Compositions containing IL-36 and / or IL-18 for use in treating eye diseases

Abstract

The present invention relates to compositions comprising IL-36 and / or IL-18 for use in the treatment of eye diseases such as age-related macular degeneration (AMD), macular edema, retinopathy, diabetic retinopathy, and glaucoma. The present invention also relates to methods for identifying patients likely to respond to anti-VEGF agents.

Description

[Technical field]

[0001] The present invention relates to compositions comprising IL-36 and / or IL-18 for use in the treatment of eye diseases such as age-related macular degeneration (AMD), macular edema, retinopathy, diabetic retinopathy, and glaucoma. The present invention also relates to methods for identifying patients likely to respond to anti-VEGF agents. [Background technology]

[0002] Increased vascular and microvascular permeability is a major contributor to organ injury in disease, and strategies to modulate endothelial cell integrity are promising therapeutic targets. Blood vessels in the central nervous system possess unique barrier properties that prevent infiltration of unwanted substances and allow precise delivery of ions, molecules, and immune cells to neural tissues.

[0003] Barrier disruption is implicated in neurological and retinal conditions, as disease-causing neovasculature often has a barrier that is incapable of maintaining vasculature integrity, thereby permitting pathological leakage. Although many agents and disease processes induce vascular permeability, only a handful of factors have been specifically discovered that enhance barrier function and make blood vessels more resistant to leakage.

[0004] In the eye, endothelial cells lining the retinal and choroidal microvasculature function to maintain the barrier between blood and tissues, thus maintaining homeostasis. Loss of endothelial barrier integrity and "de novo" blood vessel growth, termed "angiogenesis", is almost exclusively associated with adult pathology. Diseases associated with vascular leakage and angiogenesis are manifold and include blinding conditions age-related macular degeneration (AMD), diabetic retinopathy, and retinopathy of prematurity. AMD is the leading cause of central vision loss in people over 50 years of age worldwide. In Ireland alone, more than 1 million out of a population of 4.6 million are over 50 years old and more than 70,000 people have AMD, making it already the most common cause of registered blindness in Ireland. Worldwide, it is estimated that between 30 and 50 million people have AMD. There are two end-stage stages of the disease, the most severe of which is neovascular or "wet" AMD. In this form of the disease, unwanted blood vessels sprout from the underlying choroidal vasculature, disrupting the integrity of the retina and causing immediate vision loss. Approximately 10-17% of patients develop wet AMD.

[0005] Wet AMD can be acutely treated with regular monthly intraocular injections of antibodies or fusion proteins against vascular endothelial growth factor (VEGF). Such therapies have revolutionized AMD treatment, stabilizing areas of choroidal neovascularization (CNV) and slowing further neovascularization. However, there is no final stage to the treatment, and patients require regular intraocular injections to maintain efficacy. Furthermore, a significant proportion of patients, estimated at approximately 30%, become resistant to treatment after 12 months, and an additional proportion do not respond to treatment at all. There is also evidence to suggest that long-term use of anti-VEGF agents may damage the retina.

[0006] Other mechanistic approaches that do not target VEGF but are combined with anti-VEGF therapeutics such as anti-PDGF aptamers (e.g., Ophthotech's Fovista®) have proven successful in previous clinical studies but have recently failed in Phase III trials for neovascular AMD. The use of anti-PDGF aptamers administered between anti-VEGF doses has been suggested to sensitize infiltrating choroidal neovascularization to anti-VEGF therapeutics, resulting in improved efficacy, demonstrating an effective interaction between two independent mechanisms. However, these trials have proven fruitless, and better combination therapies are sorely needed.

[0007] Anti-VEGF therapy has also been utilized in proliferative diabetic retinopathy and macular edema, but has been less successful in stabilizing vascular leakage and proliferation in these diseases.

[0008] The current "state of the art" anti-VEGF therapies (Roche / Genentech's Lucentis® and Regeneron's Eylea®) are like sponges that wipe away VEGF in the local microenvironment of the eye.

