Deletion of Socs3 in LysM+ cells and Cx3cr1 resulted in age-dependent development of retinal microgliopathy

Background We generated a mouse model of primary microglial dysfunction by deleting two negative immune regulatory genes, Cx3cr1 and Socs3 (in LysM(+) cells). This study aimed to understand how primary microglial dysfunction impacts retinal neurons during aging. Methods The LysMCre-Socs3(fl/fl)Cx3cr1(gfp/gfp) double knockout (DKO), LysMCre-Socs3(fl/fl), Cx3cr1(gfp/gfp) and Socs3(fl/fl) mice were maintained up to 12 months. Eyes were collected and processed for immunohistochemistry of IBA-1, cone arrestin, secretagogin, PKC alpha and GABA. Brain microglia from DKO and WT mice were stimulated with LPS + IFN-gamma or IL-4. The expression of TNF-alpha, IL-1 beta, IL-6, iNOS, IL-12p40, IL-23p19, CCL2, CCL5, CXCL2, IL-10, CD206 and Arg1 were examined by qRT-PCR and protein production was measured by Luminex assay. Retinal explants from C57BL/6 J mice were co-cultured with microglia from DKO or WT mice for 24 h, after which the number of cone arrestin(+) cells in retinal flatmount were quantified. Results In 3-5 month old mice, the number of microglia in retinal ganglion cell layer (GCL) and inner plexiform layer (IPL) were comparable in all strains of mice. The DKO mice had a significantly higher number of microglia in the outer plexiform layer (OPL) but significantly lower numbers of cone arrestin(+), secretagogin(+) and GABA(+) cells compared to Socs3(fl/fl) and single KO mice. During aging, 57% of the DKO mice died before 12 months old. The 10-12 months old DKO mice had significantly higher numbers of microglia in GCL/IPL and OPL than age-matched Socs3(fl/fl) and single KO mice. The aged DKO mice developed retinal pigment epithelial (RPE) dysmorphology accompanied by subretinal microglial accumulation. The number of photoreceptors, bipolar cells (Secretagogin(+) or PKC alpha(+)) and GABA(+) amacrine cells was significantly lower in aged DKO mice compared to age-matched Socs3(fl/fl) and single KO mice. Microglia from DKO mice showed significantly higher levels of phagocytic activity and produced higher levels of TNF-alpha, IL-6, CCL2, CCL5, CXCL2 and CXCL10 compared to microglia from Socs3(fl/fl) mice. Co-culture of retinal explants with LPS + IFN-gamma or IL-4 pre-treated DKO microglia significantly reduced cone photoreceptor survival. Conclusions The LysMCre-Socs3(fl/fl)Cx3cr1(gfp/gfp) DKO mice displayed primary microglial dysfunction and developed age-related retinal microgliopathy characterized by aggragated microglial activation and multiple retinal neuronal and RPE degeneration.