Characterization of NLRP3 Inflammasome Activation in the Onset of Diabetic Retinopathy.

The aim of this study was to characterize the role of nucleotide-binding oligomerization domain- (NOD-) like receptor (NLR) protein 3 (NLRP3) inflammasome activation in the onset of diabetic retinopathy (DR) using retina and vitreous from donors without diabetes mellitus (CTL), with diabetes mellitus alone (DM), and with DR. Retinal expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), the key markers of retinal inflammation, connexin43 (Cx43) which is involved in upstream inflammasome regulation, as well as NLRP3 and cleaved caspase-1, the main markers of inflammasome activation, were evaluated using immunohistochemistry and Western blotting. Vitreous interleukin (IL)-1ß and IL-18, biomarkers of the activated inflammasome, were measured using a Luminex multiplex assay. Results showed a significant increase in the number and size of Iba-1+ cells and NLRP3 expression in DM, while a significant increase in GFAP, Cx43, cleaved caspase-1 and vitreous IL-18, as well as a further increase in Iba-1 and NLRP3 was found in DR. This suggests that the inflammasome is already primed in DM before its activation in DR. Furthermore, IL-18 may act as the major effector of inflammasome activation in DR while nuclear translocation of cleaved caspase-1 may play a role in gene transcription contributing to DR onset.

Connexin43 hemichannel block inhibits NLRP3 inflammasome activation in a human retinal explant model of diabetic retinopathy.

Diabetic retinopathy (DR), the most common ocular complication associated with diabetes, is a chronic vascular and inflammatory disease that leads to vision loss. The inflammasome pathway, a key part of the innate immune system, is required to activate chronic inflammation in DR. Unfortunately, current therapies for DR target pathological signs that are downstream of the inflammasome pathway, making them only partly effective in treating the disease. Using in vitro and in vivo DR models, it was discovered that connexin43 hemichannel blockers can inhibit activation of the inflammasome pathway. However, those studies were conducted using in vitro cell culture and in vivo animal disease models that are predictive but do not, of course, like any model, completely replicate the human condition. Here, we have developed an addition to our armamentarium of useful models, an ex vivo human organotypic retinal culture model of DR by exposing human donor retinal explants to a combination of high glucose (HG) and pro-inflammatory cytokines, interleukin-1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α). We hypothesized that in this model, connexin43 hemichannel block would protect against NLRP3 inflammasome complex assembly which would in turn decrease signs of inflammation characteristic of DR. To test our hypothesis, molecular changes in the inflammatory and inflammasome pathway were assessed using immunohistochemistry and a Luminex cytokine release assay. Our results showed that the human retinal explant DR model was associated with increased inflammation and activation of the inflammasome pathway, characteristic of the human condition. Furthermore, we showed that by blocking connexin43 hemichannels with the hemichannel modulator, tonabersat, we were able to prevent NLRP3 inflammasome complex assembly, Müller cell activation, as well as release of pro-inflammatory cytokines and VEGF. This further supports the possible use of connexin43 hemichannel blockers as potential new therapies for DR.