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Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6.

Liu Y, Zhang R, Yan K, Chen F, Huang W, Lv B, Sun C, Xu L, Li F, Jiang X - J Neuroinflammation (2014)

Bottom Line: We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia.In addition, we found that the activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The National Key Clinic Specialty, the Neurosurgery Institute of Guangdong Province, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, 253# Gongye Road, Guangzhou 510282, China. jiangxiao_dan@163.com.

ABSTRACT
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-α 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)-κB 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-κB 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.

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MSCs and TSG-6 suppress LPS-induced pro-inflammation gene expression of microglia. BV2 cells were co-cultured with MSCs, TSG-6-siMSCs, or control-siMSCs activated by TNF-α and different concentration of rmTSG-6 (1 ng/ml, 10 ng/ml, 100 ng/ml) and treated with or without LPS (100 ng/ml) for 6 hours. (A-D), mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were measured by qRT-PCR analyses and quantified as fold induction over the levels in unstimulated control cells. All transcriptional levels were normalized to GAPDH mRNA levels and determined by the 2-ΔΔCt method. Values in the bar graphs are presented as mean ± SD. n = 3; *P <0.05; significantly different from LPS-treated cells. Abbreviations: TSG-6-siMSCs, MSCs transfected with TSG-6 siRNA; Ctrl(control)-siMSCs, MSCs transfected with control siRNA; LPS, lipopolysaccharide; iNOS, induced nitric oxide synthase.
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Figure 1: MSCs and TSG-6 suppress LPS-induced pro-inflammation gene expression of microglia. BV2 cells were co-cultured with MSCs, TSG-6-siMSCs, or control-siMSCs activated by TNF-α and different concentration of rmTSG-6 (1 ng/ml, 10 ng/ml, 100 ng/ml) and treated with or without LPS (100 ng/ml) for 6 hours. (A-D), mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were measured by qRT-PCR analyses and quantified as fold induction over the levels in unstimulated control cells. All transcriptional levels were normalized to GAPDH mRNA levels and determined by the 2-ΔΔCt method. Values in the bar graphs are presented as mean ± SD. n = 3; *P <0.05; significantly different from LPS-treated cells. Abbreviations: TSG-6-siMSCs, MSCs transfected with TSG-6 siRNA; Ctrl(control)-siMSCs, MSCs transfected with control siRNA; LPS, lipopolysaccharide; iNOS, induced nitric oxide synthase.

Mentions: We first investigated whether the mRNA expression of the anti-inflammatory gene TSG-6 was increased in MSCs that had been stimulated by the inflammatory cytokine TNF-α. Using qRT-PCR analysis, we found that in response to TNF-α stimulation, MSCs expressed TSG-6 mRNA to a level seven-fold higher than the control level (Additional file 2: Figure S2). Next, we investigated the effect of MSCs and TSG-6 on the expression of inflammatory factors by LPS-activated microglia. To this end, BV2 microglial cells were co-cultured with MSCs and rmTSG-6 at different concentrations for 6 hours. As shown in Figure 1, we observed a significant decrease in the levels of TNF-α, IL-1β, IL-6, and iNOS mRNA expression when LPS-stimulated BV2 cells were cultured in the presence of MSCs. However, the MSCs had reduced effects when the expression of the TSG-6 gene was knocked down using TSG-6 siRNA compared with non-transfected MSCs or control siRNA transfected MSCs. To confirm the key role of TSG-6 in modulating the function and activity of the LPS-activated microglia, we added rmTSG-6 directly to the microglial cultures during LPS treatment. TSG-6 supplementation largely reproduced the effects of the MSCs in a dose-dependent manner. The results shown in Figure 1 demonstrate that in the presence of 10 ng/ml and 100 ng/ml of TSG-6, the levels of TNF-α, IL-1β, IL-6, and iNOS mRNA expression by the BV2 microglia were significantly reduced, similar to those produced by activated MSCs.


Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6.

