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

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

Plasma microRNA profiling: Exploring better biomarkers for lymphoma surveillance

<div><p>Early detection of relapsed lymphoma improves response and survival. Current tools lack power for detection of early relapse, while being cumbersome and expensive. We searched for sensitive biomarkers that precede clinical relapse, and serve for further studies on therapy response and relapse. We recruited 20 healthy adults, 14 diffuse large B-cell lymphoma (DLBCL) patients and 11 Hodgkin lymphoma (HL) patients at diagnosis. Using small-RNA sequencing we identified in DLBCL patients increased plasma levels of miR-124 and miR-532-5p, and decreased levels of miR-425, miR-141, miR-145, miR-197, miR-345, miR-424, miR-128 and miR-122. In the HL group, we identified miR-25, miR-30a/d, miR-26b, miR-182, miR-186, miR-140* and miR-125a to be up-regulated, while miR-23a, miR-122, miR-93 and miR-144 were down-regulated. Pathway analysis of potential mRNAs targets of these miRNA revealed in the DLBCL group potential up-regulation of STAT3, IL8, p13k/AKT and TGF-B signaling, and potential down-regulation of the PTEN and p53 pathways; while in the HL group we have found the cAMP-mediated pathway and p53 pathway to be potentially down-regulated. Survival analyses revealed that plasma levels of miR-20a/b, miR-93 and miR-106a/b were associated with higher mortality. In conclusion, we identified sets of dysregulated circulating miRNA that might serve as reliable biomarkers for relapsed lymphoma.</p></div

Box plots showing levels of top 10 miRNA in healthy controls' plasma and corresponding levels in paired exosome preparations.

<p>Statistically differences between plasma and exosomes are not significant, as inferred from a DESeq2 analysis presented in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187722#pone.0187722.s002" target="_blank">S2 File</a>.</p

Top 10 influential miRNA for classification; inclusion rate in 200 iterations, each retaining 80 miRNAs.

<p>Top 10 influential miRNA for classification; inclusion rate in 200 iterations, each retaining 80 miRNAs.</p

Clinical characteristics of study participants.

<p>Clinical characteristics of study participants.</p

Distributions of misclassification rates within 1000 iterations of training and validation using 'cancerclass', performed on DLBCL patients' and healthy controls' miRNA profiles (A) or HL patients' and healthy controls' profiles (B).

<p>Distributions of misclassification rates within 1000 iterations of training and validation using 'cancerclass', performed on DLBCL patients' and healthy controls' miRNA profiles (A) or HL patients' and healthy controls' profiles (B).</p

Box plots showing the distribution of composite scores based on the levels of differentially expressed miRNA between DLBCL patients and healthy volunteers (A-C) or between HL patients and healthy volunteers (D-F).

<p>Scores are composed of up-regulated miRNA (<b>A</b> and <b>D</b>), down-regulated miRNA (<b>B</b> and <b>E</b>) or all dysregulated miRNA (<b>C</b> and <b>F</b>) in the respective pairwise analysis; however, all 3 groups of patients are shown in all plots. The composite scores were calculated for each patient by standardizing miRNA levels (namely, scaling the average level of each miRNA to 0 and standard deviation of 1) and summing the respective miRNA. Upregulated miRNAs were added to the score sum, while downregulated miRNA were subtracted.</p

Discrimination value of up and down regulated plasma miRNA.

<p>Discrimination value of up and down regulated plasma miRNA.</p