Moss-derived human complement factor H modulates retinal immune response and attenuates retinal degeneration

Abstract

Background: AMD is a multifactorial progressive disease of the central retina that leads to severe vision loss among the elderly. Genome-wide association studies in AMD patients and preclinical data have identified a dysregulated complement system and aberrant microglia responses in the pathogenesis of AMD. Specifically, a genetic variant in the complement factor H (CFH) gene, an important inhibitor of the alternative complement pathway, confers the strongest risk for AMD. Here, we investigated whether moss-derived recombinant human CFH proteins, termed CPV-101 and CPV-104, can modulate microglia reactivity and limit retinal degeneration in a murine light damage paradigm mimicking important features of AMD.

Methods: Two glycosylated human recombinant CFH proteins CPV101, and CPV-104 were produced in moss suspension cultures. In addition, glycans of the CPV-104 variant are sialylated, an optimization that makes CPV-104 an analog of human CFH. BALB/cJ mice received intravitreal injections of 5 µg CPV-101 and CPV-104 or vehicle, starting 1 day prior to exposure to 10,000 lx white light for 30 min. The effects of CPV-101 and CPV-104 treatment on mononuclear phagocyte and Müller cell reactivity were analyzed by immunostainings of retinal sections and flat mounts. Gene expression of microglia markers was analyzed using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT); Blue Peak Autofluorescence (BAF); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.

Results: Light-exposed mice treated with moss-derived recombinant human full-length CFH showed reduced complement activation and MAC deposition in the retina. Concomitantly, mononuclear phagocyte and Müller cell reactivity in light-exposed retinas were also ameliorated upon CFH substitution. Moreover, attenuated light-induced retinal degeneration was detected in mice that received moss-derived CFH.

Conclusion: Modulating the alternative complement pathway using moss-derived recombinant human full-length CFH variant CPV-101 and CPV-104 counter-regulate gliosis and attenuates light-induced retinal degeneration, highlighting a promising concept for the treatment of AMD patients.

Keywords: CPV-101; CPV-104; Complement factor H; Light damage; Microglia; Moss-derived recombinant CFH; Retinal degeneration.

Fig. 1

Moss-derived recombinant human CFH variants CPV-101 and CPV-104 reduce accumulation of Iba1⁺ cells in the subretinal space. A Representative images from Iba1-stained retinal flat mounts and autofluorescence (AF) in the subretinal space (SRS) one, three and four days after light exposure of mice treated with 5 µg CPV-101, CPV-104 or vehicle control. Scale bar: 100 µm. B Quantification of Iba1⁺ area in SRS. C Quantification of Iba1⁺ cells in SRS. D Quantification of autofluorescent area. Data shown as mean ± SEM, n = 9–11 retinas. One-way ANOVA followed by Sidak’s multiple comparison test; *P < 0.05, **P < 0.01 and ***P ≤ 0.001

Fig. 2

Moss-derived recombinant human CFH variants attenuate mononuclear phagocyte reactivity in light-exposed retinas. A Representative images from the outer plexiform layer (OPL) of Iba1-stained retinal flat mounts from control and light-exposed mice one, three and four days after light exposure. Scale bar: 100 µm. B Pro-inflammatory cytokine and complement levels in the retina of control and light-exposed mice at indicated time point. C Representative Western blot of C3d positive C3 cleavage products 1d after light exposure. D Quantification of C3 cleavage products normalized to actin band intensity and no light control. Data shown as mean ± SEM, n = 3–9 retinas. One-way ANOVA followed by Sidak’s multiple comparison test; *P < 0.05, **P < 0.01 and ***P ≤ 0.001. N.d. not detected

Fig. 3

Moss-derived recombinant human CFH variants reduce formation of membrane attack complex and mononuclear phagocyte migration in light-exposed retinas. A Representative images from Iba1-, C5b-9 membrane attack complex (MAC)—and DAPI-stained cryosections from control and light-exposed mice at indicated time points. AF, autofluorescence. Scale bar: 100 µm. ONL, outer nuclear layer; INL, inner nuclear layer and GCL, ganglion cell layer. B, C Quantification of Iba1⁺ cells in ONL (B) and in SRS (C) at indicated time points. D Quantification of C5b-9 area in SRS and ONL. Data show mean ± SEM; n = 9–13 retinas. One-way ANOVA followed by Sidak’s multiple comparison test; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001

Fig. 4

Moss-derived recombinant human CFH variants CPV-101 and CPV-104 attenuate light-induced retinal degeneration. A Representative heat maps from fundus images at indicated time points from control and light-exposed mice. Lower panel shows OCT scan. Scale bar: 200 µm. ONL, outer nuclear layer. B Quantification of retinal thickness in the central (circle diameter 3 mm) and peripheral area (circle diameter 6 mm). Data show mean ± SEM; n = 18–27 eyes. One-way ANOVA followed by Sidak’s multiple comparison test; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001

Fig. 5

Moss-derived recombinant CFH variants CPV-101 and CPV-104 attenuate photoreceptor cell death in light-exposed retinas. A Representative images from TUNEL- and DAPI-stained cryosections from control and light-exposed mice at indicated time points. Scale bar: 100 µm. ONL, outer nuclear layer; INL, inner nuclear layer and GCL, ganglion cell layer. B Quantification of TUNEL⁺ cells at indicated time points. Data show as mean ± SEM; n = 9–13 retinas. One-way ANOVA followed by Sidak’s multiple comparison test; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001