Bursopentin (BP5) protects dendritic cells from lipopolysaccharide-induced oxidative stress for immunosuppression.
Qin T1, Yin Y1, Yu Q1, Yang Q1.
Key Lab of Animal Physiology and Biochemistry of China's Department of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, People's Republic of China.
Dendritic cells (DCs) play a vital role in the regulation of immune-mediated inflammatory diseases. Thus, DCs have been regarded as a major target for the development of immunomodulators. However, oxidative stress could disturb inflammatory regulation in DCs. Here, we examined the effect of bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, on the protection of DCs against oxidative stress for immunosuppression. BP5 showed potent protective effects against the lipopolysaccharide (LPS)-induced oxidative stress in DCs, including nitric oxide, reactive oxygen species and lipid peroxidation. Furthermore, BP5 elevated the level of cellular reductive status through increasing the reduced glutathione (GSH) and the GSH/GSSG ratio. Concomitant with these, the activities of several antioxidative redox enzymes, including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), were obviously enhanced. BP5 also suppressed submucosal DC maturation in the LPS-stimulated intestinal epithelial cells (ECs)/DCs coculture system. Finally, we found that heme oxygenase 1 (HO-1) was remarkably upregulated by BP5 in the LPS-induced DCs, and played an important role in the suppression of oxidative stress and DC maturation. These results suggested that BP5 could protect the LPS-activated DCs against oxidative stress and have potential applications in DC-related inflammatory responses.
BP5 efficiently suppressed NO production in DCs.
DCs (1×106 cells/ml) were incubated with the indicated concentrations of BP5 for 2 h, and then incubated with or without LPS (100 ng/ml) for 22 h. Supernatants were collected and NO production was measured using the Griess reagent. Data shown are the means ± SD of three samples. *P < 0.05 in comparison with the LPS-only group; # P < 0.05 in comparison with the untreated group. All results are representative of three independent experiments.
Bursopentin (BP5) Protects Dendritic Cells from Lipopolysaccharide-Induced Oxidative Stress for Immunosuppression
PLoS One. 2015;10(2):e0117477.
BP5 impaired the production of ROS by LPS-stimulated DCs.
(A) DCs (1×106 cells/ml) were incubated with the indicated concentrations of BP5 for 2 h, and then incubated with or without LPS (100 ng/ml) for 22 h, and then incubated with 10 μM DCFH-DA at 37°C for 20 min. ROS production was detected by fluorescence microplate reader. H2O2 (50 μM) was used as a positive control. (B-C) DCs stimulated with LPS before, at the same time as, or after BP5 (50 μg/ml) incubation. ROS production was detected by FACS. Data shown are the means ± SD of three samples. **P < 0.01, *P < 0.05 in comparison with the LPS-only group; # P < 0.05 in comparison with the untreated group. All results are representative of three independent experiments.
BP5 suppressed lipid peroxidation in the LPS-stimulated DCs.
DCs (1×106 cells/ml) were pretreated with or without BP5 for 2 h, and then exposed to LPS (100 ng/ml) or not for 22 h. The MDA contents in cell lysate supernatants were measured as described in Materials and Methods. Data shown are the means ± SD of three samples. *P < 0.05 in comparison with the LPS-only group; # P < 0.05 in comparison with the untreated group. All results are representative of three independent experiments.
BP5 modulated intracellular GSH, GSSG and the GSH/GSSG ratio in the LPS-stimulated DCs.
DCs (1×106 cells/ml) were pretreated with or without BP5. After 2 h, the cells were stimulated with LPS (100 ng/ml) or not for 22 h. The levels of (A) GSH, (B) GSSG and (C) GSH/GSSG ratio in the cells were measured as described in Materials and Methods. Data shown are the means ± SD of three samples. *P < 0.05 in comparison with the LPS-only group; # P < 0.05 in comparison with the untreated group. All results are representative of three independent experiments.
BP5 enhanced the activities of antioxidant enzymes in the LPS-treated DCs.
DCs (1×106 cells/ml) were pretreated with BP5 for 2 h, followed by stimulation with or without LPS (100 ng/ml). After 22 h, Intracellular levels of (A) GPx, (B) CAT and (C) SOD were measured using commercial kits. Data shown are the means ± SD of three samples. *P < 0.05 in comparison with the LPS-only group; # P < 0.05 in comparison with the untreated group. All results are representative of three independent experiments.
BP5 suppressed the DC maturation in the LPS-stimulated ECs/DCs coculture system.
Experimental setting to study the DC maturation in the ECs/DCs coculture system. (A) The scheme depicts: Caco-2 cells were grown on the filter to form a tight monolayer, and then DCs were cultured facing the basolateral side of Caco-2 cells on the bottom of the filter for 4 h. Pretreatment of BP5 on the basolateral DCs for 2 h, medium or LPS (300 ng/ml) was incubated on the apical side of the ECs for 22 h, then the basolateral DCs and culture supernatants were collected. (B) TEER was measured by a Millicell-ERS epithelial voltohmmeter (Millipore) at indicated time. (C-D) The filters were fixed with 4% paraformaldehyde and then processed to immunofluorescence stain for CLSM. Three-dimensional rendering of representative fields was obtained with Imaris 7.2 software, DCs (red, CD11c), tight junction of ECs (green, occludin). Submucosal DCs sent dendrites (arrows) to creep through ECs in response to LPS but not medium. (E-F) Supernatants were collected and NO production was measured using the Griess reagent. (G-H) TNF-α released from basolateral supernatants was measured by ELISA. (F, H) DCs stimulated with LPS before, at the same time as, or after BP5 (100 μg/ml) incubation. (I-L) The expressions of CD86 and MHCII on DCs were analyzed by FACS. Data shown are the means ± SD of three samples. *P < 0.05; **P < 0.01. SN, supernatants. To confirm the results, we repeated these experiments three times.
BP5 upregulated the expression of HO-1 protein in the LPS-induced DCs.
DCs (1×106 cells/ml) were treated with the indicated concentrations of BP5 for 2 h, and then incubated with or without LPS (100 ng/ml) for 22 h. (A) HO-1 levels were assessed by western blotting. (B) Quantification of the blots. (C-H) DCs were pretreatment with BP5 (100 μg/ml) in the presence or absence of Snpp (25 μM) or Copp (50 μM) for 2 h, and then incubated with or without LPS (100 ng/ml) for 22 h. (C) Supernatants were collected and NO production was measured using the Griess reagent. (D) TNF-α released from supernatants was measured by ELISA. DCs were harvested and the expressions of CD80 (E, G) and CD86 (F, H) were analyzed by FACS. Data shown are the means ± SD of three samples. *P < 0.05; **P < 0.01. All results are representative of three independent experiments.
Catalase/immunology
Catalase/metabolism
Dendritic Cells/immunology
Dendritic Cells/pathology*
Glutathione/immunology
Glutathione/metabolism
Glutathione Peroxidase/immunology
Glutathione Peroxidase/metabolism
Immune Tolerance/drug effects*
Lipopolysaccharides/toxicity*
Oligopeptides/pharmacology*
Oxidative Stress/drug effects*
Oxidative Stress/immunology
Reactive Oxygen Species/immunology
Reactive Oxygen Species/metabolism*
Superoxide Dismutase/immunology
Superoxide Dismutase/metabolism
Lipopolysaccharides
Oligopeptides
cysteinyl-lysyl-aspartyl-valyl-tyrosine
Catalase
Glutathione Peroxidase
Superoxide Dismutase