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Klotho sensitive regulation of dendritic cell functions by vitamin E

View Article: PubMed Central - PubMed

ABSTRACT

Background: Dendritic cells (DCs) are the most potent professional antigen-presenting cells for naive T cells to link innate and acquired immunity. Klotho, an anti-aging protein, participates in the regulation of Ca2+ dependent migration in DCs. Vitamin E (VitE) is an essential antioxidant to protect cells from damage and elicits its inhibitory effects on NF-κB-mediated inflammatory response. However, the roles of VitE on mouse DC functions and the contribution of klotho to those effects both are unknown. The present study explored the effects of VitE on klotho expression, maturation, ROS production and migration in DCs.

Methods: The mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were stimulated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM). RT-PCR and immunoprecipitation methods were employed to determine klotho expression, ELISA to determine cytokine release, flow cytometry to analyze number of CD86+CD11c+ cells, the intracellular expression of cytokines and reactive oxygen species (ROS) production and a transwell migration assay to trace migration.

Results: Klotho transcript level and this hormone secretion in DC supernatant were enhanced by VitE treatment and further increased in the presence of NF-κB inhibitor Bay 11-7082 (10 µM). Moreover, VitE treatment inhibited IL-12p70 protein expression of, ROS accumulation in and CCL21-dependent migration of LPS-triggered mature DCs, these effects were reversed following klotho silencing.

Conclusion: The up-regulation of klotho by VitE could contribute to the inhibitory effects of VitE on NF-κB-mediated DC functional maturation. The events might contribute to immunotherapeutic effect of VitE on the pathophysiology of klotho-related disease.

No MeSH data available.


Related in: MedlinePlus

Effect of VitE on klotho expression. a Original Western blot of DCs were either treated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM, 2 h) or left untreated (control). Protein extracts were analyzed by direct Western blotting using antibodies directed against p-IκBα and GAPDH. b Arithmetic mean ± SEM (n = 4) of the abundance of p-IκBα protein as the ratio of p-IκBα/GAPDH. c Arithmetic mean ± SEM (n = 5) of klotho transcript level is shown prior to control (white bar) and 5 h following incubation with VitE (500 µM) either in the absence (black bar) or presence of NF-κB inhibitor Bay 11-7082 (10 µM, grey bar). d Immunoprecipitation of klotho in DC supernatants is shown prior to control (1st panel) and 5 h following treatment with VitE (500 µM) either in the absence (2nd panel) or presence (3rd panel) of Bay 11-7082 (10 µM). Immunoprecipitates were stained for klotho and GAPDH antibodies. e Arithmetic mean ± SEM (n = 5) of the abundance of klotho protein as the ratio of klotho/GAPDH. *(p < 0.05) represent significant difference from control DCs, and #(p < 0.05) indicates significant difference from VitE-treated DCs (ANOVA)
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Fig1: Effect of VitE on klotho expression. a Original Western blot of DCs were either treated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM, 2 h) or left untreated (control). Protein extracts were analyzed by direct Western blotting using antibodies directed against p-IκBα and GAPDH. b Arithmetic mean ± SEM (n = 4) of the abundance of p-IκBα protein as the ratio of p-IκBα/GAPDH. c Arithmetic mean ± SEM (n = 5) of klotho transcript level is shown prior to control (white bar) and 5 h following incubation with VitE (500 µM) either in the absence (black bar) or presence of NF-κB inhibitor Bay 11-7082 (10 µM, grey bar). d Immunoprecipitation of klotho in DC supernatants is shown prior to control (1st panel) and 5 h following treatment with VitE (500 µM) either in the absence (2nd panel) or presence (3rd panel) of Bay 11-7082 (10 µM). Immunoprecipitates were stained for klotho and GAPDH antibodies. e Arithmetic mean ± SEM (n = 5) of the abundance of klotho protein as the ratio of klotho/GAPDH. *(p < 0.05) represent significant difference from control DCs, and #(p < 0.05) indicates significant difference from VitE-treated DCs (ANOVA)

Mentions: The activation of NF-κB signaling has been determined to be suppressed by treatment of cells with VitE [15]. To explore the modulation effects of VitE on NF-κB signaling in mouse DCs, bone marrow cells were cultured with GM-CSF for 8 days to attain BMDCs and subsequently treated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM) for 2 h. In this study, LPS stimulation led to enhanced level of phosphorylated IκBα, the effect was significantly suppressed when VitE was present in the cell culture (Fig. 1a, b). Next, experiments were performed to examine the roles of VitE and NF-κB signaling on klotho expression. RT-PCR disclosed the upregulation of klotho mRNA expression following treatment of DCs with VitE for 5 h (Fig. 1c). Immunoprecipitation confirmed the expression of klotho protein in culture supernatant and revealed that the abundance of klotho protein was significantly enhanced by exposure of DCs to VitE (Fig. 1d, e). The further increase of klotho transcript and protein levels were observed by using pharmacological inhibition of NF-κB signaling pathway with Bay 11-7082 (10 μM, Fig. 1c–e). Thus, VitE participated in promoting klotho expression through suppressing activation of NF-κB signaling.Fig. 1


