Agents that inhibit the binding of CFH to CD11 b/CD18 and uses thereof

Abstract

Disclosed are agents inhibiting the interaction between CFH and CD11b/18, as well as the use of such agents, in particular for treating inflammatory disorders, such as age-related macular degeneration.

Claims

1. A method for treating inflammation in a subject comprising administering to the subject an agent inhibiting the binding of Complement Factor H to CD11b/CD18, wherein said agent binds CD11b/CD18 to at least one binding site to Complement Factor H, wherein said agent is an anti-CD11b antibody selected from the group comprising the clone 5C6 and the clone ICRF44 and wherein said inflammation is atrophic age-related macular generation.

2. The method according to claim 1, wherein said inflammation is associated with mononuclear phagocytes accumulation.

3. The method according to claim 1, wherein said agent is topically administered to said subject.

4. A method for treating inflammation in a subject comprising administering to the subject an agent inhibiting the binding of Complement Factor H to CD11b/CD18, wherein said agent binds CD11b/CD18 to at least one binding site to Complement Factor H, wherein said agent is an anti-CD18 antibody selected from the group comprising erlizumab and the clone L130 and wherein said inflammation is atrophic age-related macular generation.

5. The method according to claim 4, wherein said inflammation is associated with mononuclear phagocytes accumulation.

6. The method according to claim 4, wherein said agent is topically administered to said subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a set of graphs showing that CFH deficiency prevents chronic pathogenic subretinal inflammation. A: Representative images of 12 month-old IBA-1stained RPE-flatmounts of Cx3cr1.sup.GFP/GFP and Cx3cr1.sup.GFP/GFPcfh.sup.−/− mice and quantification of subretinal IBA-1.sup.+ MPs in 2-3-month-old and 12-month-old mice of the indicated strains (n=11-17 per group, one-way Anova/Bonferroni test *p<0.0001 versus all other groups, .sup.$p<0.0001 versus Cx3cr1.sup.GFP/GFP 12-month-old). B: Micrographs, taken 1000 μm from the optic nerve of 12-month-old Cx3cr1.sup.GFP/GFP and Cx3cr1.sup.GFP/GFPCfh.sup.−/− mice. B′: Photoreceptor nuclei rows at increasing distances (−3000 μm: inferior pole, +3000 μm: superior pole) from the optic nerve (0 μm) in 12-month-old mice. B″: Quantification of the area under the curve of photoreceptor nuclei row counts of 12-month-old indicated transgenic mouse strains (one-way Anova/Bonferroni test C″: n=4-6/group, *p<0.001; D: n=7-9/group *p<0.001). Mice were taken from several (≥3) independent cages for the quantifications. ONL: outer nuclear layer. Scale bar=50 μm. C: Micrographs, taken in the superior periphery of peanut agglutinin (staining cone segments, red), cone arrestin (white), IBA-1 (green) triple stained 12 month-old Cx3cr1GFP/GFP and Cx3cr1GFP/GFP Cfh−/− mice. C′: Cone density quantifications on retinal flatmounts in peripheral and central, inferior and superior retina (−3000 μm: inferior pole, +3000 μm: superior pole, optic nerve: 0 μm) and their average (C″) in 12 month-old mice of the indicated transgenic mouse strains. (C″: one-way Anova/Bonferroni test: *p<0.0001 versus all other groups; Mann Whitney .sup.$p=0.0024 versus Cx3cr1GFP/GFP mice). N is the number of replicates indicated in the graphs; replicates represent quantifications of eyes from different mice of at least three different cages.

(2) FIG. 2 is a set of graphs showing cytokine multiplex analysis (A: IL-1α; B: IL-1β; C: TNFα; D: IL-6) of supernatants from cultured primary bone marrow monocytes (BM-Mos, 100 000 cells/well) and brain microglial cells (MCs, 200 000 cells/well), incubated for 24 h in serum free DMEM medium or stimulated with APOE3 (5 μg/ml) of Cx3cr1.sup.GFP/GFP and Cx3cr1.sup.GFP/GFP Cfh.sup.−/− mice.