[0009] Furthermore, evidence suggests that long-term use of anti-VEGF agents may damage the retina due to the role of VEGF as a survival factor for neuronal cells, and this evidence is highlighted by the observation that patients receiving long-term anti-VEGF therapy may begin to degenerate the neural retina, resulting in further irreversible vision loss. Tailoring treatment to attenuate specific signaling cascades and reduce the side effects of anti-VEGF agents has great therapeutic potential. Because VEGFR1 promotes cell survival, whereas VEGF receptor-2 (VEGFR2) signaling appears to be primarily involved in promoting vascular permeability and angiogenesis, designing treatments to specifically block VEGFR2 signaling should be most effective.

[0010] Thus, there remains a clinical need for effective therapeutic approaches that target ocular diseases. Summary of the Invention

[0011] According to a first aspect of the present invention, there is provided a composition comprising IL-36 for use in the treatment or prevention of an eye disease.

[0012] According to a second aspect of the present invention, there is provided a method for the treatment or prevention of an eye disease, the method comprising the step of administering to a subject in need thereof a pharma- ceutical effective amount of a composition comprising IL-36.

[0013] According to a third aspect of the present invention there is provided the use of a pharma- ceutically effective amount of IL-36 in the manufacture of a medicament for use in the treatment or prevention of an eye disease.

[0014] According to a fourth aspect of the present invention there is provided a composition comprising IL-18 for use in the treatment or prevention of an eye disease.

[0015] According to a fifth aspect of the present invention, there is provided a method for the treatment or prevention of an eye disease, the method comprising the step of administering to a subject in need thereof a pharma- tically effective amount of a composition comprising IL-18.

[0016] According to a sixth aspect of the invention there is provided the use of a pharma- ceutically effective amount of IL-18 in the manufacture of a medicament for use in the treatment or prevention of an eye disease.

[0017] According to a seventh aspect of the present invention there is provided a composition comprising IL-36 and IL-18 for use in the treatment or prevention of an eye disease.

[0018] According to an eighth aspect of the present invention there is provided a method for the treatment or prevention of an eye disease, the method comprising the step of administering to a subject in need thereof a composition comprising pharma- ceutical effective amounts of IL-36 and IL-18.

[0019] According to a ninth aspect of the invention there is provided the use of pharma- ceutically effective amounts of IL-36 and IL-18 in the manufacture of a medicament for use in the treatment or prevention of an eye disease.

[0020] Optionally, the use comprises administering the composition to a subject in need thereof. Further optionally, the use comprises administering a composition comprising IL-36 to a subject in need thereof. Even further optionally, the use comprises administering a composition comprising IL-18 to a subject in need thereof.

[0021] Optionally, the use further comprises administering an anti-VEGF agent to a subject in need thereof. Even more optionally, the use comprises administering an anti-VEGF agent and a composition comprising IL-36 to a subject in need thereof. Even more optionally, the use comprises administering an anti-VEGF agent and a composition comprising IL-18 to a subject in need thereof. Even more optionally, the use comprises administering an anti-VEGF agent and a composition comprising IL-36 and IL-18 to a subject in need thereof.

[0022] Optionally, the method further comprises administering a pharma- ceutical effective amount of an anti-VEGF agent to a subject in need thereof. Even more optionally, the method comprises administering a pharma- ceutical effective amount of an anti-VEGF agent and a composition comprising IL-36 to a subject in need thereof. Even more optionally, the method comprises administering a pharma- ceutical effective amount of an anti-VEGF agent and a composition comprising IL-18 to a subject in need thereof. Even more optionally, the method comprises administering a pharma- ceutical effective amount of an anti-VEGF agent and a composition comprising IL-36 and IL-18 to a subject in need thereof.

[0023] Optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-VEGF agent and IL-36 is provided. Further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-VEGF agent and IL-18 is provided. Still further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-VEGF agent, IL-36 and IL-18 is provided.

[0024] Optionally, the anti-VEGF agent and the composition are administered simultaneously. Further optionally, the anti-VEGF agent and the composition comprising IL-36 are administered simultaneously. Still further optionally, the anti-VEGF agent and the composition comprising IL-18 are administered simultaneously. Still further optionally, the anti-VEGF agent, the composition comprising IL-36, and the composition comprising IL-18 are administered simultaneously.

