The Effects And Mechanism Of Immune Microenvironment On Regulatory T Cells Function During Corneal Allograft Rejection

Background: CD4+CD25+Foxp3+ regulatory T cells(Tregs)are a subpopulation of T cells with negative immunoregulatory function,and play critical role in establishing and maintaining transplantation tolerance.However,the functional changes of Tregs during corneal allograft rejection remains unknown.Purpose: This study was mainly to investigate phenotypic and functional changes of Tregs during corneal allograft rejection,and further explore the possible mechanism,to provide theoretical guidance for prevention and treatment of corneal allograft rejection.Methods:(1)Changes in Number and Function of Regulatory T Cells during Corneal Allograft Rejection A mice model of full-thickness penetrating keratoplasty(PKP)was established.The changes in number and function of Tregs during corneal allograft rejection were analyzed through the following experiments:(1)Single cell suspension of anterior segment was prepared,and the percentage of Tregs was detected by flow cytometry;(2)The anterior segment tissue was collected,the levels of Forkhead box protein P3(Foxp3)were examined by quantitative real-time polymerase chain reaction(q RT-PCR)and flow cytometry;(3)Tregs were cocultured with effector T cells(Teffs),and the supernate of rejected anterior segment was used to simulate the allograft rejection microenvironment.The proliferation of Teffs was detected through flow cytometry.(2)The Mechanism of Immune Microenvironment Affecting Regulatory T Cells Function Tregs and Teffs were purified from normal BALB/c spleen cells using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit(Miltenyi Biotec,Auburn,CA),respectively.A coculture model of Teffs and Tregs was used to investigate the mechanism of immune microenvironment on Tregs' function:(1)Western blot(WB)and flow cytometry were performed to test the expression of caspase-8 and Foxp3;(2)After inhibiting the activity of caspase-8 by small molecular compound,functional impact of caspase-8 inhibitor on Tregs were assessed by flow cytometry;(3)WB was used to analyze the effect of caspase-8 inhibitor on Foxp3 expression of Tregs.(3)The Effects of Tumour Necrosis Factor-? Neutralizing Antibody on Foxp3 and Caspase-8 Expression during Corneal Allograft Rejection The mice after PKP received tumour necrosis factor-?(TNF-?)neutralizing antibody by subconjunctival injection,and the effects of TNF-? neutralizing antibody on corneal allograft rejection were evaluated as follows:(1)All grafts underwent slit-lamp examination,graft survival time was recorded,and graft survival curve was drawn using Kaplan-Meier statistical method;(2)Immunofluorescence staining,enzyme-linked immunosorbent assay(ELISA)and q RT-PCR were executed to test pathological changes of the grafts;(3)Protein levels of caspase-8 and Foxp3 in grafts were determined by WB after TNF-? neutralizing antibody treatment.(4)The Effects of Caspase-8 Inhibitor on Corneal Allograft Rejection After PKP,mice were suffered subconjunctival injection of caspase-8 inhibitor,to assess the effects of caspase-8 inhibitor on corneal allograft rejection:(1)Graft status was examined under a slit-lamp biomicroscope,graft survival time was statistically analyzed using Kaplan-Meier method;(2)The effects of caspase-8 inhibitor on allograft pathological changes were detected by hematoxylin and eosin staining(HE)and q RT-PCR;(3)The activity of caspase-8 and expression of Foxp3 in allografts were investigated by q RT-PCR,immunofluorescence staining and WB.Results:(1)The Relative Quantity of Regulatory T Cells during Corneal Allograft Rejection was Increased,but its Immunosuppressive Function was Impaired.Compared with the syngenic group,the relative number of Tregs in the anterior segment significantly increased in the allogenic group.Tregs in the allogenic group showed higher Foxp3 m RNA level but lower Foxp3 protein level than that in the syngenic group.In vitro coculture experiment showed impaired immunosuppressive function of Tregs in immune microenvironment,and TNF-? neutralizing antibody rescued the immunosuppressive function of Tregs.(2)Impaired Immunosuppressive Function of Regulatory T Cells was Correlated with Upregulation of Caspase-8 The findings from WB and flow cytometry test manifested the increased expression of activated caspase-8 and decreased expression of Foxp3 protein in Tregs pre-treated with supernate of rejected anterior segment.The results suggested that caspase-8 may be related to the functional changes of Tregs.Furthermore,we found that blockade of caspase-8 activity restored the immunosuppressive ability of Tregs through stabilizing Foxp3 protein.(3)TNF-? Neutralizing Antibody Treatment could Significantly Delay Corneal Allograft Rejection Immunofluorescence staining and ELISA displayed high levels of TNF-? in ocular microenvironment during corneal allograft rejection.In vivo study showed that neutralization of TNF-? could not only effectively prolong the survival of the corneal allograft,but also dramatically alleviate the inflammatory response.We also found that after treatment with TNF-? neutralizing antibody,caspase-8 level in corneal grafts decreased but Foxp3 level increased,suggesting that TNF-? neutralizing antibody may exert its anti-rejection effect by improving Tregs' immunosuppressive activity.(4)Blocking the Activity of Caspase-8 could Remarkably Prolong the Survival of the Grafts Our results revealed that both total and activated caspase-8 were significantly elevated in rejected allografts.Compared with untreated group,grafts in caspase-8 inhibitor treated group remained transparent.The infiltration of inflammatory cells and expression of inflammatory cytokines were significantly reduced,and the survival time of allografts was prolonged.We also found that the levels of Foxp3 in corneal allografts were significantly increased after caspase-8 inhibitor intervention,suggesting that the anti-rejection effect of caspase-8 inhibitor maybe related with the enhanced immunosuppressive function of Tregs.Conclusions:(1)In the case of immunological rejection after corneal transplantation,the anterior chamber microenvironment leads to the impaired immunosuppressive function of Tregs in the intraocular tissues.(2)Changes of microenvironment after corneal transplantation could reduce the stability of Foxp3 protein by enhanced caspase-8 activity in Tregs,thus damaging Tregs' immunosuppressive function.(3)TNF-? neutralizing antibody as well as caspase-8 inhibitor could substantially delay immune rejection after PKP,and the underlying mechanism maybe correlated with the restored Foxp3 expression.