sorted cells stimulated with CpG or FMDV in presence of cytokine combined with type-I and II IFN are shown. The effects of IFN-β or IFN-γ in combination with GM-CSF, Flt3-L, IL-3, IL-4 or IL-10 using concentrations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060893#pone-0060893-g002" target="_blank">Figure 2</a> were tested. Cells were either directly stimulated for 24 h (grey bars, A, C) or pre-incubated for 16 h before stimulation with CpG or FMDV for another 24 h (white bars, B, D). After stimulation, supernatants were collected and IFN-α was measured by ELISA. Values are shown as box plots representing at least 3 independent experiments with each condition performed in triplicate cultures. The black line represents the median value and the red line the mean value. The error bars represent the standard deviation. The asterisk indicates statistical significance between non-treated and treated cells (Mann-Withney Rank Sum test, P<0.05).</p

Schematic overview of the plasmid constructs used for DNA vaccination.

<p>The pcDNA6-V5-His B plasmid backbone (Vector) carrying a cytomegalovirus immediate early promoter (P_CMV), a multiple cloning site (MCS) and the transcription termination and polyadenylation sequence (pA) was used for all protein expression constructs. The monocistronic expression plasmids pcDNA6-HA and pcDNA6-chMDA5(1-483) carry the HA gene of the H5N1 AIV Yamaguchi-7/04 and a cassette encoding the N-terminal 483 amino acids of the chicken MDA5, respectively. In the bicistronic expression plasmids pcDNA6-HA-IRES-eGFP and pcDNA6-HA-chMDA5(1-483), the IRES of the encephalomyocarditis virus was used to separate the HA gene from the eGFP and the chMDA5(1-483) gene, respectively. The plasmid pcDNA6-HA-IRES carries an empty MCS downstream of the IRES.</p

Effect of chMDA5(1-483) co-expression on HA-specific antibody responses elicited by HA DNA vaccination.

<p>Groups of 6 chickens were immunized with a plasmid DNA mixture containing equal amounts of the plasmids for HA expression and for chMDA5(1-483) expression. Doses of 25 µg or 2.5 µg of each plasmid DNA per animal were applied to two groups of 6 animals/dose, as indicated. The same two doses of plasmid DNA for HA expression alone mixed with an equal amount of empty expression plasmid were applied to two other groups of 6 chickens. Two control groups of 3 chickens each received 25 µg or 2.5 µg of empty expression plasmid each (Vector). Two immunizations were applied at an interval of 23 days. 20 days after the second vaccination (<i>i.e.</i> at 43 dpi), the chicken sera were tested for the presence of HA-specific antibodies. H5-specific antibodies were detected using a commercial competition ELISA. The reactivity of HA antibodies was calculated according the formula 100-[OD(Probe)/OD(Negative)x100] (a). Alternatively, the anti-HA IgY titers were determined in an indirect ELISA using immobilized recombinant HA protein and represented as reciprocal of the highest dilution that yielded an OD greater than 2.1 x of parallel preimmune serum samples (b). The HI titer was determined as the highest serum dilution resulting in complete inhibition of aggregation of chicken red blood cells caused by 8 HAU of Vac-1/04 (H5N1) virus (c). The neutralizing antibody titers in serum samples were determined with Vac-1/04 virus and plotted as reciprocal of the highest dilution resulting in 50% virus neutralization (d). The HA-specific IgA antibody content in the serum was measured by ELISA. Results are shown as absolute OD values from which the unspecific background values were subtracted (e). Each symbol represents an individual animal. The * indicates statistical significant differences calculated with the students t-test (p<0.05).</p

Quantification of viral RNA in tracheal swabs from animals vaccinated with 25 µg of DNA.

<p>Abbreviations: dpc, days post-challenge; -, PCR negative; RNA copies per 100 µl swab, (+) <10³,+<10⁴,++<10⁵,+++<10⁶,++++<10⁷,+++++<10⁸;</p>†<p>animal dead;</p>††<p>animal euthanized.</p

Expression of chMDA5(1-483) induces type I IFN.

<p>The effect of chMDA5(1-483) expression on the activation of the chIFN-β promoter (a) and on type I IFN secretion (b) was analyzed in the DF-1 fibroblast cell line (a) and in the HD-11 macrophage-like cell line (b), respectively. DF-1 cells were transfected with the reporter plasmids for measuring chIFN-β promoter activity and with the indicated expression constructs (a). The firefly and <i>Renilla</i> luciferase activities were determined 24 h later. The IFN-β promoter-induced firefly luciferase activity was normalized with the corresponding <i>Renilla</i> luciferase activity and expressed as fold induction compared to cells transfected with the empty expression plasmid (Vector). The bars represent the mean values of five independent transfections with the error bars showing the standard deviations. The graph is representative of two independent experiments. For type I IFN induction, HD-11 cells were transfected with the indicated plasmids (b). Eighteen hours later, the cell supernatants were assayed for type I IFN using the bioassay as described. The bars represent the mean values of six independent transfections with the error bars showing the standard deviations.</p

Effect of chMDA5(1-483) co-expressed with HA on clinical symptoms and survival after challenge with HPAIV H5N1.

<p>The chickens received two intramuscular immunizations with 25 µg (a and b) or 2.5 µg (c and d) of the indicated plasmid DNA at 23 days interval. After another 23 days (or at 46 dpi), the animals were challenged by intratracheal administration of the HPAIV Yamaguchi-7/04. The chickens were observed daily for clinical signs for a period of 9 days after the challenge infection. The daily clinical index was monitored as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049952#pone.0049952-Veits1" target="_blank">[58]</a> with minor modifications: healthy (0), reduced activity (0.25), slightly ill (0.5), ill (1), severely ill (2), severely ill and euthanized (2.5) or dead (3). The daily clinical index is represented as the mean value of all chickens per group (a and c). The number of surviving animals is plotted against the time (in days) after the challenge (b and d). Note that the two groups (25 µg and 2.5 µg dose) vaccinated with control vector plasmid (Vector) consisted of only three animals each.</p

Quantification of viral RNA in cloacal swabs from animals vaccinated with 25 µg of DNA.

<p>Abbreviations: dpc, days post-challenge; -, PCR negative; RNA copies per 100 µl swab, (+) <10³,+<10⁴,++<10⁵,+++<10⁶,++++<10⁷,+++++<10⁸;</p>†<p>animal dead;</p>††<p>animal euthanized.</p