Blockade of NLRP3 inflammasome activation in diabetic retina by the ketone metabolite beta-hydroxybutyrate is mediated by GPR109A

Abstract

Purpose : NLRP3 inflammasome pathway activation has emerged as a key source of subclinical inflammation in diabetic retinopathy (DR). The ketone body beta-hydroxybutyrate (β-HB) has been shown to block the NLRP3 inflammasome pathway in chronic inflammatory states, but its utility has not been investigated in diabetic retina. Here, we evaluated whether β-HB negatively regulates NLRP3 activation through GPR109A, a G_i^-protein coupled receptor for β-HB, by assessing the effects of β-HB in streptozotocin (STZ)-induced diabetic Gpr109a^+/+ (wildtype, WT) and Gpr109a^-/- (knockout, KO) mouse retina.

Methods : Diabetes was induced in male, WT and KO mice using STZ (75 mg/kg in 0.01M sodium-citrate buffer, pH 4.5). The GPR109A cognate ligand β-HB was administered daily (40 mg/mouse, i.p.) to diabetic (DB) and age-matched, control non-diabetic (non-DB) WT and KO mice for a period of 2 weeks beginning 16-wks post-diabetes onset. WT and KO, non-DB and DB, animals injected with PBS only served as controls. Multiplex cytokine arrays (R&D systems) and immunofluorescence techniques were used to assess retinal inflammation. Western blotting was used to confirm changes in the expression of key inflammasome-associated cytokines (IL-1 beta and IL-18) and, pro-caspase 1 and cleaved caspase 1, proteins integral to NLRP3 inflammasome activation. Blood-retinal barrier breakdown and increased vascular permeability was assessed by albumin extravasation and FITC-dextran staining and, FITC-ConA perfusion to evaluate leukostasis.

Results : Basal levels of pro-inflammatory proteins and pro-inflammatory cytokines were higher in WT DB and KO non-DB compared to WT non-DB retinas. The increases were exacerbated even further in KO DB eyes. β-HB treatment significantly reduced pro-inflammatory protein expression, pro-inflammatory cytokine levels, and cleaved caspase 1 expression in WT DB mice, but had little to no effect in KO control and KO DB animals. Notably, we also observed accelerated and worsened pathology in KO DB animals, evidenced by increased vascular permeability and leukocyte adhesion.

Conclusions : These data show that β-HB reduces NLRP3-derived inflammation in the diabetic retina through GPR109A, identifying this metabolite and its receptor as a new potential therapeutic drug and target for combating inflammation in DR.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.