The Roles Of Autophagy-Interleukin-1? Signal Pathway In Corneal Allografts Immune Rejection

Objective:The aim of this study was to investigate the roles of autophagy-interleukin-1? signal pathway in corneal allografts immune rejection using a mouse corneal transplantation model.Methods:(1)Allogenic full-thickness corneal transplantation was performed in Balb/c mice and the mice were randomly divided into three groups: an allograft group(Allo),which received no therapy;an allograft group treated with rapamycin(RAPA)nano-micelle eye drops(Allo+RAPA);and an allograft group treated with 3-Methyladenine(3-MA)eye drops(Allo+3-MA).The survival of corneal allografts was observed by slit lamp,and histopathological changes of corneal allografts were examined using hematoxylin-eosin(H-E)staining.The corneal grafts were isolated at 2 weeks after keratoplasty,and used to determine the levels of autophagy-related protein and interleukin-1? in cornea by Western Blot.(2)BMDMs induced by macrophage colony stimulating factor(M-SCF)were stimulated with lipopolysaccharide(LPS)and adenosine triphosphate(ATP)after pretreatment with RAPA or 3-MA,separately.Autophagy-related protein and IL-1? in macrophage lysis were examined using Western Blot,and the levels of IL-1? in supernatants were determined by enzyme-linked immunosorbent assay(ELISA).(3)Allogenic full-thickness corneal transplantation was performed using wild-type(WT)and NLRP3-/-mouse as corneal recipients.The corneal rejection was observed using slit lamp,and histopathological changes were examined by H-E staining.The corneal allografts were isolated at 2 weeks after corneal transplantation.The levels of Interleukin-17(IL-17)in corneal allografts were detected by real-time quantitative PCR,and the levels of IL-1? were analysed using western blot.Results:(1)The mean survival times of the allografts in Allo,Allo+RAPA and Allo +3-MA were(16.83±1.21)days,(30.00±0.00)days and(12.63±3.42)days,respectively.Their statistical difference was significant(P<0.05).Compared with allografts from Allo,the corneal allografts treated with 3-MA eyedrops were rejected with obviously turbid edema and a large amount of inflammatory cells infiltrating into corneal stroma,while the corneal allografts instilled with RAPA eyedrops were shown hyaline with less inflammatory cells infiltrating into corneal stroma.These results demonstrated that RAPA could inhibit corneal allograft rejection;while 3-MA promoted corneal allograft rejection.As shown by western blot,the corneal allografts treated with RAPA showed increased expression of microtubule-associated protein 1 light chain 3(LC3)-II(membranebound type)and decresed expression of LC3-I(cytoplasmic type),as well as decreased expression of p62/SQSTM1 protein(an important substrate of autophagy),suggested the elevated autophagic activity;while 3-MA treated corneal allografts showed dereased expression of LC3-II and increased expression of LC3-I,as well as increased p62/SQSTM1 protein levels,which indicated decreased autophagic activity.The results demonstrated that autophagy induced by RAPA delayed corneal allografts rejection,while inhibition of autophagy by 3-MA promoted allograft rejection.Furthermore,we also found that the cleavaged form of pro-interleukin-1?(pro-IL-1?)in RAPA treated corneal grafts was more significantly decreased than in Allo groups,while more pronouncedly elevated in 3-MA treated corneal grafts than in corneal grafts from Allo groups.This suggested that the maturation of pro-IL-1? be regulated by autophagy and involvement of IL-1? in the pathogenesis of corneal allografts rejection.(2)We found that the matured form of IL-1? in RAPA pre-treated BMDMs was much more siginificantly declined both in supernatants and lysis after stimulation with LPS and ATP than in BMDM without RAPA treatrment,as well as pronouncedly decreased levels of p62/SQSTM1 protein;While much more increased levels of matured IL-1? both in supernatants and lysis were found in 3-MA pretreated BMDMs than in without pretreatment,alonged with increased levels of p62/SQSTM1 protein.The results above further improved that autophagy was involved in the maturation of IL-1?.(3)In order to evaleuate the pathogical roles of IL-1? implicated in corneal allografts rejection,NLRP3-/-mice were used as corneal recipients to perform keratoplasty.Compared with WT mice,the mean survival time of corneal grafts in NLRP3-/-mice was more siginificantly prolonged,with less inflammatory infiltration and decreased production of pro-inflammatory factors,interleukin-17(IL-17).Moreover,western blot analysis also found the more siginificant reduction of IL-1? protein in cornea of NLRP3-/-mice than in WT mouse,which was recorrespondance with levels of IL-1? in RAPA treated coreal allografts.The supplement of IL-1? with biological acitivity through subconjunctival injection promted the allografts rejection in NLRP3-/-mice.Based on the above results,we suggested that NLRP3 can be involved in corneal allografts immune rejection by affecting the maturation of IL-1?.Conclusion:1.Induction of autophagy can inhibit corneal allograft rejection,and prolong the corneal graft survival;while inhibition of autophagy can promote allogrft rejection.2.Autophagy involves in the pathogenesis of the corneal allograft rejection through regulating the maturation of IL-1? induced by NLRP3 inflammasome.