Complement Factor H-Related 3 induces inflammation and complosome activation in human RPE cells

Abstract

Background Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration. The non-canonical local, cellular functions of FHR-3 remained poorly understood.Methods Human retinal pigment epithelium (RPE) cells (ARPE-19 cells and primary human RPE cells (hpRPE)), cultivated in Transwell® inserts, were apically treated with either FHR‑3 alone or with the chimerized monoclonal anti‑FHR-3 antibody RETC-2-ximab, or with FHR-1, FH, Properdin or not treated for 5 – 24 h, respectively. Interaction of FHR-3 with oxidative stress epitopes was determined by ELISA. Internalization studies of FHR-3 or FH by ARPE‑19 cells was determined by immunofluorescence live cell imaging. Impact of FHR-3 on RPE cell-specific complement components and inflammation markers were analyzed on mRNA (RT-qPCR) and on protein level (Western Blot, ELISA, protein secretion assays, immunofluorescence). Results Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by senescent viable RPE cells and modulated time-dependently complement component (C3, CFB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory micro-environment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Additionally, important pattern recognition molecules of the innate immune system, Toll-like receptors 1 and 3, as well as proteasome subunits were impaired in RPE cells after FHR-3 incubation. A chimerized monoclonal anti-FHR-3 antibody, RETC‑2‑ximab, ameliorated the effect of FHR-3 on ARPE-19 cells.Conclusion Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell complosome and as a productive target for a new therapeutic approach using RETC‑2‑ximab for associated degenerative diseases.