Isolement et caractérisation des vésicules extracellulaires sécrétées par les cellules souches humaines MuStem : rôle potentiel en médecine régénératrice ?

Isolement et caractérisation des vésicules extracellulaires sécrétées par les cellules souches humaines MuStem : rôle potentiel en médecine régénératrice ?. XXXème congrès de l'AFH: les nouveaux mondes de l'histologi

MicroRNA-29b Modulates Innate and Antigen-Specific Immune Responses in Mouse Models of Autoimmunity

International audienceIn addition to important regulatory roles in gene expression through RNA interference, it has recently been shown that microRNAs display immune stimulatory effects through direct interaction with receptors of innate immunity of the Toll-like receptor family, aggravating neuronal damage and tumour growth. Yet no evidence exists on consequences of microRNA immune stimulatory actions in the context of an autoimmune disease. Using microRNA analogues, we here show that pancreatic beta cell-derived microRNA sequences induce pro-inflammatory (TNFa, IFNa, IL-12, IL-6) or suppressive (IL-10) cytokine secretion by primary mouse dendritic cells in a sequence-dependent manner. For miR-29b, immune stimulation in RAW264.7 macrophages involved the endosomal Toll-like receptor-7, independently of the canonical RNA interference pathway. In vivo, the systemic delivery of miR-29b activates CD11b+B2202 myeloid and CD11b-B220+ plasmacytoid dendritic cells and induces IFNa, TNFa and IL-6 production in the serum of recipient mice. Strikingly, in a murine model of adoptive transfer of autoimmune diabetes, miR-29b reduces the cytolytic activity of transferred effector CD8+ T-cells, insulitis and disease incidence in a single standalone intervention. Endogenous miR-29b, spontaneously released from beta- cells within exosomes, stimulates TNFa secretion from spleen cells isolated from diabetes-prone NOD mice in vitro. Hence, microRNA sequences modulate innate and ongoing adaptive immune responses raising the question of their potential role in the breakdown of tolerance and opening up new applications for microRNA-based immune therapy

Isolement et caractérisation des vésicules extracellulaires sécrétées par les cellules souches humaines MuStem : rôle potentiel en médecine régénératrice ?

Isolement et caractérisation des vésicules extracellulaires sécrétées par les cellules souches humaines MuStem : rôle potentiel en médecine régénératrice ?. XXXème congrès de l'AFH: les nouveaux mondes de l'histologi

Molecular and Functional Diversity of Distinct Subpopulations of the Stressed Insulin-Secreting Cell's Vesiculome

International audienceBeta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell's vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting toward EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response

Molecular and functional signatures of distinct subpopulations of extracellular vesicles provide a rationale for beta-cell mediated immune disease

International audienc

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 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

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