Published October 10, 2014, doi:10.4049/jimmunol.1401613 The Journal of Immunology

Identification of Factor H–like 1 as the Predominant Complement Regulator in Bruch’s Membrane: Implications for Age-Related Macular Degeneration

Simon J. Clark,*,† Christoph Q. Schmidt,‡ Anne M. White,*,† Svetlana Hakobyan,x B. Paul Morgan,x and Paul N. Bishop*,†,{

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular de- generation (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch’s membrane. Bruch’s membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch’s membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch’s membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH , associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies. The Journal of Immunology, 2014, 193: 000–000.

ge-related macular degeneration (AMD) is the leading United States it has been estimated that there will be a 50% in- cause of blindness in the developed world, affecting ∼50 crease in the number of affected individuals between 2004 and A million people worldwide. The prevalence of this con- 2020 (1). This debilitating disease can be subdivided into neo- dition is predicted to rise as the elderly population expands: in the vascular (“wet”) and atrophic (“dry”) AMD (2), both of which are usually preceded by the formation of drusen. These aggregates of lipids, , and cellular debris accumulate within Bruch’s *Centre for Hearing and Vision Research, Institute of Human Development, Univer- sity of Manchester, Manchester M13 9PT, United Kingdom; †Centre for Advanced membrane, a sheet of extracellular matrix (ECM) that separates Discovery and Experimental Therapeutics, University of Manchester and Central the retinal pigment epithelium (RPE) from the blood vessels of the Manchester University Hospitals NHS Foundation Trust, Manchester Academic choroid. The presence of complement proteins and downstream Health Science Centre, Manchester M13 9WL, United Kingdom; ‡Institute of Phar- macology of Natural Products and Clinical Pharmacology, Ulm University, 89081 inflammatory markers in drusen has led to the hypothesis that x Ulm, Germany; Complement Biology Group, Institute of Infection and Immunity, chronic local inflammation in Bruch’s membrane and surrounding School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom; and {Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS structures, resulting from inappropriate complement activation, Foundation Trust, Manchester M13 9WL, United Kingdom has a major influence on the pathogenesis of AMD (3–5). ORCID: 0000-0001-8394-8355 (S.J.C). Genetic alterations are a major risk factor for AMD. Two major Received for publication June 26, 2014. Accepted for publication September 11, loci have been identified: one is on 10 near the ARMS2/ 2014. HTRA1 and the other is on involving com- S.J.C. is a recipient of Medical Research Council Career Development Fellowship plement factor H (FH) and the FH-related (FHR) proteins (6). MR/K024418/1 and had previously been supported by a Stepping Stones Fellowship from the Faculty of Medicine and Human Sciences, University of Manchester. The Additionally, genes encoding members of the alternative comple- eye tissue holdings were initiated and supported by Medical Research Council Grants ment pathway have been implicated, including C3, complement G0900592 and MR/K004441/1. The Bioimaging Facility microscopes used in this factor I (FI), and complement factor B (FB), thereby providing study were purchased with support from the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, and the University of Manchester Strategic strong evidence that this pathway is involved in AMD pathogenesis Fund. (6–8). The locus on chromosome 1 is complex with multiple hap- S.J.C. designed the study, performed experiments, and wrote the manuscript; C.Q.S. lotypes having been identified that modify AMD risk (9). expressed the recombinant FHL-1 proteins; A.M.W. helped with the design and Within the chromosome 1 locus the Y402H polymorphism in FH execution of PCR experiments; S.H. and B.P.M. purified FH from human donors; and P.N.B. advised on experimental design and writing the manuscript. represents a major risk factor for AMD (10–14). The frequency of ∼ Address correspondence and reprint requests to Dr. Simon J. Clark, Centre for Hear- this risk allele is 35% in individuals of European descent and ing and Vision Research, University of Manchester, AV Hill Building, Oxford Road, results in a tyrosine being replaced by a histidine residue at po- Manchester M13 9PT, U.K. E-mail address: [email protected] sition 402 (using the preprotein sequence numbering) (15). The The online version of this article contains supplemental material. effects of the Y402H polymorphism appear to be mediated locally, Abbreviations used in this article: AMD, age-related macular degeneration; CCP, as the polymorphism does not promote complement activation in complement control protein; ECM, extracellular matrix; FB, complement factor B; FH, complement factor H; FHL-1, factor H–like protein 1; FHR, factor H–related; FI, the blood (14), unlike some mutations found in the C-terminal complement factor I; GAG, glycosaminoglycan; HS, heparan sulfate; RPE, retinal complement control protein (CCP) domains of FH. pigment epithelium. The central activating mechanism of the alternative pathway is Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 the covalent deposition of the protein C3b (an opsonin) on all local

