Cytokine profile in BALB/c mice serum after intravenous miRNA delivery.

<p>Cytokine content in serum from BALB/c mice was analysed by a BD Cytometric Bead Array two and seven hours following intravenous injection of miR29b, the immune-silent miR-127 or positive (R848) or negative (HBS) controls. Results are presented as mean concentration (pg/ml) ± SEM from two experiments (n = 4 total mice); nd: not detected.</p><p>Cytokine profile in BALB/c mice serum after intravenous miRNA delivery.</p

Stimulation of the TLR-7 pathway by miR-29b in murine RAW264.7 macrophages.

<p>(A) 2′-O-methyl modifications were introduced in all uracil residues of the miR-29b reverse strand as indicated. RAW264.7 cells were plated four hours before stimulation with DOTAP-embedded miR-29b, 2′-O-Me-modified miR-29b, or the control miR-127 (750 nM working concentration). TNFa was quantified in supernatants eighteen hours later. 2′-O-Me modifications were introduced in the miR-29b reverse strand before annealing to the unmodified guide strand. Results are represented as individual values of cytokine concentrations (pg/ml). Data from one representative experiment out of three is shown. *<i>P</i><0.05 (Mann-Whitney). (B) Intracellular distribution of Alexa-488-labelled miR-29b 1hour after transfection of RAW264.7 cells was observed with a confocal fluorescence microscope. Top row: IF EEA-1 on fixed cells Bottom row: lysotracker in living cells. Scale bar = 20 µm for fluorescence images and overlays with differential interference contrast (DIC) (a–d, f–i) except for enlarged single cell images scale bar = 5 µm (e, j). (C) Chloroquine (CQ) was added to the plated RAW264.7 cells, at a final concentration of 10 nM, 30 minutes before stimulation with miR-29b or the miR-127 control (750 nM). Supernatants were harvested eighteen hours later for TNFa quantification. Results are represented as individual values of cytokine concentrations (pg/ml) compiled from two independent experiments. **<i>P</i><0.01 (Mann-Whitney) (D) RAW264.7 cells were stimulated with miR-29b, miR-127 (750 nM), the positive controls TLR-7-ligand imiquimod and R848, or were left untreated (NT), and were cultured eighteen hours with or without the TLR-7 antagonist IRS661. TNFa was quantified in supernatants. Results are presented as mean cytokine concentration of replicates (pg/ml) ± SEM. Data from one representative experiment out of three is shown.</p

Cytokine secretion induced by miRNAs <i>in vitro</i> and <i>in vivo</i>.

<p>Purified mouse CD11c⁺ bmDCs or RAW264.7 mouse macrophages were stimulated <i>in vitro</i> with miRNAs complexed to DOTAP at a working concentration of 150 nM (bmDCs) or 750 nM (macrophages, positive controls (siRNA9.2 or LPS), negative controls (siRNA9.1 or DOTAP alone) or were left untreated (NT). IL-12 (A), TNFa (B) and IL-10 (C) cytokine levels were assessed by ELISA in bmDC supernatants eighteen hours after stimulation. Results are presented as mean cytokine concentration of duplicates (pg/ml) ± SEM. Data from one representative experiment out of two is shown. (D) TNFa secreted by RAW264.7 macrophages was quantified in supernatants after eighteen hours of stimulation. Results compiled from four independent experiments are shown and analysed using a Kruskal-Wallis test (***<i>P</i><0.001 and **<i>P</i><0.01). (E) Serum IFNa in BALB/c mice was quantified by ELISA seven hours following intravenous injection of miRNAs complexed to DOTAP or controls. Results are presented as mean concentration of duplicates (pg/ml) ± SEM, and are confirmed in a second independent experiment (n = 4 total mice). (F) Serum IL-6, TNFa and IL-12 in BALB/c mice was quantified two hours following intravenous injection of miR-29b, the positive control R848, or the immune-silent miR-127 using a BD Cytometric Bead Array. Results are presented as mean concentration ± SEM (pg/ml) from two experiments (n = 4 total mice). IL-6: <i>P</i><0.05 for miR-29b vs miR-127 and miR-127 vs R848; IL-12: <i>P</i><0.05 for miR-127 vs R848 (Kruskal-Wallis).</p

Splenic mDC and pDC activation by miR-29b <i>in vivo</i>.