(3) FIG. 3 is a set of graphs showing that MP-derived CFH inhibits the resolution of acute subretinal inflammation. A: Quantification of subretinal IBA-1.sup.+ MPs in non-illuminated (NI), 4d-light challenged (d4), and 4d-light challenged followed by 10 recovery days (4+10) of 2-3-month-old mice of the indicated strains. (n=6-12 per group, one way Anova/Bonferroni test *p<0.001 versus the NI groups, .sup.$p<0.0001 versus Cx3cr1.sup.GFP/GFPd4+10;). B: Quantification by ELISA of circulating plasma C3 in 2-3-month-old Cx3cr1.sup.GFP/GFP, Cx3cr1.sup.GFP/GFPCfh.sup.−/− mice before (D0), at the end (D4) and ten days after (D14) the light-challenge model. Four days before mice were injected with 100 μg of empty plasmid or expressing murine CFH or not injected. (n=4-5 per group one way Anova/Bonferroni test *p<0.001). C: Quantification of subretinal IBA-1.sup.+ MPs/mm.sup.2 in light-challenge model at day 14 of 2-3-month-old mice Cx3cr1.sup.GFP/GFP, Cx3cr1.sup.GFP/GFPCfh.sup.−/− that were injected with 100 μg of empty plasmid or expressing murine CFH (n=6-10 per group one way Anova/Bonferroni test *p<0.001). D: Quantitative RT-PCR of Cfh mRNA normalized with Rps26 mRNA of retina, choroid/RPE, circulating monocytes (cMo), bone marrow monocytes (BM-Mo), retinal microglia (MC Retina), brain microglia from the indicated strains (MC CNS; n=3 preparations from 5 pooled mice). E: Representative micrograph of CFSE.sup.+ MCs of the indicated strains, 24h after subretinal adoptive transfer to Cfh.sup.−/− mice. Quantification of CFSE.sup.+ MCs of the indicated strains, 24h after adoptive transfer to C57BL6/J wildtype or Cfh.sup.−/− mice (n=12-18/group; one way Anova/Bonferroni test *p<0.001 versus C57BL6/J wildtype CFSE.sup.+ MCs, .sup.$p<0.0001 versus Cx3cr1.sup.GFP/GFP CFSE.sup.+ MCs, †p<0.0001 versus Cx3cr1.sup.GFP/GFPCfh.sup.−/− CFSE.sup.+ MCs). Scale bar=50 μm.

(4) FIG. 4 is a set of graph and photographs showing plasma C3 concentrations and C3, and C3 fragment, and CFH immunohistochemistry in the transgenic mice. A: Complement factor C3 (C3)-ELISA measurements of plasma C3 concentrations from C57BL6/J wildtype, Cx3cr1.sup.GFP/GFP or Cx3cr1.sup.GFP/GFP Cfh.sup.−/− mice. B-D: Immunohistochemistry for C3 (B, clone 11H9 Hycult biotech), C3b/iC3b/C3c (C, clone 3/26 Hycult biotech), and CFH (D, ab8842 Abcam) in 4d light-challenged Cx3cr1.sup.GFP/GFP and Cx3cr1.sup.GFP/GFP Cfh.sup.−/− mice. ONL: outer nuclear layer; RPE retinal pigment epithelium. negative control: omitting the primary antibodies revealed no staining (not shown); the experiment was repeated three times with similar results. Scale bar=50 μm.

(5) FIG. 5 is a set of graphs showing Cd11b and Cd18 transcription in MPs. Quantitative RT-PCR of Cd11b (A-B), Cd18 (C-D) mRNA, normalized with Rps26 mRNA, in retina (1), retina without MCs (2, after sorting of MCs), liver (3), bone marrow monocytes (BM-Mo), retinal microglia (5, MC Retina) and brain microglia (6, CNS MC) from WT C57BL6/J mice (left column), and of BM-Mo, and MCs from retina and CNS of Cx3cr1.sup.GFP/GFP and Cx3cr1.sup.GFP/GFP Cfh.sup.−/− mice (middle and right column).