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401613 2 FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE surfaces. Surface-linked C3b can react with other complement against the peptide sequence CIRVSFTL (Mimotopes, Clayton, Australia). proteins to form an active enzyme, the C3 convertase that is able to Anti–FHL-1 IgG was purified using an affinity column: recombinant FHL-1 produce further (surface-attachable) C3b molecules. This is achieved was coupled to cyanogen bromide–activated Sepharose according to the manufacturer’s instructions (GE Healthcare, Buckinghamshire, U.K.). as the C3 convertase proteolytically processes C3 in blood into Briefly, rabbit antiserum was centrifuged at 12,000 3 g for 5 min at room fresh C3b molecules, producing at the same time the anaphylatoxin temperature to remove particulate matter. The antiserum was then run down C3a. Insufficient control of the C3 convertase results in massive the FHL-1 affinity column in PBS, 1 mM EDTA. The column was washed production of C3b and C3a molecules and a shift of the com- thoroughly with 5 column volumes 0.4 M NaCl to remove weakly bound material. Two column volumes 3 M MgCl2 was used to elute bound protein. plement cascade to its terminal lytic pathway. This produces the The column was then regenerated by washing with 5 column volumes 0.4 M most potent anaphylatoxin, C5a, and the cell lytic protein complex NaCl and re-equilibrating with 5 column volumes PBS, 1 mM EDTA. Eluted termed the membrane attack complex; both C5a and the membrane protein was dialyzed into 2 l Milli-Q H2Ofor16hat4˚Cusing10kDacutoff attack complex provide strong inflammatory signals. dialysis tubing before being further dialyzed into 20 mM NaH2PO4, 150mM However, the presence of FH on host tissues, and its cofactor NaCl (pH 8.0) for a further 16 h at 4˚C. Rabbit IgG was purified from the dialysate using protein A–Sepharose, following the manufacturer’s instruc- activity for FI, results in C3b breakdown (resulting in the forma- tions (Sigma-Aldrich, Poole, U.K.). Isolated IgG was run on 4–12% tion of inactive iC3b), thereby preventing inappropriate comple- NuPAGE reducing gels to check for purity and tested for specificity to ment activation and inflammation (16). FH also exerts decay- FHL-1 in solid phase assays (see Supplemental Fig. 1). accelerating activity, which can assist in the deconstruction of Eye tissue preparation already formed C3 convertases. Whereas cell surface–expressed complement regulators also prevent complement activation on Details of donor eye tissue used in this study are listed in Table I. Human cells, the blood-borne FH is currently the only known complement eyes were obtained from the Manchester Royal Eye Hospital Eye Bank after removal of the corneas for transplantation. Our research adhered to regulator to bind and confer protection to ECM such as Bruch’s the tenets of the Declaration of Helsinki. In all cases, there was prior membrane (17). The protective FH is recruited to self-surfaces, at consent for the eye tissue to be used for research, and guidelines estab- least in part, by binding specific polyanions such as glycosami- lished in the Human Tissue Act of 2004 (U.K.) were followed. Except in noglycan (GAG) chains or sialic acid groups, which are not nor- the case of donor tissue used for the staining of drusen, none of the other mally present on potential pathogens. GAGs are long, unbranched donors had a history of visual impairment or eye disease. For the donor eye pairs used for immunohistochemistry, PCR analysis, polysaccharides made up of repeating disaccharide units that can and Western blotting, one globe was designated for RPE cell isolation and be variably sulfated: the sulfation pattern can drive specific protein Bruch’s membrane enrichment. These eyes were opened by making three recruitment (18). One ubiquitously expressed GAG, and a major incisions into the eyecup and flattening out the tissue. The vitreous and ligand for FH, is heparan sulfate (HS), which comprises both low- neurosensory retina were removed, RPE cells were harvested with gentle scraping, and RNA was isolated (see below). The Bruch’s membrane was and high-sulfated regions (18). These sulfated ligands are essential enriched by removal of the sclera and choroid. The Bruch’s membrane was ECM components responsible for a range of biological processes, washed multiple times with PBS and either frozen for analysis by Western including immune homeostasis (19, 20). blotting or used in Ussing chamber diffusion experiments (both method- FH comprises 20 CCP domains and contains two main GAG- ologies are described below). binding regions, in CCP7 (with some contribution from CCPs 6 The second globe from each donor was used to prepare frozen tissue sections and was fixed in 4% (v/v) formaldehyde for 2 h. Eyes were then and 8) and CCP20 (21) (see Fig. 1A): the Y402H polymorphism processed and serial sections of 5 mm were prepared using a cryostat, as resides in CCP7 and alters FH binding to sulfated GAGs (22). The described previously (23, 34). 