June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
NLRP3 inflammasome activation in human retinal pigment epithelium under inflammation and oxidative stress
Author Affiliations & Notes
  • Yujuan Wang
    Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD
    Zhongshan Ophthalmic Center, Sun Yat-sun University, Guangzhou, China
  • Mones Abu-Asab
    Histopathology Core, National Eye Institute, National Institutes of Health, Bethesda, MD
  • DeFen Shen
    Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD
  • Xi Chu
    Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD
  • Alexander Ogilvy
    Histopathology Core, National Eye Institute, National Institutes of Health, Bethesda, MD
  • Jingsheng Tuo
    Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD
  • Chi-Chao Chan
    Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD
  • Footnotes
    Commercial Relationships Yujuan Wang, None; Mones Abu-Asab, None; DeFen Shen, None; Xi Chu, None; Alexander Ogilvy, None; Jingsheng Tuo, None; Chi-Chao Chan, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 149. doi:
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      Yujuan Wang, Mones Abu-Asab, DeFen Shen, Xi Chu, Alexander Ogilvy, Jingsheng Tuo, Chi-Chao Chan; NLRP3 inflammasome activation in human retinal pigment epithelium under inflammation and oxidative stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):149.

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      © ARVO (1962-2015); The Authors (2016-present)

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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  
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