Klotho sensitive regulation of dendritic cell functions by vitamin E
Effect of VitE on klotho expression. a Original Western blot of DCs were either treated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM, 2 h) or left untreated (control). Protein extracts were analyzed by direct Western blotting using antibodies directed against p-IκBα and GAPDH. b Arithmetic mean ± SEM (n = 4) of the abundance of p-IκBα protein as the ratio of p-IκBα/GAPDH. c Arithmetic mean ± SEM (n = 5) of klotho transcript level is shown prior to control (white bar) and 5 h following incubation with VitE (500 µM) either in the absence (black bar) or presence of NF-κB inhibitor Bay 11-7082 (10 µM, grey bar). d Immunoprecipitation of klotho in DC supernatants is shown prior to control (1st panel) and 5 h following treatment with VitE (500 µM) either in the absence (2nd panel) or presence (3rd panel) of Bay 11-7082 (10 µM). Immunoprecipitates were stained for klotho and GAPDH antibodies. e Arithmetic mean ± SEM (n = 5) of the abundance of klotho protein as the ratio of klotho/GAPDH. *(p < 0.05) represent significant difference from control DCs, and #(p < 0.05) indicates significant difference from VitE-treated DCs (ANOVA)
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Fig1: Effect of VitE on klotho expression. a Original Western blot of DCs were either treated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM, 2 h) or left untreated (control). Protein extracts were analyzed by direct Western blotting using antibodies directed against p-IκBα and GAPDH. b Arithmetic mean ± SEM (n = 4) of the abundance of p-IκBα protein as the ratio of p-IκBα/GAPDH. c Arithmetic mean ± SEM (n = 5) of klotho transcript level is shown prior to control (white bar) and 5 h following incubation with VitE (500 µM) either in the absence (black bar) or presence of NF-κB inhibitor Bay 11-7082 (10 µM, grey bar). d Immunoprecipitation of klotho in DC supernatants is shown prior to control (1st panel) and 5 h following treatment with VitE (500 µM) either in the absence (2nd panel) or presence (3rd panel) of Bay 11-7082 (10 µM). Immunoprecipitates were stained for klotho and GAPDH antibodies. e Arithmetic mean ± SEM (n = 5) of the abundance of klotho protein as the ratio of klotho/GAPDH. *(p < 0.05) represent significant difference from control DCs, and #(p < 0.05) indicates significant difference from VitE-treated DCs (ANOVA)
Mentions: The activation of NF-κB signaling has been determined to be suppressed by treatment of cells with VitE [15]. To explore the modulation effects of VitE on NF-κB signaling in mouse DCs, bone marrow cells were cultured with GM-CSF for 8 days to attain BMDCs and subsequently treated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM) for 2 h. In this study, LPS stimulation led to enhanced level of phosphorylated IκBα, the effect was significantly suppressed when VitE was present in the cell culture (Fig. 1a, b). Next, experiments were performed to examine the roles of VitE and NF-κB signaling on klotho expression. RT-PCR disclosed the upregulation of klotho mRNA expression following treatment of DCs with VitE for 5 h (Fig. 1c). Immunoprecipitation confirmed the expression of klotho protein in culture supernatant and revealed that the abundance of klotho protein was significantly enhanced by exposure of DCs to VitE (Fig. 1d, e). The further increase of klotho transcript and protein levels were observed by using pharmacological inhibition of NF-κB signaling pathway with Bay 11-7082 (10 μM, Fig. 1c–e). Thus, VitE participated in promoting klotho expression through suppressing activation of NF-κB signaling.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Dendritic cells (DCs) are the most potent professional antigen-presenting cells for naive T cells to link innate and acquired immunity. Klotho, an anti-aging protein, participates in the regulation of Ca2+ dependent migration in DCs. Vitamin E (VitE) is an essential antioxidant to protect cells from damage and elicits its inhibitory effects on NF-&kappa;B-mediated inflammatory response. However, the roles of VitE on mouse DC functions and the contribution of klotho to those effects both are unknown. The present study explored the effects of VitE on klotho expression, maturation, ROS production and migration in DCs.

Methods: The mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were stimulated with LPS (100&nbsp;ng/ml) in the presence or absence of VitE (500&nbsp;&micro;M). RT-PCR and immunoprecipitation methods were employed to determine klotho expression, ELISA to determine cytokine release, flow cytometry to analyze number of CD86+CD11c+ cells, the intracellular expression of cytokines and reactive oxygen species (ROS) production and a transwell migration assay to trace migration.

Results: Klotho transcript level and this hormone secretion in DC supernatant were enhanced by VitE treatment and further increased in the presence of NF-&kappa;B inhibitor Bay 11-7082 (10&nbsp;&micro;M). Moreover, VitE treatment inhibited IL-12p70 protein expression of, ROS accumulation in and CCL21-dependent migration of LPS-triggered mature DCs, these effects were reversed following klotho silencing.

Conclusion: The up-regulation of klotho by VitE could contribute to the inhibitory effects of VitE on NF-&kappa;B-mediated DC functional maturation. The events might contribute to immunotherapeutic effect of VitE on the pathophysiology of klotho-related disease.

No MeSH data available.


Related in: MedlinePlus