402H disease-associated variant binds significantly less well to human Bruch’s membrane (an important site in AMD pathogenesis) Fluorescent immunohistochemistry than does the 402Y form when applied exogenously to tissue sec- Tissue sections were stained for the presence of endogenous FH or FHL-1 tions (23). We have demonstrated that the GAG binding region in using methods described previously (23). Briefly, tissue sections were in- 2 CCP6–8 is responsible for surface anchoring and hence host rec- cubated with chilled ( 20˚C) histological grade acetone (Sigma-Aldrich) for 20 s before thorough washing with PBS. Tissue sections were blocked ognition in eye structures, including Bruch’s membrane, whereas with 0.1% (w/v) BSA, 1% (v/v) goat serum, and 0.1% (v/v) Triton X-100 the CCP19–20 region anchors FH to ECM in the kidney (24). in PBS for 1 h at room temperature. After washing with PBS, tissue In this study we investigated the distribution of a naturally sections were incubated with Ab combinations of either 10 mg/ml mix of occurring truncated form of FH called FH-like protein 1 (FHL-1), L20/3 and anti–FHL-1 or of OX23 and anti–FHL-1 for 16 h at 4˚C. which arises from alternative splicing of the CFH gene (25). FHL-1 Sections were washed and Ab binding was detected using fluorescently labeled secondary Abs (Life Technologies, Paisley, U.K.). An equal 1:5000 is identical to FH for the first seven CCP domains before dilution mix of Alexa Fluor 488–conjugated goat anti-rabbit (to detect terminating with a unique 4-aa C terminus (see Fig. 1A). Impor- anti–FHL-1) and Alexa Fluor 594–conjugated goat anti-mouse (against tantly, FHL-1 retains all the necessary domains for function and is OX23 or L20/3) was added to sections for 2 h at room temperature. Finally, m also subject to the Y402H polymorphism. Previous studies have DAPI was applied as a nuclear counterstain (at 0.3 M for 5 min) prior to mounting with medium (Vectashield; Vector Laboratories, Peterborough, demonstrated FHL-1 expression by RPE cells (13, 26) and it has UK) and application of a coverslip. been identified in the vitreous of the eye where FH and FHL-1 In some experiments the enzymatic pretreatment of tissue was per- were reported to be in equimolar concentrations (27). In human formed as described previously (23). Briefly, 20 U/ml each of heparinase I, II, blood the concentration of FH is ∼300 mg/ml (28) and FHL-1 and III (all from Flavobacterium heparinum, Sigma-Aldrich) in PBS was ∼50 mg/ml (29), resulting in a molar ratio of ∼2:1. applied to tissue sections for 1.5 h at 37˚C; this was performed after fixation in acetone and followed by washing with PBS prior to the blocking step. Images were collected on a snapshot widefield microscope (Olympus BX51) using a 340/0.30 Plan Fln objective. Microscopy images were Materials and Methods captured using a CoolSNAP ES camera (Photometrics) via MetaVue Primary Abs and protein reagents software (Molecular Devices). To prevent bleed-through of color from one channel to the next, specific band pass filter sets were used for DAPI, Both recombinant 402H and 402Y forms of FHL-1 were generated according FITC, and Texas Red. All images were handled using ImageJ64 (version to the methodology described previously (30). The full-length FH protein was 1.40g; http://rsb.info.nih.gov/ij). purified from human plasma, as described by Hakobyan et al. (31). Com- mercial Abs used in this study were OX23 (AbD Serotec, Kidlington, U.K.) Western blotting (32) and L20/3 (Hycult Biotech, Uden, the Netherlands), which has been published previously with the clone name C02 (33). Both of these Abs Samples were run on 4–12% NuPAGE Bis-Tris gels (Life Technologies) at recognize different epitopes on FH (see Fig. 1A). Anti–FHL-1 was generated 200 V for 60 min and transferred onto nitrocellulose membranes at 80 mA The Journal of Immunology 3 for 2.5 h using semidry transfer apparatus in transfer buffer (25 mM Tris, The density of the 68-kDa iC3b product band was measured using 192 mM glycine, 10% [v/v] methanol). The membranes were blocked in ImageJ64 (version 1.40g; http://rsb.info.nih.gov/ij) and used to track C3b PBS, 10% (w/v) milk, and 0.02% (w/v) BSA for 16 h at 4˚C before the breakdown efficiency of the FHL-1 proteins. For both forms of FHL-1, addition of an OX23 and anti–FHL-1 Ab mix, each at 0.5 mg/ml, in PBS averaged data from three separate experiments were used. with 0.2% (v/v) Tween 20 (PBST) for 2 h at room temperature. Mem- branes were washed twice for 30 min in PBST before the addition of Solid-phase plate assays a 1:2000 dilution of IRDye 680RD–conjugated goat anti-mouse and IRDye The heparin-binding characteristics of the FHL-1 (402H and 402Y variants) 800CW–conjugated goat anti-rabbit (LI-COR Biosciences U.K., Cam- proteins were analyzed using microtiter plate-based assays, where either bridge, U.K.) for 2 h at room temperature, protected from light. Mem- heparin or one of its selectively desulfated derivatives (all from Iduron) was branes were washed and protein bands were visualized using a LI-COR immobilized noncovalently on allylamine-coated heparin-binding plates Odyssey infrared imaging system and Image Studio software. (BD Biosciences, Oxford, U.K.) as described previously (22, 35). The selectively desulfated heparin samples used in this study were 2-O– RPE cell complement desulfated, 6-O–desulfated, and N-desulfated heparin. HS from either Total RNAwas isolated from human RPE cells isolated from the donors listed porcine mucosa (Iduron) or bovine kidney (Sigma-Aldrich) was also tested in Table I, using standard extraction and purification methods. Briefly, RPE under the same conditions. m cells were homogenized in 1 ml cold TRI Reagent (Life Technologies) per All GAGs were diluted in PBS and immobilized at 1 g/well in a volume m 30 mg tissue using a TissueLyser II bead mill (Qiagen). After homogeni- of 100 l/well overnight at room temperature. Plates were blocked for m zation, RNA was isolated following