The present invention relates to a method of treating age-related macular degeneration. In particular embodiments, the invention relates to methods for treating dry age-related macular degeneration.
Age-related macular degeneration (AMD) is the leading cause of central blindness in adults. Dry AMD (also called nonexudative AMD) is a broad designation, encompassing forms of AMD that are not neovascular. At present, there are no treatments for dry AMD. Genetic factors, age, diet and smoking are risk factors for AMD.
The common, coding variant Y402H in the Complement Factor H (CFH) gene is strongly associated with influencing susceptibility to AMD. In fact, the role of complement in the retina is a topic of intense investigation, as it contributes to a variety of other retinal disease pathologies in addition to AMD. Variants in other complement-related genes are associated with AMD risk, including C3, CFI and C9, all result in an overly active complement system. Congruent with this, the deposition of C3, C5, and presence of membrane attack complex (MAC), have been demonstrated in donor eyes with early AMD. Accordingly, SNPs in complement factors account for ˜75% of genetic risk of developing AMD.
However, molecular triggers that initiate complement fixation in individuals with no apparent genetic risk remain unknown. Smoking is the largest modifiable risk factor for AMD, consequently oxidative stress has been implicated in disease. Genotype and smoking have been independently related to AMD with multiplicative joint effects, however, a tangible connection between the effects of oxidative stress and complement-associated pathology remains largely unidentified.
The retina is exposed to oxidative stress, which refers to cellular damage caused by reactive oxygen species (ROS), due to its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its exposure to visible light. Excessive oxidative stress induces deleterious changes that result in visual impairment. AMD is a leading causes of visual impairment and involvement of oxidative stress has been reported. Furthermore, oxidative stress is thought to contribute to loss of cone photoreceptors in rare inherited retinopathies after degeneration of rod photoreceptors. 2-(w-Carboxyethyl) pyrrole (CEP) is an oxidative-stress modification involved in promoting angiogenesis during wound healing. Excessive ROS can damage lipids through a mechanism known as lipid peroxidation and CEP modifications are generated by oxidation of docosahexaenoate (DHA)-containing lipids, which are found at high levels in the membrane of photoreceptor cells. Of note, CEP-adducted proteins and CEP-ethanolamine phospholipids (CEP-EPs) are found in abundance in eyes and serum of patients with AMD compared with age-matched controls.
Toll-like receptors (TLRs) are a family of membrane-bound pattern recognition receptors (PRRs) located either on the cell surface or in endosomal compartments. These receptors are known to respond to host-molecules termed damage-associated molecular patterns (DAMPs) that have taken on the appearance of “non-self”. Sterile inflammation occurs in response to a growing list of DAMPs ranging from oxidized lipids or lipoproteins, to deposits of protein/lipid aggregates or particulate matter. As these stimuli are often not easily cleared, they can persist causing over-activation of the immune system and contributing to disease pathogenesis. Ten human TLRs utilize four adaptor proteins to fine tune the response required; MyD88, Mal/TIRAP, TRAM and TRIF. Activation of TLRs leads to activation of a multitude of signaling pathways and transcription factors that determine the type and duration of the inflammatory response.
According to a first aspect of the present invention there is provided a method of treating age-related macular degeneration in a subject in need thereof.
Optionally, the method comprises the step of decreasing the expression or activation of a toll-like receptor in the subject.
Optionally, the toll-like receptor is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13.
Preferably, the toll-like receptor is TLR2.
Optionally, the method of treating age-related macular degeneration in a subject in need thereof comprises the step of decreasing the expression or activation of TLR2 in the subject.
Optionally, the age-related macular degeneration is dry age-related macular degeneration. Further optionally, the age-related macular degeneration is non-exudative age-related macular degeneration. Still further optionally, the age-related macular degeneration is non-neovascular age-related macular degeneration. Still further optionally, the age-related macular degeneration is not wet age-related macular degeneration.
Optionally, the method of treating dry age-related macular degeneration in a subject in need thereof comprises the step of decreasing the expression or activation of TLR2 in the subject.
Optionally, the method comprises the step of administering an antagonist of a toll-like receptor to the subject. Further optionally, the method comprises the step of administering a pharmaceutically effective amount of an antagonist of a toll-like receptor to the subject. Still further optionally, the method comprises the step of administering a pharmaceutically effective amount of an antagonist of a toll-like receptor to the subject to decrease the expression or activation of the toll-like receptor in the subject. Still further optionally, the method comprises the step of administering a pharmaceutically effective amount of an antagonist of a toll-like receptor to the subject to decrease the expression or activation of the toll-like receptor and so treat age-related macular degeneration in the subject.
Optionally, the method comprises the step of administering an antagonist of TLR2 to the subject. Further optionally, the method comprises the step of administering a pharmaceutically effective amount of an antagonist of TLR2 to the subject. Still further optionally, the method comprises the step of administering a pharmaceutically effective amount of an antagonist of TLR2 to the subject to decrease the expression or activation of TLR2 in the subject. Still further optionally, the method comprises the step of administering a pharmaceutically effective amount of an antagonist of TLR2 to the subject to decrease the expression or activation of TLR2 and so treat age-related macular degeneration in the subject.
Optionally, the method of treating dry age-related macular degeneration in a subject in need thereof comprises the step of administering a pharmaceutically effective amount of an antagonist of TLR2 to the subject to decrease the expression or activation of TLR2 and so treat age-related macular degeneration in the subject.
Optionally, the method comprises decreasing expression of the toll-like receptor. Further optionally, the method comprises decreasing expression of TLR2.