(6) FIG. 6 is a set of graphs showing that CFH fixation to CD11b/CD18 inhibits MP elimination. A and B: Representative cytometry images of (A) sorted brain Cx3cr1.sup.GFP/GFPCfh.sup.−/− Microglial Cells (gated on GFP.sup.high) incubated with increasing dose of hCFH::Cy5.5 (15,625 μg/ml to 500 gig/ml), and (B) sorted Cx3cr1.sup.GFP/GFPCfh.sup.−/− bone marrow monocytes pre-incubated with an isotype or anti-CD11b 5C6 antibody before hCFH::cy3 (100 μg/ml) or PBS incubation. Bone marrow monocytes were gated on GFP.sup.+ CD115.sup.+ LY6G.sup.− cells (The experiments were repeated three times with similar results). C: Quantification of Cx3cr1.sup.GFP/GFPCfh.sup.−/− CFSE.sup.+ MCs with control IgG or anti-CD11b 5C6 antibody, 24h after adoptive transfer (n=12-13/group; Anova/Bonferroni test †p<0.0001 versus Cx3cr1.sup.GFP/GFPCfh.sup.−/− CFSE.sup.+ MCs, ‡p<0.0001 versus Cx3cr1.sup.GFP/GFPCfh.sup.−/−+CFH+cIgG). Scale bar=50 μm. D: Quantification of subretinal IBA-1.sup.+ MPs on the RPE counted at a distance of 0-500 μm to CD102.sup.+CNV 7 days after the laser-injury of 3-month-old Cx3cr1.sup.GFP/GFP mice injected with control IgG, or anti CD11b 5C6 at d0 (n=7-12/group MannWhitney .sup.$p=0.0298 versus cIgG). E: Quantification of CD102.sup.+CNV on the RPE/choroid at 7 days after the laser-injury of 3-month-old Cx3cr1.sup.GFP/GFP mice injected with control IgG, or anti CD11b 5C6 at d0 (n=7-12/group MannWhitney .sup.$p=0.035 versus cIgG).

(7) FIG. 7 is a set of histograms showing that the AMD-associated 402H CFH inhibits subretinal MP elimination significantly more than the common 402Y CFH. A: Quantification of subretinal Cx3cr1.sup.GFP/GFP Cfh.sup.−/− CFSE+ monocytes on RPE and retinal flatmounts 24h after adoptive transfer to WT C57BL6/J mice with and without purified CFH.sub.Y402 or CFH.sub.402H (Anova/Bonferroni test *p<0.0001). B: Quantification of Cx3cr1.sup.GFP/GFPCfh.sup.−/− CFSE.sup.+ MCs on RPE and retina flatmounts with PBS, hCFH 402Y or hCFH 402H (n=18-30 per group, one way Anova/Bonferroni test *p<0.0001). Mos: monocytes; MCs: microglial cells; n=number of replicates indicated in the graphs, replicates represent quantifications from individual mice from two (A) to three (B) experiments with three different cell preparations.

EXAMPLES

(8) The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

(9) Materials and Methods

(10) Animals

(11) Cfh.sup.−/−-mice were a generous gift. Cx3cr1.sup.GFP/GFP-mice were purchased (Charles River Laboratories, Jackson laboratories) and Cx3Cr1.sup.GFP/GFPCfh.sup.−/− mouse strains were generated. All mice were either negative or backcrossed to become negative for the Crb1.sup.rd8, Pde6b.sup.rd1, and Gnat2.sup.cpfl3 mutations. Mice were housed in the animal facility under specific pathogen-free condition, in a 12/12h light/dark (100-500 lux) cycle with water and normal diet food available ad libitum. All experimental protocols and procedures were approved by the local animal care ethics committee “Comité d'éthique en experimentation animale Charles Darwin” (No Ce5/2010/011, Ce5/2010/044, Ce5/2011/033).

