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Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE.

ABSTRACT: Recently, it has been observed that mesenchymal stem cells (MSCs) can modulate their immunoregulatory properties depending on the specific in-vitro activation of different Toll-like receptors (TLR), such as TLR3 and TLR4. In the present study, we evaluated the effect of polyinosinic:polycytidylic acid (poly(I:C)) and lipopolysaccharide (LPS) pretreatment on the immunological capacity of MSCs in vitro and in vivo.C57BL/6 bone marrow-derived MSCs were pretreated with poly(I:C) and LPS for 1 hour and their immunomodulatory capacity was evaluated. T-cell proliferation and their effect on Th1, Th17, and Treg differentiation/activation were measured. Next, we evaluated the therapeutic effect of MSCs in an experimental autoimmune encephalomyelitis (EAE) model, which was induced for 27 days with MOG35-55 peptide following the standard protocol. Mice were subjected to a single intraperitoneal injection (2?×?106 MSCs/100 ?l) on day 4. Clinical score and body weight were monitored daily by blinded analysis. At day 27, mice were euthanized and draining lymph nodes were extracted for Th1, Th17, and Treg detection by flow cytometry.Pretreatment of MSCs with poly(I:C) significantly reduced the proliferation of CD3+ T cells as well as nitric oxide secretion, an important immunosuppressive factor. Furthermore, MSCs treated with poly(I:C) reduced the differentiation/activation of proinflammatory lymphocytes, Th1 and Th17. In contrast, MSCs pretreated with LPS increased CD3+ T-cell proliferation, and induced Th1 and Th17 cells, as well as the levels of proinflammatory cytokine IL-6. Finally, we observed that intraperitoneal administration of MSCs pretreated with poly(I:C) significantly reduced the severity of EAE as well as the percentages of Th1 and Th17 proinflammatory subsets, while the pretreatment of MSCs with LPS completely reversed the therapeutic immunosuppressive effect of MSCs.Taken together, these data show that pretreatment of MSCs with poly(I:C) improved their immunosuppressive abilities. This may provide an opportunity to better define strategies for cell-based therapies to autoimmune diseases.

SUBMITTER: Vega-Letter AM

PROVIDER: S-EPMC5057482 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE.

Vega-Letter Ana María AM Kurte Mónica M Fernández-O'Ryan Catalina C Gauthier-Abeliuk Melanie M Fuenzalida Patricia P Moya-Uribe Ivón I Altamirano Claudia C Figueroa Fernando F Irarrázabal Carlos C Carrión Flavio F

Stem cell research & therapy 20161010 1

<h4>Background</h4>Recently, it has been observed that mesenchymal stem cells (MSCs) can modulate their immunoregulatory properties depending on the specific in-vitro activation of different Toll-like receptors (TLR), such as TLR3 and TLR4. In the present study, we evaluated the effect of polyinosinic:polycytidylic acid (poly(I:C)) and lipopolysaccharide (LPS) pretreatment on the immunological capacity of MSCs in vitro and in vivo.<h4>Methods</h4>C57BL/6 bone marrow-derived MSCs were pretreated ...[more]

PMID: 27724984

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Project description:BackgroundDifferentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties.MethodsThrough the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers.ResultsThe cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization.ConclusionsMSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Project description:Mesenchymal stem cells (MSCs) affect diverse aspects of tumor progression, such as angiogenesis, tumor growth and metastasis. Bone marrow MSCs (BM?MSCs) are fibroblast?like cells with multipotent differentiation ability, that localize to areas of tissue damage, including wounds and solid tumors. The tumor suppressor gene, p53, is functionally involved in cell cycle control, apoptosis and genomic stability, and is mutated and inactivated in most human cancers. The present study aimed to investigate the role of p53 in the biology of BM?MSCs. In the present study, p53 wild?type (p53+/+), knockdown (p53+/?) and knockout (p53?/?) mouse BM?MSCs (mBM?MSCs) were observed to be similar in appearance and in the expression of cell surface biomarkers, but expressed differential p53 protein levels. The p53+/? and p53?/? mBM?MSCs demonstrated an increased proliferation rate compared with mBM?MSCs derived from p53+/+ mice. mBM?MSCs from all three groups, representing distinct p53 statuses, were unable to form tumors over a 3?month period in vivo. The adipogenic and osteogenic differentiation of mBM?MSCs was increased in the absence of p53. The colony formation and migratory abilities of p53+/? and p53?/? mBM?MSCs were markedly enhanced, and the expression levels of stem cell?associated proteins were significantly increased compared with p53+/+. The expression levels of microRNA (miR)?3152 and miR?337 were significantly increased in p53+/? and p53?/? mBM?MSCs, whereas the expression levels of miR?221, miR?155, miR?1288 and miR?4669 were significantly decreased. The expression levels of tumor necrosis factor?? and interferon???inducible protein?10 were significantly upregulated in the supernatant of p53+/? and p53?/? mBM?MSCs. Ubiquitin protein ligase E3 component n?recognin 2, RING?finger protein 31 and matrix metalloproteinase 19 were highly expressed in p53+/? and p53?/? mBM?MSCs. The results of the present study indicated that p53 may serve an important role in the biology of mBM?MSCs, and may provide novel insights into the role of cells with different p53 statuses in cancer progression.

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Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE.

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Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE.

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