**3. Results**

#### *3.1. Stressed, In Vivo-Like Cultivation of ARPE-19 Cells*

We investigated cellular stress response and cell-specific complement expression in a cell line of human RPE cells, the ARPE-19 cell line. Aged ARPE-19 cells of passage 39 were cultivated under in vivo-like, unstressed conditions. This was visualized by staining zonula occludens 1 (ZO-1), an important protein for cell–cell contact, and this showed the formation of stable tight junctions and mainly mononuclear, polarized cell growth on transwell filters (Figure 1A,D). Stable transepithelial resistance (TER), a measure of the cell layer's barrier function, and the cell layer's capacitance, which is indicative of the expression of membrane protrusions such as microvilli and other membrane folding, were characteristics of the in vivo-like cultivated ARPE-19 cells (Supplementary Materials, Figure S1A,B). H2O2 treatment resulted in cellular stress, which was indicated by reduced cell–cell contact after 4 h (Figure 1B) and a time-dependent translocation of ZO-1 from the cell membrane to the cytoplasm after 24 h (Figure 1E). Evidence of induced cellular stress by H2O2 was also observed in the increased mRNA expression of vimentin (*vim*) and α-smooth muscle actin (α*-sma*), a typical mesenchymal marker indicating an epithelial–mesenchymal transition (Supplementary Materials, Figure S1C,D) [38–40]. However, the majority of the ARPE-19 cells did not undergo apoptosis under these nonlethal oxidative stress conditions, as shown by a low number of TUNEL-positive cells (Figure 1C,F), and H2O2-treated cells maintained the cell layer's barrier function as well as the cell layer's capacitance (Supplementary Materials, Figure S1E,F).

**Figure 1.** ARPE-19 cells reduced tight junctions and circumvented apoptosis under oxidative stress. (**A**,**D**) ARPE-19 cells untreated (without (w/o)) and stressed with H2O2 for (**B**,**C**) 4 h or (**E**,**F**) 24 h translocated the zonula occludens protein 1 (ZO-1, green) time-dependently from the (**A**,**D**) cell membrane to the (**B**,**E**) cytoplasm. (**C**,**F**) ARPE-19 cells treated with oxidative stress showed a minimal TUNEL-positive (light blue) apoptotic reaction after (**F**) 24 h.

#### *3.2. ARPE-19 Cells Increased Complement Receptor Expression under Oxidative Stress*

ARPE-19 cells express cellular receptors, sense the cellular environment, and can react to complement activation products. Complement receptor 3 (CR3) is a heterodimer integrin consisting of two noncovalently linked subunits (CD11b and CD18) on leukocytes/microglia, and it is activated by C3 cleavage products (iC3b, C3d, and C3dg). We detected CD11b with low expression in mRNA and low protein levels in ARPE-19 cells (Figure 2A,B). Oxidative stress increased *cd11b* mRNA expression after 4 h, which was also shown in protein levels with immunostaining (Figure 2A,C).

The activation of complement protein C5 was detected by complement receptor C5aR1, which was expressed by ARPE-19 cells (Figure 2D). H2O2 treatment increased *c5ar1* expression comparably to *cd11b* expression (Figure 2D-F). C5aR1 protein accumulation was observed after 4 h in the cell nuclei (Figure 2F), which was more distributed in/on the cell after 24 h (Figure 2G). Increased C5aR1 protein levels were also confirmed in Western blots (Figure 2H,I).

The transcription levels of complement receptor *c3aR* were not significantly changed in H2O2-treated ARPE-19 cells (Supplementary Materials, Figure S2A).