TRI Reagent/BCP disruption and phase 90 min at 37˚C with 300 l/well 1% (w/v) BSA in assay buffer (20 mM separation. RNA was further purified by absorption to an RNeasy mini spin HEPES, 130 mM NaCl, 0.05% [v/v] Tween 20 [pH 7.3]). This standard column (Qiagen) with on-column DNAse I treatment. RNA purity and assay buffer was used for all subsequent incubations, dilutions, and concentration, as measured by a NanoDrop spectrophotometer, were deter- washes, and all steps were performed at room temperature. FHL-1 protein mined by absorbance at 230, 260, and 280 nm. cDNA was synthesized using was incubated with the immobilized GAGs for 4 h. After washing, bound m m a Transcriptor High Fidelity cDNA synthesis kit (Roche Diagnostics, protein was detected by the addition of 100 l/well 0.5 g/ml OX23 Ab Burgess Hill, U.K.) with 1 mg RNA and primed with oligo(dT) primer and incubated for 30 min followed by washing and a 30-min incubation in 18 m according to the manufacturer’s instructions. 100 l of a 1:1000 dilution of alkaline phosphatase–conjugated anti-mouse m Target gene sequences were obtained from GenBank and PCR primers to IgG (Sigma-Aldrich). Plates were developed using 100 l/well 1 mg/ml specific targets were designed with Primer3 software (http://www.ncbi.nlm. disodium p-nitrophenylphosphate solution (Sigma-Aldrich) in 0.05 M Tris- nih.gov/tools/primer-blast/) and are shown in Supplemental Table I. Pri- HCl, 0.1 M NaCl (pH 9.3). The absorbance values at 405 nm were de- mers for the RPE cell–specific genes (Bestrophin-1 and RPE65) were also termined after 10 min of development at room temperature and corrected included to ensure purified cDNA was indeed obtained from RPE cells. against blank wells (i.e., those with no immobilized GAGs). cDNA was amplified using the following PCR protocol: 95˚C for 5 min Heparin affinity chromatography followed by 40 cycles of 95˚C for 10 s, 60˚C for 15 s, and 72˚C for 20 s, followed by a melting program. The integrity of the PCR reactions was The heparin-binding properties of the 402H and 402Y FHL-1 variants were verified by detection of a single band of the correct size by agarose gel compared by affinity chromatography on a heparin affinity column in which electrophoresis. The same experiments were also performed on pooled 30 mg heparin (Iduron) was coupled to 1.5 ml cyanogen bromide–activated liver cDNA (Sigma-Aldrich). Sepharose (GE Healthcare) in 0.1 M NaHCO3, 0.5 M NaCl (pH 8.3) using the manufacturer’s protocol. Before sample loading, the column was Ussing chamber diffusion experiments equilibrated in 5 ml PBS (Sigma-Aldrich). Purified recombinant protein (100 mg) was loaded onto the column in a total volume of 5 ml PBS. The The macular region of enriched Bruch’s membrane isolated from donor column was washed with 4 column volumes PBS before bound protein was eyes (described above) was mounted in an Ussing chamber (Harvard eluted with a linear salt gradient of 130 mM–1 M NaCl over 20 ml by Apparatus, Hamden, CT). Once mounted, the 5-mm-diameter macular area mixing elution buffer (PBS, 1 M NaCl) with equilibration buffer at a flow was the only barrier between two identical compartments (see Fig. 3A). rate of 1 ml/min; fractions (1 ml) were collected throughout the protocol. Both sides of Bruch’s membrane were washed with 2 ml PBS for 5 min at room temperature. Human serum (Sigma-Aldrich) was diluted 1:1 with PBS and 2 ml was added to the Ussing compartment representing the Results choroidal side of Bruch’s membrane. After 1 min when no leaks were FHL-1 is present in the human Bruch’s membrane detected into the second compartment (which would indicate a compro- To determine whether FHL-1 was present in human Bruch’s mem- mise in membrane integrity), 2 ml PBS alone was added to the second compartment and the Ussing chamber was left at room temperature for brane we employed the Abs OX23 (which recognizes both FH and 24 h with gentle stirring in each compartment to avoid generating gradients FHL-1) (32), L20/3 (previously published as clone C02 and recog- of diffusing proteins. Samples from each chamber were analyzed by gel nizes only FH) (33), and a specific anti–FHL-1 Ab that we generated electrophoresis. Gels were either stained with Instant Blue stain (Expedeon, (Fig.1A).Theanti–FHL-1andL20/3Abswereshowntobespecific Harston, U.K.) for 60 min at room temperature or subject to Western for their intended targets and exert no cross-reactivity when tested blotting (see above). In some experiments the above protocol was repeated using 100 mg/ml against recombinant FHL-1 or FH purified from human serum FH purified from human plasma (31) on three donor Bruch’s membranes (Supplemental Fig. 1A–C). Fluorescent staining of six separate human (see Table I). After 24 h the entire PBS compartment, where FH would maculae (Table I) identified FHL-1 throughout Bruch’s membrane diffuse into, was collected and StrataClean beads (Agilent Technologies, (Fig. 1B). In contrast, full-lengthFHproteinwasidentifiedonthe Cheadle, U.K.) used to pull all proteins out of solution. The entire content of the PBS compartment was analyzed by gel electrophoresis as described choroidal side of Bruch’s membrane (with particular accumulation in above. Similarly, to ascertain whether the AMD-associated polymorphism the choriocapillaris), and small amounts were present in patches on altered the diffusion properties of FHL-1, 100 mg/ml recombinant 402H or the RPE-facing side, but staining was not seen within Bruch’s mem- 402Y forms of