Lentivirus-mediated PD-L1 overexpression in bone marrow-derived dendritic cells induces immune tolerance in a rat keratoplasty model.

PURPOSE: The side effects of immune suppressants on immune rejection have become increasingly apparent after keratoplasty. To find out new alternative immunotherapy strategies, we studied the role of programmed death-1 (PD-1) and its ligand (PD-L1) co-stimulatory pathway in inducing immune tolerance of rat keratoplasty. METHODS: The PD-L1 protein was constitutively overexpressed via lentiviral transduction in bone marrow-derived dendritic cells (BMDCs) from rats, then infused via the tail vein into rats before undergoing keratoplasty. Western blot analysis of PD-L1 protein confirmed the effectiveness of lentivirus-mediated. The phenotype of immature BMDC was confirmed by flow cytometry analysis with CD80, CD86, CD11c and MHC-II antibodies. To investigate the mechanism of the immune tolerance induced by BMDCs transfusion, PD-L1, IFN-γ and IL-17 in serum and cell culture supernatant were assessed by ELISA and qPCR. RESULTS: After LPS stimulation, immature dendritic cells with over-expression of PD-L1 still showed high expression of PD-L1(p < 0.001), and low expression of IL-17 and IFN-γ (p < 0.001), which reduced neovascularization (p < 0.05), and prolonged the survival after corneal implants. CONCLUSION: Immature DC cells with overexpression of PD-L1 have low ability to activate T cells，which is a potential treatment for avoiding graft rejection by promoting natural immunosuppression. This cellular treatment is expected to reduce the use of immune suppressants and the occurrence of side effects.

Protective effect of histatin 1 against ultraviolet-induced damage to human corneal epithelial cells.

The aim of the present study was to investigate the role of histatin 1 (Hst1) in human corneal epithelial cells (HCECs) exposed to ultraviolet (UV) radiation. Prior to UV irradiation for various durations, HCECs were pre-treated with different concentrations of Hst1 and the effect on cell apoptosis and cell viability were examined by flow cytometry, alamarBlue® and MTT assays to determine the optimal concentration of Hst1 and UV dose. Cells were then subjected to quantitative PCR, ELISA and western blot analysis to determine the expression of cell damage-associated genes. HCECs exposed to UV light for 1 h displayed decreased viability when compared to that of control cells, and a 3 h UV exposure markedly increased the apoptotic rate of HECEs, while apoptosis was inhibited by pre-treatment with Hst1. UV radiation downregulated expression of insulin-like growth factor (IGF)-1 and B-cell lymphoma 2 (Bcl-2), while it upregulated Bcl-2-associated X protein (Bax) expression. Hst1 protected HCECs against UV-induced damage by upregulating the expression of IGF-1 protein and increasing the Bcl-2/Bax ratio. In conclusion, Hst1 may prevent UV-induced damage to corneal epithelial tissue injury and promote its healing.

Inhibition of RelA expression via RNA interference induces immune tolerance in a rat keratoplasty model.

RelA, the most important regulator of NF-kB activity, and its mechanisms in keratoplasty immune rejection have not been fully investigated. In the present study, lentivirus-mediated silencing of RelA expression in a bone marrow-derived dendritic cell (BMDC) model was tested. The BMDCs were transfected with RelA-shRNA to induce an immature, maturation-resistant and tolerogenic phenotype, while not significantly changing IFN-γ, IL-10 and IL-17 expression. A fully allogeneic rat cornea transplant model was established for in vivo studies. The allograft mean survival time (MST) of lv-shRelA-DC injection groups were significantly longer than the untreated BMDC group and control group. The corneal opacity and neovascularization scale of the lv-shRelA-DC injection groups were slight compared to pair control others. Postoperative flow cytometric analysis revealed that the percentage of Treg positive cells was dramatically increased in animals that received an lv-shRelA-DC injection. ELISA and qRT-PCR analyses of serum showed that IFN-γ and IL-17 expression were suppressed by lv-shRelA-DC treatement. In vivo experiments demonstrated that IL-10 induced immunosuppression was partly attributed to injection of lv-shRelA-DC throughout the experiment, differing from the general anti-inflammatory factors. Luciferase and Chromatin IP evaluation showed that RelA knockdown in BMDCs significantly reduces DNA binding to IFN-γ, IL-10 and the IL-17 promoter and inhibited of transcriptional activity. Taken together, this study illustrates a significant role of RelA in mediating the corneal neovascularization by affecting IL-17 expression. Our comprehensive analysis shows that the significant role of RelA provides a novel and feasible therapeutic approach for the prevention of corneal allograft rejection.