A Study On The Relationship Between Macrophage Autophagy And Lattice Corneal Dystrophy

Objective.Lattice corneal dystrophy(LCD)is a hereditary disease caused by mutations in the transforming growth factor-?-induced gene(TGFBI).The denatured TGFBIp expressed by mutant TGFBI deposits in the corneal stroma,resulting in damage to the structure and function of the cornea and significant loss of vision.Therefore,how to remove TGFBIp effectively is the key process to LCD treatment.Alzheimer's disease(AD)is one of the most studied diseases caused by amyloidosis.It has been found that microglia(a macrophage)in AD can degrade exogenous ?-amyloid by autophagy.Recent studies have found that the defective autophagy and degradation of TGFBIp in corneal fibroblasts with granular corneal dystrophy(GCD).The aim of this study was to investigate whether autophagy is involved in the degradation of TGFBIp in macrophages,and to investigate the relationship between this protein degradation and the pathogenesis of LCD.Methods.A genetic disease survey was conducted in a family in Hainan Province.Slit lamp microscope was used to observe the cornea of the proband and family members,and the cornea of the proband was obtained for histopathological staining.The peripheral blood of the patient was used for TGFBI sequence detection and sequence comparison to determine its genotype.The cornea of the proband was fluorescently stained to observe the fluorescence distribution relationship between TGFBIp and CD11 b.Peripheral peripheral blood mononuclear cells(PBMC)were isolated and induced into macrophages by GM-CSF.Macrophages treated with recombinant wild-type(normal)TGFBIp and mutant(denatured)TGFBIp,and the following experiments were performed.After recombinant TGFBIp was treated with macrophages for 5 h,the TGFBIp in the medium was removed and cultured for another 5 h,and detected at 0,2.5,5,7.5,and 10 h.WB was used to detect the content of TGFBIp,the expression of CD36 and CD68 and the expression of LC3-I,LC3-II and SQSTM1 in autophagy.The concentration of TGFBIp in culture medium was detected by enzyme-linked immunosorbent assay(ELISA).The macrophages were fluorescently stained,and the colocalization of TGFBIp and CD11 b was observed by confocal microscopy to determine the relationship between macrophages and TGFBIp.The colocalization of endogenous LC3 spots and TGFBIp and the colocalization of endogenous LC3 spots and LAMP1 were observed by confocal microscopy to determine autophagy and its flux.The expression levels of CD36 and CD68 in macrophages were detected by flow cytometry at indicated time points.Results.Of the 25 family members in this study,14 members were diagnosed with LCD.Gene sequencing revealed that all patients had a point mutation in the TGFBI gene,that is,c.1673 T > C,resulting the replacement of tyrosine by proline in the 558 th position(p.Leu558Pro).Significant TGFBIp aggregation and macrophage infiltration were found in the lesioned corneal tissue.Patient-derived macrophages have similar uptake capacity in both WT and MU TGFBIp,and TGFBIp levels were similar in macrophages.But macrophages treated with MU TGFBIp still have higher concentrations of TGFBIp after exogenous TGFBIp removal,whereas TGFBIp levels were not detected in cells treated with WT TGFBIp.After using inhibitor 3-MA or leupeptin,the level of TGFBIp of macrophages treated with WT TGFBIp was similar to those treated with MU TGFBIp,and no such phenomenon under the influence of MG132.High expression of CD36 and CD68 was observed in macrophages treated with WT TGFBIp,whereas CD36 and CD68 of macrophages treated with MU TGFBIp were not significantly expressed.Detecting autophagy-associated molecule LC3-II with WB,it was found that LC3-II was highly expressed in macrophages treated with WT and MU TGFBIp,but in macrophages treated with MU TGFBIp,the levels of TGFBIp remained high after TGFBIp removal,whereas LC3-II of macrophages treated with WT TGFBIp returned to normal levels of macrophages untreated with TGFBIp.The similar phenomenon was also further confirmed by confocal microscopy for detection of colocalization of LC3 and TGFBIp.The level of SQSTM1 in macrophages treated with WT TGFBIp was significantly decreased by WB,but the level of SQSTM1 in macrophages treated with MU TGFBIp remained unchanged.Confocal microscopy revealed that there was significant colocalization of LC3 and LAMP1 in macrophages treated with WT TGFBIp.In addition,After blocking autophagy with 3-MA and then detected the expression of CD68 and CD36 by WB and flow cytometry.It was found that the expression of these two molecules was significantly different from those before unblocking.The expression levels of these two molecules were significantly decreased in macrophages treated with WT TGFBIp and 3-MA,and were similar to those expressed by macrophages treated with MU TGFBIp only.Conclusions.The above experimental results show that the TGFBI gene mutation is c.1673 T > C in this family,causing protein denaturation(p.Leu558Pro).Denatured TGFBIp deposition in the cornea is an important cause of LCD lesions.Macrophages autophagic degradation of denature TGFBIp during incomplete autophagy flux(autolysosome could not form effectively).The incomplete autophagy of macrophages not only causes defective degradation of denatured TGFBIp,but also reduce the phagocytic activity of macrophage,thereby reducing the ability of macrophages to uptake and degrade TGFBIp continuously,and aggravating corneal deposition and accumulation of TGFBIp.These results also suggest that trying to restore autophagy and the effective degradation of denatured TGFBIp in macrophages may be new strategies for LCD therapy.