H5N1 infection induced marked CD34⁺ HSC and MSC apoptosis.

<p>(<b>A</b>) CD34⁺ HSCs were infected with H5N1 virus at an MOI of 10, whereas (<b>B</b>) MSCs were infected with either avian or human influenza virus at the same MOI as CD34⁺ HSCs. Parallel samples were treated with DnaseI which breaks double stranded DNA as a positive control. After 18 h, cells were fixed, permeabilized and multiple stained using Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) staining (red), FITC-labeled anti-NP (green) and DAPI (blue). Arrows in B. indicate cells that simultaneously expressed intracellular viral antigen and apoptotic signal. The samples were examined on a confocal microscopy and photographed at a ×200 magnification.</p

Infection and Replication of CD34⁺ HSCs by H5N1 virus.

<p>(<b>A</b>) CD34⁺ HSCs from CB were infected with both live and heat-killed avian (H5N1) and human influenza viruses (H1N1 and H3N2) at an MOI of 10 for 24 h. MDCK infected with H5N1 at MOI 1 for 12 hours was used as a positive control. Cells were fixed and permeabilized for intracellular staining of Influenza nucleoprotein (NP; green). CD34⁺ cells were counterstained with anti-human CD34-PE (red), and then examined by confocal microscopy and photographed at a ×200 magnification. The percentage of NP-positive (NP+) cells was obtained by counting the number of NP+ cells per total cells in five random fields. (<b>B</b>) CD34⁺ HSCs from both BM and CB sources were exposed to either a high (MOI 10) or low (MOI 1) dose of H5N1 virus. The expression of viral antigens was analyzed by flow cytometry. (<b>C</b>) Supernatants of infected CD34⁺ HSCs at MOI 1 and 10 were collected at different time points after infection and then titrated on MDCK cells to measure virus production by plaque assay. The results were obtained from three different experiments and are presented as means plus standard errors. <i>P</i> value<0.05 indicate the significance of H5N1 infection compared with human influenza viruses</p

Avian influenza could infect and replicate in MSCs.

<p>(<b>A</b>) The ability of avian influenza H5N1 to infect MSCs was compared to human influenza viruses. Cells were infected with H5N1; live or heat-inactivated, H1N1 and H3N2 at an MOI of 10. After 24 h, cells were stained and examined by confocal microscopy for the expression of viral antigens (green) with Evan's blue (red) as a counterstain. The percentages of infected MSCs at high and low MOIs of (<b>B</b>) H5N1 virus and (<b>C</b>) human influenza viruses were determined by flow cytometry. (<b>D</b>) Percent infection of MSCs infected mock, H1N1, H3N2, H5N1, and heat-killed H5N1 at MOI 10 for 24 hours. MDCK infected with H5N1 at MOI 1 for 12 hours was used as a positive control. (<b>E</b>) Real-time PCR of H5N1 M gene in MSCs after infection with H5N1 at various doses for 12 h. The amount of the virus RNA was standardized by beta actin. (<b>F</b>) Production of H5N1 virus from infected MSCs at various time points was measured by plaque assay. The results represent the means and SD of three independent experiments. <i>P</i><0.05 indicates statistically significant differences between H5N1 and human influenza viruses infections.</p

Production of IL-6, MCP-1, and MIP-1β from H5N1-infected cocultures.

<p>(<b>A</b>) IL-6, (<b>B</b>) MCP-1, and (<b>C</b>) MIP-1β levels from supernatants of infected CD14⁺ monocytes and MSCs monoculture and 1∶5 ratio of MSCs/CD14⁺ monocytes cocultures at MOI 0.04 were measured by using Bio-plex Cytokine assay. Data was analyzed via Bio-plex 5.0 Software. The two different cell types used in the experiment were derived from different donors. The results represent the means and SD of two independent experiments. Single asterisk indicates statistically significant differences between mock and infected cells with <i>P</i> values of <0.05 and double asterisks indicate statistically significant differences between cocultures and monoculture groups with <i>P</i> values of <0.05.</p

An influenza A virus agglutination test using antibody-like polymers

<p>Antibodies are commonly used in diagnostic routines to identify pathogens. The testing protocols are relatively simple, requiring a certain amount of a specific antibody to detect its corresponding pathogen. Antibody functionality can be mimicked by synthesizing molecularly imprinted polymers (MIPs), i.e. polymers that can selectively recognize a given template structure. Thus, MIPs are sometimes termed ‘plastic antibody (PA)’. In this study, we have synthesized new granular MIPs using influenza A virus templates by precipitation polymerization. The selective binding of influenza A to the MIP particles was assessed and subsequently contrasted with other viruses. The affinities of influenza A virus towards the MIP was estimated based on an agglutination test by measuring the amount of influenza subtypes absorbed onto the MIPs. The MIPs produced using the H1N1 template showed specific reactivity to H1N1 while those produced using H5N1 and H3N2 templates showed cross-reactivity.</p

P2Y₆ receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 & CXCL8 mRNA expression in respiratory epithelium

<div><p>One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-β, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (β-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y₆R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca²⁺ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y₆ purinergic receptors.</p></div

Inactivated-H5N1 increases phosphorylation of ERK1/2 and p38 MAPK.

