Anaphylatoxins Activate Ca2+, Akt/PI3-Kinase, and FOXO1/FoxP3 in the Retinal Pigment Epithelium

Abstract

PurposeThe retinal pigment epithelium (RPE) is a main target for complement activation in age-related macular degeneration (AMD). The anaphylatoxins C3a and C5a have been thought to mostly play a role as chemoattractants for macrophages and immune cells; here, we explore whether they trigger RPE alterations. Specifically, we investigated the RPE as a potential immunoregulatory gate, allowing for active changes in the RPE microenvironment in response to complement.DesignIn vitro and in vivo analysis of signaling pathways.MethodsIndividual activities of and interaction between the two anaphylatoxin receptors were tested in cultured RPE cells by fluorescence microscopy, western blot, and immunohistochemistry.Main outcome measuresIntracellular free calcium, protein phosphorylation, immunostaining of tissues/cells, and multiplex secretion assay.ResultsSimilar to immune cells, anaphylatoxin exposure resulted in increases in free cytosolic Ca2+, PI3-kinase/Akt activation, FoxP3 and FOXO1 phosphorylation, and cytokine/chemokine secretion. Differential responses were elicited depending on whether C3a and C5a were co-administered or applied consecutively, and response amplitudes in co-administration experiments ranged from additive to driven by C5a (C3a + C5a = C5a) or being smaller than those elicited by C3a alone (C3a + C5a < C3a).ConclusionWe suggest that this combination of integrative signaling between C3aR and C5aR helps the RPE to precisely adopt its immune regulatory function. These data further contribute to our understanding of AMD pathophysiology.

Properdin Modulates Complement Component Production in Stressed Human Primary Retinal Pigment Epithelium Cells

“…This can be carried out either by enzyme complexes formed by different complement proteins (convertases) or by specific proteases, e.g., cathepsin L or cathepsin B [ 11 , 69 ]. This results in high levels of anaphylatoxins C3a and C5a, which have been associated with physiological and pathological processes in the retina [ 27 , 70 , 71 ]: Eye morphogenesis [ 31 ], sub-RPE depositions [ 32 ], pro-inflammatory RPE reaction [ 33 , 34 , 35 ], PI3/Akt-pathway activation [ 29 ], and ER stress-mediated lipid accumulation [ 36 ].…”

“…Additionally, generation of complement activation products, such as anaphylatoxins and opsonins, by healthy and stressed RPE cells independent of any external complement source is described [ 21 , 24 , 26 , 27 ]. Recently, it was reported, that endogenous CFH and anaphylatoxins contribute to transcriptional and metabolic homeostasis of RPE cells [ 28 , 29 , 30 ]. In RPE cells complement anaphylatoxins receptor signaling is involved in eye morphogenesis [ 31 ], sub-RPE deposits [ 32 ], pro-inflammatory RPE reaction [ 33 , 34 , 35 ], PI3/Akt-pathway activation [ 29 ], and stress-mediated lipid accumulation in RPE cells [ 36 ].…”

“…Recently, it was reported, that endogenous CFH and anaphylatoxins contribute to transcriptional and metabolic homeostasis of RPE cells [ 28 , 29 , 30 ]. In RPE cells complement anaphylatoxins receptor signaling is involved in eye morphogenesis [ 31 ], sub-RPE deposits [ 32 ], pro-inflammatory RPE reaction [ 33 , 34 , 35 ], PI3/Akt-pathway activation [ 29 ], and stress-mediated lipid accumulation in RPE cells [ 36 ]. Together this indicates an involvement of autocrine complement reactivity in housekeeping mechanisms maintaining RPE physiology.…”

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers

“…Western Blotting was performed using previously published protocols [35], using primary antibodies (1:1,000) against annexin A2 (Abcam, ab41803) or acetyl α-tubulin (Santa Cruz Biotechnology, 6–11B-1) and β-actin (Cell Signaling Technologies, #8457S) as control. Proteins were visualized with horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology), followed by incubation with Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.) and chemiluminescent detection.…”

C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

“…Nozaki and colleagues were the first to show an impact of C3a on choroidal neovascularization in a mouse model of wet AMD 26 . More recent studies have associated C3a/C3aR function with oxidative stress and calcium mobilization in the RPE 24 , 25 . Additionally, C3a has been associated with changes in the proteolytic activity of the proteasome in a mouse model of age-related RPE atrophy 27 .…”