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

Journal of Neuroinflammation volume 22, Article number: 104 (2025) Cite this article

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.

Background

Age-related macular degeneration (AMD) is a complex and multifactorial disease and one of the leading causes of vision loss in the elderly of the western world. In early asymptomatic stages of AMD, insoluble lipid-rich deposits, so called drusen, accumulate between RPE and Bruch’s membrane. In advanced stages, AMD can be classified either as geographic atrophy (GA) (dry form) or as the neovascular form characterized by neoangiogenesis [1]. The etiology of AMD is still not fully understood due to complex interactions of environmental and genetic factors that influence the disease risk. Beside the impact of age, sex and ethnicity on the prevalence of AMD, individual lifestyle such as high fat diet and smoking play also an important role [2,3,4]. In addition, several lines of evidence including genome-wide association studies in AMD patients and animal model systems implicate dysregulated complement system and microglia reactivity as relevant pathomechanisms in the development and progression of AMD [5, 6].

Microglia, the tissue-resident immune cells of the retina, play crucial roles in both initiating innate immune responses and preserving tissue integrity. However, chronic microglia reactivity, a common hallmark in retinal degenerative diseases, may endanger the compromised tissue and negatively contribute to disease progression [7, 8]. Notably, several studies have demonstrated that targeted microglia-directed immunomodulation can improve disease outcome in a variety of retinal degenerative diseases [9,10,11,12]. Equipped with various receptors, microglia respond to different disease-related factors such as cell debris, modified cell surfaces and complement factors [10, 13, 14]. Additionally, it has become evident in recent years that microglia not only express complement receptors but also locally produce certain complement factors [15, 16]. Therefore, de novo complement production and activation by retinal microglia may contribute to photoreceptor degeneration and disease progression.

The complement system, as a part of the innate immune system, is a highly regulated protein cascade that lead upon activation to clearance of damaged cells or pathogens [17]. While the three complement pathways, classical, alternative and mannose-binding lectin pathway, differ in their initiation, they all result in complement activation and the formation of the C5b9 membrane attack complex (MAC) [18, 19]. The complement cascade, especially the alternative pathway needs to be tightly regulated to avoid excessive activation and systemic inflammation. Dysregulation and over-activation of the complement system is associated with a number of systemic and organ-specific diseases including the rare kidney diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy but also with blinding diseases such as AMD [20,21,22].

Several genetic studies have identified common [23,24,25,26] and rare [27, 28] single nucleotide polymorphisms (SNPs) within the complement factor H (CFH) gene, an important regulator of the alternative complement pathway, that are associated with significantly increased AMD risk. In particular, the CFH Y402H polymorphism (rs1061170) is one of the most studied and replicated common genetic variant that confers the strongest risk for AMD [23,24,25,26]. Subsequently, additional AMD-risk–associated genetic variants have been identified in other complement genes including C3; CFI, CFB and C9 which highlights the complement system and its components as therapeutic targets [29,30,31,32].

However, over the last years, most clinical trials targeting the complement system in patients with GA have failed or barely met the endpoint [33]. With the recent FDA approvals of avacincaptad pegol (Izervay®) and pegcetacoplan (SYFOVRE®), modulation of the complement system via binding to C5 and C3 respectively has emerged as a promising therapeutic approach for attenuating progression of GA although no significant effect on visual acuity was observed [34, 35]. Both drugs block the complement cascade at its central stages that could lead to overall increased susceptibility to infections. Thus, therapeutic approaches aiming at replacing or substituting of complement regulators such as CFH could be beneficial.

CFH regulates the alternative pathway either by inhibiting the assembly of C3 and C5 convertases via competition with factor B for C3b binding, by facilitating the disassembly of the convertases by displacing bound factor Bb (decay accelerating activity) or by acting as a cofactor for CFI mediated proteolysis in order to protect host cells from self-attack by complement [36]. Notably, the substitution of purified plasma-derived CFH have already been shown to improve the renal phenotype in a mouse model for complement-mediated C3 glomerulopathy. Here, CFH-deficient mice that received purified CFH showed normalized complement levels and activity followed by the resolution of glomerular C3 deposits [37].

In the eye, AAV-mediated expression of truncated versions of human CFH in retinal Müller cells resulted in attenuated glial reactivity and retinal degeneration following ischemic injury [38]. Transcriptomic analysis revealed reduced complement activation concomitantly with increased expression of complement regulators upon CFH treatments [38].