(12) Choroidal and Retinal Flatmounts for Mononuclear Phagocytes and Cones Quantification

(13) Eyes were enucleated, fixed in 4% PFA 30 minutes and sectioned at the limbus; the cornea and lens were discarded. The retinas were carefully peeled from the RPE/choroid/sclera. Retina and choroid were incubated with Peanut agglutinin Alexa 594 (Thermofisher; 1:50), anti-IBA-1 antibody (Wako chemicals; 1:400), and anti-mouse cone-arrestin antibody (Millipore, #AB15282; 1:10 000) followed by secondary anti-rabbit antibody coupled to Alexa 488 and Alexa 647 (Thermo Fisher; 1:500) and nuclear staining using Hoechst. Choroids and retinas were flatmounted and viewed with a fluorescence microscope DM5500B (Leica). IBA-1.sup.+ cells were counted on whole RPE/choroidal flatmounts and on the outer segment side of the retina. PA+cone arrestin+ cells were counted on oriented retinal flatmounts in the central and peripheral retina.

(14) Histology and Immunohistochemistry

(15) Eyes were fixed in 0.5% glutaraldehyde, 4% PFA for 2h, dehydrated and mounted in Historesin (Leica). 5 μm oriented sections crossing inferior pole, optic nerve and superior pole were cut and stained with toluidin blue. Rows of nuclei in the ONL were counted at different distances from the optic nerve (Sennlaub et al., EMBO Mol Med. 2013, 5: 1775-1793). For immunohistochemistry, eyes from 4d light-challenged mice were fixed for 2h in 4% PFA, incubated in 30% sucrose overnight at 4° C., embedded in OCT and sectioned (10 μm), and stained with anti-C3 antibody (clone 11H9 Hycult biotech; 1:50), anti-C3b/iC3b/C3c antibody (clone 3/26 Hycult biotech; 1:50), and anti-CFH antibody (ab8842 Abcam; 1:100) and appropriate secondary antibodies and Hoechst nuclear stain.

(16) Light Challenge Model

(17) Two- to three-month-old mice were adapted to darkness for 6 hours, pupils dilated daily and exposed to green LED light (starting at 2 AM, 4500 Lux, JP Vezon equipements) for 4 days and subsequently kept in cyclic 12h/12h normal facility conditions as previously described (Sennlaub et al, EMBO Mol Med. 2013, 5:1775-1793). MP count was assessed at the end of light exposure or 10 (d14) later.

(18) Hydrodynamic Injection

(19) Murine Complement Factor H was cloned in Plive vector (Mirus) using NheI and SacII restriction sites. The plasmid was sequenced and amplified with endotoxin-free Megaprep kits (Qiagen). 100 μg of plasmids were injected per mouse diluted in NaCl 0.9%. The volume injected in the venous tail was 10% of the body weight (around 25 g) (Rayes et al., Blood. 2010, 115:4870-4877). Four days later, mice were exposed to the light challenge model and 50 μl of blood were taken (mandibular vein) at D0, D4 and D14 to quantify C3 concentration.

(20) Reverse Transcription and Real-Time Polymerase Chain Reaction and ELISA

(21) RT-PCRs (CFH sense: 5′AAG AGA TTC ACC GCC ATT TC-3′; CFH antisense: 5′TGC ATG TGC CTT TCT AAA CA 3′; S26 sense: 5′AAG TTT GTC ATT CGG AAC ATT-3′; S26 antisense: 5′AGCAGGTCTGAATCGTGG TG-3′) using Sybr Green (Life Technologies) and ELISAs on isolated plasma using mouse C3 ELISA kit (Innovative Research) as previously described (Sennlaub et al., EMBO Mol Med. 2013, 5:1775-1793).

