Up-regulation of P2RX7 in Cx3cr1-deficient mononuclear phagocytes leads to increased interleukin-1β secretion and photoreceptor neurodegeneration

Abstract

 

Purpose

 

Inflammatory mononuclear phagocytes (MP) accumulate in geographic atrophy form of age related macular degeneration (AMD) which are associated with photoreceptor degeneration. We have previously shown that Cx3cr1-deficient mice develop age-related subretinal accumulation of MP with photoreceptor degeneration. Cx3cr1-deficient MP have been shown to lead to increased neuronal apoptosis through IL-1beta (IL-1β) in the brain. However the mechanisms for this increased IL-1β secretion from Cx3cr1-deficient MP is still unknown.

 

Methods

 

Bone marrow monocytes (BMM) were isolated from wildtype and Cx3cr1-deficient mice and analyzed by flow cytometry for P2RX7 expression. Equal numbers of wildtype and Cx3cr1-deficient BMM were co-cultured with wildtype retinal explants or purified photoreceptor outer segment (POS). After 18 hours, BMM-derived MP mRNA expression of specific pro- or anti-infammmatory markers were assayed by qPCR. ATP and IL-1β were assayed in BMM-derived supernatants. In specific experiments, TUNEL staining was performed on retinal explants to assay the neurotoxicity of wildtype and Cx3cr1-deficient BMM.

 

Results

 

Cx3cr1-deficient BMM have an increased surface expression of P2RX7 and secreted increased amount of IL-1β upon TLR stimulation in an P2RX7-dependent mechanism when compared to wildtype BMM. Culturing Cx3cr1-deficient BMM in the presence of POS enhanced the expression of inflammatory genes such as IL-1β and P2RX7 while in similar conditions wildtype BMM express higher levels of CD206 and IL-1Ra. Cx3cr1-deficient BMM have an increased photoreceptor toxicity in a BMM/retina co-culture model. Addition of IL-1Ra or Brilliant blue G efficiently reduce photoreceptor cell loss in the BMM/retina co-culture.

 

Conclusions

 

We here show that spontaneous P2RX7 activation and enhanced IL-1β secretion are responsible for subretinal CX3CR1-deficient MP photoreceptor toxicity. Our results suggest that inhibition of the inflammasome may reduce photoreceptor cell loss in AMD