Although several studies aiming at replacing or substituting CFH show promising results, the production of a recombinant full-length CFH is challenging as it is a complex, glycosylated molecule. So far, only production in human embryonic kidney (HEK) cells [39] and moss has been able to yield relevant amounts of functional CFH with human-like glycosylation [40]. However, the plant-derived complex N-glycan formations carry fucose and xylose residues that are capable of inducing adverse immune reactions as they are not present in mammals [41]. Thus, we took advantage of the glyco-engineered moss strain Physcomitrium patens, that is lacking the α1,3 fucosyltransferase and β1,2 xylosyltransferase, in order to produce two full-length recombinant human CFH variants termed CPV-101 and CPV-104. Both variants show full in vitro complement regulatory activity similar to that of serum-derived CFH [40]. Glycans of the CPV-104 variant are additionally sialylated, resulting in improved pharmacokinetics [42].

In this study, we investigated the immunomodulatory properties of two moss-derived recombinant human CFH variants during retinal degeneration. Using the light-induced acute retinal degeneration mouse model, an established system to study key aspects of dry AMD [43], we demonstrated that both CFH variants reduced not only complement activation but also attenuated retinal mononuclear phagocytes and Müller cell reactivity. The subsequent retinal degeneration was strongly diminished by intravitreal injections of both CFH variants. Collectively, our findings revealed that moss-derived recombinant human CFH variants CPV-101 and CPV-104 exert immunomodulatory and neuroprotective effects, representing a promising concept for the treatment of AMD patients.

Material and methods

Production and purification of CPV-101 and CPV-104

The amino acid sequence of CPV-101 and CPV-104 match the sequence of human CFH (UniProt ID: P08603) except position 62, where we selected the polymorphism 62I. Recombinant proteins were produced in moss suspension cultures cultivated in illuminated 500-L single-use stirred-tank reactors (Sartorius Biostat STR500) as previously described [40]. The clarified culture supernatant was concentrated by tangential flow filtration (30 kDa cut-off, regenerated cellulose) and loaded onto a CFH affinity column (Repligen) equilibrated with 20 mM Tris, 150 mM NaCl, pH 7.4. CPV-101 was eluted in a single step using 100 mM citric acid, 5% propylene glycol, 100 mM L-arginine pH 3.0. The resulting pool was either directly further purified as described below to yield CPV-101 or used for two-step in vitro sialylation using β−1,4-galactosyltransferase and α−2,6-sialyltransferase (Roche Custom Biotech, Germany) according to the manufacturer’s protocol. The resulting sialylated recombinant CFH (CPV-104) was loaded onto a CaptoDeVirs column (Cytiva, Uppsala, Sweden) equilibrated with 20 mM Tris, 50 mM NaCl, pH8.5 and eluted using step gradient with increased NaCl concentration. Fraction containing CPV-104 was loaded onto a CaptoAdhere ImpRes column (Cytiva, Uppsala, Sweden) equilibrated with 20 mM, Tris 500 mM, NaCl pH8.5. pH was lowered to 5.0 with a washing step and CPV-104 was eluted with step gradient by reduction of NaCl. Fraction containing CPV-104 was formulated in 20 mM Tris–HCl (pH 8.5) containing 150 mM NaCl and 0.02% Tween-20 at a concentration of 10–20 mg/ml. Product was stored at − 80 °C. For use in mice studies both CFH variants underwent buffer change into PBS and were diluted to approx. 5 mg/ml. 45–60% of the N-linked glycans in CPV-101 feature terminal N acetylglucosamine (GlcNac/GlcNac) residues. Due to the additional sialylation step CPV-104 variant exhibits in contrast ~ 50% diantennary sialylated glycans.

Animals

8–10-week-old albino BALB/cJ mice of both sexes purchased from Janvier were housed under specific pathogen–free conditions in individually ventilated caging (IVC) systems (GM 500, Tecniplast® Greenline) with a maximum cage density of five adult mice per cage. Light was adjusted to a 12 h/12 h light/dark cycle with light on at 6 a.m., temperature and relative humidity were regulated to 22 ± 2 °C and 45–65% relative humidity. Mice were fed irradiated phytoestrogen-free standard diet for rodents (Altromin 1314; 59% carbohydrates, 27% protein, 14% fat) and had access to food and acidified water ad libitum. All experimental procedures complied with the German law on animal protection and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animal protocols used in this study were reviewed and approved by the governmental body responsible for animal welfare in the state of Nordrhein-Westfalen (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany, Application No. 81-02.04.2021.A277).