FHL-1 were separately tested on three donor Bruch’s brane (Fig. 1C, 1D). The choroidal stroma stained for (full-length) FH, membranes (see Table I). The protein content of each Ussing chamber andtherewasweakstainingforFHL-1, especially near Bruch’s compartment was analyzed by gel electrophoresis and band densities were normalized and compared with a 0 h, 100 mg/ml FHL-1 sample. membrane. FHL-1 staining could be ablated by preabsorbing the Ab with recombinant protein (Supplemental Fig. 1D, 1E), thereby provid- FHL-1 fluid-phase cofactor activity ing further evidence that the staining identified endogenous protein. The fluid-phase cofactor activity of the FHL-1 402H and 402Y variants Additionally, as expected, we demonstrated colocalization of anti– were measured by incubating FHL-1 (either form), C3b, and FI together in FHL-1 and OX23 Abs throughout Bruch’s membrane (Supplemental a total volume of 20 ml PBS for 15 min at 37˚C. For each reaction, 2 mg Fig. 1F). Analyses of drusen from four donors (Table I) demonstrate C3b and 0.04 mg of FI were used with varying concentrations of FHL-1 staining for endogenous FH in the periphery of the lesions whereas ranging from 0.0125 to 0.8 mg per reaction. The assay was stopped with the addition of 5 ml53 SDS reducing sample buffer and boiling for 10 min staining with FHL-1 appeared throughout the lesions (Fig. 1E). at 100˚C. Samples were run on a 4–12% NuPAGE Bis-Tris gel at 200 V for We performed Western blots on extracts from isolated human 60 min to maximize the separation of the C3b breakdown product bands. Bruch’s membrane and probed them with the anti–FHL-1 and OX23 4 FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE

FIGURE 1. FHL-1 rather than FH is the predominant complement regulator in human Bruch’s membrane. (A) Schematic indicating the CCP regions of FH and FHL-1 recognized by the OX23, anti–FHL-1, and L20/3 Abs. The AMD-associated Y402H polymorphism is located in CCP7 of both FH and FHL-1. (B) Gray-scale fluorescent staining of a human macula with anti–FHL-1. (C) Gray-scale staining of the same donor as shown in (B) but with the Ab L20/3. (D) Fluorescent staining of human macula with an equal mix of both anti–FHL-1 (green) and L20/3 (red). (E) Labeling of a druse with FHL-1 (green) and FH (red) Abs. (F) Western blot of solubilized Bruch’s membrane from four donors stained with anti–FHL-1 (green) and OX23 (red): yellow staining is indicative of colocalization of both Abs. Blue color represents DAPI staining of cell nuclei. Images in (B)–(D) are representative of six individual donors. Scale bars, 10 mm. Image in (E) is representative of four donors. Scale bar, 5 mm.

Abs. We identified an ∼49-kDa band that migrated to the same The experiments were repeated using purified FH, and after 24 h position as recombinant FHL-1 protein control (Fig. 1F). This was the entire protein content of the diffusate chamber was concen- identified by both OX23 and anti–FHL-1, thereby confirming that trated and analyzed, and we were unable to detect any FH protein this band was FHL-1 and not one of a number of known tryptic having crossed Bruch’s membrane (Fig. 3C). To ascertain whether fragments of FH (25, 36). The OX23 Ab was, however, unable to the Y402H AMD-associated polymorphism affected FHL-1 detect a 155k-Da species corresponding to FH in the extracts. diffusion, we repeated the experiments using purified 402H and 402Y forms of FHL-1 (Fig. 3D, 3E). In the case of both Human RPE transcription of FHL-1, FH, and genes associated variants, equilibrium was reached, although a reduction in the with the alternative complement pathway calculated protein recovery for the 402Y form was observed (an We investigated mRNA expression of a number of complement average of 73% over three separate experiments opposed to 92% genes by RPE cells isolated from donor tissue (for a list, see for the 402H form). Supplemental Table I). We found that most genes involved in an The AMD-associated 402H polymorphism does not affect C3b alternative pathway complement response were transcribed, in- cofactor activity of FHL-1 cluding C3, FB, FI, FHL-1, and FH (Fig. 2A). We assessed whether the AMD-associated Y402H polymorphism FHL-1 can diffuse across the Bruch’s membrane would alter the ability of FHL-1 to regulate complement activation Next we investigated whether FH and FHL-1 could contribute to on host surfaces by catalyzing the FI-mediated breakdown of C3b the protection of the Bruch’s membrane/RPE interface by dif- to iC3b. When run on reducing gradient gels, the a- and b-chains fusing across Bruch’s membrane from the blood supply. Isolated of C3b can be clearly separated (Supplemental Fig. 3A). When Bruch’s membrane was sandwiched between two compartments C3b is incubated with FI and either the 402Y or 402H form of of an Ussing chamber (see Fig. 3A) as previously described (37), FHL-1, the proteolytic cleavage of the C3b a-chain by FI yeilds and the diffusion of FH and FHL-1 from human serum across the bands at 68 and 43 kDa (38), the appearance of which can be used membrane was investigated. After 24 h only an ∼49-kDa band as a measure of FHL-1 cofactor activity. In the present study, we was identified in the diffusate by Western blot using OX23 noted no difference in the ability of the two FHL-1 forms to break (Fig. 3B). Dual staining of this band with OX23 and anti–FHL-1 down C3b (see Supplemental Fig. 3B). This result was predictable confirmed that this was indeed FHL-1 traversing the Bruch’s because the C3b/FI binding regions and the Y402H polymorphism membrane (Fig. 3B). In contrast, FH (155 kDa) was not detected. site are at opposite ends of the FHL-1 protein (Fig. 1A) (39, 40). The Journal of Immunology 5

