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Functional and molecular characterization of exosomes secreted from mesenchymal stromal cells exposed to IFNg in the presence of autophagy modulators tamoxifen and chloroquine

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272209 | GEO | 2025/12/23

REPOSITORIES: GEO

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Project description:Mesenchymal stromal cells (MSCs) can be utilized clinically for treatment of conditions that result from excessive inflammation. In a pro-inflammatory environment, MSCs adopt an anti-inflammatory phenotype resulting in immunomodulation. A sub-type of MSCs referred to as “marrow-isolated adult multilineage inducible” (MIAMI) cells, which were isolated from bone marrow, were utilized to show that the addition of autophagy modulators, tamoxifen (TX) or chloroquine (CQ), can alter how MIAMI cells respond to IFNg exposure in vitro resulting in an increased immunoregulatory capacity of the MIAMI cells. Molecularly, it was also shown that TX and CQ each alter both the levels of immunomodulatory genes and microRNAs which target such genes. However, the role of other non-coding RNAs (ncRNAs) such as long non-coding RNAs (lncRNAs) in regulating the response of MSCs to inflammation has been poorly studied. Here, we utilized transcriptomics and data mining to analyze the putative roles of various differentially regulated lncRNAs in MIAMI cells exposed to IFNg with (or without) TX or CQ. The aim of this study was to investigate how the addition of TX and CQ alters lncRNA levels and evaluate how such changes could alter previously observed TX- and CQ-driven changes to the immunomodulatory properties of MIAMI cells. Data analysis revealed 693 long intergenic non-coding RNAS (lincRNAs), 480 pseudogenes, and 642 antisense RNAs that were differentially regulated with IFNg, IFNg+TX and IFNg+CQ treatments. Further analysis of these RNA species based on the existing literature data revealed 6 antisense RNAs, 2 pseudogenes, and 5 lincRNAs that have the potential to modulate MIAMI cell’s response to IFNg treatment. Functional analysis of these genomic species based on current literature linking inflammatory response and ncRNAs indicated their potential for regulation of several key pro- and anti-inflammatory responses, including NFkB signaling, cytokine secretion and auto-immune responses. Overall, this work found potential involvement of multiple pro-and anti-inflammatory pathways and molecules in modulating MIAMI cells’ response to inflammation.

Project description:The main goal of this study is to characterize the transcriptional modulations induced by antiviral compounds derived from chloroquine on Huh7 cells infected with HCV. And thus to identify among the genes modulated by infection, those who are regulated by chloroquine and potentially involved in the antiviral activity of the compound.Two HCV cell models were used: a non-infectious HCV replicon and the infectious HCV cell culture (HCVcc). In addition, this study aimed to highlight the characteristics of derivatives of antiviral chloroquine, compared with chloroquine itself. Abstract from the associated publication: Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portion of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Indeed, infection with hepatitis C virus (HCV) was shown to induce autophagy through ER stress signaling and subsequent Unfolded Protein Response (UPR) activation. Moreover, a role of autophagy in promoting HCV infection has been suggested and chloroquine (CQ), a lysosomal protease inhibitor, has been seen blocking autophagy as well as inhibiting HCV replication. In the present report, mechanisms accounting for these inhibitory effects were investigated. Gene expression profiling was performed on CQ treated JFH-1-infected Huh7 cells to identify the host cellular genes that are transcriptionally regulated by infection, and silenced by CQ-based treatment. Herein, we demonstrate that CQ reduces the expression of genes induced by the viral infection such as those encoding autophagic key factors triggering the turn-off of mTOR activity as well as factors involved in p53 and NF-KappaB activities. However, we present several lines of evidence demonstrating that the CQ repressive effect observed on the HCV-induced pathways results from a decrease of ER stress due to the upstream pH-dependent inhibitory effect of CQ on viral replication. Gene expression modulations were measured in Huh7 harboring replicon cells stimulated with a 10M-BM-5M of a Chloroquine derivative compound called CQd, during 6h, 12h and 24h. Two independent experiments were performed at each time. An untreated control condition was performed in each experiment, expression was measured at the 24h time point. For the HCVcc model, gene expression analysis was analyzed in two conditions: infection and treatment of infected cells. To characterize modulations in time course of infection, Huh7 naM-CM-/ve cells were infected with JFH1/CS-N6-A4 viral stock and gene expression was measured at 6h, 24h and 48h postinfection. For each kinetic time point, an uninfected condition was performed and gene expression was measured. An additional control testing the interferon (IFN) induced modulations on infected cells was included and gene expression was measured after a 48h stimulation with 100 IU of IFN (alpha 2 b). To characterize the antiviral modulations resulting from treatment with Chloroquine (CQ) or a derivative compound (CQd), gene expression was measured in JFH1/CS-N6-A4 infected Huh7 cells treated with 40M-BM-5M of CQ or CQd during 12h or 48h post-treatment and infection. For each kinetic time point 12h and 48h, an untreated infected control was also performed and gene expression measured.

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Functional and molecular characterization of exosomes secreted from mesenchymal stromal cells exposed to IFNg in the presence of autophagy modulators tamoxifen and chloroquine

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