Intravitreal injections

Animal cages were randomly allocated to the experimental groups. The following compounds (all diluted in 1 × PBS) were injected intravitreally (ivt.) one day before light exposure: 1 μl of either 5 µg CPV-101; 5 µg CPV-104 or PBS as vehicle control. For this, eyes were treated with Conjuncain® EDO® 0.4 mg/ml (Bausch & Lomb) eye drops to anesthetize the ocular surface and a 34-gauge needle was inserted into the vitreous space approximately 1.5 mm below the limbus and the compounds were administered bilaterally with a NanoFil syringe (Word Precision Instruments). Afterwards, eyes were covered with Bepanthen® eye and nose ointment (Bayer HealthCare).

Light exposure regimen

Mice were dark-adapted for 16 h before light exposure. After pupil dilatation with a topical drop of phenylephrine 2.5%–tropicamide 0.5% under dim red light, the mice were exposed to bright white light with an intensity of 10,000 lx for 30 min. After light exposure, the mice were housed under normal light conditions for the remaining experimental period.

Spectral domain optical coherence tomography (SD-OCT) and BluePeak autofluorescence (BAF)

Mice were anesthetized with a mixture of ketamine (100 mg/kg body weight, Ketavet; Pfizer Animal Health) and xylazine (5 mg/kg body weight, 2% Rompun; Bayer HealthCare) diluted in 0.9% sodium chloride by intraperitoneal (i.p.) injection and their pupils dilated with a topical drop of phenylephrine 2.5%–tropicamide 0.5% before image acquisition. Spectral domain optical coherence tomography (SD-OCT) and BluePeak autofluorescence was performed on both eyes with a Spectralis™ HRA + OCT device (Heidelberg Engineering) to investigate structural changes in the retina after light exposure. Retinal thickness measurements were performed using the Heidelberg Eye Explorer Software using a circular ring scan (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (μm) in a certain field.

Immunohistochemistry

Mice were euthanized by cervical dislocation and the eyes enucleated and fixed in 4% ROTI®Histofix (Carl Roth) for 3 h at room temperature (RT). Eyes were embedded in Tissue-Tek® optimal cutting temperature (O.C.T) compound (Sakura Finetek) or dissected for flat mount analysis as described before [44]. The dissected retinal flat mounts were permeabilized and blocked overnight in Perm/Block buffer (5% normal donkey serum (NDS), 0.2% BSA, 0.3% Triton X-100 in PBS) at 4 °C while 10 µm cryosections were rehydrated in 1 × PBS and blocked in BLOTTO (1% milk powder, 0.01% Triton X-100. The flat mounts or cryosections were subsequently incubated with a polyclonal rabbit anti-Iba1 antibody (1:500 diluted in Perm/Block or BLOTTO, 019–19741, Wako) for 48 h at 4 °C. After washing three times with PBST-X (0.3% Triton X-100 in 1 × PBS), the flat mounts were incubated for 1 h with donkey anti-rabbit AlexaFluor™ 488 (1:1000 diluted in Perm/Block; A21206, Invitrogen). After several washing steps, flat mounts were mounted on a microscope slide. Flat mounts and cryosections were embedded with fluorescence mounting medium (Vectashield HardSet H-1400, Vector Labs) or Fluoromount-GTM with DAPI, 00–4959-52, Thermo Scientific), respectively. Glial fibrillary acidic protein (GFAP) was stained accordingly with a polyclonal rabbit anti-GFAP antibody (1:400 diluted in Perm/Block; OG9269, Sigma-Aldrich) and a donkey anti-rabbit AlexaFluor™ 647 (1:1000 diluted in Perm/Block; A-31573, Invitrogen). Glutamine synthetase (GS) was stained with monoclonal mouse anti-GS antibody (1:200 diluted in Perm/Block; MAB302, Sigma-Aldrich) and a goat anti-mouse AlexaFluorTM488 (1:1000 diluted in Perm/Block; A-11001, Invitrogen). Membrane attack complex (MAC, C5b9) was stained with a monoclonal mouse anti-MAC antibody (1:100 diluted in Perm/Block; FGI-10–1801, Biozol) and a goat anti-mouse AlexaFluor™ 488 (1:1000 diluted in Perm/Block; A-11001, Invitrogen).

