Influenza virus infection decreases CLEC7A (Dectin1) mRNA and reduces phagocytosis of zymosan by AM.

<p>Panel A. Human AM were cultured and infected by PR/8 at a MOI of 0.5. The total RNA from infected and non-infected cells was evaluated for the expression of macrophage receptor genes by real-time RT-PCR at 24 hpi. The data show the relative expression levels of each gene in virus-infected cells compared to that of non-infected cells after normalized to the expression of constitutive probe from 4 to 14 donors. Each symbol indicates one donor. * indicates there was a significant difference between control and virus-infected cells (P<0.05). Panel B. PR/8 infection induced a dose-dependent decrease of uptake of zymosan. Isolated AM were cultured and infected by PR/8. At 24 hpi, fluorescent FITC-labeled zymosan was added without serum for 2 h and then the cells were washed and fixed with paraformaldehyde. Uptake of zymosan was measured as percent of cells containing zymosan evaluated under fluorescent microscopy. The data represent mean+SE of percent cells uptaking zymosan. N = 4. ** indicates P<0.01, *** indicates P<0.001 vs. non-infected cells.</p

Inhibition of TNF and/or IL-1 pathways decreases release of CXCL8 and CCL5 but not CXCL10 and IFNs induced by influenza infection.

<p>Human AM isolated from donor lungs were cultured and infected by PR/8 at a MOI of 0.5. Soluble TNF p55 receptor and IL-1Ra were added to the cultures at 10 µg/ml 45 min before the infection and added back to the cultures after viral inoculation. Secretion of chemokines and cytokines was measured by ELISA at 24 hpi. The data represent mean+SE of each released cytokine and chemokine (pg/ml). N = 6. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001 vs. virus-infected cells.</p

Kinetics of influenza infection with live and UV-inactivated PR/8.

<p>Primary AM were cultured and infected by live PR/8 at a MOI of 0.5 or the equal amount of UV-inactivated PR/8, and cells were harvested at designated time post inoculation. Panels A–F. Kinetics of viral antigen synthesis and infectious virus release. Panels A–C show representative immunofluorescence staining for influenza HA from live PR/8-infected AM culture at 6, 24, and 48 hpi. Panel D shows the quantitation of these experiments. The data represent mean±SE of percentage of positive-stained cells from 6 donors. Panel E. Representative staining of viral antigen in UV-inactivated PR/8 infection at 48 hpi. Panel F. Representative release of infectious viral particles from both live and UV-inactivated PR/8-infected AM from 6 donors. Panels G–K. Time course of cytokine and chemokine response in PR/8-infected AM. The supernatant from cultured cells were collected at 1, 6, 24, and 48 hpi. Secretion of TNF-α (Panel G), IFN-α (Panel H), CXCL10 (Panel I), CXCL8 (Panel J), and CCL5 (Panel K) was measured by ELISA. Data show representative release of each cytokine from infected AM of 6 donors that all showed similar response.</p

Verification of virus-induced secretion of chemokines and cytokines by ELISA.

<p>Human AM isolated from donor lungs were cultured and infected by PR/8 at a MOI of 0.5. Secretion of chemokines and cytokines from infected and non-infected cultures was measured by ELISA at 24 hpi. The data represent mean+SE of each released cytokine and chemokine (pg/ml). The number of individual donors ranged from 8 to 16. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001 vs. non-infected cells.</p

Innate immune response of both live and UV-inactivated contemporary H3N2 influenza viruses-infected AM.

<p>Human AM isolated from donor lungs were cultured and infected by live NY/238 virus at a MOI of 0.1 or the equal amount of UV-inactivated NY/238. Cells were harvested at designated times for evaluation of their innate immune response. Panel A. Alterations in mRNAs of innate immune response-related genes at 3 and 24 hpi by realtime RT-PCR. The data represent mean+SE of the relative expression levels of each gene in infected cultures compared to that of non-infected controls after normalization to the level of the constitutive probe cyclophilin B, N = 4. * indicates P<0.05 and ** indicates P<0.01 between live and UV-inactivated cells. Panel B. Kinetics of cytokine and chemokine response by ELISA. The data show representative release of TNF-α, IFN-α, CXCL10, CXCL8, and CCL5 from both live and UV-inactivated NY/238 virus-infected AM from one of 6 donors that all showed similar response.</p

The top 25 genes up-regulated or down-regulated by PR/8 infection in human AM at 24 hpi.

<p>Human AM from 3 non-smoking donors were isolated, cultured, and infected by PR/8 virus at a MOI of 0.5. The gene profiling of infected and non-infected cells at 24 hpi from each donor was examined by microarray experiments using Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA). The filtered gene list was generated as described in the Section of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029879#s4" target="_blank">Methods</a>. The data show the top 25 genes up-regulated or down-regulated altered by viral infection.</p><p>*indicates similar results from multiple probes.</p

Serologic responses in vaccinated recruits as measured by microneutralization assay, LAIV-vaccinated recruits (n = 78).

<p>CI, confidence interval; GMT, geometric mean titer; LAIV, live attenuated influenza vaccine.</p>1<p>Seroconversion is defined as a 4-fold increase in titer from pre-vaccine to post-vaccine titer.</p>2<p>Fold change adjusted for pre-vaccine seroprotection levels.</p

Post-vaccination microneutralization titer distribution responses in vaccinated recruits based upon vaccine type.

<p>Post-vaccination microneutralization titer distribution responses in vaccinated recruits based upon vaccine type.</p

Estimate of odds of a 4-fold conversion relative to conversions against the H3N2 virus.

<p>Estimate of odds of a 4-fold conversion relative to conversions against the H3N2 virus.</p

Affinity measurements isotyping and total anti-HA1 binding antibody in human sera following immunization.

<p><b>(A–B) Correlation between in vitro MN titers and rHA1 binding in human sera following immunization with H1N1 vaccine in TIV and LAIV groups.</b> Steady-state equilibrium analysis of the binding of vaccine serum IgG to properly folded functional HA1 oligomers was measured using surface plasmon resonance (SPR). Ten-fold diluted post-H1N1 vaccination sera from vaccine groups (LAIV in A, TIV in B) were injected simultaneously onto HA1 immobilized on a sensor chip, free of peptide. Binding was recorded in resonance units (RU) values. The maximum RU values for HA1 binding by serum antibodies obtained from vaccinated individuals with either LIAV (A) or TIV (B) vaccination is shown on the Y-axis. The MN titer is expressed as end-point neutralizing antibody titer of post-H1N1 vaccine sera and is depicted on the X-axis. <b>(C) The isotype of serum antibodies bound to rHA1 for the two vaccine groups.</b> Data shown are the means for serum from two independent experiments. <b>(D) Antibody avidity measurements in polyclonal serum by off-rate constants using SPR.</b> Antibody off-rate constants, which describe the stability of the complex were determined directly from the serum/plasma sample interaction with rHA1 protein using SPR in the dissociation phase. For accurate measurements, parallel lines in the dissociation phase for the 10-fold and 100-fold dilution for each post-vaccination human sera were required. The off-rate constants were determined from two independent SPR runs. SPR analysis of post-vaccinated human sera with LAIV (left) or TIV (right) from the vaccine trial was performed with properly folded H1N1pdm09 HA1 (A/CA/7/2009) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034581#pone.0034581-Khurana7" target="_blank">[60]</a>. Serum antibody off-rate constants for vaccinees (each symbol is one individual) were plotted. Correlation statistics of affinity measurement and off-rate constants of sera binding to rHA1 between LAIV and TIV vaccinees were statistically significant with <i>p</i><0.05 (T-test).</p