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

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

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

Nanovesicular miRNome of the lymphocyte extracellular milieu is not a mere representation of the intracellular miRNome from parental cells.

<p>A. Bio-analyzer qualitative analysis of total RNA extracted 72 hours after activation with Phytohemagglutinin (PHA) from CD4⁺ T lymphocytes (upper panel) and released nanovesicles purified by ExoMir (lower panel). A representative sample is reported. B. Fold change (96 hours compared to 6 hours upon activation with PHA) and SEM of miRNome relative quantities of nanovesicle samples (in biological triplicate) released by CD4⁺ T lymphocyte. Two profiling platforms were used (as indicated, Applied Biosystems Stem-loop RT-qPCR and Exiqon Locked Nucleic Acid (LNA)-based RT-qPCR) and only miRNAs with a Ct<35 were considered. p values of a Wilcoxon matched-pairs signed rank test comparing 6 hours and 96 hours upon activation are reported. C. Hierarchical clustering of Z score values for miRNAs significant (p<0.01) upon a paired t test considering normalized relative quantities of all co-expressed miRNAs with a Ct<35 in CD4⁺ T cellular and nanovesicular compartments (NV) at the indicated time points upon activation with PHA <i>in </i><i>vitro</i>. Biological triplicates are reported. Distance for hierarchical clustering on significant miRNA: Pearson correlation with average linkage. D. Hierarchical clustering of Z score values for normalized relative quantities of miRNome from CD4⁺ T and B lymphocytes and their relative released extracellular nanovesicles. Only the 101 co-expressed miRNAs with a Ct<35 were considered. Biological triplicates are reported. Distance: Pearson correlation with average linkage.</p

miRNA serum compartmentalization.

<p>A. Heatmap for miRNAs significant (p<0.05) upon an ANOVA test (based on F distribution) considering the three reported groups: nanovesicles purified by differential centrifugation (pellet at 110000Xg), total serum and supernatants from the centrifugation at 110000Xg (soluble fraction) from sera of 3 different individuals. Data are representative of two independent experiments. Hierarchical clustering was performed considering Log-transformed normalized relative quantities -Log₁₀(NRQs)- of all co-expressed miRNAs with a Ct<35. Distance: Pearson correlation with complete linkage. B. Ranking analysis for miR-150 and miR-126 (upper panels) and for miR-19b and miR-92a (lower panels) in 10 paired samples of total serum (TS) and purified nanovesicles (NV) (7 purified by differential centrifugation and 3 by ExoMiR). Lower ranking position=higher representation. C. miR-19b and miR-150 relatives quantities (2^^-(^{specific compartment Ct – total serum Ct)}) by single RT-qPCR assays in nanovesicles compared to soluble fractions from 3 healthy donors sera (mean of the three samples and SEM are reported) processed by differential centrifugation. p value for a 2-way ANOVA analysis showing an extremely significant effect of serum compartmentalization for different miRNAs is reported.</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

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

Differential centrifugation and microfiltration for the identification of nanovesicle associated miRNome in human serum.

<p>Schematic view of the two nanovesicle purification methods used in this work: differential centrifugation (left) and microfiltration (ExoMir, right). For the latter procedure, serum or cellular medium is passed through 2 filters connected in series. The Top Filter has a larger pore size of approximately 200 nanometers to effectively capture larger particles while the Bottom Filter has a smaller pore size of approximately 20 nanometers for capturing exosomes and other nanovesicles of similar size. The filters are then disconnected and separately flushed by an RNA extraction reagent to lyse the captured particles and release their contents with no preservation of their integrity. B. Venn diagram showing the intersection (25 miRNAs, see list in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075348#pone-0075348-t001" target="_blank">Table 1</a>) of miRNAs highly expressed (Ct<31 in all samples) in nanovesicles isolated by either differential centrifugation (33 total) or ExoMir (30).</p