Four images with each a total area of 0.15mm2 from the central part of the retina were taken with a Zeiss Imager.M2 equipped with an ApoTome.2 In retinal flat mounts, the Iba1+ area was analyzed using FIJI. For this, the images were converted to 16-bit grayscale, the threshold adjusted and the Iba1 area analyzed. The values from the four images per flat mount were averaged and displayed as one data point. Morphometric parameters in retinal flat mounts were analyzed using MotiQ, a fully automated analysis software. MotiQ was developed as an ImageJ plugin in Java and is publicly available (https://github.com/hansenjn/MotiQ). All segmentation and quantification were performed on 2D mean intensity projections (MIPs) of 3D image data.

In retinal cryosections, the C5b9+ area was analyzed using FIJI only in the SRS and ONL. For this the images were converted to 16-bit grayscale, the threshold adjusted and the C5b9 area analyzed only in the region of interest that covered the SRS and ONL. The values from the four images per cryosection were averaged and displayed as one data point.

TUNEL assay

Retinal sections were labeled with an in situ cell death detection kit (TMR red, Roche) according to the manufacturer’s instructions. Fluoromount-G™ with DAPI was used to mount the sections and to counterstain the nuclei. Four images from the central part of each retina were taken with a Zeiss Imager.M2 equipped with an ApoTome.2 and analyzed by counting all DAPI+ nuclei in the outer nuclear layer (ONL) and the TUNEL+ cells in the ONL. The percentage of TUNEL+ photoreceptors in the ONL was calculated as the ratio of TUNEL+ nuclei to the total number of photoreceptor nuclei in the ONL, multiplied by 100. For each mouse the four values were averaged and displayed as one data point.

ELISA

The concentration of cytokines and chemokines in total retinal and RPE lysates were measured by ELISA as described before [45]. In brief, tissue samples were sonicated in 1 × PBS supplemented with protease and phosphatase inhibitors (Complete protease inhibitor cocktail, Roche). Ccl2/JE/Mcp-1 (DY479), Il-1beta/IL-1F2 (DY401) and Il-6 (DY406) were purchased from R&D Systems. ELISA for C3 (ab263884) and C5a (ab193718) were purchased from abcam. Each ELISA was used accordingly to manufacturer's instructions. Absorbance was measured with a TECAN infinite M1000.

Western blot

Retinas were lysed by sonication in 1 × PBS supplemented with protease and phosphatase inhibitors (Complete protease inhibitor cocktail, Roche). Protein concentration was determined by BCA Protein Assay according to the manufacturer’s instructions (Thermo Scientific). A total of 40 µg of samples were heated for 5 min at 95 °C in Laemmli buffer supplemented with β-mercaptoethanol and then loaded onto 12% tris–glycine polyacrylamide gels and run under standard conditions. For immunoblotting, proteins were transferred on 0.45 µm nitrocellulose membrane (Bio Rad) at 25 V and max. 1 A for 30 min. Membranes were blocked with 5% nonfat dried milk powder in TBS‐T before incubation with primary antibody against C3d (1:2500 dilution, polyclonal goat, AF2655, R&D systems) or Actin (1:2500 dilution, monoclonal mouse, clone AC‐74, A2228, Sigma‐Aldrich). After washing with TBS-T, membranes were incubated with HRP-conjugated secondary antibodies (1:5000 dilution, polyclonal rabbit anti-goat, P0449, Dako or polyclonal goat anti-mouse P0447, Dako) and blot was imaged with ECL chemostar, Intas with ChromoStarTS software. A volume of 5 µl Thermo Scientific™ PageRuler™ Prestained Protein ladder was used for identification of protein size. C3b cleavage product amounts were analyzed with FIJI. Uncropped Western Blot are shown in supplement Fig. 1.