<p>Immunoblot analysis of ERK1/2 <i>(A)</i> and p38 MAPK <i>(B)</i> protein expression in 16HBE14o- cells treated with inactivated-H5N1. Cells were untreated or treated with PD98059 (50 μM) or SB203580 (10 μM) as appropriate. One hour later, cells were treated for 30 min with vehicle, allantoic fluid or inactivated-H5N1 before being harvested for immunoblot analysis for the presence of ERK1/2, phospho-ERK1/2 (P-ERK1/2), p38 MAPK or phospho-p38 MAPK (P-p38). Upper panel, representative gels for immunoblot analysis of ERK1/2 and phosphorylated ERK1/2 (P-ERK1/2) <i>(A)</i> or p38 MAPK and phosphorylated p38 MAPK (P-p38) <i>(B)</i>. Lower panel, quantitative analysis of the amount of P-ERK1/2 (<i>A</i>) or P-p38 MAPK <i>(B)</i>, expressed as fold change, using ImageJ software (NIH). Values are means ± SEM from at least 3 sets of experiments. * and *** indicates <i>p</i> < 0.05 and <i>p</i> < 0.001, respectively (one-way ANOVA with Student-Newman-Keuls post-hoc test).</p

The cellular signaling mechanism by which inactivated-H5N1 increases mRNA for IL-6 and CXCL8 involves intracellular Ca²⁺, PLC, PKC, ERK1/2 and p38 MAPK.

<p>16HBE14o- cells were incubated with BAPTA-AM (50 μM), U73122 (10 μM), BIM (1 μM), Gö6976 (10 μM) <b><i>(</i></b><i>A and B)</i>, or with SN-50 (10 μM), PD98059 (50 μM), SB203580 (10 μM) or SP600125 (10 μM) <i>(C and D)</i>, for 1 hr before being exposed to inactivated-H5N1. Three hours later, mRNA expression of IL-6 <i>(A and C)</i> or that of CXCL8 <i>(B and D)</i> was analyzed. All data were normalized with corresponding control groups treated with inactivated-H5N1 (open bars) and reported as a percentage. <i>(E)</i> Cells were treated with SB203580 (10 μM) for 1 hr before incubation with inactivated-H5N1 (20 μg/ml hemagglutinin). Thirty-six hours later, the cell-free culture medium was harvested and analyzed for the presence of IL-6 and CXCL8 proteins as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176974#pone.0176974.g001" target="_blank">Fig 1</a>. Data are presented as a percentage of control (treated with inactivated-H5N1, open bar). Values are means μ SEM from at least 3 sets of experiments. *, ** and *** indicates <i>p</i> < 0.05, <i>p</i> < 0.01 and <i>p</i> < 0.001 compared with control, respectively (unpaired Student’s <i>t</i>-test).</p

Effect of inactivated-H5N1 is mediated via P2Y₆ purinoceptors.

<p>The effect of inactivated-H5N1 on IL-6 <i>(A and C)</i> and CXCL8 <i>(B and D)</i> mRNA expression. 16HBE14o- cells were incubated with vehicle (dimethyl sulfoxide (DMSO) or H₂O as appropriate) as control, 1 U/ml sialidase, 2 μM cytochalasin D (cytoD) <i>(A and B)</i> or with apyrase (2 U/ml), suramin (100 μM), MRS2578 (10 μM) or MRS2179 (20 μM) <i>(C and D)</i>, for 1 hr before exposure to inactivated-H5N1. Three hours after treatment, cells were harvested for qRT-PCR. Data were normalized with corresponding control groups from the same batch of cells treated with inactivated-H5N1 (open bars) and reported as a percentage. <i>(A and B)</i> cells were treated (+) or untreated (-) with inactivated-H5N1 as indicated. <i>(E)</i> 16HBE14o- cells were transfected with siRNA directed against P2Y₆R or a scrambled-siRNA (100 pmol). Forty-eight hours after transfection, cells were exposed to inactivated-H5N1 for 3 hr before being harvested and the mRNA expression of P2Y₆R analyzed. <i>(F)</i> Cells in <i>(E)</i> were incubated with inactivated-H5N1 for 3 hr before being harvested and analyzed for mRNA expression of IL-6 and CXCL8. Data were normalized with a group of cells treated with allantoic fluid, as described, and reported as a percentage of normalized scrambled-siRNA treated group. Values are means ± SEM from at least 3 sets of experiments. *, ** and *** indicate <i>p</i> < 0.05, <i>p</i> < 0.01 and <i>p</i> < 0.001, respectively (unpaired Student’s <i>t</i>-test).</p

Inactivated-H5N1 increases mRNA of IL-6 & CXCL8.

<p><b><i>(</i></b><i>A)</i> Fold changes (log scale) in mRNA level of TNF-α, IL-6, CXCL8, CCL5 or CXCL10 in 16HBE14o- cells treated with inactivated-H5N1 (20 μg/ml hemagglutinin) or untreated cells (control) for 3 hr, 6 hr or 12 hr. <i>(B)</i> Fold changes in mRNA expression level of IL-6 and <i>(C)</i> CXCL8 in untreated cells (control), allantoic fluid, inactivated-H5N1 or inactivated-H1N1. <i>(D)</i> Fold changes in protein concentrations of IL-6 and CXCL8 in cell-free culture medium in untreated cells (control) or treated for 36 hr with allantoic fluid or inactivated-H5N1 were analyzed by Bio-plex assay. Data were normalized against control untreated groups. Values are means ± SEM from at least 3 sets of experiments. *, ** and *** indicates <i>p</i> < 0.05, <i>p <</i> 0.01 and <i>p</i> < 0.001, respectively (one-way ANOVA with Student-Newman-Keuls post-hoc test), compared to untreated control <i>(A)</i> or allantoic fluid <i>(B</i>, <i>C</i>, and <i>D)</i>.</p