(22) Monocyte and Microglial Cell Preparations, Analysis, and Culture

(23) Blood samples were collected for determination of plasma C3 (Innovative Research) levels by ELISA. Bone marrow monocytes, circulating monocytes, central nervous MC and retinal MC were purified. Mos were isolated by negative selection (EasySep Mouse Enrichment Kit, Stemcell Technologies). MCs were prepared from dissociated PBS-perfused brains or retinas (Neural Dissociation Kit, Miltenyi Biotec). After dissociation, 70 μm filtered cell suspensions were washed and myelin was eliminated Percoll density gradient centrifugation. Then cells labeled with anti-CD11b microbeads (clone M1/70.15.11.5, Miltenyi Biotec) were purified by MS Columns (Miltenyi Biotec) and washed. No serum was used in any step of the purification to avoid cell contamination with serum derived CFH. The cells were used for adoptive transfer experiments, analyzed by RT-qPCR or cultured for 24h in serum free DMEM, in presence of recombinant human APOE3 (Interchim; 10 μg/mL), and finally their supernatants were analyzed by cytokine multiplex array (MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel, Merck Millipore).

(24) Subretinal Adoptive MP Transfer and Clearance

(25) According to Levy et al. (EMBO Mol Med. 2015, 7:211-226), brain microglia of the indicated mouse strains were sorted as describe above, labeled in 10 μM CFSE (Life Technologies), washed and resuspended in PBS. 12000 cells (4 μL) were injected using glass microcapillaries (Eppendorf) and a microinjector in the subretinal space of anesthetized 10-14 weeks old wildtype or Cfh.sup.−/−-mice. A hole was pierced with the glass capillary prior to the subretinal injection to avoid intra-ocular pressure increase and to allow retinal detachment with 4 μl of solution. The subretinal injection was verified by fundoscopy. In specific experiments, cells were co-injected with human CFH (500 μg/ml) from donors (Tecomedical), purified 402H and 402Y CFH (Claire Harris) or synthetized (LFB Biotechnologies, Lille), anti-CD11b (10 μg/ml, clone 5C6 Abd Serotec), anti-C3b/iC3b/C3c antibody (10 μg/ml, clone 3/26 Hycult biotech), isotype control rat IgG2b (10 μg/ml, Invivogen), and isotype control mouse IgM (10 μg/ml, Invivogen). Eyes were enucleated after 24 hours, fixed in PFA 4% 30 minutes and labeled with DAPI. Eyes with hemorrhages were discarded. CFSE+ cells in the subretinal space were quantified on flatmounts on the RPE side of the retina and on the apical side of the RPE.

(26) CFH Binding Assay by Flow Cytometry

(27) Human CFH was conjugated to Cyanin 5.5 (Abcam conjugation kits). MCs from Cx3Cr1.sup.GFP/GFPCfh.sup.−/− were stained 30 minutes with conjugated human CFH of the indicated concentrations (with or without preincubation of IgG and anti CD11b 10 μg/ml) in PBS at 37° C. and washed two times before acquisition on a BD Fortessa flow cytometer (BD biosciences). Analysis was performed using Flowjo. Briefly, doublets were eliminated with FSC-H, FSC-W, SSC-H and SSC-W and microglial cells were analyzing as GFP.sup.high cells. Bone marrow monocytes incubated with hCFH::Cy5, were labeled with rat anti-CD115-PE (Abd Serotec), rat anti-LY6G AlexaFluor 700 (BD Biosciences) to be defined as GFP.sup.+, CD115.sup.+, LY6G.sup.− cells. Note that the anti-CD11b clone M1/70.15.11.5 used for MC purification does not interfere with CFH binding as shown in FIG. 6A and the fact that recombinant CFH is capable to reverse the accelerated elimination of Cx3cr1.sup.GFP/GFP Cfh.sup.−/− MCs and TRE2Cfh−/− MCs sorted using clone M1/70 (FIG. 3). This difference is likely due to the fact clone M1/70 recognizes a distinct epitope of CD11b compared to the 5C6 clone (Rosen and Gordon, J Exp Med. 1987, 166, 1685-1701).