**Figure 2.** Oxidative stress increased the expression of complement receptor subunit CD11b and C5aR1 in ARPE-19 cells. (**A**) *Cd11b* mRNA expression was increased 4 h after H2O2 treatment. This effect was confirmed on a protein level by immunohistochemistry using (**B**,**C**) anti-CD11b (red) antibodies. (**D**) *C5ar1* mRNA also increased on (**D**) mRNA and (**E**–**G**) protein level (anti-C5aR1, green) in H2O2 treated cells. (**H**) Western blots of ARPE-19 cell lysates detected C5aR1 between 40 and 60 kDa after 4–24 h H2O2 treatment (full immunoblots are shown in the Supplementary Materials, Figure S3A,B; *n* = 1) (**I**) Quantitatively, C5aR1 expression was increased in H2O2-treated cells in the Western blots. (**A**,**D**) Mean with standard deviation is shown, \* *p* ≤ 0.05, \*\* *p* ≤ 0.01. The dotted line depicts the untreated control; (**B**,**E**,**H**,**I**) w/o untreated control.

#### *3.3. Complement Proteins Accumulated in ARPE-19 Cells under Oxidative Stress*

Complement proteins, which can modulate the activity of complement receptors at the RPE, are locally produced in the retina [26,41] and by ARPE-19 cells (Figure 3; Supplementary Materials, Figure S2B–I). The mRNA expression and cellular protein levels of the stabilizing complement regulator, properdin, were increased after 24 h of H2O2 treatment (Figure 3A,C–E), but properdin secretion was

not detected (Figure 3B, Supplementary Materials, Figure S4G). This indicated properdin storage in the stressed ARPE-19 cells (Figure 3C–E).

**Figure 3.** Oxidative stress induced complement component accumulation in ARPE-19 cells. (**A**) *Properdin* mRNA levels were increased 24 h following H2O2 treatment. This did not affect (**B**) apical properdin secretion, but was confirmed in the protein level by immunohistochemistry using an (**C**–**E**) anti-properdin (red) antibody. (**F**) *C3* mRNA and (**G**) apical C3 protein secretion were not altered in stressed ARPE-19 cells. Immunohistochemistry using (**H**–**J**) anti-C3 (green) antibodies showed an increase of cell-associated (**I**,**J**) C3 after oxidative stress treatment. (**K**) *Cfh* mRNA and (**L**) CFH apical protein concentration were decreased following H2O2 treatment. (**M**–**O**) Immunohistochemistry using anti-complement factor H (CFH, purple) antibodies showed an increase in cell-associated (**N**,**O**) CFH after oxidative stress treatment. Mean with standard deviation is shown, \*\* *p* ≤ 0.01, \*\*\*\* *p* ≤ 0.0001; dotted line depicts untreated control (**A**,**F**,**K**); w/o untreated control (**G**,**G**,**L**); ELISA control standard curves and protein concentrations in the basal supernatants are shown in the Supplementary Materials, Figure S4D–I.

The transcription levels of additional complement components (*c3*, *c4a*, *c4b*, *cfb*, *cfd*, and *c5*) and soluble (*cfh*, *cfi*) and membrane-bound complement regulators (*cd46*, *cd59*) did not significantly change under oxidative stress conditions (Figure 3F,K; Supplementary Materials, Figure S2B–I).

However, we observed a change in cellular accumulation and the modulated secretion of complement components in the protein level through oxidative stress (Figure 3H–J,L–O). Central complement component *c3* was not regulated in mRNA and the protein secretion level by oxidative stress (Figure 3F,G; Supplementary Materials, Figure S4H), but we detected an increase in cellular C3 in immunostainings of ARPE-19 cells (Figure 3H–J). The secretion of C3 was more observable in younger compared to older ARPE-19 cells treated with H2O2 (Supplementary Materials, Figure S4B). A similar effect of cellular complement component accumulation and associated reduced secretion was detectable for complement regulator CFH (Figure 3L–O; Supplementary Materials, Figure S4I). However, *cfh* mRNA expression was not changed under oxidative stress (Figure 3K).

