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Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton

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Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton

Annalisa Adamo et al. Front Immunol.

Abstract

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches.

Keywords: B cells; PI3K-AKT signaling pathway; actin cytoskeleton; extracellular vesicles; high-throughput analysis; mesenchymal stromal cells; miRNA-155-5p.

Figures

Figure 1
Figure 1
Size and surface marker characterization of MSC-derived EVs. Histograms represent hydrodynamic diameter distribution plots measured on EVs freshly isolated from resting (A) and primed MSC (B) (cEVs exosomes peak 40.43 ± 15.63 nm, percentage 46.6%; cEVs microvesicles peak 207.7 ± 53.95 nm, percentage 53.4%; pEVs exosomes peak 51.17 ± 23.23 nm, percentage 75.1%; pEVs microvesicles peak 200.9 ± 63.57 nm, percentage 24.9%). (C) Hydrodynamic diameter distribution of cEV stored at −80°C (cEVs exosomes peak 51.08 ± 21.07 nm, percentage 59%; cEVs microvesicles peak 196.9 ± 65.34 nm, percentage 41%). (D) Hydrodynamic diameter distribution of pEV stored at −80°C (pEVs exosomes peak 55.91 ± 21.99 nm, percentage 83.6%; pEVs microvesicles peak 201.1 ± 76.45 nm, percentage 16.4%). Error bars represent mean ± SD obtained from at least five measurements of three independent samples. All experiments were performed in PBS at 25°C. (E) Background corrected median fluorescence intensity of CD9, CD63, CD81 markers and corresponding isotype controls on cEVs and pEVs (n = 5). (F) Background corrected median fluorescence intensity of 34 surface epitopes on cEVs and pEVs (n = 5). (G) Immunoblot analysis of CD44, CD146, CD105, and CD63 expression in cEVs and pEVs. This blot is representative of three independent experiments showing the same trends.
Figure 2
Figure 2
Incorporation of MSC-EVs and RNA transfer in activated B lymphocytes. (A) Percentage of Vibrant DiI+ Syto RNA Select+ B cells co-cultured for 24, 48, and 72 h with double stained resting or primed MSCs (n = 5) *p < 0.05. (B) Vybrant Dil Geometric Mean of Fluorescence Intensity (GMFI) of B cells co-cultured with double stained resting or primed MSCs. (C) Syto RNA Select GMFI of B cells co-cultured with double stained resting or primed MSCs. (D) Representative gating strategy on the final gated population. (E) MSC-EVs were double-stained for membrane in red (Vybrant Dil) and for RNA in green (Syto RNA Select). Labeled EVs were incubated for 24 h on activated B lymphocytes. The four panels show (from the left to the right) B cells stained with DAPI (blue), the internalization of membrane components of cEVs and pEVs (red), the distribution of Syto RNA Select carried by MSC-EVs inside B cells (green), and a merge between the three previous panels (original magnification 400x). The images are representative for three independent experiments with similar results. (F) Representative FACS analysis of Vibrant DiI+ Syto RNA Select+ B cells co-cultured with double stained (right) or not (left) resting or primed MSCs. (G) Percentage of Vibrant DiI+ Syto RNA Select+ B cells co-cultured for 24 h with double stained resting or primed MSC-EVs (n = 5) *p < 0.05. (H) Representative FACS analysis of Vibrant DiI+ Syto RNA Select+ B cells co-cultured with double stained (right) or not (left) resting or primed MSC-EVs.
Figure 3
Figure 3
Overall variation of differentially expresses proteins in MSCs and EVs following inflammatory priming and PLS-DA. Score plot of the first two PCs calculated after the application of PCA to EVs (A) and MSCs (B). Score plot of the first two LVs calculated after the application of PLS-DA to the 181 proteins selected by Ranking-PCA on the EVs dataset (C) and on the first 200 proteins selected by Ranking-PCA on the MSCs dataset (D). Score plot of the first two LVs calculated after the application of PLS-DA to the 55 proteins selected by univariate statistics on the EVs dataset (E) and on the 39 proteins selected by univariate statistics on the MSCs dataset (F).
Figure 4
Figure 4