Optionally, the method comprises the step of administering an agent capable of decreasing expression of the toll-like receptor. Further optionally, the method comprises the step of administering an agent capable of decreasing expression of the toll-like receptor gene. Still further optionally, the method comprises the step of administering an agent capable of decreasing transcription of the toll-like receptor gene.
Optionally, the method comprises the step of administering an agent capable of decreasing expression of TLR2. Further optionally, the method comprises the step of administering an agent capable of decreasing expression of the TLR2 gene. Still further optionally, the method comprises the step of administering an agent capable of decreasing transcription of the TLR2 gene.
Optionally, the agent is selected from the group consisting of antisense oligonucleotides, ribozymes, small interfering RNAs (siRNA), microRNA (miRNA), small/small hairpin RNA (shRNA), and nucleic acid aptamers.
Optionally, the agent is a nucleic acid aptamer. Further optionally, the agent is a deoxyribonucleic acid aptamer. Further optionally, the agent is the deoxyribonucleic acid aptamer AP177 (as dislcosed in Y. C. Chang, W. C. Kao, W. Y. Wang, W. Y. Wang, R. B. Yang, K. Peck “Identification and characterization of oligonucleotides that inhibit Toll-like receptor 2-associated immune responses” FASEB J., 23 (2009), pp. 3078-3088).
Optionally, the agent is a vector. Further optionally, the agent is a viral vector. Still further optionally, the agent is a virally-delivered vector. Still further optionally, the agent is a retrovirus-, adenovirus-, herpes simplex-, vaccinia-, or adeno-associated virus-delivered vector.
Optionally, the agent is delivered by injection of naked DNA, electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes, dendrimers, or inorganic nanoparticles.
Optionally, the method comprises decreasing the activation of the toll-like receptor. Further optionally, the method comprises decreasing the activation of TLR2.
Optionally, the method comprises administering a toll-like receptor antagonist selected from a competitive toll-like receptor antagonist, a non-competitive toll-like receptor antagonist, an uncompetitive toll-like receptor antagonist, a silent toll-like receptor antagonist, and an inverse toll-like receptor agonist.
Optionally, the method comprises administering a toll-like receptor antagonist selected from a reversible toll-like receptor antagonist, and an irreversible toll-like receptor antagonist.
Optionally, the method comprises administering a toll-like receptor antagonist selected from a selective toll-like receptor antagonist, and a non-selective toll-like receptor antagonist.
Optionally, toll-like receptor antagonist is selected from a chemical compound toll-like receptor antagonist, a small molecule toll-like receptor antagonist, an immunoglobulin toll-like receptor antagonist, and a lipid-A analogue toll-like receptor antagonist.
Optionally, the toll-like receptor antagonist is the small molecule toll-like receptor antagonist 2-ethoxy-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1H-1,3-benzodiazole-7-carboxylic acid (candesartan cilexetil, “Atacand”).
Optionally, the immunoglobulin toll-like receptor antagonist is an antibody toll-like receptor antagonist or antibody fragment toll-like receptor antagonist. Further optionally, the immunoglobulin toll-like receptor antagonist is a murine antibody toll-like receptor antagonist or murine antibody fragment toll-like receptor antagonist. Still further optionally, the immunoglobulin toll-like receptor antagonist is a humanised antibody toll-like receptor antagonist or humanised antibody fragment toll-like receptor antagonist. Still further optionally, the immunoglobulin toll-like receptor antagonist is a monoclonal antibody toll-like receptor antagonist or monoclonal antibody fragment toll-like receptor antagonist.
Optionally, the immunoglobulin toll-like receptor antagonist is selected from Tomaralimab (“OPN-305” as disclosed in U.S. Pat. No. 8,734,794) and T2.5 (as disclosed in U.S. Pat. No. 8,623,353).
Optionally, the lipid-A analogue toll-like receptor antagonist is OM-174 (as disclosed in WO2006095270).
Optionally, the method comprises administering the toll-like receptor antagonist to the retinal pigment epithelium. Further optionally, the method comprises administering the toll-like receptor antagonist to the plasma membrane of the retinal pigment epithelium. Still further optionally, the method comprises administering the toll-like receptor antagonist apically and/or basolaterally to the plasma membrane of the retinal pigment epithelium.
Optionally, the method comprises administering the toll-like receptor antagonist to the retinal immune cells. Further optionally, the method comprises administering the toll-like receptor antagonist to the plasma membrane of the retinal glia and/or mononuclear phagocytes. Still further optionally, the method comprises administering the toll-like receptor antagonist to the retinal microglia cells, muller glia cells and/or mononuclear phagocytes.
Optionally, the method at least reduces photoreceptor cell death. Further optionally, the method reduces photoreceptor cell death. Still further optionally, the method inhibits photoreceptor cell death.
Optionally, the method at least reduces oxidative-stress-induced photoreceptor cell death. Further optionally, the method reduces oxidative-stress-induced photoreceptor cell death. Still further optionally, the method inhibits oxidative-stress-induced photoreceptor cell death.
Optionally, the method at least reduces photoreceptor cell death in at least one row of photoreceptors. Further optionally, the method reduces photoreceptor cell death in at least one row of photoreceptors. Still further optionally, the method inhibits photoreceptor cell death in at least one row of photoreceptors.
Optionally, the method at least reduces oxidative-stress-induced photoreceptor cell death in at least one row of photoreceptors. Further optionally, the method reduces oxidative-stress-induced photoreceptor cell death in at least one row of photoreceptors. Still further optionally, the method inhibits oxidative-stress-induced photoreceptor cell death in at least one row of photoreceptors.
