Fig 2: Neutralization of TLR2 in a Photo-Oxidative Model of Retinal Degeneration and in Mononuclear Cells Decreases C3 Expression and Deposition(A–D) Three micrograms of anti-TLR2 or anti-IgG was injected IVT into C57Bl6 mice that were then exposed to 100K lux light for 7 days.(A) Quantification of photoreceptor cell rows in anti-IgG versus anti-TLR2 groups.(B and C) Immunofluorescence (IF) of C3 (green) and nuclear DAPI (blue) in mice injected with (B) anti-IgG versus (C) anti-TLR2 (scale bars represent 20 µm).(D) Quantification of outer retinal C3 positive cells/deposits detected in ONL and subretinal space. Data shown are mean ± SEM. The p value was determined by nonparametric t test, p < 0.05. n = 9–10 per experiment, *p < 0.05.(E and F) BMDMs from WT, TLR2–/–, Mal–/–, or MyD88–/–mice were treated for 3, 6, and 24 h with 20 nM of Pam3Cys4; expression of (E) C3 and (F) CFB was assayed by RT-PCR.(G and H) HEK293-TLR2 cells were transfected for 24 h with C3 promoter-luciferase (100 ng), Renilla-luciferase (40 ng), and empty vector (EV) or plasmid expressing (G) Mal or (H) MyD88 at 10, 50, and 80 ng. Results are normalized for Renilla luciferase activity and represented as relative stimulation over the non-stimulated EV control, mean ± SD for triplicate determinations p value determined by one-way ANOVA and Tukey post test: *p < 0.05, **p < 0.01, ***p < 0.001.(I and J) Secreted C3 expression in (I) BMDMs and (J) primary mouse microglia treated with 20 nM of Pam3Cys4 for 6 and 24 or 48 h.

Fig 3: Oxidative Stress Products Drive AP Activation and MAC Formation in a TLR2-Dependent Manner(A–E) Polarized hfRPE cells (A and B) or ARPE-19 cells (C–E) were treated with 10% Hi-NHS or NHS in combination with HSA or CEP-HSA for 24 h, phase transmission, presence of MAC (red), Phalloidin (green), and DAPI (blue), with representative images from three separate experiments.(B and E) Quantification of MAC+ specks in three 303 frames, data mean ± SD, one-way ANOVA followed by Tukey post-test to determine significance between groups; ***p < 0.001.(F–I) IF of MAC (red), Phalloidin (green), and DAPI (blue) in ARPE-19 cells treated with (F and G) 0.1 µg of IgG or anti-TLR2 antibody for 1 h or (H and I) with DMSO or 40 µm of Mal peptide inhibitor for 2 h prior to CEP-HSA and 10% NHS for 24 h.(G and I) quantification of MAC+ specks in four 20× frames, mean ± SD; p value determined by nonparametric t test; p < 0.05.(J–O) WT and TLR2–/– mice injected IV, with NaIO3 (50 mg/kg).(J) Quantification of MAC fluorescent intensity (scale bars represent 20 µm).(K–O) Representative IF images of MAC at 72 h, (K and O) depicts MAC staining in the whole retina section, (L–N) show higher magnifications, (L) shows MAC in the IS and OS, (M) shows MAC in OS and on RPE, (N) shows MAC on RPE.(P and R) Lysed tissue was assayed by western blot for expression of CFB(Bb). C9 and C9b at (P) 24 h and (R) 72 h.(Q and S) Mean pixel density for C9b was quantified at (Q) 24 h and (S) 72 h using the software ImageJ.See also Figures S6 and S7. *p < 0.05, **p < 0.01, ***p < 0.001.