Quantitative real-time PCR

RNA was isolated from retinas and RPE/choroidal tissue using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized from the total mRNA using the RevertAid™ H Minus First strand cDNA Synthesis Kit (Thermo Scientific). Transcript levels of Tspo, Il-6, Ccl2, and C3 were analyzed by quantitative real-time PCR performed in LightCycler® 480 II (Roche) with SYBR® Green (Takyon No Rox SYBR Master Mix dTTP blue, Eurogentec). Amplification of Atp5b served as a control. Measurements were performed in technical duplicates and ΔΔCT threshold calculation was used for relative quantification of results. All primers were ordered from IDT. Primer sequences used: mAtp5b forward primer 5’-ggcacaatgcaggaaagg-3’, reverse primer 5’-tcagcaggcacatagatagcc-3’, mC3 forward primer 5’-accttacctcggcaagtttct-3’, reverse primer 5’-ttgtagagctgctggtcagg-3’, mCcl2 forward primer 5’-catccacgtgttggctca-3’, reverse primer 5’-gatcatcttgctggtgaatgagt-3’, mIl-6 forward primer 5’-gatggatgctaccaaactggat-3’, reverse primer 5’-ccaggtagctatggtactccaga-3’, mLgals3 forward primer 5’-cccaacgcaaacaggattgt-3’, reverse primer 5’-gaagcgggggttaaagtgga-3’ and mTspo forward primer 5’-ggaacaaccagcgactgc-3’, reverse primer 5’-gtacaaagtaggctcccatgaa-3’.

Flow cytometry

Two retinas were pooled and dissociated in Collagenase D for 15 min at 37 °C. Following this, 750 U/ml DNase was added, and the sample was incubated for an additional 7 min at 37 °C. The resulting cell suspension was filtered through a 70 µm cell strainer and washed with 10 ml of HBSS+/+ containing 10% FBS. Live/dead staining was performed using Zombie Aqua, following the manufacturer’s instructions. For blocking, cells were incubated with Ultra-LEAF™ Purified anti-mouse CD16/32 antibody (Biolegend, 101330). Compensation was performed using single-stained UltraComp eBeads™ Compensation Beads (Invitrogen, 01–2222-41) and FMOs. Used antibodies (Brilliant Violet 421™ anti-mouse/human CD11b Biolegend 101251, APC anti-mouse CD45.2 Biolegend 109814, PerCP/Cyanine5.5 anti-mouse Ly-6C Biolegend 128012 and PE anti-mouse F4/80 Biolegend 111704) were incubated for 30 min at RT. Afterwards cells were washed twice with FACS buffer (PBS, 5% FCS) and resuspended in 150 µl FACS buffer for measurement. Samples were measured using a BD FACSDiva™. Analysis was done using FlowJo 10.

Statistical analysis

Data were analyzed using GraphPad Prism 9 software (v 9.4.1, GraphPad software Inc.). Significance of difference between means was determined by one-way ANOVA followed by Sidak’s multiple comparison test. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01 and ***P ≤ 0.001.

Results

Moss-derived recombinant human CFH variants reduce gliosis and complement activation in light-exposed mice

To investigate if substitution with recombinant human full-length CFH has beneficial effects on retinal degeneration, we used the light-induced retinal degeneration mouse model mimicking key features of dry AMD including gliosis and photoreceptor cell death [43]. Since the production of recombinant human full-length CFH remains challenging due to its complex glycosylation pattern, we used the glyco-engineered moss strain Physcomitrium patens and produced two different full-length CFH variants, CPV-101 and CPV-104. Both variants showed full in vitro complement regulatory activity similar to that of serum-derived CFH [40], while glycans of the CPV-104 variant are additionally sialylated, resulting in improved pharmacokinetics [42].

Next, we assessed whether treatment with moss-derived recombinant human full-length CFH variants modulates microglia reactivity. While in healthy control mice, only few Iba1+ cells are found in the subretinal space (SRS), vehicle-treated light-exposed mice revealed an increased accumulation of amoeboid-shaped Iba1+ cells and autofluorescent cell debris in the SRS starting one day post light exposure (Fig. 1). Mice that received CPV-101 or CPV-104 showed significantly reduced accumulation of reactive mononuclear phagocytes and autofluorescent material the SRS (Fig. 1). To further evaluate whether the Iba1+ cells are monocyte-derived macrophages or microglia, we analyzed the cells using flow cytometry. Here total number of both microglia and macrophages increased after light exposure (Supplementary Fig. 2). However, the majority of CD11b+ cells were microglia.