Optionally, the method at least reduces photoreceptor cell death in at least two rows of photoreceptors. Further optionally, the method reduces photoreceptor cell death in at least two rows of photoreceptors. Still further optionally, the method inhibits photoreceptor cell death in at least two rows of photoreceptors.
Optionally, the method at least reduces oxidative-stress-induced photoreceptor cell death in at least two rows of photoreceptors. Further optionally, the method reduces oxidative-stress-induced photoreceptor cell death in at least two rows of photoreceptors. Still further optionally, the method inhibits oxidative-stress-induced photoreceptor cell death in at least two rows of photoreceptors.
Optionally, the method at least reduces retinal degeneration. Further optionally, the method reduces retinal degeneration. Still further optionally, the method inhibits retinal degeneration.
Optionally, the method at least reduces oxidative-stress-induced retinal degeneration. Further optionally, the method reduces oxidative-stress-induced retinal degeneration. Still further optionally, the method inhibits oxidative-stress-induced retinal degeneration.
Optionally, the method at least reduces retinal pigment epithelium fragmentation. Further optionally, the method reduces retinal pigment epithelium fragmentation. Still further optionally, the method inhibits retinal pigment epithelium fragmentation.
Optionally, the method at least reduces oxidative-stress-induced retinal pigment epithelium fragmentation. Further optionally, the method reduces oxidative-stress-induced retinal pigment epithelium fragmentation. Still further optionally, the method inhibits oxidative-stress-induced retinal pigment epithelium fragmentation.
Optionally, the method comprises parenteral administration of the toll-like receptor antagonist.
Optionally, the method comprises injection of the toll-like receptor antagonist. Further optionally, the method comprises retinal injection of the toll-like receptor antagonist. Still further optionally, the method comprises sub-retinal injection of the toll-like receptor antagonist.
Optionally, the method comprises intravitreal administration of the toll-like receptor antagonist.
Optionally, the method comprises topical administration of the toll-like receptor antagonist. Further optionally, the method comprises topical administration of the toll-like receptor antagonist to one or both eyes. Still further optionally, the method comprises topical administration of the toll-like receptor antagonist to the surface of one or both eyes.
Optionally, the method comprises the further step of decreasing the expression or activation of Myeloid differentiation primary response 88 (MYD88) in the subject.
Optionally or additionally, the method comprises the further step of decreasing the expression or activation of MyD88-adapter-like (Mal) in the subject.
Optionally, the method comprises the further step of decreasing the expression or activation of MyD88 and Mal in the subject.
Embodiments of the present invention will be described with reference to the appended non-limiting examples and the accompanying drawings in which:
All experiments were conducted in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research, and approved by the Trinity College Dublin Animal Research Ethics Committee or the Australian National University (ANU) Animal Experimentation Ethics Committee. Mice used were C57BL/6J mice and Tlr2−/− (JAX stock #004650) at 8-12 week old. DBA/2J CS-deficient mice (JAX stock #000671) were 10 months old and matched to 10 month old C57BL/6J mice. Comparator WT vs KO mice were sex matched, as sex has a measurable effect on the NaIO3 model.
A single intravenous injection, via tail vein, of NaIO3 (50 mg/kg) was administered to C57Bl/6J and TLR2−/− mice. Mice were euthanized 8 hours post injection, eyes fixed in ice-cold methanol for 15 minutes and the choroid/RPE dissected into flatmounts. Flatmounts were blocked and permeabilised in 5% NGS, 0.05% Triton X100 for 1 hour and incubated with ZO-1 (1:100, Invitrogen) overnight at 4° C. Flatmounts were washed with PBS and incubated with goat anti-rabbit 488 for 2 hours at room temperature.
A single intravenous injection, via tail vein, of NaIO3 (50 mg/kg) was administered to C57Bl/6J and TLR2−/− mice. Mice were euthanized 72 hours post injection, eyes fixed in 4% paraformaldehyde for 90 minutes and after a 30% sucrose gradient, eyes were embedded in optimal cutting temperature compound (OCT). 12 um sections were blocked and permeabilised in 5% NGS, 0.05% Triton X100 for 1 hour and incubated with C3 (Abcam ab11887, 1:100), MAC (Biozol, FGI-10-1801, 1:100) or Iba1 (Wako 019-19741, 1:500) overnight at 4° C. Sections were washed with PBS and incubated with Alexa Fluor® goat anti-rabbit 488 or Alexa Fluor® goat anti-mouse 488 1:500 in 5% NGS for 2 hours at room temperature and counterstained with Hoechst. To quantify the numbers of Iba1+ cells, a minimum of eight 20× objective frames were counted per eye and counts were averaged per mouse. Images were cropped to only include the RPE and photoreceptor layers for MAC staining and the mean fluorescence intensity was measured.
A single intravenous injection, via tail vein, of NaIO3 (50 mg/kg) was administered to C57Bl/6J mice. Mice were euthanized 24 hours post injection, eyes fixed in 4% paraformaldehyde for 90 minutes and after a 30% sucrose gradient, eyes were embedded in OCT. 12 um sections were stained using anti-CEP ab 1:1000 (Kindly provided by Sheldon Rowan, Tufts University, USA) using the Vectastain ABC Kit following manufacturers protocol and detected using DAB (Vector Laboratories).