<p>BALB/c mice were injected intravenously with miR-29b, miR-127, or siRNA9.1. Spleens were harvested eighteen hours after injection and CD40, CD86, and H-2Kd expression was evaluated by flow cytometry, on CD11c⁺CD11b⁺B220⁻ mDC (A) or CD11c^lowCD11b⁻B220⁺ pDC (B) subsets. Histogram plots show the results of CD40, CD86 and H-2K^d staining for one mouse out of two in one experiment representative of four independent experiments. Grey shading indicates isotypic controls. For each marker, graphs represent the relative fluorescence intensity (RFI) of individual mice in two independent experiments (n = 3 mice for miR-29b and siRNA9.1, n = 4 mice for miR-127), and are representative of two other independent experiments. *<i>P</i><0.05 (Mann-Whitney).</p

Systemic delivery of miR-29b protects against adoptive transfer of T1D <i>in vivo</i>.

<p>Ins-HA mice were treated intravenously with miR-29b, miR-127, HBS buffer or DOTAP alone, eighteen hours before receiving HA-specific CTLs from CL4-TCR mice. (A) Recipients were monitored for diabetes development for at least one month. The survival curves and table summarize the results of five independent experiments after transfer of 1 to 10×10⁵ cells, with miR-29b -injected mice as filled symbols, and HBS-injected mice as empty symbols. The table indicates, for each group, the percentage of final cumulative diabetes incidence and the number of diabetic mice among all mice in the group in brackets. A logrank test was performed for statistical significance of differences between Kaplan-Meier incidence curves. (B) Eighteen hours after miRNA injection, Ins-HA recipient mice received 5×10⁵ activated HA-specific CTLs, followed 48 h later by the intravenous administration of HA-pulsed «CFSE_high » and non-pulsed «CFSE_low » target cells mixed at a 1∶1 ratio. Splenocytes from recipient Ins-HA mice were analysed by flow cytometry, sixteen hours after target cell injection. The bar chart shows the compiled results of three independent experiments (n = 4–5 mice/group) as mean specific lysis ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 (Mann-Whitney). (C–E) Eighteen hours after miRNA injection, Ins-HA were transferred with 8×10⁵ activated HA-specific Thy1.1⁺ CTLs from CL4-TCR⁺Thy1.1⁺ mice. Four days later, spleens (C) and PLNs (D) were harvested from Ins-HA recipient mice and analysed by flow cytometry. Compiled results of two independent experiments are presented as the percentage of Thy1.1⁺ cells in individual mice gated on the CD3⁺ CD8⁺ T-cell population (n = 3–5 mice), and were confirmed in a third experiment. *<i>P</i><0.05 (Mann-Whitney). (E) Histological analysis of insulitis of pancreata: 0 = islet free of mononuclear cell infiltration (unfilled bars); 1 = peri-insular infiltration involving <10% of the islet area (punctuated bars); 2 = infiltration involving between 10% and 50% of the islet area (hatched bars); 3 = infiltration involving >50% of the islet area (black bars). The stacked vertical bar graph indicates the percentage of islets in each category described above. Results are presented as the mean percentage of n = 5 mice for miR-29b, n = 3 for miR-127, and n = 4 mice in the HBS group from three independent experiments. For each pancreas, an average insulitis score was calculated by adding up the score of each islet and dividing it by the total number of islets counted. Results show the individual insulitis scores for each group of recipient mice. *<i>P</i><0.05 (Kruskal-Wallis).</p

Stimulation of immune cells with exosomes <i>in vitro</i>.

<p>(A–B, D) Cytokine concentration measured by cytometric bead analysis in supernatants from splenocytes of NOD mice at 48 h of culture (A) with 20 µg/ml of exosomes with n = 7 (NT) and n = 10 (EXO) samples per group from two independent experiments. <i>*P</i><0.05, <i>**P</i><0.01 and <i>***P</i><0.001 (Mann-Whitney) (B) after transfection with 750 nM of miR-29b or 2′-OMe-miR-29b. Data are representative of two independent experiments (n = 5–6 mice per group). <i>***P</i><0.001 (Kruskal Wallis) (C) TNFa concentration in supernatants of RAW264.7 macrophages stimulated for 48 h with various concentrations of MIN6 exosomes. Results from TNFa ELISA analysis are representative of four independent experiments (n = 12 to 15 wells per group). <i>**P</i><0.001 and <i>****P</i><0.0001(Kruskal-Wallis) (D) treatment with exosomes transfected with LNA-miR-29 family inhibitor or control (CT). Data were obtained from n = 7–8 replicates from two independent experiments. <i>**P</i><0.01 (Mann-Whitney). All bar graphs are presented as mean ± SEM. NT: no treatment.</p