(28) Laser-Injury Model

(29) Laser-coagulations were performed with a 532 nm ophtalmological laser mounted on an operating microscope (Vitra Laser, 532 nm, 450 mW, 50 ms, and 250 μm). Intravitreal injections of 2 μl of PBS, isotype control rat IgG2b (50 μg/ml, Invivogen), or rat anti-mouse CD11b (50 μg/ml, clone 5C6 Abd Serotec) were performed using glass capillaries (Eppendorf) and a microinjector, and mice were sacrificed at day 10. RPE and retinal flatmounts were stained and quantified as previously described (Sennlaub et al., EMBO Mol Med. 2013, 5, 1775-1793) using polyclonal rabbit anti-IBA-1 (Wako) and rat anti-mouse CD102 (clone 3C4, BD Biosciences) appropriate secondary antibodies and counterstained with Hoechst if indicated. Preparations were observed under a fluorescence microscope (DM5500, Leica) and IBA-1+ MPs on the RPE were counted in a diameter of 500 μm around the CD102+ neovascularizations.

(30) Statistical Analysis

(31) Sample sizes for experiments were determined according to previous studies (Combadiere et al., J Clin Invest. 2007, 117, 2920-2928; Sennlaub et al., EMBO Mol Med. 2013, 5, 1775-1793). Severe hemorrhage secondary to subretinal injection interferes with MP clearance and was used as exclusion criteria. Graph Pad 6 (GraphPad Software) was used for data analysis and graphic representation. All values are reported as mean+/−SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test (for multiple comparison) or Mann-Whitney U-test (2-group comparison) among means depending on the experimental design. The n- and P-values are indicated in the figure legends.

Example 1: CFH Deficiency Prevents Chronic Pathogenic Subretinal Inflammation

(32) The subretinal space does not contain significant numbers of mononuclear phagocytes (MPs) under normal conditions. This is likely the result of physiologically low levels of chemoattractants along with strong immunosuppressive RPE signals that quickly eliminate infiltrating MPs (Sennlaub et al, EMBO Mol Med. 2013, 5:1775-1793; Levy et al., EMBO Mol Med. 2015, 7:211-226). Cx3cr1.sup.GFP/GFP-mice do not develop drusen and RPE atrophy, but do model MP accumulation on the RPE, as well as the associated photoreceptor degeneration and excessive CNV observed in AMD (Combadière et al., J Clin Invest. 2007, 117:2920-2928; Levy et al., EMBO Mol Med. 2015, 7:211-226). To evaluate a potential role of CFH in subretinal MP accumulation, Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice were generated. All mice were free of the Crb1.sup.rd8 contamination, that can lead to AMD like feature, and raised under 12h light/12h dark cycles at 100-500 lux at the cage level, with no additional cover in the cage. Quantification of subretinal IBA-1.sup.+ MPs on retinal and RPE/choroidal-flatmounts of 2-3-month and 12-month-old mice revealed that the age-related increase in subretinal MPs in Cx3cr1.sup.GFP/GFP-mice was nearly completely prevented in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice (FIG. 1 A). We have previously shown that the accumulation of subretinal MPs observed in 12-month-old Cx3cr1.sup.GFP/GFP-mice is associated with significant photoreceptor degeneration (Sennlaub et al, EMBO Mol Med. 2013, 5:1775-1793). Micrographs, taken at equal distance from the optic nerve of 12-month-old mice also show that the thinning of the outer nuclear layer (ONL that harbors the nuclei of the photoreceptors) observed in Cx3cr1.sup.GFP/GFP-mice is not observed in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice (FIG. 1B). Photoreceptor nuclei row counts (FIG. 1B′) and calculation of the area under the curve (FIG. 1B″) showed that Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice were significantly protected against the photoreceptor cell loss observed in Cx3cr1.sup.GFP/GFP-mice and not significantly different to Cfh.sup.−/−- and wildtype-mice.

(33) Similarly, Cfh deficiency completely protected against cone loss observed on peanut agglutinin/cone arrestin stained retinal flatmounts from 12m-old Cx3cr1.sup.GFP/GFP mice (FIGS. 1C, C′ and C″). It had no effect on key pathogenic cytokine secretion of MPs in vitro (FIG. 2A-D), suggesting that the numerical increase rather than differences in polarization provokes the degeneration.

(34) Therefore, these results show that CFH significantly contributes to the chronic, age-related subretinal MP accumulation and associated photoreceptor degeneration observed in inflammation-prone Cx3cr1.sup.GFP/GFP-mice.