Fig. 1
figure 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

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Furthermore, less amoeboid and more ramified mononuclear phagocytes were detected in the outer plexiform layer (OPL) of CPV-101- and CPV-104-treated mice after light exposure compared to the vehicle group (Fig. 2A). In line with this, analysis of mononuclear phagocytes morphology revealed a significant increase in total and spanned area, outline, ramification index as well as in the number of junctions, branches and endpoints in light-exposed mice that received CPV-101 or CPV-104 compared to the vehicle group (Supplementary Fig. 3). While, light damage-induced expression of Ccl2, ll-6; Tspo and C3 was reduced after CPV-101 or CPV-104 treatment, expression of Cfh did not change upon light exposure or CPV treatment (Supplementary Fig. 4). In addition, transcript levels of Lgals3, a marker that was recently shown to be upregulated in subretinal microglia during retinal degeneration, were analyzed [46]. Here increased levels of Lgals3 were observed after light exposure, while treatment with CPV-101 or CPV-104 resulted in reduced expression (Supplementary Fig. 4).

Fig. 2
figure 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

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Also, increased chemokine and cytokine levels upon light exposure, were decreased upon treatment with both recombinant CFH variants (Fig. 2B). Increased levels of C3 and C5a were also detected in retinas of light-exposed mice, while treatment with CPV-101 or CPV-104 did not affect C3 protein levels (Fig. 2B). Although not significant, decreased C5a levels were detected upon treatment with both recombinant human full-length CFH variants (Fig. 2B).

Also, C3b cleavage products like the α chain of C3b, the β chain of C3b and iC3b as well as C3dg/ C3d were slightly increased in after light exposure (Fig. 2C, D). However, this increase in C3 cleavage products, although not significant, was more pronounced in mice-treated with CPV-101 or CPV-104.

In addition to morphology of Iba1+ cells, we also assessed the effect of both CFH variants on Iba1+ cell localization and migration upon light exposure. While control mice displayed Iba1+ cells mainly in the plexiform layers, vehicle-treated light-exposed retinas revealed an increased migration and accumulation of Iba1+ cells in the ONL and SRS starting one day post light exposure (Fig. 3A–C). This strong retinal degeneration associated with accumulation of activated mononuclear phagocytes was reduced in light-exposed mice treated with CPV-101 or CPV-104 (Fig. 3A–C).

Fig. 3
figure 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

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Complement activation results in MAC formation and deposition, producing pores in the cell membrane that finally leads to lysis of the target cells. Thus, we analyzed the effect of CPV-101 and CPV-104 on MAC formation in light-exposed retinas. Here, retinas from light-exposed vehicle-treated mice revealed increased MAC deposition in the SRS and ONL, while treatment with recombinant CFH variants CPV-101 and CPV-104 resulted in reduced MAC deposition (Fig. 3A, D).

In addition to the analysis of Iba1+ cell reactivity, we also analyzed the effect of CPV-101 and CPV-104 on Müller cell activation. For this, the expression of glutamine synthetase (Gs), a constitutively expressed Müller cell protein and glial fibrillary acid protein (Gfap), a marker for astrocytes and Müller cell reactivity, was analyzed. While no differences in Gs staining were observed, the expression of Gfap was significantly increased and extended to the ONL in light-exposed retinas. However, mice that received CPV-101 or CPV-104 showed decreased Gfap levels and stress fibers compared to the vehicle group (Supplementary Fig. 5).

Collectively, these data indicate that treatment with moss-derived recombinant human CFH variants CPV-101 and CPV-104 potently reduce not only complement activation but also retinal mononuclear phagocyte and Müller cell reactivity.

Moss-derived recombinant human CFH variants delay light-induced retinal degeneration

Next, we examined the effects of CPV-101 and CPV-104 on retinal disease progression using in vivo spectral domain optical coherence tomography (SD-OCT). While no differences in retinal thickness were detected one day after light exposure (Fig. 4A, B), strong and progressive retinal thinning were observed in light-exposed vehicle-treated mice after three and four days (Fig. 4C–F). However, mice that received CPV-101 or CPV-104 revealed a preservation of retinal thickness in the central area as well as periphery (Fig. 4). Enlarged OCT images can be found in supplementary Fig. 6. Additionally, BluePeak autofluorescence (BAF) was performed in order to examine accumulation of autofluorescent material within the light-exposed eyes. In contrast to the control, light-exposed vehicle-treated mice showed a strong accumulation of autofluorescent foci, while CPV-101 and CPV-104 treatment resulted in significantly less accumulation (Supplementary Fig. 7). Additionally, retinal thickness was also analyzed up to day 7 after light exposure to assess whether treatment with recombinant hCFH is attenuating degeneration over time (Supplementary Fig. 8). Here, we found that the degeneration of the retina is indeed attenuated and less intense upon CPV treatment compared to PBS-treated mice which showed a continuous progression of retinal degeneration over the time course.

