Study On The Mechanism Of Macrophages Participating In Repairing After Lens Injury

Purpose Emerging evidence challenges the concept of the lens as an immune privileged organ;however,the underlying mechanism remains elusive.Here,we provide a direct mechanism supporting a role of macrophage recruitment in immuno-privileged lens during capsule rupture repair,fiber cell removal and fibrosis.Methods A double knockout(d KO)mouse model deficient in connexin(Cx)50 and Aquaporin 0(AQP0)was generated and this d KO mice exhibits a phenotype of posterior lens capsule rupture.We used morphology under light microscopy,histochemical analysis,immuno-labeling,RNA sequencing(RNA-seq)analysis and real-time quantitative RT-PCR(q RT-PCR)to determine macrophages,hyaloid vessels,necrotic lens fiber cells,epithelial mesenchymal transition(EMT)and fibrosis at various developmental stages of lenses from WT and d KO mice.Results Our previous studies show that d KO mice exhibit small eyes and lens with severe cataracts and morphological defects including posterior capsule rupture.We found here that lens fiber tissues were subsequently extruded from ruptured posterior capsule to the vitreous body at postnatal age of 15 days(P15)(95.24%,n=21).By histochemical analysis,we observed a“tail-like”tissue consisting of disorganized lens fiber cells,hyaloid vessels,fibrotic tissues and macrophages.The delayed regression of the hyaloid vessel system and increased fibrosis were detected with anti-SMA antibody after P15.Immunofluorescence with anti-CD68 and anti-Cx46 antibodies showed the presence of Cx46-positive cells inside the macrophages,suggesting the phagocytic process of macrophages.Co-labeling with anti-CD68 and receptor interacting protein kinase-3(RIP3)antibodies staining,we observed that RIP3+cells were detected at lens fiber protrusion region from posterior capsule,which coincided with the presence of CD68+macrophages at P15.Macrophages continued migrating towards the interior part of the lens after P30,coinciding with an increase in necrotic fiber cells.By P90,the lens fiber protrusion disappeared and posterior capsule rupture was sealed(90%,n=20).Moreover,fibrosis increased at P15 and with aggravation of fibrosis,capsule thickness increased and the epithelial cells imitated EMT process after P30.Furthermore,M1 and M2macrophages were detected in d KO lenses at P15 and P30 by immunofluorescence.And the number of CD68~+and Arg1~+(M2)macrophages inside the P30 lens was significantly increased(p=0.002 and p=0.006,respectively).We used colony stimulating factor-1(CSF-1)and antagonist(AFS98)for intravitreal injection to further validate the specific effect of M2 macrophages in sealing process.Fibrotic tissues were significantly increased in the CSF-1-injected group when compared with non-injected(p=0.0012),saline-injected(p=0.0013)and AFS98-injected(p<0.0001)groups.Conclusions Taken together,these results suggest that necrotic fiber cells associated with lens posterior rupture lead to the recruitment of macrophages,which are delivered by the regression delayed hyaloid vessel system.Additionally,administration of CSF-1 activated polarization of macrophages to M2 and accelerated the repairing process,while inhibition of CSF-1 receptor delayed the process.To summarize,the M2macrophages activated by CSF-1 play a key role in capsule rupture repairing,necrotic fiber cell removing and developing of fibrosis.