The Role Of Interleukin-22 And Interleukin-33 In The Immune Rejection Response Of Corneal Transplantation In Rat

BackgroundAs we all know, corneal disease has has ranked second in all causes leading to blindness in China. Currently, keratoplasty is the most efficient or even the only treatment of blindness, which is also the most successful solid organ transplantation owning to ’immune privilege’ and ’ACAID’.However, the immune rejection response of corneal transplantation is the important reason for the failure of corneal transplantation. Studies suggest that the corneal transplantation immunology is a multifactorial process with participation of a variety of immune cells and activated factor. Along with the development in immunology, people are gradually getting profound understanding of the mechanism of the immune rejection response at cellular and molecular levels. To understand the role and mechanism of a variety of immune cells and cytokines may contribute to the clinical diagnosis, prevention and treatment of corneal allograft rejection.Recently,some novel cytokines have been discovered and proven to participate in the immunologic process of corneal graft rejection.Interleukin-22(IL-22) and interleukin-33(IL-33) are newly found cytokines, belonging to IL-10 and IL-1 family respectively. IL-22 binding to IL-22R1-IL10R2 complex leads to the activation of a cascade of downstream signal pathways, including Jenus kinase (JAK)1,Tyrosine kinase (TYK)2, Signal Transducer And Activator Of Transcription 3(STAT3) and mitogen-activated protein kinase (MAPK) pathways (MEK-ERK-RSK,JNK/SAPK and p38 kinase).As a newly cytokine, IL-22 involves in the immunopathogenesis of a variety of diseases extensively, including chronic inflammatory, autoimmune diseases, tumor and transplantation immunology.IL-33 binding to ST2L leads to the agglomeration of MyD88, IRAKI, IRAK4 and TRAF6 and subsequent activation of NF-κB and MAPK pathways (ERK1, ERK2, JNK1 and p38 kinase). In addition,IL-33 binding to ST2L can also induce upregulation of MHCⅡ and CD86 on DCs, which contribute to enhance the antigen presenting function of DCs. Current studies have demonstrated that IL-33 is related to a variety of diseases, most notably autoimmune diseases, allergic and cardiovascular disease.As novel members of cytokine family, IL-22 and IL-33 both have an important role to play in innate and adaptive immunity and have been widely researched in a variety of diseases, the effect and significance of which on organ transplantation have received significant attention. So far,the potential role of IL-22 and IL-33 in corneal allograft rejection has not been reported at home and abroad. Therefore, this study is trying to observe the expression of IL-22mRNA and IL-33mRNA in corneas and clarify their potential role and mechanism through establishing penetrating keratoplasty models,which may provide new insights into clinical prevention and treatment of corneal rejection in the future.Part Ⅰ The role of IL-22 in the corneal allograft rejection in ratObjectiveTo explore the expression of IL-22mRNA in the corneal graft and clarify the role of IL-22 in the allograft rejection after keratoplasty in rat.Methods1.Modeling and grouping:SPF SD rats and Wistar rats were selected as donors and recipients respectively to establish penetrating keratoplasty models.We distributed normal Wistar rats into group A, the rats for autologous corneal transplantation into group B, rats for allograft corneal transplantation into group C, rats for allograft corneal transplantation with TobraDex treatment into group D.Allograft in group D were treated with TobraDex after the operation,2 times a day, each 1 drop, a total of 2 weeks.2.Clinical observation and scoring criteria:All grafts were examined by slit-lampmicroscopy daily to calculate the rejective indexes (RIs) according to opacity, edema, and neovascularization within 14 days after transplantation and once every other day after 14 days, a total of 30 days.Corneal opacity(0-4),edema(0-2), and neovascularization(0-4) were graded according to Larkin standard:Opacity (0: completely transparent; 1:mild loss of transparency; 2:moderative loss of transparency, but iris vessels is visible on retroillumination; 3:iris vessels not visible,but pupil outline visible; 4:pupil outline not visible); Edema(0:no edema; 1:moderate edema; 2:marked edema with obvious graft thickening); Neovascularization (0:without vascularization of graft; 1:vessel growth to 25% of graft radius in any quadrant; 2:vessel growth to 50% of graft radius; 3:vessel growth to 75% of graft radius; 4:vessel growth to centre of graft radius).When the RI grade was 5 or opacity grade reached 3, rejection was acknowledged.3.Histopathological examination:Three eyeballs in each group were randomly selected at 5 and 14 days after keratoplasty for histopathological examination, the specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into serial 5μm-thick slices with HE staining, then observed and photographed them under optical microscope.4. RT-PCR:Five corneal grafts in each group were randomly selected at 5 and 14 days after keratoplasty stored at -80℃ for RT-PCR to detect the expression of IL-22mRNA and AhRmRNA.Results1.Clinical observation:During the first 3 days after keratoplasty, mild edema was observed in corneal grafts of the 3 operation groups, neovascularization was observed at the corneal limbus in group B and C but