Dataset Information

Reciprocal interactions of mouse bone marrow-derived mesenchymal stem cells and BV2 microglia after lipopolysaccharide stimulation.

ABSTRACT:

Introduction

Mesenchymal stem cells (MSCs) are immunosuppressive, but we lack an understanding of how these adult stem cells are in turn affected by immune cells and the surrounding tissue environment. As MSCs have stromal functions and exhibit great plasticity, the influence of an inflamed microenvironment on their responses is important to determine. MSCs downregulate microglial inflammatory responses, and here we describe the mutual effects of coculturing mouse bone marrow MSCs with BV2 microglia in a lipopolysaccharide (LPS) inflammatory paradigm.

Methods

Mouse MSCs were cultured from femoral and tibial bone marrow aspirates and characterized. MSCs were cocultured with BV2 microglia at four seeding-density ratios (1:0.2, 1:0.1, 1:0.02, and 1:0.01 (BV2/MSC)), and stimulated with 1 μg/ml LPS. In certain assays, MSCs were separated from BV2 cells with a cell-culture insert to determine the influence of soluble factors on downstream responses. Inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) were measured in cocultures, and MSC and BV2 chemotactic ability determined by migration assays.

Results

We demonstrated MSCs to increase expression of NO and IL-6 and decrease TNF-α in LPS-treated cocultures. These effects are differentially mediated by soluble factors and cell-to-cell contact. In response to an LPS stimulus, MSCs display distinct behaviors, including expressing IL-6 and very high levels of the chemokine CCL2. Microglia increase their migration almost fourfold in the presence of LPS, and interestingly, MSCs provide an equal impetus for microglia locomotion. MSCs do not migrate toward LPS but migrate toward microglia, with their chemotaxis increasing when microglia are activated. Similarly, MSCs do not produce NO when exposed to LPS, but secrete large amounts when exposed to soluble factors from activated microglia. This demonstrates that certain phenotypic changes of MSCs are governed by inflammatory microglia, and not by the inflammatory stimulus. Nonetheless, LPS appears to "prime" the NO-secretory effects of MSCs, as prior treatment with LPS triggers a bigger NO response from MSCs after exposure to microglial soluble factors.

Conclusions

These effects demonstrate the multifaceted and reciprocal interactions of MSCs and microglia within an inflammatory milieu.

SUBMITTER: Rahmat Z

PROVIDER: S-EPMC3706938 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundPeriodontal tissue regeneration is the ultimate goal of periodontitis treatment. Exosomes are nanoscale vesicles secreted by cells that participate in and regulate the physiological activities between cells. However, the relationship between inflammatory macrophage-derived exosomes and osteoblast differentiation in periodontitis has not been thoroughly reported. Here, we attempt to explore the role of inflammatory macrophage-derived exosomes in crosstalk with osteoblasts.MethodsPorphyromonas gingivalis lipopolysaccharide was used to stimulate macrophages and inflate their inflammatory cellular state. Exosomes were extracted from inflammatory macrophages using supercentrifugation, and their characteristics were detected by transmission electron microscopy, particle size analysis, and Western blotting. Exosome uptake bybone marrow mesenchymal stem cells (BMSCs) was observed by fluorescence microscopy. The effects of exosomes on the BMSC inflammatory response and on osteogenic differentiation were detected by quantitative polymerase chain reaction and Western blot analysis. Alkaline phosphatase activity was tested for verification.ResultsWe successfully extracted and identified inflammatory macrophage-derived exosomes and observed that BMSCs successfully took up exosomes. Inflammatory macrophage-derived exosomes upregulated the expression levels of the inflammatory factors interleukin-6 and tumour necrosis factor-alpha in BMSCs and mediated inflammatory stimulation. Additionally, they inhibited the transcription levels of the osteogenic genes alkaline phosphatase, type I collagen, and Runt-related transcription factor 2 as well as the alkaline phosphatase activity, while the use of the exosome inhibitor GW4869 attenuated this effect.ConclusionOur study shows that macrophages in periodontitis can mediate inflammatory stimulation and inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells through the exosome pathway. Interference with exosome secretion is likely to be a promising method for bone tissue regeneration in inflammatory states.

Project description:Microglia are the primary immunocompetent cells in brain tissue and microglia-mediated inflammation is associated with the pathogenesis of various neuronal disorders. Recently, many studies have shown that mesenchymal stem cells (MSCs) display a remarkable ability to modulate inflammatory and immune responses through the release of a variety of bioactive molecules, thereby protecting the central nervous system. Previously, we reported that MSCs have the ability to modulate inflammatory responses in a traumatic brain injury model and that the potential mechanisms may be partially attributed to upregulated TNF-? stimulated gene/protein 6 (TSG-6) expression. However, whether TSG-6 exerts an anti-inflammatory effect by affecting microglia is not fully understood. In this study, we investigated the anti-inflammatory effects of MSCs and TSG-6 in an in vitro lipopolysaccharide (LPS)-induced BV2 microglial activation model. We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia. However, MSC effects on microglia were attenuated when TSG-6 expression was silenced. In addition, we found that the activation of nuclear factor (NF)-?B and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6. Furthermore, we found that the presence of CD44 in BV2 microglial cells was essential for MSC- and TSG-6-mediated inhibition of pro-inflammatory gene expression and of NF-?B and MAPK activation in BV2 microglial cells. The results of this study suggest that MSCs can modulate microglia activation through TSG-6 and that TSG-6 attenuates the inflammatory cascade in activated microglia. Our study indicates that novel mechanisms are responsible for the immunomodulatory effect of MSCs on microglia and that MSCs, as well as TSG-6, might be promising therapeutic agents for the treatment of neurotraumatic injuries or neuroinflammatory diseases associated with microglial activation.

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.