Human influenza induces lung endothelial permeability.

<p>(A) Influenza induces an increase in permeability to dextran in a dose-dependent fashion. Cells were infected at the indicated multiplicity of infection (MOI) for 24 hours before permeability to dextran was analyzed. Results (mean, SD) are experiments and are normalized to control, *p<0.05 versus control. (B) A clinical H3N2 influenza isolate was used to infect lung microvascular endothelium for 24 hours. Permeability to dextran was measured as in A. Results (mean, SD) are from 3 experiments and are normalized to control, *p<0.05. (C) Similar to A, except the transendothelial electrical resistance (TEER) was measured before and after infection. Results (mean, SE) are from 3 experiments and are normalized to control, *p<0.05. (D) Influenza induces endothelial permeability even after infection from the basal aspect. Human lung microvascular endothelium seeded on transwells was infected from the basal aspect of the transwell (MOI 8) and the change in TEER was measured 24 hours later. Results (mean, SD) are from 2 experiments and are normalized to control, *p<0.05.</p

Human influenza replicates in primary lung microvascular endothelial cells.

<p>(A) Viral titer increases over time as shown by TCID₅₀ assay. This assay quantitates the ability of influenza to agglutinate red blood cells after viral replication (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047323#s2" target="_blank">Materials and Methods</a> section for further details). The initial influenza dose was 25 HAU/100 000 cells. The control group had no cells, received the same influenza dose, and was analyzed at 24 hours. Results are representative of 3 experiments. (B) qPCR showing the fold change in viral RNA for the influenza A M1 protein (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047323#s2" target="_blank">Materials and Methods</a> for primers) over time. Results are representative of 3 experiments. (C–D) Immunofluorescent images (C) and quantitation (D) showing the percentage of cells infected by influenza after 24 hours. Influenza was given at 40 HAU/100 000 cells. Nuclei are stained with DAPI and viral nucleoprotein is shown in green. Images are representative of 3 experiments; data are mean and standard error, *p<0.05 for flu vs. control (uninfected cells).</p

Replication-deficient virus induces lung endothelial permeability.

<p>(A) Replication-deficient influenza induces endothelial permeability to a lesser degree than live virus over time. Lung endothelium was infected with either live or replication-deficient (UV flu) influenza and the change in TEER was measured every 4 hours for 24 hours. Data for live flu (mean, SE) are from 4 experiments and for UV flu are from 5 experiments. All data are normalized to control, *p<0.05 for live vs UV flu. (B) Binding of influenza to endothelial cells is insufficient to produce leak. Lung endothelial cells were treated with indicated doses of hemagglutinin (HA) (Immune Technology Corp.) and the change in TEER was measured after 24 hours. Data (mean, SE) are from 3 experiments and are normalized to control.</p

Influenza-induced loss of claudin-5 is due to cleavage by matrix metalloproteases.

<p>(A) The reduction in claudin-5 levels is not due to decreased transcription. Levels of claudin-5 mRNA were assessed by quantitative PCR 24 hours after infection with replication-deficient influenza (MOI 4). Results are presented using the comparative C_T method using 18S rRNA as a control. Results are from 3 experiments, p>0.05 for control versus infected. (B) Claudin-5 does not co-localize with lysosomes. Cells were infected with UV-irradiated influenza (MOI 8) for 16, 20, and 24 hours and were co-immunostained for LAMP1 and claudin-5. Images were from the 20-hour timepoint, but are reflective of all timepoints. Asterisks indicate cell nucleus, while arrows indicate cell membrane. Images are representative of 3 experiments. (C) Claudin-5 does not interact with poly-ubiquitin. The right-most blot shows the efficacy of the poly-ubiquitin antibody (FK1) since inhibition of the proteasome by MG-132 (MG) increases the expression of poly-ubiquitinated proteins in whole cell lysates. Cells were incubated with UV-influenza (MOI 8) for 24 hours. Whole cell lysates were immunoprecipitated for claudin-5 and blotted for poly-ubiquitin. IgH denotes the heavy chain of the IP antibody. Blots are representative of 3 experiments. (D) Inhibition of MMPs by marimastat blocks influenza-induced loss of claudin-5. Whole cell lysates were probed for claudin-5. Cells were treated with marimastat and infected with UV-flu (MOI 8) for 24 hours. Blot and quantitation are representative of 3 experiments. (E-F) Over-expression of claudin-5 blocks influenza-induced leak. Cells were transfected with either GFP or claudin-5 GFP. Arrow denotes membrane expression of claudin-5-GFP (A). Monolayers were then infected with UV-irradiated influenza (MOI 8) for 24 hours and the TEER was measured pre- and post-infection. Data are mean, SE for 3 experiments, *p<0.05.</p

Replication-deficient influenza does not induce apoptosis.