[0025] Optionally, the anti-VEGF agent and the composition are administered sequentially. Further optionally, the anti-VEGF agent and the composition comprising IL-36 are administered sequentially. Still further optionally, the anti-VEGF agent and the composition comprising IL-18 are administered sequentially. Still further optionally, the anti-VEGF agent, the composition comprising IL-36 and the composition comprising IL-18 are administered sequentially.

[0026] Optionally, the anti-VEGF agent is administered prior to the composition. Further optionally, the anti-VEGF agent is administered prior to the composition comprising IL-36. Even further optionally, the anti-VEGF agent is administered prior to the composition comprising IL-18. Even further optionally, the anti-VEGF agent is administered prior to the composition comprising IL-36 and the composition comprising IL-18.

[0027] Optionally, the composition is administered prior to the anti-VEGF agent. Further optionally, the composition comprising IL-36 is administered prior to the anti-VEGF agent. Even further optionally, the composition comprising IL-18 is administered prior to the anti-VEGF agent. Even further optionally, the composition comprising IL-36 and the composition comprising IL-18 are administered prior to the anti-VEGF agent.

[0028] Optionally, the use further comprises administering an anti-IL-1 agent to a subject in need thereof. Still further optionally, the use comprises administering an anti-IL-1 agent and a composition comprising IL-36 to a subject in need thereof. Still further optionally, the use comprises administering an anti-IL-1 agent and a composition comprising IL-18 to a subject in need thereof. Still further optionally, the use comprises administering an anti-IL-1 agent and a composition comprising IL-36 and IL-18 to a subject in need thereof. Still further optionally, the use comprises administering an anti-IL-1 agent and an anti-VEGF agent to a subject in need thereof. Still further optionally, the use comprises administering an anti-IL-1 agent, an anti-VEGF agent and a composition comprising IL-36 to a subject in need thereof. Still further optionally, the use comprises administering an anti-IL-1 agent, an anti-VEGF agent and a composition comprising IL-18 to a subject in need thereof. Still further optionally, the use comprises administering to a subject in need thereof an anti-IL-1 agent, an anti-VEGF agent, and a composition comprising IL-36 and IL-18.

[0029] Optionally, the method further comprises administering a pharma- ceutical effective amount of an anti-IL-1 agent to a subject in need thereof. Still further optionally, the method comprises administering a pharma- ceutical effective amount of an anti-IL-1 agent and a composition comprising IL-36 to a subject in need thereof. Still further optionally, the method comprises administering a pharma- ceutical effective amount of an anti-IL-1 agent and a composition comprising IL-18 to a subject in need thereof. Still further optionally, the method comprises administering a pharma- ceutical effective amount of an anti-IL-1 agent and a composition comprising IL-36 and IL-18 to a subject in need thereof. Still further optionally, the use comprises administering a pharma- ceutical effective amount of an anti-IL-1 agent and an anti-VEGF agent to a subject in need thereof. Still further optionally, the method comprises administering a pharma- ceutical effective amount of an anti-IL-1 agent, an anti-VEGF agent, and a composition comprising IL-36 to a subject in need thereof. Still further optionally, the method includes administering to a subject in need thereof a pharma- ceutical effective amount of a composition comprising an anti-IL-1 agent, an anti-VEGF agent, and IL-18. Still further optionally, the method includes administering to a subject in need thereof a pharma- ceutical effective amount of an anti-IL-1 agent, an anti-VEGF agent, and a composition comprising IL-36 and IL-18.

[0030] Optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-IL-1 agent and IL-36 is provided. Further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-IL-1 agent and IL-18 is provided. Still further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-IL-1 agent, IL-36 and IL-18 is provided. Still further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-IL-1 agent and an anti-VEGF agent is provided. Still further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-IL-1 agent, an anti-VEGF agent, and IL-36 is provided. Still further optionally, in the manufacture of a medicament for use in treating or preventing an ocular disease, a pharmacologic effective amount of an anti-IL-1 agent, an anti-VEGF agent, and IL-18 is provided. Still further optionally, there is provided a pharma- ceutically effective amount of an anti-IL-1 agent, an anti-VEGF agent, IL-36 and IL-18 in the manufacture of a medicament for use in treating or preventing an ocular disease.