Example 2: MP-Derived CFH Inhibits the Resolution of Acute Subretinal Inflammation

(35) Next the effect of CFH in an acute light-induced model of subretinal inflammation was evaluated. The intensity of our light-challenge model used herein was calibrated to induce substantial subretinal inflammation in inflammation-prone Cx3cr1.sup.GFP/GFP-mice but not in C57BL6/J control mice (Sennlaub et al, EMBO Mol Med. 2013, 5:1775-1793). Quantification of subretinal IBA-1.sup.+ MPs on retinal and RPE/choroidal-flatmounts after a four-day light-challenge revealed that acute subretinal MP accumulation, observed at 4d, was similar in Cx3cr1.sup.GFP/GFP- and Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice. However, after an additional 10d in normal light conditions the MP accumulation in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice had subsided significantly more quickly than in Cx3cr1.sup.GFP/GFP-mice (FIG. 3A).

(36) Similarly to Cfh.sup.−/− mice (Pickering et al., Nat Genet. 2002, 31:424-428), Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice are characterized by low circulating levels of complement factor C3 (FIG. 4A-D), likely due to un-inhibited plasma complement activation and exhaustion, which might interfere with subretinal MP recruitment. To test whether the systemic lack of C3 were responsible for the accelerated elimination of subretinal MP in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice, hepatic Cfh were replaced by hydrodynamic injection of a plasmide encoding Cfh under an albumine promoter four days prior to the light-challenge. ELISA analysis of plasma C3 showed that the Cfh-transfection significantly restored circulating C3 concentrations in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice to 40-60% of the Cx3cr1.sup.GFP/GFP levels over the 14 day experimental protocol compared to control plasmid transfected and non-transfected Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice (FIG. 3B). However, the significant increase of circulating C3 levels did not increase the number of subretinal MPs (quantified on IBA-1-stained retinal and RPE/choroidal-flatmounts) in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice at d14 (FIG. 3C).

(37) The comparable subretinal MP counts at the beginning of the acute inflammation at d4 (FIG. 3A) and the lack of influence of circulating C3 levels on the MP count in the resolution phase (FIG. 3C) suggested that systemic CFH or C3 was not involved in the recruitment of MPs or responsible for their accelerated elimination in Cx3cr1.sup.GFP/GFPCfh.sup.−/−-mice. RT-PCRs of relative Cfh expression of retinal and RPE/choroid tissue homogenates, bone marrow- and circulating-monocytes, and brain- and retina-microglial cells (MCs) showed that the RPE/choroid and MCs expressed the highest levels of Cfh mRNA in WT and Cx3cr1.sup.GFP/GFP-mice (FIG. 3D), in accordance with CFH protein localization around subretinal MPs in vivo (FIG. 4A-D). To evaluate if CFH from MCs or from the RPE influenced subretinal MP elimination we next adoptively transferred CFSE-labeled brain MCs from the different mouse strains into the subretinal space of either wildtype- or Cfh.sup.−/− mice and counted the surviving CFSE.sup.+ MCs 24h after injection. We previously showed that wildtype MPs, that may be monocytes, MCs or Mφs, are very quickly eliminated from the subretinal space and that the clearance is significantly delayed in Cx3cr1-deficient MPs (Levy et al., EMBO Mol Med. 2015, 7:211-226). Here, results show that Cfh-deficiency significantly increases the rate of elimination of Cx3cr1.sup.GFP/GFP MCs and that this difference is reversed by purified human CFH (FIG. 3E). Interestingly, the recipient derived-CFH only had a very minor effect on the MC elimination rate.

(38) Therefore, these results show that MP derived CFH inhibits the elimination of subretinal MCs, which is likely responsible for the observed inhibitory effect of local CFH on the resolution of acute subretinal inflammation observed in vivo.