Table I. Details of donor eyes used in this study Heparin is, however, a convenient highly sulfated model of the HS that is found in ECM. HS contains large regions with low Donor Sex Age (y) sulfated disaccharides and overall has much less sulfation than Donors without known eye diseasea heparin (Fig. 4D) (18). The experiments with selectively desul- M17000 M 72 fated heparin demonstrated that the disease-associated 402H var- M17055 F 73 iant of FHL-1 relies heavily on highly sulfated GAG sequences M17119 F 89 and therefore is likely to bind less well to HS than heparin. To M17126 M 83 M17130 M 92 confirm this, solid-phase binding experiments with two sources of M17143 M 78 HS (porcine mucosa and bovine kidney) were performed and these Dual FHL-1/FH staining of drusen confirmed that the 402H form of FHL-1 binds significantly less M17142 F 82 well than does the 402Y form to HS (Fig. 4E, 4F). Conversely, the M12106 M 84 402H form of FHL-1 bound better than did the 402Y form to M12109 F 70 M14557 M 86 highly sulfated heparin (Supplemental Fig. 2), which agrees with Ussing chamber experiments the influence of sulfation availability on 402H binding, but also Whole serum highlights the unsuitability of heparin as a physiological model for M20053 M 67 FHL-1/HS interactions. M14479 F 69 M14507 F 78 The removal of HS from macula tissue sections by pretreatment Recombinant FHL-1 402H and 402Y variants with a heparinase I/II/III mix pretreatment reduced the signal seen for M20854 F 58 endogenous FHL-1 (Fig. 5B), indicating that HS is indeed one of the M20860 M 75 main ligands anchoring FHL-1 to the Bruch’s membrane. Interest- M20866 M 63 ingly, enzymatic treatment of drusen did not appear to alter detect- FH M20879 M 65 able levels of FHL-1 within the druse, but did remove the distinctive M20882 F 78 staining of FH around the edges of the lesions (Fig. 5C, 5D). M20884 M 84 aDonors without known eye disease used for immunohistochemistry, Western Discussion blot analysis, and RPE gene transcription. Since 2005, a large body of work has focused on alterations in FH as major genetic contributors to AMD pathogenesis (6, 41–44) and the importance of the alternative complement pathway. This has 402H and 402Y forms of FHL-1 differentially bind sulfated been supported by evidence of complement proteins being present self-surface markers in AMD macular tissue, including markers of dysregulation (3, 22, Although the Y402H polymorphism does not affect the ability of 23, 45). Previous studies have concluded that FH, as a blood-borne FHL-1 to inactivate C3b, it does reside in the protein’s only surface protein, is the only complement regulator that would confer pro- recognition domain in CCP7 (Fig. 1A). Previously using FH and tection to ECM such as Bruch’s membrane. Genetic studies have recombinant proteins representing the CCP6–8 region of FH, the not distinguished between FH and FHL-1 (given that they share Y402H polymorphism has been shown to affect GAG binding (22, the same gene), and although FHL-1–specific Abs have been made 23). In this study, we investigated the binding of the FHL-1 402H in the past (46) they have not been used to probe eye tissue (27). and 402Y proteins to selectively desulfated heparin. The heparin In this study, we show that FHL-1 is likely to be conferring was desulfated at the 6-O,2-O, and N positions (see Fig. 4A), and greater protection to Bruch’s membrane than does FH, whereas the although this did not affect the binding of the 402Y form of latter is the predominant form protecting the ECM of the choroid FHL-1, desulfation did significantly affect the binding of the (Fig. 1C, 1D). Furthermore, we confirm that the RPE cells tran- AMD-associated 402H form (Fig. 4B, 4C). scribe genes involved in complement activation and regulation on Bruch’s membrane, and our data demonstrate that FHL-1 is lo- cally transcribed (Fig. 2). As well as a local contribution to FHL-1 accumulation in the Bruch’s membrane, we have demonstrated that the 49-kDa protein could passively diffuse across Bruch’s membrane from the choroidal vasculature, whereas the 155-kDa glycosylated FH protein cannot (Fig. 3B, 3C). This latter obser- vation supports the previous finding that the Bruch’s membrane confers a size limit on proteins able to diffuse passively across it, the size of which decreases with age (37). The AMD-associated Y402H polymorphism does not affect the diffusion of FHL-1 across Bruch’s membrane (Fig. 3D, 3E), but differences in the amount of recovered protein between the 402H and 402Y forms may be indicative of more 402Y FHL-1 binding to Bruch’s membrane in the Ussing chamber as it passes through compared with the 402H form. Small patches of FH were observed on the RPE-facing side of FIGURE 2. Detection of complement gene transcription by human RPE Bruch’s membrane (Fig. 1D), which are likely to originate from cells. Pooled RNA from five donors’ RPE cells was used to detect com- RPE cells. Local synthesis would also explain the distinctive FH plement gene transcription. (A) RNA for a number of genes central to the alternative pathway of complement were detected in both liver and RPE labeling on the surface of drusen, which may be unable to pene- cells, including FHL-1, FH, C3, FB, and FI. (B) Two RPE-specific genes, trate into them, whereas the smaller FHL-1 can penetrate into the Best-1 and RPE65, were used as positive controls for RPE cell RNA, and drusen (Fig. 1E). Interestingly, the FH staining around drusen is b-actin and TATA-binding protein (TBP) were selected as housekeeping removed by heparinase treatment (Fig. 5), suggesting that the gene controls. protein is retained on the surface of drusen by binding HS chains. 6 FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE

