Abstract
Purpose: :
Age-related macular degeneration (AMD) is a major cause of blindness in the elderly, and cigarette smoke (CS), a powerful oxidant and potent inducer of the innate immune response, is a strong epidemiologic risk factor. CS causes direct oxidative damage to the fundus, and may facilitate an uncontrolled immune response via complement and Toll-like receptor (TLR) activation, therefore exacerbating tissue damage. Recent studies have shown that complement cross-talks and amplifies the effects of TLRs, possibly through the C5a receptor, and together, they induce tissue injury. The purpose of our studies was to determine whether CS triggers the C5a-C5aR pathway in RPE cells. Since AMD may be linked to a decline in Nrf2 signaling, which regulates the inducible expression of antioxidant genes, we also examined whether Nrf2 deficiency magnifies the CS induced immune response.
Methods: :
ARPE19 cells were transfected with siRNA to Nrf2 or scrambled control, after reaching 70% confluence, starved in serum free medium for 24h and treated with 125 ug/ml CS extract (Murty Pharmacueticals) for another 24h. Cells were subjected to DHE staining to measure superoxide, a measure of oxidative stress. Total RNA was extracted for quantitative RT-PCR. Whole cell lysates were prepared for western blot analysis.
Results: :
ARPE19 cells exposed to 125 ug/ml CS extract showed high levels of intraceullar superoxide. Nrf2 knockdown decreased the expression of antioxidant genes (Nqo1, Gclm and HO1), and caused an additional 1.2 fold increase in superoxide levels in CS extract treated cells (p=0.015). 125 ug/ml CS extract also increased C5 mRNA expression, which was elevated by Nrf2 knockdown, but attenuated by the synthetic triterpenoid CDDO-Im, a potent activator of Nrf2 signaling. C5aR expression was detected in ARPE19 cells, both at the mRNA and proteins levels, and its expression was increased by CS extract or Nrf2 knockdown. The expressions of IL-1beta, IL6, IL8, MCP-1 mRNAs were induced by C5a, indicating an active C5a-C5aR pathway in the RPE cells, and the pro-inflammatory cytokines were increased by CS extract or Nrf2 knockdown.
Conclusions: :
CS extract not only triggers oxidative stress but also activates complement via the C5aR in ARPE19 cells, and Nrf2 deficiency further magnifies the complement activation. These factors may incrementally contribute to the onset of AMD.
Keywords: age-related macular degeneration • inflammation • oxidation/oxidative or free radical damage