#### *3.4. Autocrine Complement Receptor Activation Following Oxidative Stress Was Correlated with the Release of Proinflammatory and Proangiogenic Factors*

Intracellular complement proteins and cellular complement receptors have previously been associated with the autocrine regulation of cell differentiation and cell physiology in T-cells as well as lung epithelial cells [20,42]. In line with this, we found a colocalization of CD11b and C3 in ARPE-19 cells (Figure 4A,B) and activated C3 fragments (C3b α', C3d) in the ARPE-19 cells (Figure 4C), without adding any exogenous complement source.

**Figure 4.** C3 and complement receptor CD11b were colocalized in ARPE-19. (**A**) Unstressed (w/o) and (**B**) H2O2-treated ARPE-19 cells were stained with anti-C3 (green) and anti-CD11b (red) antibodies. Overlapping staining signals (yellow) suggested a colocalization of C3 and CD11b. (**C**) C3 and activation products (C3b α' and C3d) were detected in untreated and H2O2-treated ARPE-19 cells using a Western blot under reducing conditions (controls: native C3, C3b, human serum (NHS), and C3-depleted human serum (NHS C3dpl)). Full immunoblots are shown in the Supplementary Materials, Figure S3C,D; immunoblots were repeated twice.

The intracellular cleavage of complement proteins into active fragments (independently of the systemic complement cascade) can be mediated by intracellular proteases such as cathepsin B (CTSB) and cathepsin L (CTSL) [17,18]. Both proteases were expressed by ARPE-19 cells, and they were upregulated following oxidative stress (Figure 5). The mRNA expression of *ctsb* and *ctsl* was increased after 24 h of H2O2 treatment (Figure 5A,B). We confirmed a higher concentration of CTSL in ARPE-19 cells under stress conditions also on the protein level (Figure 5C,D).

**Figure 5.** The expression of intracellular proteases was increased by oxidative stress in ARPE-19. (**A**) *Ctsb* and (**B**) c*tsl* mRNA expression increased 24 h after H2O2 treatment. This effect was confirmed on the protein level in immunostainings using an (**C**,**D**) anti-CTSL (green) antibody. (**A**,**B**) Mean with standard deviation is shown, \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, dotted line depicts untreated control; (**C**) w/o untreated control.

The activation of complement receptor signaling regulates the pro- and anti-inflammatory response in T- and RPE cells [24,43]. This can induce inflammasome activation and regulate the mammalian target of rapamycin (mTOR)-pathway, involving the FOXP3 transcription factor [24,25,44]. After the detection of H2O2-dependent regulation of complement receptors (Figure 2), cellular complement protein accumulation (Figure 3), cell-derived C3 colocalized with CD11b, and C3 activation products C3b and C3d in ARPE-19 cells (Figure 4), we hypothesized that the NLRP3 inflammasome and FOXP3 also play an autocrine, complement-dependent role in ARPE-19 cells treated with H2O2. This regulation would be independent of blood-derived complement components and would involve the release of cytokines and growth factors in stressed ARPE-19 cells (Figure 6).

**Figure 6.** Increased *nlrp3* and *foxp3* mRNA expression correlated with proinflammatory and proangiogenic factor secretion. (**A**) *Nlrp3*, (**B**) *foxp3*, and (**C**) *il1*β mRNA levels increased either (**A**,**B**) 4 h or (C) 24 h and 48 h following H2O2 treatment. The proinflammatory cytokine release of (**D**) Interleukin (IL)-1β and (**E**) IL-6 was detected in stressed ARPE-19 cells. This was correlated with an enhanced secretion of the proangiogenic factors (**F**) IL-8 and (**G**) vascular endothelial growth factor (VEGF)-α in H2O2-treated cells. MFI: mean fluorescence intensity. Mean with standard deviation is shown, \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001, \*\*\*\* *p* ≤ 0.0001; (**A**,**B**,**C**) dotted line depicts untreated control; (**D**–**G**) w/o untreated control; protein concentrations in the basal supernatants are shown in the Supplementary Materials, Figure S4J–L.

