Mesenchymal Stromal Cell-Derived Exosomes Affect mRNA Expression and Function of B-Lymphocytes

Abstract

Background: Bone marrow mesenchymal stem cells (bmMSC) may play a role in the regulation of maturation, proliferation, and functional activation of lymphocytes, though the exact mechanisms are unknown. MSC-derived exosomes induce a regulatory response in the function of B, T, and monocyte-derived dendritic cells. Here, we evaluated the specific inhibition of human lymphocytes by bmMSC-derived exosomes and the effects on B-cell function. Methods: Exosomes were isolated from culture media of bmMSC obtained from several healthy donors. The effect of purified bmMSC-derived exosomes on activated peripheral blood mononuclear cells (PBMCs) and isolated B and T lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester assay. Using the Illumina sequencing platform, mRNA profiling was performed on B-lymphocytes activated in the presence or absence of exosomes. Ingenuity® pathway analysis software was applied to analyze pathway networks, and biological functions of the differentially expressed genes. Validation by RT-PCR was performed. The effect of bmMSC-derived exosomes on antibody secretion was measured by ELISA. Results: Proliferation of activated PBMCs or isolated T and B cells co-cultured with MSC-derived exosomes decreased by 37, 23, and 18%, respectively, compared to controls. mRNA profiling of activated B-lymphocytes revealed 186 genes that were differentially expressed between exosome-treated and control cells. We observed down- and up-regulation of genes that are involved in cell trafficking, development, hemostasis, and immune cell function. RNA-Seq results were validated by real time PCR analysis for the expression of CXCL8 (IL8) and MZB1 genes that are known to have an important role in immune modulation. Functional alterations were confirmed by decreased IgM production levels. Consistent results were demonstrated among a wide variety of healthy human bmMSC donors. Conclusion: Our data show that exosomes may play an important role in immune regulation. They inhibit proliferation of several types of immune cells. In B-lymphocytes they modulate cell function by exerting differential expression of the mRNA of relevant genes. The results of this study help elucidate the mechanisms by which exosomes induce immune regulation and may contribute to the development of newer and safer therapeutic strategies.

Keywords: B-lymphocytes; CXCL8; MZB1; bmMSC-derived exosomes; ingenuity pathway analysis; mesenchymal stem cells; next generation sequencing.

Figure 1

Inhibition of peripheral blood mononuclear cell (PBMC) proliferation by mesenchymal stem cells (MSCs). Carboxyfluorescein succinimidyl ester (CFSE) labeled 1 × 10⁵ PBMCs were activated with 2 μg/ml PHA and incubated for 4 days with and without MSCs at a ratio of 1:20, 1:10, and 1:5. (A) A representative FACS analysis of CFSE-labeled PBMCs, activated and non-activated, with and without MSCs at different ratios. (B) A graphic presentation of the percent suppression of activated PBMCs with different MSCs at a ratio of 1:20, 1:10 (p < 0.003), and 1:5 (p < 0.001). Data are expressed as the mean of tested samples. Activated condition counts were set to represent 100%.

Figure 2

Mesenchymal stem cell (MSC)-derived exosome characterization. (A) Electronic microscopy of MSC-derived exosomes. Right image—no primary antibody (Ab). Right image—exosome stained with gold-conjugates, which are secondary antibody to anti-CD63. (B) Zeta sizer measurement of MSC-derived exosomes. (A representative measurement of 4 extractions is shown). (C) Anti-CD63 labeled exosomes bound to aldehyde sulfate beads. Right image—a representative graph of the percentage of CD63 staining in eight exosome batches, isolated from 8 MSC donors. The difference in CD63 expression might reflect the variability between donors and protein expression in their MSCs.

Figure 3

Exosome suppression of activated peripheral blood mononuclear cell (PBMCS) and lymphocytes and internalization assays. (A) Thymidine incorporation assays. Mesenchymal stem cell (MSC)-derived exosomes co-cultured with: (1) PHA-activated PBMCs. The results summarize 3 experiments with 6 batches of exosomes derived from 0.5 × 10⁶ MSCs and 9 batches of exosomes derived from 1 × 10⁶ MSCs (batches no. 1–9). Control—Activated condition counts set to represent 100%. P < 0.001. (2) CD3/CD28 activated T-lymphocytes. The results summarize 5 experiments with 11 batches of exosomes derived from 1 × 10⁶ MSCs (batches no. 1–11). Control—Activated condition counts were set to represent 100%, p < 0.001. (3). R-848/IL2 activated B-lymphocytes. The results summarize 3 experiments with 6 batches of exosomes derived from 1 × 10⁶ MSCs (batches no. 4–9). Control—Activated condition counts were set to represent 100%, p < 0.004. (B) The internalization of exosomes by activated lymphocytes was assessed by confocal microscopy: Blue color, nucleus; Red color, cytoplasmic CD45; Green color, exosomes. (C) FACS analysis of PKH positive peripheral blood mononuclear cell, gated to different cell populations. The results summarize 3 experiments with 6 batches of exosomes. Percent uptake of 0.04%, 5.22%, 15.89%, 65.49% in PKH only, T cell, B cell, monocyte group, respectively.

Figure 4

Differentially expressed genes in activated B-lymphocytes, with or without incubation with exosomes. All 5 replicates of cells exposed to exosomes (batches no. 6, 7, 8), samples B5, B6, B8, B9, and B10 (green), were compared to the duplicate samples of cells without exosome exposure, B3 and B4 (blue). The normalized expression of significantly expressed genes, padj <0.05, is shown as a heatmap, after scaling the values for each gene. The color scale is indicated on the top right corner (blue, below average; red, above average). Genes are ordered by hierarchical clustering. Only the 50 genes that were most differentially expressed are shown, with their official symbols.

Figure 5

Real time PCR analysis. Expression levels of mRNA depicted for (A) MZB1 (B) CXCL8, were measured in activated B cells with or without exosomes. The graphs show the results of 6 exosome batches (MZB1 exosome batches 6, 8, 10, 12, 9, 3, respectively; CXCL8 exosome batches 6, 9, 10, 8, 12, 3, respectively). GAPDH was used as internal controls for targeting mRNA expression, and data are expressed as the mean of triplicate samples ± S.E.

Figure 6

Immunoglobulin estimation by ELISA. Expression levels of immunoglobulin M were measured by ELISA, in cell culture supernatant of activated B-lymphocytes cultured with or without exosomes. The graphs show the results of 6 exosome batches (batches no. 9, 8, 3, 10, 6, 12), cultured with the same single B lymphocyte donor. Data are expressed as the mean of triplicate samples ± S.E.