Dataset Information

Upregulated TSG-6 Expression in ADSCs Inhibits the BV2 Microglia-Mediated Inflammatory Response.

ABSTRACT:

Objectives

The microglial cells are immune surveillance cells in the central nervous system and can be activated during neurological disorders. Adipose-derived stem cells (ADSCs) were reported to inhibit the inflammatory response in microglia by secreting proteins like tumor necrosis factor-inducible gene 6 protein (TSG-6). We aim to explore the mechanisms and the associated microRNAs.

Methods

ADSCs were cultured and TSG-6 expression was evaluated. ADSCs were cocultured with lipopolysaccharide- (LPS-) induced BV2 microglia and the supernatant was harvested for detecting cytokines. The total RNA was extracted and sequenced by high-throughput sequencing. MicroRNA profiles were compared between two treatment groups of ADSCs. A comprehensive bioinformatics analysis and confirmation experiments were performed to identify the microRNAs targeting at TSG-6.

Results

We found that ADSCs could secrete TSG-6 to inhibit the proinflammatory cytokines, including interleukin-1 beta and interleukin-6, and tumor necrosis factor alpha (TNF?), produced by LPS-induced microglia-mediated inflammatory response. Bioinformatics analysis showed a total of 35 microRNAs differentially expressed between the two groups of ADSCs, and miR-214-5p was identified as a regulator of TSG-6 mRNA.

Conclusion

Following a treatment with TNF?, ADSCs can regulate the inflammatory response in LPS-activated BV2 microglia by upregulating TSG-6 expression, which itself is under the negative control of miR-214-5p.

SUBMITTER: Hu Y

PROVIDER: S-EPMC6280241 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Upregulated TSG-6 Expression in ADSCs Inhibits the BV2 Microglia-Mediated Inflammatory Response.

Hu Yang Y Li Gaigai G Zhang Ye Y Liu Na N Zhang Ping P Pan Chao C Nie Hao H Li Qi Q Tang Zhouping Z

BioMed research international 20181121

<h4>Objectives</h4>The microglial cells are immune surveillance cells in the central nervous system and can be activated during neurological disorders. Adipose-derived stem cells (ADSCs) were reported to inhibit the inflammatory response in microglia by secreting proteins like tumor necrosis factor-inducible gene 6 protein (TSG-6). We aim to explore the mechanisms and the associated microRNAs.<h4>Methods</h4>ADSCs were cultured and TSG-6 expression was evaluated. ADSCs were cocultured with lipop ...[more]

PMID: 30584538

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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:Neurodegenerative disorders are known to be associated with neuroinflammation caused by microglia. Therefore, regulation of microglia activation and polarization to inhibit neuroinflammatory reactions seems to hold promise as a therapeutic approach in neurodegenerative disorders. Spatholobus suberectus Dunn (SSD) has been utilized as a traditional Chinese medicine remedy for brain diseases for thousands of years. SSD possesses various pharmacological activities, such as circulation invigoration, neuroprotection, and anti-inflammatory. The objective of this research was to examine the anti-neuroinflammatory effects and molecular mechanisms of an active fraction from SSD (ASSD) in vitro culture BV2 cells, a type of mouse microglia cell line. The inflammatory responses in BV2 cells were induced by stimulating them with 1 μg/mL lipopolysaccharide (LPS) and the effects of ASSD on LPS-stimulated inflammation were monitored. Besides, by using the methods of Western blot, immunofluorescence, and RT-PCR, the mechanisms of ASSD on microglia activation, M1/M2 polarization, and the TLR4/MyD88/NF-κB pathway were investigated. Our findings demonstrate that the treatment doses of ASSD neither induce cytotoxicity nor promote the production of inflammatory cytokines. In addition, immunofluorescence analysis show that ASSD inhibited the expression of ionized calcium-binding adapter molecule 1(Iba1) and inducible nitricoxide synthase (iNOS), and induced arginase 1 (Arg1) expression. Moreover, Western blot analysis indicated that ASSD significantly down-regulated TLR4, MyD88, p-IκB, NF-κB p65, and NF-κB p-p65 protein expression levels. Furthermore, RT-qPCR assay show that ASSD significantly down-regulated iNOS, TLR4, MyD88, and NF-κB mRNA expression levels, and up-regulated Arg1 mRNA expression level. According to the findings, ASSD can suppress microglia-mediated inflammatory responses by modulating microglia activation, inducing a shift from M1 to M2 polarization, and inhibiting the TLR4/MyD88/NF-κB signaling pathway.

Project description:Background: Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders. These cells survey the CNS and, under inflammatory conditions, become "activated" through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. In the present study, we examined the roles of the related TAM receptors, Mer and Axl, and of their ligand, Gas6, in the regulation of microglial pro-inflammatory TNF-α production and microglial morphology. Methods: Primary cultures of murine microglia of wild-type (WT), Mer-/- and Axl-/- backgrounds were stimulated by the TLR4 agonist, lipopolysaccharide (LPS) with or without pre-treatment with Gas6. Gene expression of TNF-α, Mer, and Axl was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) was used to measure TNF-α release from microglia. Immunofluorescence staining of β-actin and the microglial marker Iba1 was performed to reveal microglial morphological changes, with cellular characteristics (area, perimeter, Feret's diameter, minimum Feret, roundness, and aspect ratio) being quantified using ImageJ software. Results: Under basal conditions, TNF-α gene expression was significantly lower in Axl-/- microglia compared to WT cells. However, all microglial cultures robustly responded to LPS stimulation with the upregulation of TNF-α expression to similar degrees. Furthermore, Mer receptor expression was less responsive to LPS stimulation when in Axl knockout cells. The presence of Gas6 consistently inhibited the LPS-induced upregulation of TNF-α in WT, Mer-/- and Axl-/- microglia. Moreover, Gas6 also inhibited LPS-induced changes in the microglial area, perimeter length, and cell roundness in wild-type cells. Conclusion: Gas6 can negatively regulate the microglial pro-inflammatory response to LPS as well as via stimulation of other TLRs, acting through either of the TAM receptors, Axl and Mer. This finding indicates an interaction between TLR and TAM receptor signaling pathways and reveals an anti-inflammatory role for the TAM ligand, Gas6, which could have therapeutic potential.

Dataset Information

Upregulated TSG-6 Expression in ADSCs Inhibits the BV2 Microglia-Mediated Inflammatory Response.

Objectives

Methods

Results

Conclusion

Publications

Upregulated TSG-6 Expression in ADSCs Inhibits the BV2 Microglia-Mediated Inflammatory Response.

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