A single intravenous injection was administered via the tail of NaIO3 (50 mg/kg) in NaCl. Control mice received NaCl. In tandem, mice received a single subretinal injection of either anti-CFD antibody (R&D) antibody or IgG control (0.5 μg per eye). Mice were euthanized 72 hours post injection. Eyes were fixed in Davidson's fixative overnight, washed 3 times in PBS and embedded in paraffin wax. 5 μm sections were cut with a microtome (Leica) and subject to xylene deparaffinising and ethanol rehydration. For histology slides were stained with haematoxylin and eosin. To detect cell death sections were stained using in situ Cell Death Detection kit, TMR red (Roche) following manufacturer's protocol and nuclei counterstained with Hoechst.
A single intravenous injection was administered via the tail of NaIO3 (50 mg/kg) in NaCl. Control mice received NaCl. In tandem, mice received a single subretinal injection of either anti-TLR2 blocking (Invivogen) antibody or IgG control (3 μg per eye). Mice were euthanized 3 days' post injection eyes were fixed in Davidson's fixative overnight, washed 3 times in PBS and embedded in paraffin wax. 5 μm sections were cut with a microtome (Leica) and subject to xylene deparaffinising and ethanol rehydration. For H&E histology slides were stained with haematoxylin and eosin. TUNEL staining—To detect cell death sections were stained using in situ Cell Death Detection kit, TMR red (Roche) for 1 hour at 37° C. following manufacturer's protocol and nuclei counterstained with Hoechst. The number of photoreceptor rows was calculated by counting the number of nuclei spanning the height of the outer nuclear layer (ONL) at three individual points per 20× frame (eg. ˜12 nuclei height in ONL of wildtype mice) and an average was taken per 20× frame. To quantify the numbers of ONL rows or TUNEL+ cells, a minimum of eight 20× objective frames were counted per eye and counts were averaged per mouse.
C57BL/6J mice (8 weeks old) received a single intravitreal injection of either an anti-TLR2 antibody or IgG control (3 μg per eye). Animals were then exposed to 100 Klux light for 7 days to induce photo-oxidative damage, as described previously (NATOLI, R., JIAO, H., BARNETT, N. L., FERNANDO, N., VALTER, K., PROVIS, J. M. & RUTAR, M. 2016a. A model of progressive photo-oxidative degeneration and inflammation in the pigmented C57BL/6J mouse retina. Exp Eye Res, 147, 114-27). Following photo-oxidative damage, animals were euthanized and eyes collected for histological analysis. Retinal cryosections were stained with TUNEL (Roche) to detect photoreceptor cell death. C3 immunohistochemistry was performed using α-C3 antibody (1:100, Abcam), and C3+ cells/deposits in the outer retina (between the ONL and RPE) were counted per retinal section.
Human donor eyes obtained from the Iowa Lions Eye Bank (Iowa City, Iowa, USA) eyes were processed within 8 hours of death (Table 1). Macular punches which had been fixed in 4% paraformaldehyde and embedded in sucrose-optimal cutting medium were sectioned on a cryostat and stained for TLR2 (Abcam) and C3d (Dako) using VIP and Vectastain ABC Kit (Vector Laboratories). Characteristics of donor tissue used for immunohistochemistry are displayed in Table 2.
ARPE-19 cells (ATCC CRL 2302) 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 Ham with L-glutamine, 15 mM HEPES, sodium bicarbonate. THP-1 cells RPMI 1640 medium Immortalized bone marrow derived macrophages wildtype MyD88−/− and Mal−/− mice (Kind gift Prof. Golenbock, UMass Medical School) DMEM. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood RPMI 1640. All medium was supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Sigma-Aldrich). YFP+Cx3cr1-expressing microglia were isolated from mouse retinas according to previously described methods (FERNANDO, N., NATOLI, R., VALTER, K., PROVIS, J. & RUTAR, M. 2016. The broad-spectrum chemokine inhibitor NR58-3.14.3 modulates macrophage-mediated inflammation in the diseased retina. J Neuroinflammation, 13, 47) and were sorted into a 48-well plate at 1500 cells per well. Isolated primary microglia were cultured for 3 weeks in DMEM-F12 supplemented with 10% FBS, 1% antibiotic-antimycotic (Thermo Fisher Scientific), 3% L-glutamine, 0.25 ng/ml GM-CSF (Stem Cell Technologies) and 2.5 ng/ml M-CSF (Miltenyi Biotec) prior to TLR2 stimulation. Cells were maintained at 37° C., 5% CO2, 95% air.
Cells, provided by Dr. Arvydas Maminishkis from the National Eye Institute (NEI), Bethesda, USA, were received as a confluent monolayer of P-0 cells assays were conducted using cells at P-1. Primary human fetal RPE cells were isolated from human donor eyes as previously described and cultured in MEM-α containing 5% FCS (MAMINISHKIS, A., CHEN, S., JALICKEE, S., BANZON, T., SHI, G., WANG, F. E., EHALT, T., HAMMER, J. A. & MILLER, S. S. 2006. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci, 47, 3612-24).
Bone marrow derived macrophages (BMDMs), human monocytic cell like THP1s, PBMCs ARPE-19 cells, primary human fetal RPE cells or primary retinal microglia were stimulated with the generic TLR2/1 ligand Pam3Cys4 (Invivogen) or with CEP-HSA (kindly provided by Prof. Robert G Salomon (Case Western Reserve University, Cleveland Ohio) at indicated concentrations. Where indicated RPE cells were pre-treated with 0.1 μg/ml TLR2 antibody for 1 hour (T2.5 Invivogen), corresponding IgG control 0.1 μg/ml (Invivogen), Mal peptide inhibitor resuspended in DMSO 40 μM (Calbiochem) or an equal volume of DMSO was used in the control treatment.
