Abstract
Purpose :
The nucleotide-binding oligomerisation domain-like receptor protein 3 (NLRP3) inflammasome is a multiprotein complex that forms part of the innate immune system. Recent studies have shown that the inflammasome pathway is upregulated in diabetic retinopathy (DR) resulting in the breakdown of the retinal pigment epithelium (RPE) barrier. It has been hypothesised that small extracellular vesicles (sEVs) may transport inflammasome-related cargos between cells which in turn contributes to DR pathogenesis. This study aimed to characterise sEVs secreted by retinal pigment epithelial cells (ARPE-19) grown under normal and DR-like conditions and assess their effects on inflammasome activation.
Methods :
sEVs were isolated from ARPE-19 cell culture medium (basal sEVs) by ultracentrifugation at 100,000 g for 70 min at 4oC. To mimic DR-like conditions, ARPE-19 cells were exposed to high glucose and pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) for 72 h (HG+Cyt sEVs). sEVs were characterized for size, concentration and morphology using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Western blotting was used to determine the sEV cargo (NF-κB, IL-1β, NLRP3, cleaved caspase-1). Immunocytochemistry was performed to assess inflammasome markers (NLRP3, cleaved caspase-1) in response to sEV addition onto ARPE-19 cells grown under normal and DR-like conditions.
Results :
HG+Cyt treatment increased sEV concentration relative to basal conditions. While sEV markers (CD9 and TSG101) were equally expressed in both groups, inflammasome markers (NLRP3 and cleaved caspase-1) were increased in HG+Cyt sEVs relative to basal sEVs. When added to normal ARPE-19 cells, HG+Cyt sEVs induced inflammasome activation, while basal sEVs added to injured ARPE-19 cells reduced inflammasome activation.
Conclusions :
These findings support the hypothesis that sEVs can mediate inflammasome activation through their immunomodulatory cargo depending on the disease state and culture conditions. Further studies are necessary to determine whether other sEV cargo, such as miRNA, may also play a role in mediating the inflammasome.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.