[0031] Optionally, the anti-VEGF agent and the anti-IL-1 agent are administered simultaneously. Optionally, the anti-VEGF agent and the anti-IL-1 agent are administered sequentially. Optionally, the anti-VEGF agent is administered prior to the anti-IL-1 agent. Optionally, the anti-IL-1 agent is administered prior to the anti-VEGF agent.

[0032] Optionally, the IL-1 is selected from IL-1α and IL-1β.

[0033] Optionally, the anti-IL-1 agent is selected from IL-1Ra or a fragment thereof, an anti-IL-1 antibody or a fragment thereof, an anti-IL-1 polyclonal antibody or a fragment thereof, an anti-IL-1 monoclonal antibody or a fragment thereof, an anti-IL-1 inhibitor, a small molecule anti-IL-1 inhibitor, a fusion protein comprising IL-1 or a fragment thereof, a recombinant fusion protein comprising IL-1 or a fragment thereof, a fusion protein comprising IL-1 receptor accessory protein (IL-1RAcP) or a fragment thereof, a recombinant fusion protein comprising IL-1 receptor accessory protein (IL-1RAcP) or a fragment thereof, and each combination thereof.

[0034] Optionally, the anti-IL-1 agent is selected from anakinra, canakinumab, rilonacept, and respective combinations thereof.

[0035] Optionally, the use is in compensatory proliferation, vascular maturation, mesenchymal activation, mesenchymal contraction, and / or wound healing. Further optionally, the use is in compensatory proliferation, vascular maturation, mesenchymal activation, mesenchymal contraction, and / or wound healing in ocular diseases. Still further optionally, the use is in wound healing. Further optionally, the use is in wound healing in ocular diseases.

[0036] Optionally, the method is a method of compensatory proliferation, vascular maturation, mesenchymal activation, mesenchymal contraction, and / or wound healing. Further optionally, the method is a method of compensatory proliferation, vascular maturation, mesenchymal activation, mesenchymal contraction, and / or wound healing in an ocular disease. Further optionally, the method is a method of wound healing. Further optionally, the method is a method of wound healing in an ocular disease.

[0037] Optionally, the use is in the manufacture of a medicament for use in compensatory proliferation, vascular maturation, mesenchymal activation, mesenchymal reduction, and / or wound healing. Further optionally, the use is in the manufacture of a medicament for use in compensatory proliferation, vascular maturation, mesenchymal activation, mesenchymal reduction, and / or wound healing in ocular diseases. Still further optionally, the use is in the manufacture of a medicament for use in wound healing. Still optionally, the use is in the manufacture of a medicament for use in wound healing in ocular diseases.

[0038] Optionally, the use is in microvascular remodeling. Still optionally, the use is in microvascular remodeling in ocular diseases.

[0039] Optionally, the use is in endothelial cell migration. Still optionally, the use is in endothelial cell migration in ocular disease.

[0040] Optionally, the use is in enhancing monolayer integrity. Still optionally, the use is in enhancing monolayer integrity in ocular disease.

[0041] Optionally, the use is in reducing vascular permeability. Still optionally, the use is in reducing vascular permeability in ocular disease.

[0042] Optionally, the use is in increasing expression of components of tight junctions and / or cadherin junctions. Still optionally, the use is in increasing expression of components of tight junctions and / or cadherin junctions in ocular diseases. Still further optionally, the use is in increasing expression of components of tight junctions and / or cadherin junctions selected from VE-cadherin, ZO-1, tricellulin, occludin, claudin-5, and respective combinations thereof. Still further optionally, the use is in increasing expression of components of tight junctions and / or cadherin junctions selected from VE-cadherin, ZO-1, tricellulin, occludin, claudin-5, and respective combinations thereof in ocular diseases.

[0043] Optionally, the use is in enhancing barrier function in the microvasculature of the CNS. Still optionally, the use is in enhancing barrier function in the microvasculature of the CNS in ocular diseases.

[0044] Optionally, the use is in stabilizing pathological vascular permeability. Still optionally, the use is in stabilizing pathological vascular permeability in an ocular disease.

[0045] Optionally, the use is in reducing microvascular leakage. Still optionally, the use is in reducing microvascular leakage in ocular diseases.

[0046] Optionally, the method is a method of microvascular remodeling. Further optionally, the method is a method of microvascular remodeling in ocular disease.

