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Ali M. Saeed, Fernando Cruz-Guilloty, Stephanie J. Duffort, Robert G. Salomon, Victor L. Perez; Polarization Of Macrophages Toward M1 Pro-inflammatory Phenotype By Oxidative Stress-induced Modifications Associated With Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2716.
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Oxidative damage and inflammation have been widely recognized to play a pathological role in the development of age-related macular degeneration (AMD). Elevated retinal levels of carboxyethylpyrrole (CEP), a fatty-acid oxidation fragment, have been correlated with AMD disease state. Additionally, our previous studies have demonstrated CEP as the molecular link between oxidative damage and inflammation; such that immunization of mice with CEP-adducts leads to AMD-like lesions, anti-CEP antibody production, and localized infiltration of macrophages. The impact of CEP on macrophage physiology is unknown and we therefore sought to determine if CEP-adducts can lead to macrophage activation and polarization (distinct production of inflammatory cytokines).
Primary macrophage cultures were obtained by collecting the bone marrow of C57BL/6 and BALB/c mice and culturing the marrow cells in L-929 cell-conditioned media for seven days. Macrophages were treated with CEP-MSA or appropriate controls. Macrophage activation state was determined by measuring cytokine production by ELISA and gene expression of cytokines and other relevant genes by quantitative real-time PCR.
Macrophages become activated in response to stimulation with CEP-adducted proteins. Specifically, we found that CEP-adducts lead to upregulation of inflammatory cytokines such as IL-12, TNF-α,and IL-1β, which indicate macrophage activation and polarization to an M1 state. Upregulation of M2-macrophage markers (like IL-10 and arginase) were not observed in response to CEP-stimulation.
Our work demonstrates for the first time that CEP-adducts can lead to macrophage activation and production of inflammatory cytokines. Specifically, the cytokine profile we observed (IL-12, TNF-α, but not IL-10 production) characterizes these macrophages as M1 macrophages, which are known to have a pathological tissue destructive role. Importantly, the observed M1 phenotype from this in vitro study mirrors our in vivo findings of retina-infiltrating M1 (but not M2) macrophages in CEP-immunized mice. Overall, our findings suggest that CEP triggers an inflammatory response and may play an initiating (or at least supporting) role in retinal inflammation that leads to AMD.
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