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-Socs3Cx3cr1 double knockout (DKO), LysMCre-Socs3, Cx3cr1 and Socs3 mice were maintained up to 12 months. Eyes were collected and processed for immunohistochemistry of IBA-1, cone arrestin, secretagogin, PKCα and GABA. Brain microglia from DKO and WT mice were stimulated with LPS + IFN-γ or IL-4. The expression of TNF-α, IL-1β, 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 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 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 PKCα) and GABA amacrine cells was significantly lower in aged DKO mice compared to age-matched Socs3 and single KO mice. Microglia from DKO mice showed significantly higher levels of phagocytic activity and produced higher levels of TNF-α, IL-6, CCL2, CCL5, CXCL2 and CXCL10 compared to microglia from Socs3 mice. Co-culture of retinal explants with LPS + IFN-γ or IL-4 pre-treated DKO microglia significantly reduced cone photoreceptor survival.

CONCLUSIONS

The LysMCre-Socs3Cx3cr1 DKO mice displayed primary microglial dysfunction and developed age-related retinal microgliopathy characterized by aggragated microglial activation and multiple retinal neuronal and RPE degeneration.

TRIAL REGISTRATION

Not applicable. The article does not contain any results from human participants.