NLRP3 inflammasome activation in human retinal pigment epithelium under inflammation and oxidative stress

Abstract

Purpose: Inflammation and oxidative stress are closely involved in age-related macular degeneration (AMD).The NLRP3 inflammasome, a key mediator of neuroinflammation, is linked to AMD pathogenesis. Inflammasomes promote the maturation of certain pro-inflammatory cytokines such as IL-1β and IL-18. In this study, we aimed to evaluate the NLRP3 inflammasome-mediated effects on human retinal pigment epithelium (RPE) under inflammation and oxidative stress.

Methods: A human RPE cell line (ARPE-19) and cultured primary adult human RPE (hRPE) were starved for 24 h and stimulated with 10 ng/ml tumor necrosis factor-alpha (TNFα), 10 μg/ml lipopolysaccharide (LPS)+10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 200 μM H2O2 for 24 h to mimic inflammatory and oxidative stress status. Expression of NOD-like receptor family, pyrin domain containing 3 (NLRP3), interleukin-1 beta (IL-1β), and IL-18 transcripts was measured by quantitative reverse transcript polymerase chain reaction (qRT-PCR); and expression of NLRP3 and cleaved caspase-1 proteins was evaluated by immunohistochemistry. In ARPE-19 cells, ultrastructural changes under the stimuli were evaluated by transmission electron microscopy (TEM). The effects of NLRP3 inflammasome on IL-1β, IL-18 and cleaved caspase-1 expressions under inflammatory and oxidative stress status were also assessed by transfecting NLRP3-targeting siRNA in ARPE-19 cells.

Results: In both ARPE-19 and hRPE, stimulation with TNFα, LPS+TCDD, or H2O2 significantly enhanced NLRP3, IL-1β and IL-18 transcripts (all p<0.05); ARPE-19 produced a higher level of IL-18 mRNA than hRPE under all stimulation. Immunohistochemistry illustrated elevated NLRP3 protein expression and caspase-1 cleavage in ARPE-19 cells under inflammatory or oxidative stress. Conversely, knockdown of NLRP3 expression in ARPE-19 showed less caspase-1 cleavage under all stimulation and reduced IL-1β and IL-18 transcripts with TNFα stimulation (all p<0.05). TEM illustrated presence of autophagosomes, cytoplasmic vesicles, mitochondrial damage, cytoskeleton disruption, and nuclear membrane separation in all stimulated ARPE-19 cells as compared with controls.

Conclusions: Inflammation and oxidative stress activates NLRP3 inflammasome in human RPE cells. The pro-inflammatory cytokine production and cellular damage of RPE caused by NLRP3 inflammasome activation could play an important role in AMD pathogenesis.

Keywords: 412 age-related macular degeneration • 557 inflammation • 634 oxidation/oxidative or free radical damage