Original Contribution
Nrf2 signaling modulates cigarette smoke-induced complement activation in retinal pigmented epithelial cells

https://doi.org/10.1016/j.freeradbiomed.2014.01.015Get rights and content

Highlights

  • CSE specifically induces C3a and C3b and reduces complement regulators in RPE cells.

  • C3a induces IL-1b through its C3a receptor.

  • Nrf2 deficiency magnifies this specific proinflammatory C3a and C3b generation.

  • Nrf2 labeling is decreased in diseased RPE overlying drusen in human AMD.

Abstract

Whereas cigarette smoking (CS) and dysregulated complement are thought to play central roles in age-related macular degeneration (AMD), their exact roles are unknown. The aim of this study was to determine if CS activates complement and if the antioxidant transcription factor Nrf2 modulates this response. In AMD specimens, Nrf2 immunolabeling was strong in the cytoplasm, with scattered nuclear labeling of macular retinal pigmented epithelial (RPE) cells that appeared normal, but was decreased and without nuclear labeling in dysmorphic cells overlying drusen, a hallmark AMD lesion. Cigarette smoke extract (CSE) induced Nrf2 nuclear translocation in RPE cells with increased antioxidant and complement gene expression. Whereas CFH protein was not altered by CSE, the cell membrane regulator proteins CD46, CD55, and CD59 were decreased, and C3a and C3b, but not iC3b, were increased compared to controls. C5b-9 was increased by CSE, but at sublytic levels, only after addition of normal human serum. Nrf2 knockdown enhanced the increase in C3a and C3b from CSE, but not iC3b, C5a, or C5b-9. CSE also increased IL-1b expression and secretion after C3a generation and was reduced by a C3aR antagonist. In contrast, the Nrf2 activator CDDO-Im restored complement gene expression in RPE cells exposed to CSE. We provide evidence of altered Nrf2 in human AMD and that CSE induces a proinflammatory environment specifically by generating C3a and C3b, and Nrf2 deficiency magnifies this specific complement response.

Section snippets

Immunohistochemistry

Human autopsy eyes (n=20) were obtained from the Ocular Pathology Division at the Wilmer Eye Institute after obtaining Institutional Review Board approval. Donors were classified as “unaffected” (n=8) if they had no history of AMD or histopathologic evidence of drusen. Early AMD donors (n=12) had an AMD history, and macular drusen (n=9), but no late-stage disease such as geographic atrophy or choroidal neovascularization. Eyes were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 4 

Nrf2 immunolabeling corresponds with AMD histopathology

To demonstrate the relevance of Nrf2 signaling in AMD, we assessed the distribution of Nrf2 immunolabeling in the RPE of unaffected controls from a wide age range (n=8; mean, 56 years; range, 8 months–84 years) and in early AMD (n=12; mean, 80 years, range, 60–96 years; Table 1). In the absence of oxidative stress, Nrf2 is tethered to Kelch-like ECH-associated protein 1 in the cytoplasm and is degraded by the proteasome. With oxidative stress, Nrf2 expression is induced and, importantly, Nrf2

Discussion

Cigarette smoking is a powerful risk factor for AMD, and Nrf2 signaling has been hypothesized to play a protective role against this complex chemical oxidant. Evidence linking impaired Nrf2 signaling with human AMD has been lacking. Our study addresses this deficiency by observing Nrf2 immunolabeling in human maculas that correlated with AMD histopathology. Our work starts to unravel the complex protective effect of Nrf2 signaling in RPE cells against the potential toxicity of a complex

Acknowledgments

We thank Mike Sporn, M.D., Dartmouth School of Medicine, for providing CDDO-Im. This work was supported by EY14005 (J.T.H.), EY019904 (J.T.H.), P50HL107169 (S.B.), R01CA140492 (S.B.), a Thome Foundation Award in AMD (J.T.H.), a Research to Prevent Blindness Senior Scientist Award (J.T.H.), a Wilmer Core Grant, EY001765, an unrestricted grant from Research to Prevent Blindness, and gifts from the Merlau family and Aleda Wright. J.T.H. is a Robert Bond Welch Professor.

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    These authors contributed equally to this work.

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