Fig. 4
figure 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

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To validate these findings, we also analyzed cell death in retinal cryosections via deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Here, increased TUNEL+ cells, especially in the ONL, were observed in vehicle-treated light-exposed mice compared to control (Fig. 5A, B). Treatment with CPV-101 and CPV-104 resulted in significantly less TUNEL+ cells compared to the vehicle group (Fig. 5A, B). Taken together, treatment with moss-derived recombinant human CFH variants CPV-101 and CPV-104 reduce light-induced retinal degeneration.

Fig. 5
figure 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

Full size image

Discussion

There is strong genetic and biological evidence that dysregulation of the complement system, especially the alternative pathway, is a major driver of AMD pathogenesis.

Interestingly, substitution of CFH has had promising results in patients with complement-mediated kidney diseases, indicating a valuable treatment option for complement-related disorders [47, 48]. In this study, we demonstrate that modulation of the complement system via two moss-derived recombinant human full-length CFH variants improved disease outcome in the light-induced retinal degeneration mouse model, an established system that recapitulates key pathological features of dry AMD.

Our data show that substitution with moss-derived recombinant CFH variants significantly reduce mononuclear phagocyte reactivity and migration into the ONL and SRS upon light exposure. Anaphylatoxins are potent chemoattractants that recruit mononuclear phagocytes to the site of inflammation thus play important roles in the activation and regulation of innate immune cells [49]. Our analysis also revealed reduced levels of the anaphylatoxin C5a in light-exposed retinas of mice treated with moss-derived recombinant full-length CFH variants. In line with this, a previous study showed that complement C5a receptor knockout diminish microglia reactivity and migration after light exposure [50]. Of note, a recent report showed reduced microglia reactivity in post-ischemic murine retinas treated with two truncated CFH variants [38]. Also, in PMA-differentiated THP-1 macrophages it was shown that CFH increases the expression of anti-inflammatory genes by enhancing ApoE binding to CR3 (complement receptor 3) [51]. In contrast, others reported that binding of CFH to CR3 inhibits CD47-mediated resolution of inflammation resulting in retention of reactive mononuclear phagocytes in the subretinal space [52]. Although, we showed that the majority of CD11b+ cells in light-exposed retinas were microglia, we cannot fully delineate whether treatment with CFH affects a specific cell type in the retina. Thus, the precise role of CFH on microglia reactivity or mononuclear phagocytes deserves further studies.

Besides attenuated mononuclear phagocyte reactivity, our data also demonstrate reduced Müller cell gliosis in light-exposed retinas of mice treated with moss-derived recombinant full-length CFH variants. However, treatment with truncated CFH versions in the murine ischemia/reperfusion model, resulted only in a slight but non-significant decrease in Müller cell reactivity. This could be due to their post-damage therapy approach, as they linked the truncated CFH transgene expression to Müller cell gliosis [38].

As previously reported, the complement system is among the most affected pathways upon light-induced retinal degeneration [11, 53,54,55]. Here, we also found increased levels of C3 and C5a in retinas of light-exposed mice, whereas treatment with the two moss-derived recombinant full-length CFH variants resulted in decreased C5a levels. However, levels of C3 remained unaltered, suggesting that C3 turnover and cleavage is reduced in CFH-treated retinas. Indeed, reduced C3 turnover and complement activation were also observed in ischemic retinas that were treated with truncated CFH variants [38]. Interestingly, the cleavage cascade of C3b to C3d was slightly increased in CPV-101 and CPV-104-treated mice 1d after light exposure. This could be explained by the CFH dependency of CFI mediated C3b cleavage and an accumulation of cleavage products. Furthermore, it was shown before, that CFH functions also as a co-factor for lysosomal cysteine protease cathepsin L (CTSL) mediated intercellular cleavage of C3 [56].

Complement activation leads to the formation and insertion of MAC into cell membranes resulting in a direct loss of membrane integrity and ultimately in cell death [57]. Increased MAC deposition in the retina upon light exposure was described before [58]. In addition, our data also revealed that both moss-derived recombinant CFH variants reduce light-induced MAC formation in the retina.