[0047] Optionally, the method is a method of endothelial cell migration. Further optionally, the method is a method of endothelial cell migration in an ocular disease.

[0048] Optionally, the method is a method of enhancing monolayer integrity. Further optionally, the method is a method of enhancing monolayer integrity in an ocular disease.

[0049] Optionally, the method is a method of reducing vascular permeability. Still optionally, the method is a method of reducing vascular permeability in an ocular disease.

[0050] Optionally, the method is a method of increasing expression of components of tight junctions and / or cadherin junctions. Still optionally, the method is a method of increasing expression of components of tight junctions and / or cadherin junctions in an ocular disease.

[0051] Optionally, the method is a method of enhancing barrier function in the microvasculature of the CNS. Still optionally, the method is a method of enhancing barrier function in the microvasculature of the CNS in an ocular disease.

[0052] Optionally, the method is a method of stabilizing pathological vascular permeability. Still optionally, the method is a method of stabilizing pathological vascular permeability in an ocular disease.

[0053] Optionally, the method is a method of reducing microvascular leakage. Further optionally, the method is a method of reducing microvascular leakage in an ocular disease.

[0054] Optionally, the use is in the manufacture of a medicament for use in microvascular remodeling.Optionally, the use is in the manufacture of a medicament for use in microvascular remodeling in ocular disease.

[0055] Optionally, the use is in the manufacture of a medicament for use in endothelial cell migration. Still optionally, the use is in the manufacture of a medicament for use in endothelial cell migration in ocular disease.

[0056] Optionally, the use is in the manufacture of a medicament for use in enhancing monolayer integrity. Still optionally, the use is in the manufacture of a medicament for use in enhancing monolayer integrity in ocular disease.

[0057] Optionally, the use is in the manufacture of a medicament for use in reducing vascular permeability. Still optionally, the use is in the manufacture of a medicament for use in reducing vascular permeability in ocular disease.

[0058] Optionally, the use is in the manufacture of a medicament for use in increasing expression of components of tight junctions and / or cadherin junctions, and further optionally, the use is in the manufacture of a medicament for use in increasing expression of components of tight junctions and / or cadherin junctions in ocular diseases.

[0059] Optionally, the use is in the manufacture of a medicament for use in enhancing barrier function in the microvasculature of the CNS. Still optionally, the use is in the manufacture of a medicament for use in enhancing barrier function in the microvasculature of the CNS in ocular disease.

[0060] Optionally, the use is in the manufacture of a medicament for use in stabilizing pathological vascular permeability. Still optionally, the use is in the manufacture of a medicament for use in stabilizing pathological vascular permeability in an ocular disease.

[0061] Optionally, the use is in the manufacture of a medicament for use in reducing microvascular leakage. Still optionally, the use is in the manufacture of a medicament for use in reducing microvascular leakage in ocular disease.

[0062] Optionally, the use or method comprises administration of cells expressing IL-36 and / or IL-18, and / or their respective receptors. Further optionally, the use or method comprises administration of stem cells expressing IL-36 and / or IL-18, and / or their respective receptors. Even further optionally, the use or method comprises administration of mesenchymal stem cells expressing IL-36 and / or IL-18, and / or their respective receptors.

[0063] Optionally, the use or method comprises administration of cells adapted to express IL-36 and / or IL-18, and / or their respective receptors. Further optionally, the use or method comprises administration of stem cells adapted to express IL-36 and / or IL-18, and / or their respective receptors. Even further optionally, the use or method comprises administration of mesenchymal stem cells adapted to express IL-36 and / or IL-18, and / or their respective receptors.

[0064] Optionally, the use or method comprises administration of cells adapted to overexpress IL-36 and / or IL-18, and / or their respective receptors. Further optionally, the use or method comprises administration of stem cells adapted to overexpress IL-36 and / or IL-18, and / or their respective receptors. Even further optionally, the use or method comprises administration of mesenchymal stem cells adapted to overexpress IL-36 and / or IL-18, and / or their respective receptors.

[0065] Optionally, the eye disease is a disease associated with or caused by edema and / or neovascularization.