0.4 μM polyester transwell inserts (VWR) were coated with 100 μg/ml Collagen IV (Sigma-Aldrich C5533) for 4 hours. ARPE-19 cells were seeded at a density of 1.7×105 cells per cm2 in DMEM F-12 Ham containing 10% (FBS). Two days later medium was replaced with complete medium containing 1% FBS and replenished twice weekly for 4-6 weeks.
Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4° C. Polyvinylidene fluoride (PVDF) membranes were wash 3 times with TBS-T and incubated with horseradish peroxidase conjugated anti-rabbit, anti-mouse or anti-goat 1:2000 (Sigma-Aldrich) for 1 hour at room temperature and developed using enhanced chemiluminescence. Densitometry was used to determine relative quantity of MAC and iC3b protein relative to actin loading control using Image J software. Scanned images were converted to 8-bit images. Each protein band was measured to obtain the area and mean value. The area was multiplied by the mean to obtain a measurement for each lane. The value obtained for the protein of interest was divided by the value obtained for the actin loading control for the corresponding well.
Total RNA was extracted from BMDMs, THP1s, ARPE-19 or hfRPE cells using Isolate II RNA extraction kit (Bioline) as per manufacturer's instructions. RNA was reverse transcribed using MMLV Reverse Transcriptase (Promega). Target genes were amplified by real time PCR with SensiFast SYBR Green (Bioline) using the ABI 7900HT system (Applied Biosystems). The cycling threshold method was used for relative quantification after normalisation to the ‘housekeeping’ gene βActin. The primers used were:
MCP-1 (TebuBio), and Soluble MAC (Abbexa) was detected in cell supernatants by sandwich ELISA according to manufacturer's instructions. Absorbance was read at 450 nM on a 96 well plate spectrophotometer.
Polarised ARPE-19 cells grown on transwell filters were maintained in serum free DMEM F-12 Ham for 48 hours. Cells were stimulated with 10% Normal human serum or heat inactivated normal human serum (Hi) (56° C. 30 minutes) either alone or with human serum albumin (HSA) or CEP-HSA for 24 hours. Where indicated cells were pre-treated for 1 hour with anti-TLR2 blocking antibody (T2.5 Invivogen), IgG control (0.1 μg/ml) or Mal peptide inhibitor (Calbiochem). Supernatants were harvested and assessed for soluble MAC formation by ELISA (Abbexa). Transwell inserts were fixed with 4% Paraformaldehyde for 10 minutes at room temperature, blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with anti-mouse-05b-9 1:25 (Santa cruz) overnight at 4° C. Transwells were washed 3 times in PBS and incubated with goat anti-mouse 647 1:500 (Invitrogen) and Phallodin 1:500 (Invitrogen) for 2 hours at room temperature. Cells were counted stained with Hoechst. Transwells inserts were carefully cut with a sterile blade and mounted on to polysine coated slides (Thermo Scientific) using Mowiol® 4-88. Staining was analysed using a confocal laser scanning microscope Axio Observer Z1 inverted microscope equipped with a Zeiss LSM 700 T-PMT scanning unit and a 40× plan.
An LDH cytotoxicity kit (Pierce) was used to detect cell death following MAC formation as per manufacturer's instructions absorbance was read at 490 nm and background absorbance at 680 nm.
HEK293-TLR2 cells were transfected for 24 hours with C3 promoter-luciferase (100 ng), Renilla-luciferase (40 ng) and empty vector (EV) or plasmid expressing Mal or MyD88 in increasing doses (10, 50 and 80 ng). Results are normalised for Renilla luciferase activity and represented as relative stimulation over the non-stimulated EV control and are expressed as mean+/−SD for triplicate measurements.
PBMCs were labelled for the investigation of monocytes with the following fluorochrome-labelled antibodies: anti-CD16 (3G8) anti-CD86 (FM95) anti-CD45 (2D1); anti-CD66b (G10F5); CD14 (Tuk4); CD80 (2D10); (Biolegend or Miltenyi). Each staining well contained 4×105 cells; cells were stained with LIVE/DEAD Aqua (Molecular Probes) followed by staining for 20 min on ice, washed, and analyzed by flow cytometry immediately. Flow cytometry was carried out on a BD LSR Fortessa cell analyzer and analyzed using FlowJo software (Tree Star).
Statistical analysis was carried out using Prism Graphpad and details of each test used can be found in the figure legends.
This study did not generate/analyse datasets/code Example 1.
