Proinflammatory IL-1? Promotes Corneal Allograft Rejection Through Th17 Immune Response

Object:To evaluate the pathological role of interleukin-1?(IL-1?)during corneal allograft rejection using a mouse model of penetrating keratoplasty(PKP),and further investigate the mechanism of IL-1? involved in the pathogenesis of corneal allograft rejection.Methods:(1)The mice with PKP were randomly divided into four groups:a normal control group with no procedure performed,a syngeneic group,an allogeneic group,and an allogeneic group combined with anti-IL-1? antibody treatment.The effect of anti-IL-1? antibody on corneal allograft rejection was observed by slit lamp biomicroscopy.Real?time PCR and Western blot were used to quantify the expression of IL-1? and its receptor IL-1R1.Furtheremore,the expression of IL-1R1 and its distribution in corneal allografts were assessed by immunofluoerescence staining.(2)The wildtype mice and NLRP3 knockout mice were used for penetrating keratoplasty,respectively,and randomly seperated into three groups:a normal control group with no procedure performed,a syngeneic transplantation group,and an allogeneic transplantation group,respectively.Thereafter,the effect of NLRP3 inflammasome on corneal allograft rejection was evaluated by slit lamp biomicroscopy.The expression of NLRP3 in grafts were determined by Western Blot and Immunofluoerescence staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-17(IL-17)and Interferon-?(IFN-?)in corneal grafts.In addition,the NLRP3 inflammaome inhibitor Glubide was used to verify the effect of NLRP3 inflammasome on corneal allograft rejection.(3)Bone-marrow derived macrophages(BMDMs)were isolated and induced by macrophage-colony stimulating factor(M-SCF).The BMDMs were stimulated with ipopolysaccharide(LPS)and adenosine triphosphate(ATP),and then the level of IL-1? in the lysates or in the supernatant was analyzed through Western Blot or ELISA.(4)Splenocytes were isolated from Balb/c mice,and divided into three groups:a normal group without stimulation,a group with IL-6 and TGF-?1 treatment,and a group stimulated with IL-6,TGF-?1 and IL-1?.At same time,treated with Dynabeads(?)Mouse T-Activator CD3/CD28 for 96 hours.The percentage of CD4+IL-17+T cells was analyzed with flow cytometry,and the protein levels of IL-17 supernatant were tested using ELISA.(5)Mouse penetrating keratoplasty model was established and randomly divided into three groups,namely an allogeneic transplantation group and an allograft group treated with anti-IL-17 antibody.The effect of anti-IL-17 anibody on the survival of corneal grafts was observed by slit lamp;Hematoxylin-eosin staining(HE)was performed to evaluate the pathological changes of grafts in different groups.Results:(1)Compared with syngeneic group,the levels of IL-1? mRNA in allogeneic group were significantly elevated.The results from Western blot revealed the more matured IL-1? in allogeniec group than in syngeneic group.The receptor IL-1R1 in allogeneic group was also higher than that in syngeneic group.The immunofluoerescence staining confirmed the increased expression of IL-1R1 and showed that IL-1R1 was located at corneal epithelium,stromal and endothelium.Neutralization of IL-1? using anti-IL-1? antibody could siginificantly delayed the corneal allograft rejection.The results indicated that proinflammatory IL-1? contributed the pathogenesis of corneal allograft rejection.(2)The results from western blot showed the more elevated NLRP3 level in corneal allografts than in sygeneic grafts,and the results of immunofluoerescence revealed that NLRP3 was expressed in the corneal epithelium,stroma layer and endothelial layer.Furthermore,we found that NLRP3 knockout could significantly prolong the survival of corneal allografts,and IL-1? transfusion aggrevatged the corneal allograft rejection.The level of interleukin-17(IL-17)in NLRP3 knockout mice was lower than that in wild type mice,while there was no significant change in IFN-? levels.Moreover,the NLRP3 inflammasome inhibition using Glubide was confirmed the alleviated corneal allograft rejection.The results indicated that NLRP3 inflammasome derived IL-1? involved the pathogenesis of corneal allograft rejection.(3)Compared with wildtype BMDMs,the matured IL-1? in NLRP3-deficent BMDMs was more pronouncedly reduced.When sitmulated naive CD4+ T cells using supernant from NLRP3-deficent BMDMs,the percentage of Th17 cells was much lower than that treated with supernants from wildtype BMDMs.Furthermore,the IL-17 protein level in the supernats from NLRP3-deficent BMDMs was also reduced.The results clearly indicated that IL-1? promoted Th17 immune response.(4)Compared with the control group,IL-17 neutrolizing antibody could delayed the corneal allograft rejection,with prolonged corneal allograft survival time and less inflammatory infiltration.The results suggested the involvement of Th17 immune response during corneal allograft rejection.Conclusion:(1)During corneal allograft rejection,the enhanced IL-1(3 was observed in rejected corneal grafts,neutralization of IL-1? with antibody could siginificantly delayed the crneal allograft rejection.(2)Increased NLRP3 contributed the pathogenesis of corneal allograft rejection,disrupted NLRP3 inflammasome signaling alleviated the corneal allograft rejection(3)IL-1? promoted corneal allograft rejection through Th17 immune response,neutralization of IL-17 using antibody prolonged the survival of corneal allografts.