The Pathological,functional Changes Of Iris During Corneal Allograft Rejection And Its Related Mechanism Study

Objective: To investigate the pathological and functional changes of the iris during corneal allograft rejection as well as the mechanism underlying them,and further reveal the possible mechanism of implanting Cyclosporine A drug delivery system(Cs A DDS)into the anterior chamber implicated in the management of corneal allograft rejection.Methods:(1)The pathological changes of iris during corneal allograft rejection Syngenic(donor BALB/C to recipient BALB/C mice)and allogenic(donor C57BL/6 to recipient BALB/C mice)full-thickness corneal transplantations were performed.When allograft rejection occurred at 2 weeks after post-surgery,the pathological changes were evaluated as follows: the vascular permeability of iris was detected by angiography using FITC-Dextran and Evans Blue,and the pathological changes of aqueous humor was inspected by aqueous humor smear Wright Giemsa staining;quantitative real-time PCR(q RT-PCR)was performed to quantify the levels of pro-inflammatory cytokines(such as IL-1β,IL-6).Furthermore,the infiltration of immune cells and NF-κB activity(the levels and localization of p65,and phosphorylated p65)were evaluated by immunofluorescence staining.(2)The influence of immune microenvironment on the iris pigment epithelial cells(IPEs)and its possible mechanism The functional change of IPEs was investigated using a coculture model of mouse IPEs and spleen lymphocytes stimulated with interferon-γ(IFN-γ).The IPEs were randomly divided into four groups: a normal group without stimulation,an IFN-γ group,an IFN-γ+Cs A group,and an IFN-γ+NF-κB inhibitor(JSH23)group.The IPEs were pre-treatred for 48 h,and then harvested and cocultured with spleen cells pre-labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester(CFSE)for 96 h.The flow cytometry was performed to analyze the proliferation of CD4+ T cells.The protein levels of IFN-γ and IL-2 in supernatant were tested by enzyme linked immunosorbent assay(ELISA).The phosphorylation of p65 in IPEs was determined by Western blot to evaluate the NF-κB activity.(3)The effect of Cs A DDS implantation on iris and its possible mechanism for the prevention of corneal allograft rejection Corneal neovascularization was induced in the right eye of the rabbit.The New Zealand white rabbits were randomly divided into a control group receiving PKP without treatment(Con),and a Cs A DDS group receiving PKP and Cs A DDS implanting into their anterior chambers.The clinical outcomes of corneal grafts and irises were evaluated,and the testing procedures were as follows.Grafts survival was monitored routinely by slit-lamp biomicroscopy,and the pathological changes of corneal grafts were assessed by the in vivo laser scanning confocal microscopy,anterior segment optical coherence tomography(AS-OCT)and Hematoxylin Eosin(HE)staining.The iris HE staining and aqueous humor Wright Giemsa staining were executed to evaluate the pathological changes of the iris and aqueous humor,respectively.The expressions of cytokines and costimulatory molecules in the iris were quantified by q RT-PCR.The levels of IL-2 in aqueous humor were determined by ELISA.Immunofluorescence staining was performed to assess the immune cell infiltration and NF-κB activity in iris.Results:(1)Aggravated inflammatory response of iris was observed in corneal allograft rejection Compared with the Syngenic(Syn)group,the vascular lumen and vascular permeability of iris in allogenic(Allo)group were significantly increased.The levels of pro-inflammatory cytokines such as IL-1β,IL-6,IL-12 and IL-15 were pronouncedly elevated both in the irises and the grafts from the Allo group than in Syn group,as well as much more CD4+ T cells’ infiltration.The immunofluorescence staining showed increased levels of p65 and phosphorylated p65 in the Allo group,as well as their nuclear translocation,which indicated an increased NF-κB activity.(2)Increased NF-κB activity in IPE cells impaired its immunosuppressive function.IPEs pre-treated with IFN-γ displayed impaired immunosuppressivity,characterized by increased CD4+ T cells proliferation and higher levels of IFN-γ and IL-2 in supernatant when co-cultured with spleen cells.While Cs A could significantly restore its impaired immunosuppressivity caused by IFN-γ,as indicated by decreased CD4+ T cells proliferation,as well as declined levels of IFN-γ and IL-2 in supernatant.Mechanically,Western blot revealed increased NF-κB activity(increased phosphorylated p65)in IFN-γ conditioned IPEs.However,Cs A could significantly suppress its NF-κB activity(decreased phosphorylated p65).Furthermore,blocking NF-κB activity using NF-κB inhibitors(JSH23)could also significantly counterbalance the impaired immunosuppressivity caused by IFN-γ.The results indicated Cs A could enhance the IPEs’ immunosuppressive ability via suppressing NF-κB activity.(3)Implantation of Cs A DDS into anterior chamber could effectively inhibit the inflammatory response in iris Compared with the control group,Cs A DDS implanted into the anterior chamber could significantly inhibit the graft’s immune response,as well as the pathological changes in iris and aqueous humor.The iris HE staining and aqueous humor Wright Giemsa staining showed that the vascular lumen and the immune cells in iris and aqueous humor were both significantly decreased in the Cs A DDS group than in Con group.The decreased expression of pro-inflammatory cytokines(such as IFN-γ,IL-6,and IL-2)and costimulatory molecules(CD80,and CD86)were found in the iris of the Cs A DDS group,as well as less CD4 positive cells in the iris.We also found that the IL-2 level in aqueous humor was much lower in the Cs A DDS group than in Con group by ELISA(P<0.01).Moreover,compared with Con group,the expression and nuclear translocation of p65 in iris were both significantly reduced after treatment with Cs A DDS.The results suggested that Cs A DDS could significantly alleviate the iris and aqueous humor’s pathological changes through inhibiting NF-κB activation.Conclusion:(1)The aggravated inflammatory responses and increased NF-κB activity in iris were observed in corneal allografts rejection.(2)Increased NF-κB activity impaired its immunosuppressivity in IFN-γ-conditioned IPEs.Cs A counterbalanced its impaired immunosuppressivity via suppressing the NF-κB activity.(3)In addition to effectively alleviating the immune responses in the corneal graft,Cs A DDS could also significantly prevent allograft rejection through mitigating the inflammatory responses in iris.