Concomitantly with reduced complement activation and gliosis, the subsequent light-induced retinal degeneration due to loss of photoreceptors is attenuated by both moss-derived CFH variants. To date, no other data on CFH substitution in light-induced retinal degeneration exist, however there are some promising results from the laser-induced CNV model that recapitulates key pathological features of neovascular AMD, available. Here it was shown that siRNA-mediated knockdown of CFH aggravated disease outcome in the laser-induced CNV model, resulting in exaggerated MAC deposition and CNV formation. Conversely, intravitreal injection of CFH that was isolated from human serum resulted in reduced MAC and CNV formation after laser damage [59, 60].

Of note most clinical trials targeting the complement system in patients with GA have failed or barely met the endpoint [33]. The recent FDA-approved RNA aptamer avacincaptad pegol and the peptide pegcetacoplan targeting C5 or C3 respectively, have been shown to attenuate GA progression in patients although no significant effect on visual acuity was observed so far [34, 35]. Both substances make use of PEGgylation which is a common way of enhancing pharmacokinetics and therapeutic potential [61]. However, PEGylated substances were also discussed to induce anti-PEG antibody production and hypersensitivity in some cases [62]. Additionally, it was shown that subretinal injections of PEG induce CNV in mice [63]. In line with this, patients treated with avacincaptad pegol or pegcetacoplan were more likely to develop neovascular AMD than sham-treated control patients [64, 65]. Of note, the current available therapies block the complement pathway altogether that could lead to increased susceptibility of infections. One attempt to modulate only the alternative pathway via intravitreal injection of GEM103, a full-length recombinant human CFH protein [39], has been halted as no beneficial effect on GA progression in patients was observed (Phase II ReGAtta study, NCT04643886) [33, 66]. Therefore, future research should aim to decipher in detail the precise role of CFH and the effect of its substitution on AMD pathogenesis.

Conclusion

Our findings demonstrate that substitution with moss-derived recombinant human full-length CFH variants reduced light-induced complement activation and ameliorated the overall retinal immune activation status. Concomitantly, targeting the alternative complement system reduced photoreceptor cell death and delayed light-induced retinal degeneration. These findings highlight a broad but specific potential new therapeutic strategy for slowing down AMD disease progression. Further studies and optimization of the delivery system and mode of applications are needed to enhance efficacy.

Availability of data and materials

All data supporting the findings of this study are available within the paper and its Supplementary Information.

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Acknowledgements

We thank Eva Scheiffert for excellent technical assistance. We also thank Saskia Scheider, Jule Meseke and Berivan Ates for support in animal caretaking. We also thank Dr. Thomas Clahsen for his valuable support and expertise in flow cytometry.

Funding

This project was supported by Eleva GmbH.

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Authors and Affiliations

  1. Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, 50931, Cologne, Germany

    Mandy Hector, Verena Behnke, Thomas Langmann & Anne Wolf

  2. Eleva GmbH, Hans-Bunte-Straße 19, 79108, Freiburg, Germany

    Paulina Dabrowska-Schlepp, Andreas Busch & Andreas Schaaf

  3. Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931, Cologne, Germany

    Thomas Langmann & Anne Wolf

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Contributions

M.H.; V.B and A.W. conducted and analyzed all experiments. T.L. obtained the funding, and together with A.W.; P.D.; A.B.; A.S. conceived the study, and designed the experiments. A.W., M.H. and T.L. wrote the manuscript and all authors read and approved the final manuscript.

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Correspondence to Anne Wolf.

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All animal experiments were reviewed and approved by the government office responsible for animal welfare in North-Rhine-Westphalia (Landesamt für Natur, Umwelt und Verbraucherschutz, Application No. 81-02.04.2021.A277). All experimental procedures complied with the German law on animal protection and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

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Competing interests 

Paulina Dabrowska-Schlepp, Andreas Busch and Andreas Schaaf are employees of Eleva GmbH and are co-authors of a patent for recombinant CFH (CPV-104).

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Hector, M., Behnke, V., Dabrowska-Schlepp, P. et al. Moss-derived human complement factor H modulates retinal immune response and attenuates retinal degeneration. J Neuroinflammation 22, 104 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12974-025-03418-2

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Journal of Neuroinflammation

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