Example 3: CFH Fixation to CD11b/CD18 Inhibits MP Elimination

(39) The integrins CD11b (ITGAM) and CD18 (ITGB2) form a heterodimer (MAC-1, CR3) that has been shown to bind surface CFH on monocytes and neutrophils. CD11b/CD18 is strongly expressed in MPs with no detectable differences in our different mouse strains (FIG. 5A-N). Flow cytometry of Cx3cr1.sup.GFP/GFPCfh.sup.−/−-MCs, incubated with Cy5 conjugated purified CFH showed that CFH also fixes on the surface of purified MCs (FIG. 6A). Next, negatively sorted Cx3cr1.sup.GFP/GFPCfh.sup.−/− bone marrow monocytes were pre-incubated with a control IgG2 or the anti-CD11b 5C6 antibody clone prior to Cy3 conjugated CFH incubation. Flow cytometry showed that the anti-CD11b 5C6 clone strongly inhibited the cell surface CFH fixation (FIG. 6B). Interestingly, the anti-CD11b M1/70 clone that is used to purify the MCs did not interfere with CFH binding, demonstrated by the flow cytometry in FIG. 6A and the fact that recombinant CFH is capable to reverse the accelerated elimination of M1/70-sorted Cx3cr1.sup.GFP/GFPCfh.sup.−/−-MCs (FIG. 3). This difference is likely due to the fact that the M1/70 clone recognizes a distinct part of CD11b compared to the 5C6 clone 48. Indeed, blocking the CFH fixation using the anti-CD11b 5C6 IgG in subretinally adoptively transferred Cx3cr1.sup.GFP/GFPCfh.sup.−/−-MCs (wildtype recipients) completely prevented the inhibitory effect of added CFH protein on the elimination rate compared to the control IgG (FIG. 6C). To test whether it is possible to therapeutically speed-up subretinal inflammation resolution, subretinal inflammation in 3-month-old Cx3cr1.sup.GFP/GFP-mice was induced using a laser burn, which induces significant subretinal MP accumulation peaking at d4 and resolving thereafter (Lavalette et al., Am J Pathol. 2011, 178:2416-2423). Results show that the local, intravitreal injection at d0 of the anti-CD11b 5C6 IgG (to inhibit CFH fixation) significantly reduced the number of subretinal IBA.sup.+ MPs around the impact (FIG. 6D) on retinal/RPE flatmounts and the associated neovascularization (FIG. 6E; quantified as CD102 positive area) compared to the control IgG at d7.

(40) These results show that CFH fixes to MC surfaces and that the fixation of CFH to MC CD11b/CD18 is necessary for CFH to inhibit MC elimination. They also demonstrate that the inhibition of the binding of CFH to CD11b/CD18 speeds up MP elimination on the site of inflammation.

Example 4: The AMD-Associated 402H CFH Inhibits Subretinal MP Elimination Significantly More than the Common 402Y CFH

(41) The Y402H polymorphism that is strongly associated with soft drusen and AMD is located in the SCR7 of CFH, which mediates its binding to polyanionic cell surfaces, but also to CD11b/CD18 (Losse et al., J Immunol. 2010, 184:912-921; Kang et al., Immunobiology. 2012, 217:455-464). To evaluate whether the disease associated 402H CFH differed in its capacity to inhibit subretinal MP elimination from the common 402Y CFH, CFSE-labeled Cx3cr1.sup.GFP/GFPCfh.sup.−/−-MCs were adoptively transferred into the subretinal space of wildtype recipients and co-injected either form of CFH at 500 jtg/ml, which corresponds to the plasma concentration. Subretinal CFSE.sup.+ MC counts on RPE/retinal flatmounts 24h after the injection revealed that both isoforms significantly inhibited the MC elimination compared to cells injected without CFH (FIG. 7) and similar to commercially available purified CFH, which likely contains a mixture of both isoforms (see FIG. 6). However, CFSE.sup.+ MCs injected with the disease-associated 402H CFH form resisted the clearance significantly more than those injected the common 402Y CFH form (FIG. 7).

(42) Therefore, these results show that the AMD-associated 402H CFH inhibits subretinal MP elimination more strongly than the common 402Y CFH. It might thereby inhibit inflammation resolution and contribute to the chronification of subretinal inflammation in AMD, evidenced by the presence of subretinal MPs observed in patients with soft drusen and late AMD.