Abstract
Purpose :
Uptake of lipofuscin (LF) in microglial cells has been reported in subretinal microglia in older mice, which suggests a link to AMD pathology where microglia also accumulate in the subretinal space. The detailed reaction of the microglia towards LF is not known yet. We therefore studied effects of LF on microglial cells in vitro.
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Methods :
Human LF was extracted from human donor eyes. Immortalized BV-2 microglial cells and primary cells (PC) from murine CNS were incubated with a mixture of medium and lipofuscin in different concentrations for 24 hours. Further samples were additionally treated with hydrocortisone, minocycline and the tripeptide TKP. We analyzed the phagocytosis of LF by detecting its autofluorescence. Immunohistochemical stainings for microglial surface markers (CD-11b, F-4/80 and Iba-1) and also for VEGF were performed. In order to analyze cytokine release, we used a multiplex immunoassay for 17 pro-inflammatory cytokines and also an ELISA for VEGF.
Results :
Apart from morphological changes in the microglial cells (all CD-11b+, F-4/80+, Iba-1+) treated with LF, autofluorescence signals from inside these cells demonstratedphagocytosis of the LF. Immunohistochemical staining revealed elevated VEGF expression in BV-2 cells treated with LF. Highly elevated levels of pro-inflammatory cytokines in the supernatants for both, BV-2 cells and primary cells, were found by the multiplex assay, in particular IL-6, IL-1α and TNF-α (Fig. 1). Highly elevated VEGF levels in the supernatants of BV-2 cells after LF stimulation correlating with the LF concentration were found by ELISA (Fig. 2). However, in our primary cell culture, VEGF-levels were not significantly elevated (p=0.06).
Hydrocortisone significantly inhibited the immune reaction suppressing, among others, the cytokines that had the greatest increase after the exposure to LF (i.a. IL-6, IL-1 and TNF-α; all at least p<0.05). TKP and minocycline suppressed microglial proliferation but had less impact on cytokine levels.
Conclusions :
We were able to demonstrate for the first time that microglial cells phagocytose fast and completely human LF in vitro. Our results demonstrate the inflammatory potency of the microglia upon interaction with LF and hence, point to their potential role in degeneration. Elevated VEGF levels more specifically present a link to neovascularization in AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.