Complement factor H binds malondialdehyde epitopes and protects from oxidative stress

Abstract

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

Figure 1. CFH specifically binds to MDA modifications

a, b, Immunoblot for CFH (molecular weight: 150 kDa) using eluates from either polylysine (PL) or MAA-polylysine (MAA-PL) beads incubated with Ldlr^−/−Rag^−/− mouse plasma (a) or human plasma (b). c, ELISA for binding of purified CFH (5 μg ml⁻¹) to coated native LDL, MAA-LDL, BSA and MAA-BSA. Values are mean ± s.d. relative light units (RLU) per 100 ms of triplicate determinations. d, ELISA for binding of purified CFH or CRP (5 μg ml⁻¹) to coated BSA, MAA-BSA and PC-BSA. Values are mean ± s.d. RLU per 100 ms of triplicate determinations. e–h, Competition immunoassays. e, f, Binding of purified CFH (e) or binding of plasma CFH (f) to coated MAA-BSA in the presence of increasing concentrations of LDL, MDA-LDL, MAA-LDL and Cu²⁺-oxidized (CuOx)-LDL, or BSA and MAA-BSA. g, Binding of biotinylated MDA-LDL to coated CFH in the presence of increasing concentrations of LDL, MDA-LDL, MAA-LDL and CuOx-LDL. h, Binding of biotinylated MDA-LDL to coated CFH in the presence of increasing concentrations of monoclonal antibodies specific for ApoB100 (MB24 and MB47) or MDA (MDA2). Data are expressed as a ratio of binding in the presence of competitor divided by the binding in the absence of competitor (B/B₀) and represent the mean ± s.d. of triplicate determinations. As an estimate for the affinity, the dissociation constants K_d were calculated as 6.4 × 10⁻⁸ mol l⁻¹ for the binding of CFH to coated MAA-BSA and 1.6 × 10⁻⁹ mol l⁻¹ for the binding of MAA-BSA to coated CFH.

Figure 2. The SCR7 domain of CFH is critical for MDA binding

a, ELISA for binding of CFH and recombinantly expressed CFH fragments to coated BSA (white bars) or MAA-BSA (black bars). The length of CFH fragments is indicated by schematic representations with each circle depicting one SCR. Values are mean ± s.d. RLU per 100 ms of triplicate determinations. b, ELISA for binding of purified CFH variant Y402 and CFH variant H402 (both at 1 μg ml⁻¹) to coated MAA-BSA. Values are mean ± s.d. RLU per 100 ms of triplicate determinations. c, ELISA for binding of plasma CFH to coated MDA-LDL in plasma of subjects homozygous for the H402 risk allele (CC, n = 38), heterozygous for the H402 risk allele (CT, n = 88) or homozygous for the Y402 allele (TT, n = 45). The association of rs1061170 with CFH binding to MDA was calculated with P = 1.29⁻⁴⁰ using an additive model. Symbols represent individual subject samples with horizontal bars indicating the mean of each group. Values are mean ± s.d. RLU per 100 ms of triplicate determinations (***P < 0.001).

Figure 3. CFH binds to MDA epitopes present in AMD lesions, on necrotic cells and apoptotic blebs

a–i, Immunohistochemistry of MDA (left), CFH (middle) and C3d (right) localization in human maculas. a–c, Eye of a 72-year-old subject heterozygous for the H402 SNP without AMD. d–f, Eye of a 93-year-old subject homozygous for the H402 SNP with AMD. g–i, IgG control immunostains. Red arrows indicate positive labelling of choriocapillaris basement membrane and arrowheads indicate labelling of Bruch’s membrane (BrM). Scale bar, 25 μm. Sections are representative for 7 donors (5 AMD, 2 controls). j, Confocal immunofluorescent photograph of necrotic RPE cells stained with the MDA-specific IgM natural antibody EO14 (green) and CFH (red), respectively. The right panel shows a merged picture indicating co-localization of CFH binding with the presence of MDA epitopes (yellow). k, Competition assay for the binding of CFH to apoptotic blebs from Jurkat T cells either alone or in the presence of BSA or MAA-BSA assessed by flow cytometry. Values are expressed as mean ± s.e.m. CFH binding (B/B₀) based on mean fluorescence intensities of four independent experiments (**P < 0.01).

Figure 4. CFH inactivates complement on MDA-bearing surfaces

a, Immunoblot of C3b cleavage products induced by three concentrations of CFH variants bound to coated MAA-BSA. α41/43 indicates iC3b cleavage products of the C3b α-chain. β indicates the C3b β-chain that remains uncleaved and served as a loading control. α41/43 was densitometrically quantified, and data are presented as percentage of iC3b generation achieved with 5 μg ml⁻¹ CFH Y402 (= 100%). Shown is the mean ± s.e.m. of three independent experiments. b, Immunoblot of C3b degradation products induced by CFH bound to coated MAA-BSA in the presence of either CFH 18–20 or CFH 15–19.

Figure 5. CFH neutralizes proinflammatory effects of MDA

a, Expression of indicated genes in ARPE-19 cells stimulated for 24 h with 50 μg ml⁻¹ BSA compared to MAA-BSA as determined by quantitative RT–PCR. Data represent the mean ± s.e.m. of three independent experiments. b, Cell-based ELISA for the binding of biotinylated MAA-LDL to thioglycollate-elicited macrophages in the presence of BSA or CFH. Data are expressed as B/B₀ and represent mean ± s.d. of triplicate determinations. c, Secretion of IL-8 by THP-1 cells stimulated for 12 h with BSA or MAA-BSA in the absence or presence of CFH. Numbers below indicate concentrations of CFH, BSA and MAA-BSA in μg ml⁻¹. Error bars represent mean ± s.e.m. of three independent experiments. d, e, Intravitreal injection of BSA, MAA-BSA and/or CFH in mice (n = 4–5 per group). Six hours after injection, RPE/choroid was isolated. d, RT–PCR for Rpe65 and Rho in RPE/choroid fractions. cDNA isolated from neurosensory retina was used as a control (Ct). e, Expression of KC in RPE/choroid as assessed by quantitative RT–PCR. Error bars represent mean ± s.e.m. expression normalized to the BSA-injected group (*P < 0.05, **P < 0.01, ***P < 0.001).