Proteomic profile of resting and primed MSCs and corresponding EVs. (A) Differentially expressed proteins in pEVs compared to cEVs obtained both from univariate and multivariate approach (adj p < 0.05, Fold Change (FC) > 1.5 and FC < 0.667) (n = 7). (B) Differentially expressed proteins in pMSCs compared to cMSCs obtained both from univariate and multivariate approach (adj p < 0.05, FC > 1.5, and FC < 0.667) (n = 7). (C) Venn diagram representing modulated proteins both in pMSCs and pEVs. (D) Immunoblot analysis of LG3BP, MOES, PTX3, and S10A6 expression in cEVs and pEVs. This blot is representative of three independent experiments showing the same trends.
Figure 5
Figure 5
GO protein annotation and Pathway Enrichment Analysis. Cellular component GO category annotation of differentially expressed proteins in pEVs [(A), the top 10 terms are shown] and pMSCs (B). Pathway enrichment analysis on differentially expressed proteins in pEVs [(C), the top 10 terms are shown] and pMSCs (D). Terms with adjusted p-value < 0.05 were considered significantly enriched. Cytoscape platform based ClueGO/CluePedia pathway analysis and visualization of differentially expressed proteins in pEVs (E) and pMSCs (F). Terms are grouped based on shared genes (kappa score) showed in different colors. The size of nodes indicates the degree of significance. The most significant term defines the name of the group. (G) Modulation of differentially expressed proteins in pEVs enriched in “PI3K-AKT signaling pathway,” “regulation of actin cytoskeleton,” “focal adhesion,” and “leukocyte trans-endothelial migration” KEGG pathway (FC < 0.0667 or FC > 1.5, p < 0.05).
Figure 6
Figure 6
PI3K-AKT signaling pathway expression in activated B lymphocytes co-cultured with MSC-EVs. (A) Relative expression of PAN AKT, AKT pS473, GSK3b pS9, p70S6K, S6 pS240, S6 pS235pS236 in activated B lymphocytes analyzed by flow cytometry. (B) Relative expression of PAN AKT, AKT pS473, GSK3b pS9, p70S6K, S6 pS240, S6 pS235pS236 in activated B lymphocytes treated or not with resting or primed EVs. Wilcoxon test *p < 0.05. (C) Representative histograms showing the levels of expression of PAN AKT, AKT pS473, GSK3b pS9, p70S6K, S6 pS240, S6 pS235pS236 in activated B lymphocytes treated or not with resting or primed EVs.
Figure 7
Figure 7
B cell spreading inhibition mediated by MSC-EVs. (A) Cell area of B cells plated on coverslips coated or not with F(ab′) 2 anti-human IgM/IgA/IgG (n = 42–204). (B) Double-stained B cells with DAPI (blue) and rhodamine phalloidin (F-actin, red) before and after the induction of cell spreading (60 min). (C) Cell area of B cells pre-treated with cEVs or pEVs and then plated on coated coverslips (n = 212–398). (D) Percentage of spread B cells treated with cEVs or pEVs after 60 min of incubation on coated coverslips. (E) Double-stained B cells with DAPI (blue) and rhodamine phalloidin (F-actin, red) after 60 min of incubation on coated coverslips. The images are representative for three independent experiments with similar results (Original magnification 400x). Error bars represent mean ± SEM of three independent experiments. t-test *p < 0.05, ***p < 0.001.
Figure 8
Figure 8
miRNA expression profile of MSCs and EVs following inflammatory priming. PCA plot calculated using regularized-logarithm transformation of miRNA counts on EVs (A) and MSCs (B) samples. Heatmap summarizing differentially expressed miRNAs in pEVs (C) and pMSCs (D). Bar plot representing the log2-fold change of differentially expressed miRNAs in pEVs (E) and pMSCs (F). (G) Venn diagram representing common differentially expressed miRNAs in pEVs and pMSCs. (H) RT-qPCR validation of differentially expressed miRNA in pEVs (n ≥ 3). Wilcoxon test *p < 0.05.
Figure 9
Figure 9
Effect of miR-155-5p on B cell activity. (A) Relative cell viability of B cells transfected with double-stranded RNA mimic miR-155-5p (n = 7). (B) Relative PI3K-AKT signaling pathway expression in B cells transfected with double-stranded RNA mimic miR-155-5p (n = 5). Wilcoxon test *p < 0.05.
Figure 10
Figure 10
Combination of miRNA and proteomic profiles of MSCs and EVs. Score plot of the first two PCs calculated after the application of PCA to EVs (A) and MSCs (B). Chord diagrams representing proteins belonging to “PI3K-AKT signaling pathway” (C), “regulation of actin cytoskeleton” (D), “focal adhesion” (E), and “leukocyte trans-endothelial migration” (F) KEGG pathways targeted by miRNAs differentially expressed in pEVs.

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