To confirm a role for TLR2 in initiating the complement cascade, gene expression levels of key AP components Complement Factor B (CFB) and Complement factor 3 (C3) in response to TLR2 activation over time with generic TLR2 ligand, Pam3Cys4, a synthetic triacylated lipopeptide PAMP, were measured. Upregulation of CFB and C3 in TLR2 activated bone marrow derived macrophages (BMDMs) (
AMD-Associated Oxidative Stress Products Induce AP Complement Secretion from hfRPE Cells
It was next assessed whether a physiologically relevant DAMP generated by oxidative stress could induce the same response in primary human fetal RPE (hfRPE) cells. The retina is one of the most highly metabolically active tissues in the body. This oxidative burden, results in generation of lipid oxidation products such as CEP (
Overexpressing C3 in the retina can promote many features of AMD, while inhibiting various complement factors can protect against photoreceptor cell death in models of retinal degeneration. To define a role for TLR2 in bridging oxidative stress to complement activation and assessing its function in retinal degeneration, a well-characterized light-induced photo-oxidative stress model of retinal degeneration was utilized, in which locally produced C3 is known to contribute causally to retinal degeneration. In this model, there is a significant increase in C3+ macrophage/microglia in the photoreceptor layer and a decrease in outer nuclear layer (ONL) thickness. The ONL is made up of the nuclei of the rod and cone photoreceptors, and a decrease in the ONL thickness is indicative of photoreceptor cell death and retinal degeneration. An anti-TLR2 neutralizing antibody or control anti-IgG was injected intravitreally (IVT) into both eyes of each animal. Animals were subsequently exposed to 100K lux light for 7 days continuously. 1-2 more photoreceptor cell rows present in TLR2-neutralised retinas were observed compared to IgG controls, indicating that TLR2 blockade confers protection from oxidative stress induced photoreceptor cell loss in this model (
The NaIO3 mouse model of oxidative stress mimics some features of human retinal disease albeit in an acute manner; notably complement deposition, RPE fragmentation and photoreceptor cell degeneration. In vitro, NaIO3 dose dependently induced the expression of TLR2, CFB and C3 in RPE cells (
Having established that oxidative stress induced amplification of the AP is particularly damaging to the RPE in vivo, it was investigated whether TLR2 deficiency would modify this oxidative damage induced RPE fragmentation using TLR2 knockout (TLR2−/−) mice. Marked degradation of the RPE was observed in the NaIO3 group compared with vehicle NaCl group (
H&E staining of WT and TLR2−/− eyes post NaIO3 treatment indicated that the structure of the neural retina was better preserved in the TLR2−/− mice (
At this point, the data indicated that TLR2 plays a role in mediating C3 activation in response to oxidative stress resulting in the deposition of C3 and its opsonizing cleavage products in the outer retina. It was next investigated whether TLR2 can promote formation of the terminal complement complex MAC. CFB is the key rate limiting AP complement factor, as such, small increases in CFB expression, leads to formation of a C3 convertase (C3bBb) that amplifies the proteolytic cascade leading to C5 cleavage and ultimately formation of terminal complement MAC. During bacterial infection MAC usually leads to formation of a pore on the cell membrane, lysis and death of the bacteria. However, MAC is rarely lytic for nucleated cells and is reported to induce signaling pathways resulting in pro-inflammatory and pro-angiogenic gene expression on the RPE. Of particular interest is that known consequences of sub-lytic MAC formation are the release of chemokine monocyte chemoattractant protein CCL2/MCP-1 and vascular endothelial growth factor (VEGF), both cytokines are believed to have fundamental roles in promotion of dry and wet AMD respectively. It was suspected that the protective effect of TLR2 deficiency observed in the NaIO3 model of retinal degeneration may be partially attributed to blockade of sub-lytic MAC formation and signaling in the RPE. To determine whether the presence of CEP with provision of complete complement was sufficient to drive the proteolytic complement cascade to completion in vitro, hfRPE were cultured on transwell membranes for >4 weeks prior to stimulation with either HSA or CEP-HSA in the presence of heat-inactivated (Hi) or normal human serum (NHS) for provision of complete complement. Culture of hfRPE cells in the presence of 10% NHS and HSA resulted in the appearance of visible MAC (
To confirm a role for TLR2 in the recognition of CEP-HSA and promotion of MAC in the RPE, the observation that ARPE-19 cells formed membrane-embedded MAC only in the presence of 10% NHS and CEP-HSA, with no visible membrane-embedded MAC formed in the presence of 10% NHS or 10% NHS+HSA alone (
To interrogate whether CEP/NHS induced MAC formation on the RPE was lytic or sub-lytic, a lactate dehydrogenase (LDH) assay was used as an indicator of cell death and an MCP-1/CCL2 ELISA as an indicator of sub-lytic MAC signaling. The RPE supernatant was harvested from the MAC-assay (
TLR2 deficiency has been reported to reduce macrophage infiltration in the CNS in response to spinal nerve injury. The retina is an extension of the CNS and MCP-1/CCL2 is a potent chemoattractant and the major chemokine responsible for macrophage and microglial infiltration in the retina. The implication is that TLR2 deficiency may result in reduced macrophage and microglial infiltration to the retina in response to oxidative stress. A recent report has demonstrated that photoreceptor cell death in the NaIO3 model is correlative with activated macrophage accumulation in the outer retina following RPE degeneration. The extent to which absence of TLR2 might influence macrophage/microglial cell migration into the outer retina was next assessed in response to oxidative stress. IHC for Iba1 72 hours post NaIO3 was assessed. Iba1 stains both macrophages and microglia and by 72 hours large Iba1+ cells were found both in the ONL (
Anti-TLR2 neutralizing antibodies had preserved photoreceptor degeneration in the focal photo-oxidative stress induced model of retinal degeneration. It was next investigated whether pharmacological blockade of TLR2 signaling, through use of the same anti-TLR2 neutralizing antibody would rescue RPE fragmentation and photoreceptor degeneration in the NaIO3 model and could therefore broadly present TLR2 as a therapeutic target for oxidative stress induced retinal degeneration. WT mice were injected IV with NaIO3 or with vehicle NaCl and with sub-retinal anti-IgG or anti-TLR2 antibodies and retinal histology was assessed 72 hours after NaIO3. As expected, marked fragmentation of the RPE was observed in mice injected IV with NaIO3 and sub-retinal anti-IgG compared with mice injected IV with vehicle NaCl and sub-retinal anti-IgG (
With progressive age, increased oxidative damage occurs in many tissues, including the retina, and is thought to contribute to the progression of multiple forms of retinal degeneration most notably AMD. This is highlighted in a variety of experimental models where increased oxidative stress leads to a dry AMD-like pathology, including immunization with CEP, knockdown of SOD2 and NaIO3 injection. In addition to excessive oxidative stress an accumulation of complement factors in the retina and choroid is a pathological hallmark of AMD. It has been suggested that products of photo-oxidation of bis-retinoid lipofuscin pigments could serve to activate complement. However, the underlying mechanisms that trigger complement fixation in response to oxidative stress remain unknown. CEP has been shown to act as a ligand for TLR2 promoting angiogenesis in response to oxidative stress and indeed blocking TLR2 signaling in two mouse models of choroidal neovascularization (CNV) was recently shown to be efficacious in reducing CNV lesion size. This indicates that inhibitors of TLR2 have potential therapeutic utility for wet AMD.