[0066] Optionally, the eye disease is selected from any one or more of age-related macular degeneration (AMD), nascent geographic atrophy, incomplete retinal pigment epithelium and outer retinal atrophy (iRORA), wet AMD, geographic atrophy, dry AMD, macular edema, retinopathy, diabetic retinopathy, and glaucoma.

[0067] Optionally, the anti-VEGF agent is selected from an anti-VEGF antibody or fragment thereof, an anti-VEGF polyclonal antibody or fragment thereof, an anti-VEGF monoclonal antibody or fragment thereof, a VEGF inhibitor, a small molecule VEGF inhibitor, a tyrosine kinase inhibitor, a small molecule tyrosine kinase inhibitor, a fusion protein comprising VEGF or a fragment thereof, a recombinant fusion protein comprising VEGF or a fragment thereof, and each combination thereof.

[0068] Optionally, the anti-VEGF agent is selected from aflibercept, axitinib, bevacizumab, cabozantinib, lapatinib, lenvatinib, pazopanib, ponatinib, ramucirumab, ranibizumab, regorafenib, sorafenib, sunitinib, vandetanib, and each combination thereof.

[0069] Preferably, the anti-VEGF agent is selected from aflibercept, bevacizumab, ranibizumab, and respective combinations thereof.

[0070] Optionally, the use or method comprises administration by a route selected from topical, parenteral, intraarterial, intravenous, intraocular, intravitreal, and each combination thereof.

[0071] Preferably, the use or method comprises administration by a route selected from intraocular, intravitreal, and each combination thereof.

[0072] Optionally, the use or method comprises administration of the composition by a route selected from topical, parenteral, intra-arterial, intravenous, intraocular, intravitreal, and each combination thereof. Further optionally, the use or method comprises administration of a composition comprising IL-36 by a route selected from topical, parenteral, intra-arterial, intravenous, intraocular, intravitreal, and each combination thereof. Still further optionally, the use or method comprises administration of a composition comprising IL-18 by a route selected from topical, parenteral, intra-arterial, intravenous, intraocular, intravitreal, and each combination thereof.

[0073] Preferably, the use or method comprises administration of a composition by a route selected from intraocular, intravitreal, and respective combinations thereof.More preferably, the use or method comprises administration of a composition comprising IL-36 by a route selected from intraocular, intravitreal, and respective combinations thereof.Even more preferably, or in addition, the use or method comprises administration of a composition comprising IL-18 by a route selected from intraocular, intravitreal, and respective combinations thereof.

[0074] Optionally, the use or method comprises administration of the anti-VEGF agent by a route selected from topical, parenteral, intra-arterial, intravenous, intraocular, intravitreal, and each combination thereof.

[0075] Preferably, the use or method comprises administration of the anti-VEGF agent by a route selected from intraocular, intravitreal, and each combination thereof.

[0076] Optionally, the use or method comprises administering at a dose of at least 0.3 ng / ml. Even more optionally, the use or method comprises administering at a dose of at least 3 ng / ml. Even more optionally, the use or method comprises administering at a dose of at least 30 ng / ml.

[0077] Optionally, the use or method comprises administering at a unit dose of at least 0.3 ng. Even more optionally, the use or method comprises administering at a unit dose of at least 3 ng. Even more optionally, the use or method comprises administering at a unit dose of at least 30 ng.

[0078] Optionally, the use or method comprises administering at a unit dose of at least 0.3 ng / eye. Even more optionally, the use or method comprises administering at a unit dose of at least 3 ng / eye. Even more optionally, the use or method comprises administering at a unit dose of at least 30 ng / eye.

[0079] Optionally, the use or method includes administering the composition at a dose of at least 0.3 ng / ml. Even more optionally, the use or method includes administering the composition at a dose of at least 3 ng / ml. Even more optionally, the use or method includes administering the composition at a dose of at least 30 ng / ml.

[0080] Optionally, the use or method includes administering the composition in a unit dose of at least 0.3 ng. Even more optionally, the use or method includes administering the composition in a unit dose of at least 3 ng. Even more optionally, the use or method includes administering the composition in a unit dose of at least 30 ng.

