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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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TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; **P <0.01; *P <0.05; significantly different from LPS-treated cells. Abbreviations: LPS, lipopolysaccharide; NF-κB, nuclear factor (NF)-κB.
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Figure 2: TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; **P <0.01; *P <0.05; significantly different from LPS-treated cells. Abbreviations: LPS, lipopolysaccharide; NF-κB, nuclear factor (NF)-κB.

Mentions: The above results indicate that TSG-6 was involved in reducing the level of LPS-mediated expression of inflammatory mediators in BV2 cells. Thus, we investigated whether TSG-6 played a role in the inflammatory signaling events triggered by LPS stimulation. Because NF-κB is a major transcription factor mediating pro-inflammatory gene expression in the LPS-TLR4 signaling pathway [22], we examined possible alterations in NF-κB signaling induced by TSG-6 in BV2 microglia. First, we analyzed NF-κB p65 nuclear translocation in BV2 cells using immunofluorescence staining and confocal laser-scanning microscopy. After a 1-hour treatment, the cells were fixed for immunostaining. NF-κB p65 largely remained in the cytoplasm of the control group under basal (unstimulated) conditions. Upon LPS stimulation, nuclear translocation of NF-κB p65 occurred in BV2 cells, but to a lesser extent in the TSG-6-treated group (Figure 2A, B).We next evaluated the DNA-binding activity of NF-κB using a chemiluminescence-based EMSA assay. Nuclear extracts were prepared 0.5, 1, and 2 hours after LPS stimulation of control and TSG-6-treated cells, and were used to compare the binding of a biotin-labeled NF-κB-specific DNA probe . The increase in DNA-protein complex formation was less prominent as early as 0.5 hours after LPS stimulation in the TSG-6-treated cells than in the LPS-treated cells (Figure 2C).Finally, we evaluated NF-κB transcriptional activity using luciferase reporter assays. pNF-κB-Luc containing five tandem repeats of an NF-κB consensus binding site or pRL-TK as the normalization control were transiently expressed for 24 hours before LPS stimulation. Measurements of the luminescence intensity showed that TSG-6 inhibited LPS-induced NF-κB activation (Figure 2D). Taken together, the results suggested that TSG-6 was actively involved in the regulation of TLR4-mediated NF-κB signaling.


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)

TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; **P <0.01; *P <0.05; significantly different from LPS-treated cells. Abbreviations: LPS, lipopolysaccharide; NF-κB, nuclear factor (NF)-κB.
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Related In: Results  -  Collection

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Figure 2: TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; **P <0.01; *P <0.05; significantly different from LPS-treated cells. Abbreviations: LPS, lipopolysaccharide; NF-κB, nuclear factor (NF)-κB.
Mentions: The above results indicate that TSG-6 was involved in reducing the level of LPS-mediated expression of inflammatory mediators in BV2 cells. Thus, we investigated whether TSG-6 played a role in the inflammatory signaling events triggered by LPS stimulation. Because NF-κB is a major transcription factor mediating pro-inflammatory gene expression in the LPS-TLR4 signaling pathway [22], we examined possible alterations in NF-κB signaling induced by TSG-6 in BV2 microglia. First, we analyzed NF-κB p65 nuclear translocation in BV2 cells using immunofluorescence staining and confocal laser-scanning microscopy. After a 1-hour treatment, the cells were fixed for immunostaining. NF-κB p65 largely remained in the cytoplasm of the control group under basal (unstimulated) conditions. Upon LPS stimulation, nuclear translocation of NF-κB p65 occurred in BV2 cells, but to a lesser extent in the TSG-6-treated group (Figure 2A, B).We next evaluated the DNA-binding activity of NF-κB using a chemiluminescence-based EMSA assay. Nuclear extracts were prepared 0.5, 1, and 2 hours after LPS stimulation of control and TSG-6-treated cells, and were used to compare the binding of a biotin-labeled NF-κB-specific DNA probe . The increase in DNA-protein complex formation was less prominent as early as 0.5 hours after LPS stimulation in the TSG-6-treated cells than in the LPS-treated cells (Figure 2C).Finally, we evaluated NF-κB transcriptional activity using luciferase reporter assays. pNF-κB-Luc containing five tandem repeats of an NF-κB consensus binding site or pRL-TK as the normalization control were transiently expressed for 24 hours before LPS stimulation. Measurements of the luminescence intensity showed that TSG-6 inhibited LPS-induced NF-κB activation (Figure 2D). Taken together, the results suggested that TSG-6 was actively involved in the regulation of TLR4-mediated NF-κB signaling.

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