It was chosen to study the effect of neutralizing TLR2 in two different experimental models of retinal degeneration. While both models utilized are oxidative stress induced models of retinal degeneration known to deposit complement and result in loss of photoreceptor cells, the major cell types effected in each model differ. In the photo-oxidative stress model, C3 is microglia/macrophage derived, deposited in the outer segments, and has been shown to contribute causally to photoreceptor loss. However, despite reports of C3 accumulation, no causative role for the AP had been implicated in retinal degeneration in the NaIO3 model. In order to determine whether AP activation was a driver of the pathology observed in the NaIO3 model or simply a bystander effect, an immunoprecipitating blocking antibody for Complement factor D (CFD) was used. CFD is a serine protease that cleaves CFB once bound to C3b, resulting in the assembly of the AP C3 convertase. An interesting observation relating to the use of anti-CFD in the NaIO3 model was the resulting protection of the RPE, with no significant loss of photoreceptor numbers. CFD binds to C3 only after it has bound CFB, at which point it cleaves CFB and enables amplification of the AP. For this reason, introduction of anti-CFD will block the amplification step of the AP, inhibiting MAC formation, but its inhibition of C3 cleavage into opsonizing fragments is less effective. With this in mind the anti-CFD data indicates that photoreceptors are sensitive to C3 deposition/opsonisation whereas the RPE may be more sensitive to the effects of amplifying the AP. Indeed, others have shown that complement regulators Cd55/Cd59 are reduced specifically in the photoreceptors in a model of retinal detachment, making photoreceptors especially sensitive to opsonization and complement-mediated death. By contrast, the fact that TLR2 deficiency protected both photoreceptor numbers and the RPE implies that, in response to oxidative stress, TLR2 signalling promotes both C3 opsonisation and the amplification of the AP. Indeed, CFB is exclusive to the AP and is the key rate limiting protein in AP activation. Simply increasing CFB expression can lead to the formation of the C3 convertase, activating the AP by cleaving C3. TLR2 activation consistently induced gene expression of both CFB and C3 to significant levels in all cell types tested, implying that TLR2 activation can universally activate the AP in vitro. Likewise, in vivo, we observed C3 opsonin fragment deposition in response to oxidative stress was lessened in the absence of TLR2. It is worth noting that, CFH functions to inhibit the amplification of the AP by competing with CFB for binding with C3. In this way variants in CFH that heighten risk for dry AMD and progression to GA are less efficient at preventing the amplification of the AP, again indicating a sensitivity of the RPE to the effects of the amplification of the AP. Amplification of the AP leads to terminal complement activation; whereupon its individual components C5b, C6, C7, C8, and C9 combine to form a lytic pore (C5b-9/MAC) on the surface of target cell membranes, capable of inducing cell lysis and inflammatory processes, as well as activating various cell signaling pathways. In the human retina, the MAC complex is identified in Bruch's membrane in eyes as young as 5 years of age. The presence of MAC increases with normal ageing, but it accumulates at higher levels in individuals with risk-associated AMD genotypes and has been identified in AMD patients within drusen in Bruch's membrane surrounding the choriocapillaris, and on RPE overlying drusen in vivo. The fact that the RPE is more intact in the absence of TLR2, despite being subjected to oxidative stress, implies that amplification of the AP has been inhibited due to the loss of TLR2 signalling. Indeed, the lack of active MAC formation in response to oxidative stress in the retina, in the absence of TLR2 was marked when compared to WT mice. Previous reports demonstrate that inhibition of TLR2 reduces C3 deposition in ischemia-reperfusion injury, these data demonstrate that TLR2 can also directly trigger the proteolytic complement cascade to completion with formation of the terminal complement complex, MAC.
