Differences in Western Blotting and human miRNA analyses between immunoprecipitated HBsAg particles and control immunoprecipitations.

<p>Left-most: Western blotting analysis of protein lysates from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs) for detection of Ago2 protein. IP+ lanes contain pooled protein lysates from samples # 1–13 and single lysates from samples # 1 and 9 respectively. Ctrl-IPs lanes contain protein lysates from immunoprecipitates obtained from HBsAg positive sera with mouse monoclonal anti-human c-myc antibody (unrelated-IP+) and HBsAg negative sera using anti-HBs monoclonal antibody (HBs-IP−). HEK is the protein lysate from HEK cells used as positive control for Ago2 protein. The figure is representative of 3 independent experiments. Right-most: HCL is unsupervised hierarchical cluster analysis of detected miRNAs (DC_T values) in both HBs-immunoprecipitated fractions from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs). GDM is supervised gene distance matrix correlating DCt values of HBs-IP+ vs ctrl-IP samples.</p

Identification of human miRNAs associated with serum HBsAg.

<p>(a) The circulating HBsAg-miRNA signature: average RQs were obtained from the comparative DDC_T analysis. Values are reported in a bar plot as a logarithmic scale base 10 along with SD. (b) Differentially detected miRNAs between HBsAg positive immunoprecipitates (HBs-IP+) samples (left-most, n = 11; right-most, n = 4) and the group of control HBsAg negative immunoprecipiates (ctrl-IPs) (n = 4) were selected by Mann-Whitney test on –DCt values (left-most, p<0.1; right-most, p<0.05), and an unsupervised hierarchical cluster analysis was finally performed. Venn diagrams indicate the comparison among the pool of HBsAg-associated miRNAs obtained from the comparative DDC_T analysis of panel a (blu circle) and the HBsAg-associated miRNAs obtained from the Mann-Whitney tests (red circle).</p

Demographic and Virologic Characteristics of Individuals and Sera.

<p>Abbreviations: VP = viral particles, virion; SVP = subviral HBsAg particles; IU = International Units; ng = nanograms; ALT = serum alanine amino trasferase.</p><p>Quantitative values for VP and SVP were obtained as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Gerlich1" target="_blank">[14]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Desire1" target="_blank">[15]</a>: 1 IU of HBsAg corresponds to 1,1E+06 IU HBV DNA and1 ng of HBsAg corresponds to 2,08E+08 SVP or 5,0E+07 VP.</p><p>HBV infection and disease phases were characterized as previuosly reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Brunetto1" target="_blank">[8]</a>: IC = Inactive HBsAg Carriers with serum HBV-DNA persistently below 2000 IU and without liver disease; AC1 = Active HBsAg Carriers with serum HBV-DNA fluctuating below 20.000 IU with normal liver histology; AC2 = Active HBsAg carriers with serum HBV-DNA fluctuating above 20.000 IU with chronic active hepatitis at histology, patients with chronic hepatitis B (CHB).</p

Canonical pathways enriched by predicted targets of HBsAg-associated miRNAs.

<p>Target genes of HBsAg-associated miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3a</a>) were predicted (TargetScan) and ranked. Functional analysis (IPA) was then applied on predicted target genes, and canonical pathways were finally ranked according to their significance in a Fisher exact test (p-value≤0.05, horizontal threshold line in the plot). Left axis: –log(p-values) as histogram boxes; the asterisk (*) marks the three pathways that resulted as significantly enriched by the analyzed gene-list; right axis: for each pathway, black dots indicate the ratio of pathway-defining genes, which is calculated as the number of involved target genes over the total number of genes in that pathway reference dataset.</p

HBs-immunoprecipitation led to a significant change of detection of some human miRNAs in examined sera.

<p>Heatmap of differentially detected miRNAs in whole HBsAg sera (S+) and HBsAg positive flowthroughs after immunoprecipitation (HBs-F+) was obtained by Mann-Whitney test (p<0.05) followed by hierarchical clustering (-DC_T are represented). Venn diagram: the 157 differentially abundant serum miRNAs between whole HBsAg positive sera (S+) and HBsAg positive flowthroughs (HBs-F+) were compared to miRNAs of clusters 1-to-5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3b</a> (right panel).</p

Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

<div><p>Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation <i>in vitro</i>, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. <i>In vivo</i>, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.</p> </div

Circulating miR-150 modulation in human serum upon flu vaccination.