FIGURE 3. FHL-1 is able to diffuse through Bruch’s membrane from human serum. An Ussing chamber was used to compare the diffusion of FH and FHL-1 from human serum across enriched Bruch’s membrane from human donor eyes. (A) Schematic of the Ussing chamber layout where (a) the Bruch’s membrane, (b) sampling access points, and (c) magnetic stirrer bars are shown. (B) Fluorescent Western blot of one representative experiment from a total of three showing 0 and 24 h samples from the PBS compartment: the left-hand lane shows a positive control sample containing 1 mg each of FH and FHL-1. Red bands are recognized by OX23 alone, green by anti–FHL-1 alone, and yellow by both Abs. The Odyssey protein markers used are visualized in the red channel. The Western blot shown is representative of three separate experiments. (C) Purified FH was placed in one compartment and after 24 h the entire protein content of the other “PBS” compartment was concentrated, subjected to SDS-PAGE, and the resultant gel was stained with Coomassie blue. (D and E) Diffusion experiments with purified recombinant FHL-1 proteins examined potential differences in the ability of the 402H and 402Y variants to cross Bruch’s membrane. Both 402H and 402Y forms were tested separately using three donor Bruch’s membranes, and data are shown as percentage protein detected in each chamber after 24 h at room temperature: the 50% mark is shown as a dashed line. The donor tissues used in (C)–(E) are listed in Table I.

The lack of heparinase effect on FHL-1 staining within drusen CCPs 19–20 of FH are more important in the kidney (24, 33, 48). suggests that the protein is either interacting with a currently The absence of full-length FH in Bruch’s membrane (and thus unidentified ligand, or it is trapped among the plethora of com- the CCPs 19–20) may have resulted in evolutionary pressure to ponents that make up these hallmark lesions of AMD. Similarly, it express HS species capable of recruiting FHL-1 via its CCP7 may be possible that the heparinase itself is not able to penetrate domain. the tight matrix of the druse. Whether FHL-1 retains activity in Although our work suggests that FHL-1 is a major regulator of this environment is unclear. complement in Bruch’s membrane, there is genetic evidence that We show that the FHL-1 within Bruch’s membrane is immo- FH is also important. A highly penetrant mutation R1210C in the bilized there largely through interactions with HS. In other recent C terminus of FH is a strong risk factor for AMD and is thought to work we have shown that there is a marked decrease in the levels coincide with a 6-y earlier onset of the disease (9). The R1210C of HS GAGs in Bruch’s membrane with age (47). This decrease in form of FH is exclusively found covalently bound to albumin in HS levels in Bruch’s membrane coupled with the poorer binding plasma (49), which would affect the proteins mobility, and al- of the 402H form of FHL-1 could explain why AMD is a disease though albumin binding has no effect on GAG binding, it does of aging. Individuals with the 402H polymorphism may, as they alter FH binding to C3b (49, 50). As such, it is likely this mutation age, be unable to localize sufficient FHL-1 to Bruch’s membrane hampers FH tissue penetration and surface protection. Further- so that its protective effects are lost and the complement cascade is more, most AMD patients with the R1210C haplotype have the activated with damaging consequences that ultimately lead to FH/FHL-1 402H polymorphism on the other allele (9). It may be AMD. Furthermore, the dominant role of FHL-1 in complement the case that any contribution conferred by FH to immune regu- regulation at this site provides, to our knowledge for the first time, lation in the macula is perturbed by the R1210C mutation and an explanation of why the GAG-binding region in CCP7 was amplifies an already imbalanced immune homeostasis (conferred found to be vital for protein localization in the eye (23), whereas by linked genetic factors such as the Y402H polymorphism). The Journal of Immunology 7

FIGURE 4. The 402H form of FHL-1 shows greater dependency on GAG sulfa- tion for binding. Heparin is a highly sulfated model of HS. (A) Schematic showing the basic iduronic acid–glucosamine backbone disaccharide of heparin where all four pos- sible sulfation positions are listed as fol- lows: R1,6-O sulfation; R2, N sulfation; R3, 2-O sulfation; and R4,3-O sulfation. (B and C) Plate assays demonstrating the binding activities of FHL-1 402Y and 402H forms for selectively desulfated heparin. (D)Sche- matic diagram demonstrating the different disaccharide regions of an HS chain. GlcNAc, N-acetylated glucosamine; GlcNS, N-sul- fated glucosamine; GlcUA, glucuronic acid; IdoUA, iduronic acid; 2S, sulfation in the 2-O position; 6S, sulfation in the 6-O posi- tion. (E and F) Plates assays demonstrating the AMD-associated 402H form of FHL-1 binding relatively poorly to two forms of HS compared with the 402Y form. Data in (B), (C), (E), and (F) are n = 6, averaged from two independent experiments 6 SEM.