[0081] Optionally, the use or method comprises administering the composition at a unit dose of at least 0.3 ng / eye. Even more optionally, the use or method comprises administering the composition at a unit dose of at least 3 ng / eye. Even more optionally, the use or method comprises administering the composition at a unit dose of at least 30 ng / eye.

[0082] Optionally, the use or method comprises administering a composition comprising IL-36 at a dose of at least 0.3 ng / ml. Even more optionally, the use or method comprises administering a composition comprising IL-36 at a dose of at least 3 ng / ml. Even more optionally, the use or method comprises administering a composition comprising IL-36 at a dose of at least 30 ng / ml.

[0083] Optionally, the use or method comprises administering a composition comprising IL-36 at a unit dose of at least 0.3 ng. Even more optionally, the use or method comprises administering a composition comprising IL-36 at a unit dose of at least 3 ng. Even more optionally, the use or method comprises administering a composition comprising IL-36 at a unit dose of at least 30 ng.

[0084] Optionally, the use or method comprises administering a composition comprising IL-36 at a unit dose of at least 0.3 ng / eye. Even more optionally, the use or method comprises administering a composition comprising IL-36 at a unit dose of at least 3 ng / eye. Even more optionally, the use or method comprises administering a composition comprising IL-36 at a unit dose of at least 30 ng / eye.

[0085] Optionally, the use or method comprises administering a composition comprising IL-18 at a dose of at least 0.3 ng / ml. Even more optionally, the use or method comprises administering a composition comprising IL-18 at a dose of at least 3 ng / ml. Even more optionally, the use or method comprises administering a composition comprising IL-18 at a dose of at least 30 ng / ml.

[0086] Optionally, the use or method comprises administering a composition comprising IL-18 at a unit dose of at least 0.3 ng. Even more optionally, the use or method comprises administering a composition comprising IL-18 at a unit dose of at least 3 ng. Even more optionally, the use or method comprises administering a composition comprising IL-18 at a unit dose of at least 30 ng.

[0087] Optionally, the use or method comprises administering a composition comprising IL-18 at a unit dose of at least 0.3 ng / eye. Even more optionally, the use or method comprises administering a composition comprising IL-18 at a unit dose of at least 3 ng / eye. Even more optionally, the use or method comprises administering a composition comprising IL-18 at a unit dose of at least 30 ng / eye.

[0088] Optionally, the medicament comprises a dose of at least 0.3 ng / ml. Even more optionally, the medicament comprises a dose of at least 3 ng / ml. Even more optionally, the medicament comprises a dose of at least 30 ng / ml.

[0089] Optionally, the medicament comprises a unit dose of at least 0.3 ng of the composition. Even more optionally, the medicament comprises a unit dose of at least 3 ng of the composition. Even more optionally, the medicament comprises a unit dose of at least 30 ng of the composition.

[0090] Optionally, the medicament comprises a composition comprising IL-36 at a unit dose of at least 0.3 ng. Further optionally, the medicament comprises a composition comprising IL-36 at a unit dose of at least 3 ng. Even further optionally, the medicament comprises a composition comprising IL-36 at a unit dose of at least 30 ng.

[0091] Optionally, the medicament comprises a composition comprising IL-18 in a unit dose of at least 0.3 ng. Further optionally, the medicament comprises a composition comprising IL-18 in a unit dose of at least 3 ng. Even further optionally, the medicament comprises a composition comprising IL-18 in a unit dose of at least 30 ng.

[0092] Optionally, the IL-36 is selected from IL36α, IL36β, or IL36γ.

[0093] Preferably, the IL-36 is selected from IL36β.

[0094] According to a further aspect of the invention there is provided a method for identifying a subject likely to respond to an anti-VEGF agent comprising the steps of: (a) providing or obtaining a biological sample from a subject; and (b) determining a quantitative or qualitative level of IL-18 in the biological sample; and (c) identifying subjects likely to respond to an anti-VEGF agent based on the quantitative or qualitative level of IL-18 in the biological sample.

[0095] Optionally, the method is an in vitro method. Further optionally, the method comprises: (a) providing a biological sample from a subject; (b) determining a quantitative or qualitative level of IL-18 in the biological sample; and (c) identifying a subject likely to respond to an anti-VEGF agent based on the quantitative or qualitative level of IL-18 in the biological sample.

