CX3CL1-CX3CR1 axis in diabetic retinopathy (DR) in STZ-diabetic mice.

Abstract

Purpose: Elevated chemokines have been observed in the vitreous and serum of DR patients and in a rodent model of retinal ischemia-reperfusion injury. CX3CL1 is lower in retinal neurons as well as cells of the iris and retina. Its receptor CX3CR1 is expressed by CNS microglia and by circulating cells. In the retina this pathway has been implicated in inflammatory ocular diseases. The purpose of this study was to ascertain effects of CXCR1 deficiency on retinal and systemic monocyte/macrophage populations in DR in an STZ-induced diabetes model.

Methods: Hyperglycemia was induced in CX3CR1^−/− and ^+/− mice by intraperitoneal STZ injection for 5 days and maintained up to 4 months. Control mice were injected with vehicle only. To confirm diabetes HbA1c was measured before euthanasia. Acellular capillaries were counted in trypsin digests of retinas. Immunohistochemistry was performed on whole eye cryosections. Cells from fresh retina, brain, spleen, bone marrow (BM) and blood were counted (flow cytometry) for lineage markers, Ly6C & CD11b. One-way ANOVA followed by the Tukey post hoc test were performed (P-value <0.05 for significance).

Results: Hyperglycemia elevated acellular capillaries in all groups vs. controls. CX3CR1 deletion further elevated this compared to STZ-WT. Diabetes increased monocytes in blood & spleen vs. control WT. Control CX3CR1^-/- mice showed lower total blood monocytes, but higher splenic and BM monocytes compared to WT. Diabetes resulted in fewer monocytes in CX3CR1^-/- mice and shifted the monocyte population toward Ly6C^hi in the BM & spleen but not blood of WT mice. Diabetic CX3CR1^-/- mice had the opposite: more blood Ly6C^hi & less in spleen & BM. Lack of CX3CR1 in control & STZ-WT increased monocytes in retinas. Diabetes did not affect monocyte movement into CX3CR1^-/- retinas but did show increased differentiation of infiltrating monocytes into Ly6C^hi subset.

Conclusions: CX3CL1-CX3CR1 interferes with development &/or slows egress & homing of monocytes. Since CX3CL1 reduces microglial activation in the CNS, we hypothesize this occurs in retinas. Our studies show CX3CR1 deficiency is associated with rapid development of acellular capillaries. Thus CX3CR1 may be needed for monocyte release from BM & retention in spleen in hyperglycemia. Its lack may cause a shift of the monocyte pool to an inflammatory subset. Therefore CX3CR1 may be a good target for early vasodegenerative stages of DR (currently no treatment).