Our findings also have implications for understanding how the with alterations in AMD risk (41, 42), and the FHR proteins can five FHR proteins contribute to immune homeostasis in the eye. compete with FH binding to C3b and/or GAGs, and even to form Variations in the genes encoding the FHR proteins are associated a novel C3 convertase (51). As such, they are fast being considered

FIGURE 5. Heparinase pretreatment alters the pattern of FHL-1 and FH staining in the macula. Both FHL-1 and FH localization were visualized in eye tissue before and after enzymatic pretreatment with a heparinase I/ II/III mix using the six normal donor eyes and four drusen containing AMD (eyes as listed in Table I). In each case green staining rep- resents FHL-1 and red staining FH. (A)Dis- tribution of FHL-1 and FH before removal of HS, and (B) after enzymatic treatment in macular tissue without AMD pathology. FHL-1 and FH labeling of a druse without (C) and with heparinase treatment (D). Blue staining represents DAPI staining of cell nuclei. Scale bars, 10 mm. 8 FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE newly identified positive regulators of the alternative pathway of ment factor H variant increases the risk of age-related macular degeneration. Science 308: 419–421. complement (41, 52–54). However, many of these biochemical 12. Klein, R. J., C. Zeiss, E. Y. Chew, J.-Y. Tsai, R. S. Sackler, C. Haynes, studies have assumed that the FHR proteins would be competing A. K. Henning, J. P. SanGiovanni, S. M. Mane, S. T. Mayne, et al. 2005. with FH as the main regulator, which in the context of Bruch’s Complement factor H polymorphism in age-related macular degeneration. Sci- ence 308: 385–389. membrane is not the case, and so their competitive properties with 13. Hageman, G. S., D. H. Anderson, L. V. Johnson, L. S. Hancox, A. J. Taiber, FHL-1 may well differ. L. I. Hardisty, J. L. Hageman, H. A. Stockman, J. D. Borchardt, K. M. Gehrs, The unique FHL-1 C terminus means that it is necessary to et al. 2005. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc. investigate the binding characteristics of known FH ligands such Natl. Acad. Sci. USA 102: 7227–7232. as C-reactive protein (55, 56) and markers of oxidative stress (such 14. Hecker, L. A., A. O. Edwards, E. Ryu, N. Tosakulwong, K. H. Baratz, as malondialdehyde) (57) in relationship to the Y402H polymor- W. L. Brown, P. Charbel Issa, H. P. Scholl, B. Pollok-Kopp, K. E. Schmid- Kubista, et al. 2010. Genetic control of the alternative pathway of complement in phism. Furthermore, the results shown in the present study may humans and age-related macular degeneration. Hum. Mol. Genet. 19: 209–215. have implications for immune homeostasis in other tissues and in 15. Day, A. J., A. C. Willis, J. Ripoche, and R. B. Sim. 1988. Sequence polymor- other diseases where FH struggles to access ECM structures or phism of human complement factor H. Immunogenetics 27: 211–214. 16. Walport, M. J. 2001. Complement. First of two parts. N. Engl. J. Med. 344: extracellular debris, whereas FHL-1 can. An example is the brain 1058–1066. lesions of Alzheimer disease patients (58), and it is of note that 17. Day, A. J., S. J. Clark, and P. N. Bishop. 2011. Understanding the molecular changes in HS sulfation are also associated with this condition basis of age-related macular degeneration and how the identification of new mechanisms may aid the development of novel therapies. Expert Rev. Oph- (59). The identification of FHL-1 as the main complement regu- thalmol. 6: 123–128. lator at a key site in AMD pathogenesis shapes our understanding 18. Sarrazin, S., W. C. Lamanna, and J. D. Esko. 2011. Heparan sulfate proteo- of the molecular biology of the disease. Only by elucidating the glycans. Cold Spring Harb. Perspect. Biol. 3: a004952. 19. Clark, S. J., P. N. Bishop, and A. J. Day. 2013. The proteoglycan glycomatrix: precise biochemistry behind complement regulation at the macula a sugar microenvironment essential for complement regulation. Front. Immunol. alongside understanding genetic regulation of the implicated 4: 412. proteins can we hope to design successful therapeutic strategies 20. Park, P. J., and D. Shukla. 2013. Role of heparan sulfate in ocular diseases. Exp. Eye Res. 110: 1–9. for this debilitating disease. 21. Schmidt, C. Q., A. P. Herbert, D. Kavanagh, C. Gandy, C. J. Fenton, B. S. Blaum, M. Lyon, D. Uhrı´n, and P. N. Barlow. 2008. A new map of glycosaminoglycan and C3b binding sites on factor H. J. Immunol. 181: 2610–2619. Acknowledgments 22. Clark, S. J., V. A. Higman, B. Mulloy, S. J. Perkins, S. M. Lea, R. B. Sim, and We thank Prof. Paul Barlow (University of Edinburgh, Edinburgh, U.K.) and A. J. Day. 2006. His-384 allotypic variant of factor H associated with age-related Prof. Anthony Day (University of Manchester, Manchester, U.K.) for ad- macular degeneration has different heparin binding properties from the non- disease-associated form. J. Biol. Chem. 281: 24713–24720. vice, support, and direction during the early stages of this work. Also, 23. Clark, S. J., R. Perveen, S. Hakobyan, B. P. Morgan, R. B. Sim, P. N. 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