Pharmacological depletion of microglia alleviates neuronal and vascular damage in the diabetic CX3CR1-WT retina but not in CX3CR1-KO or hCX3CR1I249/M280-expressing retina

Abstract

Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. There is no cure for DR, and medical interventions marginally slow the progression of disease. Microglia-mediated inflammation in the diabetic retina is regulated via CX3CR1-FKN signaling, where FKN serves as a calming signal for microglial activation in several neuroinflammatory models. Polymorphic variants of CX3CR1, hCX3CR1I249/M280, found in 25% of the human population, result in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in CX3CR1-KO and FKN-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of hCX3CR1I249/M280-expression in microglia-mediated inflammation in the diseased retina are relevant to identify mechanisms by which microglia contribute to disease progression. Our results show that hCX3CR1I249/M280 mice are significantly more susceptible to microgliosis and production of Cxcl10 and TNFα under acute inflammatory conditions. Inflammation is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to CX3CR1-WT mice. Therefore, to further investigate the role of hCX3CR1I249/M280-expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1+ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic CX3CR1-WT prevented TUJ1+ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate TUJ1+ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in CX3CR1-KO mice but not in hCX3CR1I249/M280 mice, suggesting that hCX3CR1I249/M280 expressing microglia influences vascular pathology differently compared to CX3CR1-KO microglia. Currently CX3CR1-KO mice are the most commonly used strain to investigate CX3CR1-FKN signaling effects on microglia-mediated inflammation and the results in this study indicate that hCX3CR1I249/M280 receptor variants may serve as a complementary model to study dysregulated CX3CR1-FKN signaling. In summary, the protective effects of microglia depletion is CX3CR1-dependent as microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate retinal degeneration nor microglial morphological activation as observed in CX3CR1-WT mice.

Fractalkine isoforms differentially regulate microglia-mediated inflammation and enhance visual function in the diabetic retina

“…In contrast, repopulation of CX3CR1 KO or hCX3CR1 I249/M280 microglia conferred worse retinal pathology in STZ models of diabetes. These data, supports the rationale that CX3CR1/FKN signaling axis is an important inhibitor of microglia hyperinflammation [ 3 , 23 ]. Therefore, this study aims to understand the contribution of mFKN and sFKN in inflammation and vascular damage in the diabetic retina.…”

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice

“…Our MACS approach has the limitation that Müller cells may be slightly contaminated with astrocytes, which express high levels of GFAP in the homeostatic retina. Thus, the increase in GFAP levels detected in the MACS-purified glial population may also be due to expression changes in astrocytes that has consistently been documented in db/db mice [ 37 , 72 , 74 ] or may be too low to be detected in Müller cells by immunostaining as also described by others [ 75 ]. However, other molecular markers of Müller cell gliosis were found to be dysregulated including Kir4.1, Stat3 , S100a1 and Cd44 [ 47 , 48 ].…”

Microglia depletion/repopulation does not affect light-induced retinal degeneration in mice

“…Thus, microglia depletion prevented breakdown of the blood retinal barrier, subretinal fluid accumulation, and release of pro-inflammatory cytokines in LPS induced chronic inflammation ( 43 ). CSF1R antagonist treatment also alleviated neuronal and vascular abnormalities in CX3CR1 diabetic mice ( 44 ). However, our findings are in agreement with recent studies where microglia depletion with either PLX3397 or PLX5622, caused functional impairment of rods and cones in a rat model of retinitis pigmentosa ( 45 ) and reduced visual acuity and RPE function in aged mice ( 46 ).…”