not group D. Edema and opacity of the allografts in group B and D became decreased over time, on the contrary became aggravated and until entirely opaque with obvious graft thickening in group C. In addition, neovascularization extended progressively to the centre of grafts in group B and C by day 14.Nevertheless,neovascularization was limited to the corneal bed in group D.2.Mean survival time(MST):There was apparent discrepancy in MST between the grafts in group C (10.13±0.44) d and D (18.00±0.66) d (P<0.01).3.The corneal endothelium in normal rat cornea (A) was composed of 5-6 layers of cells,the stroma fibers were orderly,neither edema nor infiltration of inflammatory cells.However, a typical inflammatory lesion of grafts was observed in group C,such as marked edema with obvious graft thickening,disorderly arrangement of the stroma fibers,which was apparent at 14 day after operation.While the corneal structure was roughly normal in both group B and D,with stromal infiltration of a few inflammatory cells.4.The expression of IL-22mRNA was observed in both normal corneas(A) and isografts(B). There was a marked augment in the expression of IL-22mRNA in group C, but a notable decrease in group D (P<0.05).In addition, the expression of AhRmRNA followed a similar trend.ConclusionThe results suggested that IL-22 could take part in the development of graft rejection after corneal transplantation; Moreover, AhR was shown to be crucial for IL-22 expression.Part Ⅱ The role of IL-33 in the immune rejection response of corneal transplantation in ratObjectiveTo study the expression level of IL-33 in the corneal grafts and clarify the effect of IL-33 on the immune rejection response of corneal transplantation.Methods1.Modeling and grouping:SPF SD rats and Wistar rats were selected as donors and recipients respectively to establish penetrating keratoplasty models.We distributed normal Wistar rats into group A, the rats for autologous corneal transplantation into group B, rats for allograft corneal transplantation into group C, rats for allograft corneal transplantation with TobraDex treatment into group D.Allograft in group D were treated with TobraDex after the operation,2 times a day, each 1 drop, a total of 2 weeks.2.Clinical observation and scoring criteria:All grafts were examined by slit-lampmicroscopy daily to calculate the rejective indexes (RIs) according to opacity, edema, and neovascularization within 14 days after transplantation and once every other day after 14 days, a total of 30 days.Corneal opacity(0-4),edema(0-2), and neovascularization(0-4) were graded according to Larkin standard:Opacity (0: completely transparent; 1:mild loss of transparency; 2:moderative loss of transparency, but iris vessels is visible on retroillumination; 3:iris vessels not visible,but pupil outline visible; 4:pupil outline not visible); Edema(0:no edema; 1:moderate edema; 2:significant edema with obvious graft thickening); Neovascularization (0:without vascularization of graft; 1:vessel growth to 25% of graft radius in any quadrant; 2:vessel growth to 50% of graft radius; 3:vessel growth to 75% of graft radius; 4:vessel growth to centre of graft radius).When the RI grade was 5 or opacity grade reached 3, rejection was acknowledged.3.Histopathological and Immunohistochemistry:Three eyeballs in each group were randomly selected at 5 and 14 days after keratoplasty for histopathological and immunohistochemistry examination, the specimens were fixed with 4% paraformaldehyde, embedded in paraffin and cut into serial 5μm-thick slices with HE staining; Besides, the expression of ST2 was detected by immunohistochemical staining, then observed and photographed them under optical microscope.4. RT-PCR:Five corneal grafts in each group were randomly selected at 5 and 14 days after keratoplasty stored at -80℃ for RT-PCR to detect the expression of IL-33mRNA and ST2mRNA.Results1.Clinical observation:During the first 3 days after keratoplasty, slight edema was observed in corneal grafts of the 3 operation groups,neovascularization was observed at the corneal limbus in group B and C but not group D. Edema and opacity of the allografts in group B and D became decreased over time, on the contrary became aggravated and until entirely opaque with obvious graft thickening in group C. In addition, neovascularization extended progressively to the centre of grafts in group B and C by day 14.Nevertheless,neovascularization was limited to the corneal limbus in group D.2.Mean survival time(MST):There was apparent discrepancy in MST between the grafts in group C (10.13±0.44)d and D (18.00±0.66)d(χ2=16.442, P=0.000).3.The corneal endothelium in normal rat cornea (A) was composed of 5-6 layers of cells,the stroma fibers were orderly, neither edema nor infiltration of inflammatory cells.However, a typical inflammatory lesion of grafts was observed in group C,such as marked edema with obvious graft thickening,disorderly arrangement of the stroma fibers, which was apparent at 14 day after operation. While the corneal structure was roughly normal in both group B and D,with stromal infiltration of a few inflammatory cells. 4.The expression of IL-33mRNA、ST2mRNA and ST2 protein were detected in both group A and B. There was a significant augment in group C,but a notable decrease in group D (P<0.05)ConclusionIt is concluded that IL-33 may play an important role in the development of immune rejection after corneal transplantation.