<p>(A) Lung endothelium was infected with live or replication-deficient influenza (MOI 8) for 24 hours and then visualized by phase contrast microscopy. Cells exposed to replication-deficient influenza appear much healthier than cells infected with live virus and similar to infected cells. Images were captured as above and are representative of 3 experiments. (B–C) Endothelial apoptosis after exposure to replication-deficient influenza at the indicated MOI was assessed by binding of Annexin V 24 hours later. Histogram (B) is representative of 5 experiments. The percentage of Annexin V-positive cells (C) was similar in all groups. Results (mean, SE) are from 5 experiments. (D) The caspase-inhibitor ZVAD-FMK does not attenuate endothelial permeability induced by replication-deficient influenza. Cells were infected with UV flu (MOI 8) with or without 80 µM ZVAD-FMK for 24 hours. Permeability to dextran was measured. Results (mean, SD) are representative of 3 experiments, *p<0.05.</p

Human influenza causes lung vascular leak in a murine model, which is abrogated by formoterol.

<p>(A) C57BL/6 mice were infected nasally with UV influenza (128 HAU/mouse and 256 HAU/mouse), and live human influenza (128 HAU/mouse). Some mice infected with live influenza were given formoterol (0.2 mg/kg) or vehicle by intraperitoneal injection immediately afterwards and daily for 3 days. Lung vascular leak was measured by injection of Evans Blue (EB). Results are means normalized to serum EB levels (to control for unequal administration) and to the mean control EB of the experiment, *p<0.05. (B) Replication of influenza (MOI 8) in mouse lung endothelial cells (with or without 0.1 µM formoterol) was assessed by immunofluorescence for viral nucleoprotein as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047323#pone-0047323-g001" target="_blank">Figure 1</a>. Data are mean and SE from 3 experiments. (C) The reduction in lung permeability from formoterol is not due to decreased endothelial apoptosis. Mouse endothelial cells infected with live influenza (MOI 8) with or without formoterol (0.1 µM) were probed with Annexin V 24 hours later. Histogram is representative of 3 experiments.</p

Human influenza induces lung endothelial apoptosis.

<p>(A) Influenza induces loss of endothelial cell viability, detected by phase contrast microscopy. Cells were infected for 24 hours at the indicated multiplicity of infection (MOI). Images were captured using a Nikon Eclipse and are representative of 3 experiments. (B) Influenza induces lung endothelial apoptosis as shown by flow cytometry. Cells were infected with influenza at the indicated MOI for 4 hours and binding of annexin V was measured. (C) The number of annexin V-positive cells increased significantly in a dose-dependent fashion. Y-axis is the percentage of cells that are positive for annexin V. Results (mean, SE) are from 4 experiments, *p<0.05. (D) ZVAD-FMK partially prevents the induction of influenza-mediated lung endothelial permeability. Endothelial cells on transwells were infected with influenza (80 HAU/100 000 cells) for 24 hours with or without 80 µM ZVAD-FMK. Permeability to dextran was measured. Results (mean, SE) are from 4 experiments and are normalized to control, *p<0.05.</p

Replication-deficient virus induces loss of claudin-5 while VE-cadherin and F-actin are unaffected.

<p>(A,C) Replication-deficient virus induces a loss of claudin-5 as shown by immunofluorescence (A) and western blot (C). In A, cells were infected (MOI 8) for 24 hours. Images are randomly selected and representative of 3 experiments. In C, cells were infected with influenza at the indicated MOI (control  = 0). Image is representative of 4 experiments; histogram is the quantitation and shows mean and standard deviation and is normalized to control, *p<0.05. (B) In contrast, replication-deficient virus (MOI 8 in B) does not affect levels or distribution of VE-cadherin as shown by immunofluorescence (B) and western blot (C). Immunofluorescence images are representative of 3 experiments and western blot is from 4 experiments. The histogram is normalized to control. (D) Replication-deficient virus (MOI 8) does not alter the actin cytoskeleton as shown by immunostaining of F-actin with phalloidin. Images are representative of 3 experiments.</p

Characterization of replication-deficient virus.

<p>(A) Influenza irradiated with ultraviolet light (UV flu) cannot replicate. Influenza (40 HAU/100 000 cells) was irradiated with UV light for 10 minutes before viral titer was measured using the TCID₅₀ assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047323#pone-0047323-g001" target="_blank">Figure 1A</a>. Results are representative of 2 experiments. (B) Viral proteins are expressed in endothelial cells infected with UV-irradiated influenza. Influenza was given at an MOI of 8 for 12 hours. Nuclei are stained with DAPI and viral nucleoprotein is shown in green. Images are representative of 2 experiments. Immunofluorescent images (C) and quantitation (D) showing the ratio of nuclear to cytosolic p65, a measure of NFκB activation, in cells infected by influenza after 12 hours. Influenza was given at an MOI of 8. Arrows indicate infected cells, while asterisks denote their nuclei. P65 is shown in green. Infected cells were identified by immunofluorescence for influenza viral protein M1 (not shown). Images are representative of 4 experiments; data are mean and standard error, *p<0.05 for live flu and UV flu vs. control (uninfected cells).</p