Liu Y, Zhang R, Yan K, Chen F, Huang W, Lv B, Sun C, Xu L, Li F, Jiang X - J Neuroinflammation (2014)

MSCs and TSG-6 suppress LPS-induced pro-inflammation gene expression of microglia. BV2 cells were co-cultured with MSCs, TSG-6-siMSCs, or control-siMSCs activated by TNF-α and different concentration of rmTSG-6 (1 ng/ml, 10 ng/ml, 100 ng/ml) and treated with or without LPS (100 ng/ml) for 6 hours. (A-D), mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were measured by qRT-PCR analyses and quantified as fold induction over the levels in unstimulated control cells. All transcriptional levels were normalized to GAPDH mRNA levels and determined by the 2-ΔΔCt method. Values in the bar graphs are presented as mean ± SD. n = 3; *P <0.05; significantly different from LPS-treated cells. Abbreviations: TSG-6-siMSCs, MSCs transfected with TSG-6 siRNA; Ctrl(control)-siMSCs, MSCs transfected with control siRNA; LPS, lipopolysaccharide; iNOS, induced nitric oxide synthase.
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Related In: Results  -  Collection

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Figure 1: MSCs and TSG-6 suppress LPS-induced pro-inflammation gene expression of microglia. BV2 cells were co-cultured with MSCs, TSG-6-siMSCs, or control-siMSCs activated by TNF-α and different concentration of rmTSG-6 (1 ng/ml, 10 ng/ml, 100 ng/ml) and treated with or without LPS (100 ng/ml) for 6 hours. (A-D), mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were measured by qRT-PCR analyses and quantified as fold induction over the levels in unstimulated control cells. All transcriptional levels were normalized to GAPDH mRNA levels and determined by the 2-ΔΔCt method. Values in the bar graphs are presented as mean ± SD. n = 3; *P <0.05; significantly different from LPS-treated cells. Abbreviations: TSG-6-siMSCs, MSCs transfected with TSG-6 siRNA; Ctrl(control)-siMSCs, MSCs transfected with control siRNA; LPS, lipopolysaccharide; iNOS, induced nitric oxide synthase.
Mentions: We first investigated whether the mRNA expression of the anti-inflammatory gene TSG-6 was increased in MSCs that had been stimulated by the inflammatory cytokine TNF-α. Using qRT-PCR analysis, we found that in response to TNF-α stimulation, MSCs expressed TSG-6 mRNA to a level seven-fold higher than the control level (Additional file 2: Figure S2). Next, we investigated the effect of MSCs and TSG-6 on the expression of inflammatory factors by LPS-activated microglia. To this end, BV2 microglial cells were co-cultured with MSCs and rmTSG-6 at different concentrations for 6 hours. As shown in Figure 1, we observed a significant decrease in the levels of TNF-α, IL-1β, IL-6, and iNOS mRNA expression when LPS-stimulated BV2 cells were cultured in the presence of MSCs. However, the MSCs had reduced effects when the expression of the TSG-6 gene was knocked down using TSG-6 siRNA compared with non-transfected MSCs or control siRNA transfected MSCs. To confirm the key role of TSG-6 in modulating the function and activity of the LPS-activated microglia, we added rmTSG-6 directly to the microglial cultures during LPS treatment. TSG-6 supplementation largely reproduced the effects of the MSCs in a dose-dependent manner. The results shown in Figure 1 demonstrate that in the presence of 10 ng/ml and 100 ng/ml of TSG-6, the levels of TNF-α, IL-1β, IL-6, and iNOS mRNA expression by the BV2 microglia were significantly reduced, similar to those produced by activated MSCs.

Bottom Line: We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia.In addition, we found that the activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The National Key Clinic Specialty, the Neurosurgery Institute of Guangdong Province, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, 253# Gongye Road, Guangzhou 510282, China. jiangxiao_dan@163.com.

ABSTRACT
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-α 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)-κB 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-κB 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.

Show MeSH
Related in: MedlinePlus