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:IntroductionThe extracellular deposition of β-amyloid (Aβ) is a pathological hallmark in Alzheimer's disease (AD), which induces microglial activation in the pathology of AD. The expression of serine/threonine-protein kinase 2 (SRPK2) is increased in the brain tissues of patients with AD. In this study, we examined the effect of SRPK2 in the activation of microglia.MethodsMicroglia (BV2) cells were cultured and the expression of SRPK2 was enhanced by transfection of SRPK2 recombinant vectors or knockdown by SRPK2 small interfering RNA (siRNA). The cells were stimulated by lipopolysaccharide (LPS) + interferon-γ (IFN-γ) or Aβ in vitro, generating inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-10, and IL-6], which were investigated by real-time quantitative PCR (qPCR) and ELISA. The proliferation ability of the BV2 cells with/without SRPK2 expression was evaluated by WST-1 under pressure in the presence of Aβ. The effects of SRPK2 on microglia polarization were evaluated by investigating the expression of CD16/32 and CD206 by western blot and the expression of ionized calcium-binding adapter molecule-1 (IBA-1) and arginase-1 (Arg-1) by immunofluorescence. Hippocampal cells HT-22 were cultured with a BV2 cell (with/without SRPK2 expression)-derived medium stimulated by Aβ or LPS + IFN-γ, prior to the evaluation of HT-22 cytotoxicity by assessment of cell viability. Possible relationships between Akt and SRPK2 in the BV2 cells were investigated by western blot.ResultsThe expression of SRPK2 was related to the phenotype polarization changes of microglia with increased expression of CD16/32 and IBA-1. The expression of proinflammatory cytokines IL-6 and TNF-α was increased, whereas the expression of anti-inflammatory cytokine IL-10 was decreased in the BV2 cells with SRPK2 overexpression. Moreover, with the expression enhancement of SRPK2, the BV2 cells had a higher proliferation rate. Aβ treatment can promote SRPK2 expression in BV2 cells. Aβ or LPS + IFN-γ promoted the production of cytokines IL-6 and TNF-α but decreased cytokine IL-10 in the BV2 cells. SRPK2 deficiency alleviated the cytotoxic effects of Aβ or LPS + IFN-γ exposed microglia on HT22 cells. In addition, the activated Akt pathway promoted the expression of SRPK2 in the BV2 cells.ConclusionOur data have found that enhanced SRPK2 expression contributed to the proinflammatory activation of microglia. Thus, SRPK2 may be a key modulating pathway of inflammatory mediators in AD pathology.

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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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