Indeed, we detected an increased expression of *nlrp3* and *foxp3* mRNA after 4 h of H2O2 treatment (Figure 6A,B). Subsequent enhanced expression of *il1*β mRNA after 24 h and 48 h was associated with increased *nlrp3* levels in stressed ARPE-19 cells (Figure 6C). However, the mRNA expression of *il18* was not changed (Supplementary Materials, Figure S2J). Further, we found higher proinflammatory cytokine levels in the H2O2-treated ARPE-19 cell supernatants compared to the untreated controls (Figure 6D,E). IL-1β was slightly increased after treatment, while IL-6 was significantly elevated in the supernatant of stressed ARPE-19 cells.

Increased *foxp3* expression is an attribute of anti-inflammatory regulatory T-cells, which secrete mainly transforming growth factor (TGF)-β and IL-10. We did not detect a change in *tgf-*β expression (Supplementary Materials, Figure S2K) or IL-10 secretion in H2O2-treated ARPE-19 cells. Therefore, we assumed a proangiogenic function of *foxp3* in the cells, as previously reported [22,23]. In line with this, we observed an increase in IL-8 and VEGF-α concentration in the apical supernatant of stressed ARPE-19 cells (Figure 6F,G). This correlation between complement components, *foxp3* expression, and proangiogenic reactions in RPE cells needs to be further investigated.

As a side note, IL-17, interferon (IFN)-γ, IL-18, IL-2, and tumor necrosis factor (TNF)-α were not detected in the apical or basal supernatant of 4-, 24-, and 48-h untreated and H2O2-treated ARPE-19 cells (data not shown).

#### *3.5. Olaparib Boosted the Proinflammatory Response of ARPE-19 Cells to Oxidative Stimuli*

Oxidative stress-induced cellular reactions have been previously ameliorated by an approved anticancer drug, olaparib, which is an inhibitor of poly(ADP-ribose) polymerase (PARP) [45–47]. We investigated the effects of olaparib on H2O2-dependent mRNA expression changes of complement receptors, components, and inflammation-related transcripts (Figure 7, Supplementary Materials, Figure S5). Oxidative stress increased the expression of *cd11b*, *c5ar1*, and *nlrp3* after 4 h of H2O2 treatment. This was further enhanced by olaparib treatment (Figure 7A–C). An increase in *properdin* and *ctsb* transcripts was observed after 24 h following oxidative stress alone (Figures 3A and 5A). A combination of H2O2 and olaparib accelerated this reaction, with a significant increase in *properdin* and *ctsb* mRNA expression after only 4 h (Figure 7D,E). The expression of *cfd* (Supplementary Materials, Figure S2E) was not altered under oxidative stress; however, H2O2 and olaparib together increased *cfd* transcript levels (Figure 7F). Olaparib did not interfere with the transcription of *foxp3* (Figure 7G) and other transcripts (*c3*, *c4a*, *c5*, *cfb*, *cfh*, *cfi*, *c3ar*, and *ctsl*) (Supplementary Materials, Figure S5) in ARPE-19 cells treated with H2O2.

**Figure 7.** Olaparib enhanced oxidative stress-dependent expression changes in ARPE-19 cells**.** ARPE-19 cells were treated for 4 h with H2O2, and the effect of simultaneously added olaparib on transcription was investigated. (**A**) *Cd11b*, (**B**) *c5aR1*, and (**C**) *nlrp3* transcripts were significantly increased in olaparib-treated, stressed cells compared to unstressed cells. Olaparib also increased the expression of (**D**) *properdin*, (**E**) *ctsb*, and (**F**) *cfd*. (**G**) *Foxp3* mRNA levels were not changed in stressed ARPE-19 cells following olaparib addition. Mean with standard deviation is shown, \* *p* ≤ 0.05; dotted line depicts untreated control.
