Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1–48 h to H2O2 had reduced cell–cell contact and increased markers for epithelial–mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration.

The retinal pigment epithelium (RPE) maintains visual function and preserves structural integrity of the retina. Chronic dysfunction of the RPE is associated with retinal degeneration, including age-related macular degeneration (AMD). The AMD pathogenesis includes both increased oxidative stress and complement dysregulation. Physiological sources of oxidative stress in the retina are well known, while complement sources and regulation are still under debate. Using human primary RPE (hpRPE) cells, we have established a model to investigate complement component expression on transcript and protein level in AMD-risk and non-risk hpRPE cells. We evaluated the effect of properdin, a complement stabilizer, on the hpRPE cell-dependent complement profile exposed to oxidative stress. hpRPE cells expressed complement components, receptors and regulators. Complement proteins were also stored and secreted by hpRPE cells. We associated AMD-risk single nucleotide polymorphisms with an increased secretion of complement factors D (CFD) and I (CFI). Furthermore, we detected hpRPE cell-associated complement activation products (C3a, C5a) independent of any extracellularly added complement system. Exogenous properdin increased the mRNA expression of CFI and CFD, but decreased levels of complement components (C1Q, C3), receptors (C3AR, C5AR1, CD11B) and inflammation-associated transcripts (NLRP3, IL1B) in hpRPE cells exposed to oxidative stress. This properdin effect was time-dependently counter regulated. In conclusion, our data unveiled a local, genotype-associated complement component production in hpRPE cells, regulated by exogenous properdin. The local complement production and activation via blood-independent mechanisms can be a new therapeutic target for AMD.

15 16 Page 2 of 35 HIGHLIGHTS 17  Oxidative stress accumulates complement proteins and receptors in RPE cells 18  Oxidative stress activates the RPE inflammasome without external complement proteins 19  Oxidative stress increases foxp3 expression and IL-8/VEGF secretion in RPE cells 20  Olaparib enhances pro-inflammatory response of RPE 21 22 ABSTRACT 23Oxidative stress-induced damage of the retinal pigment epithelium (RPE) together with chronic 24 inflammation has been suggested as major contributors to retinal diseases. Here, we examine the 25 effects of oxidative stress and endogenous complement components on the RPE and its pro-26 inflammatory and -angiogenic responses. 27The RPE cell line, ARPE-19, treated with H 2 O 2 reduced cell-cell contacts, increased marker for 28 epithelial-mesenchymal transition but showed less cell death. Stressed ARPE-19 cells increased the 29 expression of complement receptors CR3 and C5aR1. CR3 was co-localized with cell-derived 30 complement protein C3, which was observed in its activated form in ARPE-19 cells. C3 as well as its 31 regulators CFH and properdin accumulated in ARPE-19 cells after oxidative stress independent from 32 external complement sources. This cell-associated complement accumulation promoted nlrp3 and 33 foxp3 expression and subsequent increased secretion of pro-inflammatory and pro-angiogenic factors. 34The complement-associated ARPE-19 reaction to oxidative stress, independent from external 35 complement source, was increased by the PARP-inhibitor olaparib. 36Our results indicated that RPE cell-derived complement proteins and receptors are involved in RPE cell 37 homeostasis following oxidative stress and should be considered as targets for treatment 38 developments for retinal degeneration. 39 40 Page 4 of 35 GRAPHICAL ABSTRACT 41 42We show a functional link between oxidative stress, complement receptors, endogenous complement 43 proteins, pro-angiogenic and -inflammatory responses in ARPE-19 cells. These effects are independent 44 from extracellularly added complement proteins or receptor ligands. We suggest an oxidative stress-45 associated autocrine mechanism of complement receptor regulation in ARPE-19 cells in connection 46 with upregulated intracellular proteases. 47