Functional and molecular characterization of exosomes secreted from mesenchymal stromal cells exposed to IFNg in the presence of autophagy modulators tamoxifen and chloroquine
Bone marrow mesenchymal stromal cells (MSCs) are therapeutic cells which adopt an immunomodulatory phenotype when exposed to pro-inflammatory cytokines and thus can be used for treatment of conditions associated with excessive inflammation. It was suggested recently that many therapeutic benefits of MSCs can be attributed to the secretion of exosomes, small membrane vesicles of endocytic origin. Exosome formation and release are impacted by the autophagy pathway, a homeostatic quality control mechanism that recycles proteins and organelles via lysosomal degradation. We hypothesized that the exposure of MSCs to autophagy modulators in the presence of a pro-inflammatory cytokine IFN? may impact on exosome RNA content and immunomodulatory properties. To address this hypothesis, we treated MSCs with either tamoxifen (TX) or chloroquine (CQ) to stimulate or inhibit autophagy, respectively, in the presence of pro-inflammatory cytokine, IFN?. Subsequently, we characterized exosomes from such cells and determined their functional and molecular properties. Exosomes' immunoregulatory properties were evaluated ex-vivo using activated CD4 T cells and subsequent flow cytometry analysis to assess the effects of exosomes on CD4 T cell proliferation. We also performed small RNA sequencing to determine exosomal microRNA content. Subsequently, we performed functional analysis to determine potential effects of exosomal microRNAs on the expression of genes in target cells. Our data indicate that the exosomes isolated from MSCs treated with CQ+IFN? possess higher capacity to decrease T cell proliferation. Small RNA sequencing of exosomal RNA revealed that, although similar microRNAs were found to be expressed in exosomes isolated from all treated cells, the treatments did affect the levels of multiple microRNAs. Analysis of putative gene targets revealed that most microRNA-target genes belong to either cancer or inflammation-related biological pathways. Overall, we conclude that the pre-treatment of MSCs with small molecules, in addition to pro-inflammatory cytokines, has a potential to additionally modulate biological targets of exosomes and thus biological effects of the latter. Overall design: MSCs were grown in serum-free media and treated with IFNgamma in the presence or absence of tamoxifen (5 microM) or chloroquine (10 microM) for 48 h. Subsequently serum-free media was used to harvest MSC-secreted exosomes which were used to extract microRNAs which were subjected to deep sequencing. microRNA were then counted and following contrasts were created: 1. untreated vs. IFNgamma treated; 2. untreated vs. tamoxifene treated; 3. untreated vs chloroquine treated; 4. untreated vs. IFNgamma+tamoxifene treated; 5. untreated vs. IFNgamma+chloroquine treated.