<p>A. miR-150 quantities relative to exogenous spike-in ath miR-159a in sera of 50 H1N1-MF59 vaccinated children (samples collected at time of first dose, T0, at time of second dose 30 days after, T1 and 30 days after the second dose, T2) (left) and 46 pairs of samples (time of vaccination, T0 and 30 days after, T1) from H1N1-MF59 vaccinated healthy adults (right). Data were centered on the mean at T0 and mean values, SEM and two-tailed paired t test p values are reported. B. Box plot of miR-150 quantities relative to exogenous spike-in ath miR-159a (whiskers: 10-90 percentile) in the indicated serum compartments of 17 pairs of H1N1-MF59 at T0 (white) and T1 (grey). Two-tailed paired t test p values are reported. C. Receiver Operating Characteristic (ROC) curves for total serum, nanovesicular and microvesicular miR-150 increment in H1N1-MF59 vaccinated adults compared to pre-vaccination level (SE=Sensitivity; SP= Specificity). Area under the curve (AUC) and p value (calculated with χ² test) for nanovesicular miR-150 are reported.</p

miR-150 expression in human resting lymphocytes and tissues.

<p>A. Box plot of miRNome relative quantities in 17 different lymphocyte subsets, as indicated (blue, B; red, CD4⁺ T; green, CD8⁺ T; grey, NK). Only co-expressed miRNAs with a Ct<35 were considered. White circles indicate miR-150 expression level. B. miR-150 level in a panel of 20 different human tissues (as indicated) by RT-qPCR, relative to the internal control MammU6, and reported in percentage relative expression among tissues.</p

miR-150 intracellular down-modulation and release upon <i>in vitro</i> activation of CD4⁺ T lymphocytes.

<p>A. Heatmap showing the expression fold change of the indicated miRNAs at the indicated time points upon activation with Phytohemagglutinin (PHA) of CD4⁺ T lymphocytes compared to Time 0 (T0=1) (left panel); and Log₁₀ transformed relative expression of the same miRNAs in samples of nanovesicles collected at the indicated time points (right panel). Values are mean of a biological triplicate. The down-regulated (all 5) and the up-regulated (representative 5/56) miRNAs were selected by an ANOVA test (based on F distribution). B. Column chart plotting mean and SEM (of a biological triplicate) of fold change of CD4⁺ T lymphocyte intracellular miR-150 down-regulation and parallel c-Myb up-regulation (normalized by expression of internal control MammU6 and relative to Time 0) at the indicated time points upon activation with PHA. Asterisks indicate a t test resulting in a p value<0.05. C. Column charts plotting mean and SEM (of a biological triplicate) of fold change of CD4⁺ T lymphocyte intracellular miR-150 modulation (normalized by expression of internal control MammU6 and relativized to control, i.e. treatment with IL-2 alone) and nanovesicular accumulation (expressed as relative quantities of extracellular nanovesicular miR-150 upon activation compared to control IL-2 alone treated cells) 72 hours upon starting the indicated treatments (PMA for Phorbol 12-Myristate 13-Acetate; PHA for Phytohemagglutinin; SEB for <i>Staphylococcus aureus</i> enterotoxin B). Two profiling platforms were used (as indicated, Applied Biosystems Stem-loop RT-qPCR and Exiqon Locked Nucleic Acid (LNA)-based RT-qPCR) to validate results obtained treating cells with either PHA or SEB (right panel). D. Correspondence between RT-qPCR (upper panel) and Northern Blot (lower panel) for MammU6 snRNA (used as endogenous control, left) and miR-150 (right) expression level at time 0 and 72 hours upon starting the indicated treatments.</p

Correlation between circulating miR-150 modulation and immune response.

<p>A. Box plot of indicated miRNA quantities at T1 (30 days after vaccination) relative to exogenous spike-in ath miR-159a (whiskers: 10-90 percentile) in 46 flu vaccinated individuals stratified for having developed an antibody response lower (white) or higher (grey) than 1:320, as assessed by hemagglutination inhibition test assay. The p value of a Mann Whitney test is reported. B. Column chart plotting mean and SEM (of a biological triplicate) of mature miR-150 relative quantities in the indicated mouse lymphocytes: intracellular level 72 hours upon activation was normalized first by expression of internal control MammU6 snRNA and then by level at T0. Extracellular accumulation was calculated as 2^-^{{Ct(intracellular)-Ct(nanovesicles)}miR-150} / 2^-^{{Ct(intracellular)-Ct(nanovesicles)}MammU6}. Data are representative of two independent experiments. C. miR-150 quantities relative to exogenous spike-in ath miR-159a in wild type and MHCII^-/- mice vaccinated with ovalbumin (OVA) adjuvanted with alpha-galactosylceramide (αGalCer) 2 days before vaccination (-2, or T0) and 7 days after vaccination (each treatment normalized to miR mean relative quantity at T0). p value for a paired t test is reported. Four wild type mice and four MHCII^-/- mice were used for vaccination experiment. D. Correlation between anti-OVA total Ig concentration (assessed by ELISA) at T=7 days after vaccination in mice vaccinated with αGalCer + OVA (black) or Alum + OVA (grey) and serum circulating miR-150 fold change T1/T0 (T1=7 days after vaccination). Spearman r and p value are reported. miR-150 fold changes values for mice vaccinated with non-adjuvanted OVA are also reported (white).</p