MAC activation on choroidal endothelial cells induces lysis but studies describe how RPE cells are resistant to MAC mediated lysis and efficiently remove MAC before lysis can take place; instead, sub-lytic MAC induces inflammatory signaling pathway activation. In support of these reports, RPE cell death under TLR2 induced MAC-forming culture conditions were not observed in vitro, indicating that TLR2-induced MAC formation on RPE cells is sub-lytic. Sub-lytic MAC is characterized by secretion of MCP-1/CCL2, a key monocyte chemoattractant that also signals for monocyte differentiation into macrophages. In line with this characteristic, a synergistic effect on MCP-1/CCL2 secretion was observed under culture conditions where TLR2 induced MAC is formed, above the induction observed in response to CEP alone, suggesting that MAC formed on RPE cells in response to TLR2 activation is sub-lytic and has the potential to create an environment that attracts phagocytes to the outer retina. Interestingly, MCP-1 secretion from the RPE was highly polarized favoring a role for resident microglia activation in the neural retina. From a mechanistic stand point, support for MCP-1/CCL2 as a major factor in recruiting phagocytes in retinal degeneration comes from reports that genetic deletion of MCP-1/CCL2 prevents inflammatory monocyte recruitment, accumulation and photoreceptor degeneration in vivo in mouse models. The decreased Iba1+ staining and photoreceptor degeneration we observed in TLR2 deficient mice after treatment with NaIO3 supports the existence of a TLR2-driven chemokine gradient, attracting these cells to the outer retina and contributing to photoreceptor cell death, which may be a consequence of sub-lytic MAC signaling, although this remains to be definitively tested.
These data indicate that TLR2 mediates complement deposition in response to oxidative stress that is pathological in nature, and that blocking TLR2 signaling preserves both photoreceptor and RPE integrity in vivo under conditions of acute oxidative stress. However, the respective contributions of the different cells in the retina that can respond to TLR2 and their individual contributions to oxidative stress-induced TLR2 promotion of retinal degeneration requires further examination. It cannot yet be distinguished between relative contributions to pathology made by TLR2-activated RPE and TLR2-activated mononuclear phagocytes. The observation that neutralizing TLR2 in the photo-oxidative damage model of retinal degeneration, significantly reduced complement deposition and preserved photoreceptor cell layers, indicates that in addition to RPE-originating signals, blocking TLR2 signaling in the macrophage/microglia cells is also likely to contribute a significant aspect to the prevention of TLR2-mediated retinal degeneration. Furthermore, given these data, and supporting literature, that MAC formation on the RPE is sub-lytic, it remains to be understood how oxidative stress results in RPE fragmentation in vivo and following this why blocking TLR2 signaling in response to oxidative stress delays the RPE from this degeneration. Others have demonstrated that merely overexpressing C3 alone in vivo with C3-expressing adenovirus exhibited similarly significantly increased RPE death, in addition to loss of photoreceptor outer segments, and reactive gliosis. So it appears that, in vivo, unregulated complement activation results in an environment that promotes RPE death, be it as a result of experimental AAV-inducible C3 overexpression, or in our case oxidative damage-induced TLR2-mediated C3/MAC activation. Importantly, during the course of this study, it was also discovered TLR2 effects on the RPE that are independent of its role in inducing complement but undoubtedly contribute to RPE degeneration. Specifically, oxidative stress induced TLR2 signalling can also reduce tight junction expression, likely contributing to weakening the RPE and consequently the outer blood retinal barrier.
In conclusion, we show that TLR2 deficiency reduces complement activation, delays oxidative damage induced RPE fragmentation, delays migration of microglia/macrophages to the RPE and outer neural retina, and delays photoreceptor degeneration. These data contribute towards understanding the mechanisms underlying oxidative stress induced retinal degeneration and pinpoints TLR2 as a PRR bridging the detection of oxidative damage to activation of the complement response providing new targets for the prevention of oxidative stress induced pathology.
TLR2 heterodimerises with either TLR1 or TLR6 and recognizes diacyl and triacylated lipopeptides. TLR2 and TLR4 protect against infection in the anterior region of the eye. However, investigations into roles for TLRs in outer retinal disease are sparse, and mainly confined to genetic investigations, including several contradicting reports of associations between various SNPs in TLRs and risk of AMD. TLR signaling and the complement system have been linked in intestinal ischemia-reperfusion injury, where C3 deposition was markedly decreased in mice deficient in TLR4, and in a renal transplant ischemia-reperfusion injury model, where inhibition of TLR2 led to a decrease in C3 deposition. Furthermore, activation of TLR4 and TLR2 increases C1-13 expression in macrophages. TLR function has not been explored in outer retinal degenerative disease and RPE pathology. Here, it was sought to explore whether TLR2 might act as a bridge between effects of oxidative stress and complement activation in the retina. In doing so, it was discovered that TLR2 inhibition provides striking protection to the retina in response to oxidative stress. It is shown that oxidative stress activates TLR2 to trigger the proteolytic alternative pathway (AP) to completion with generation of the terminal complement complex, that forms sub-lytic MAC on the RPE and induces the pro-inflammatory chemokine MCP-1/CCL2. It is demonstrated that inhibition of TLR2 reduces complement activation, C3 opsonization and MAC deposition, ameliorates RPE fragmentation, prevents Iba1+ve macrophage/microglial cell infiltration to the outer retina, and preserves photoreceptor cells in response to acute oxidative stress. These data suggest that TLR2 signaling promotes an environment that drives a retinal degenerative phenotype, and presents TLR2 as a possible link between oxidative damage and excessive complement activation in retinal degenerative disease.
Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-Like Receptors (TLRs) are primary components of the innate immune system involved in signal transduction. The present invention shows that TLR2 induces complement factors C3 and CFB, the common and rate limiting factors of the Alternative Pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralisation of TLR2 reduces opsonising fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2 deficient retina. These data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.
This application claims benefit of priority of U.S. Provisional Patent Application No. 63/150,177 entitled, “A METHOD OF TREATING AGE-RELATED MACULAR DEGENERATION”, filed Feb. 17, 2021. The entire contents and disclosures of these patent applications are incorporated herein by reference in their entirety.
Number | Date | Country | |
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63150177 | Feb 2021 | US |