[0096] Optionally, the determining step (b) includes comparing the quantitative or qualitative level of IL-18 in the biological sample with the quantitative or qualitative level of IL-18 in a normal sample.

[0097] Optionally, the normal sample is a biological sample from a subject not treated with an anti-VEGF agent. More optionally, the normal sample is a biological sample from a subject prior to treatment with an anti-VEGF agent. Still more optionally, the normal sample is a biological sample from a subject who is a responder to an anti-VEGF agent.

[0098] Optionally, the anti-VEGF agent is selected from an anti-VEGF antibody or fragment thereof, an anti-VEGF polyclonal antibody or fragment thereof, an anti-VEGF monoclonal antibody or fragment thereof, a VEGF inhibitor, a small molecule VEGF inhibitor, a tyrosine kinase inhibitor, a small molecule tyrosine kinase inhibitor, a fusion protein comprising VEGF or a fragment thereof, a recombinant fusion protein comprising VEGF or a fragment thereof, and each combination thereof.

[0099] Optionally, the anti-VEGF agent is selected from aflibercept, axitinib, bevacizumab, cabozantinib, lapatinib, lenvatinib, pazopanib, ponatinib, ramucirumab, ranibizumab, regorafenib, sorafenib, sunitinib, vandetanib, and each combination thereof.

[0100] Preferably, the anti-VEGF agent is selected from aflibercept, bevacizumab, ranibizumab, and respective combinations thereof.

[0101] Optionally, a quantitative or qualitative level of IL-18 that is higher than the quantitative or qualitative level of IL-18 in a normal sample is indicative of a responder.

[0102] Optionally, a quantitative or qualitative level of IL-18 that is lower than the quantitative or qualitative level of IL-18 in a normal sample is indicative of a non-responder.

[0103] Optionally, the biological sample is selected from whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, cerebrospinal fluid, and aqueous humor.

[0104] Preferably, the biological sample is aqueous humor.

[0105] The present invention will now be described with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0106] [Figure 1A] Isolectin staining in Z-stacks highlighting the vasculature of retinal flat mounts from 12-month-old C57BL / 6J wild-type and IL-36rn− / − mice (representative of N=2 wild-type and N=4 IL-36rn− / − mice), showing that overt IL-36 signaling leads to increased retinal vascular density. [Figure 1B] Showing that overt IL-36 signaling leads to increased retinal vascular density. Schematic of quantifiable measurements of retinal flat mounts using REAVER analysis, showing vessel length, branching points, and retinal labyrinthiness (distance required to go from point to point). [Figure 1C] Figure 2 shows that overt IL-36 signaling leads to increased retinal vascular density and vessel length. [Figure 1D] Overt IL-36 signaling leads to increased retinal vascular density. Number of branch points measured using the REAVER program on representative flat Z-stacks taken at 20x magnification of isolectin-stained retinal flat mounts from wild-type (WT) and IL-36rn- / - (IL-36RAKO). [Figure 1E] Figure 1 shows that overt IL-36 signaling leads to increased retinal vascular density. MFI of isolectin-stained retinal flatmount flattened Z-stack images taken at 20x magnification from WT or IL-36RAKO mice. [Figure 1F]Figure 1 shows that overt IL-36 signaling leads to increased retinal vascular density. Representative flattened Z-stacks of isolectin-stained retinal flatmounts from WT and IL-36RAKO mice were taken at 20x magnification and retinal labyrinthiness was measured using the REAVER program. All statistical analyses were performed using t-tests, p<0.05=*. [Figure 2A] FIG. 13 is a brain endothelial cell heatmap showing expression of actively translated IL-1 family receptors at baseline and in response to in vivo LPS challenge. [Figure 2B] Figure 1 shows expression and activation of IL-36 receptor in microvascular endothelial cells. Relative expression of IL-36R and IL-1RAcP in HRMEC, demonstrated by qPCR using β-actin as housekeeping gene and normalized to IL-18R expression, combined N=4, statistical analysis performed using t-test comparing treated groups with untreated controls, *=p<0.05, **=p<0.01***=p<0.001. [Figure 2C] IL-36 receptor expression and activation in microvascular endothelial cells. Localization of IL-36R in HRMEC, representative images of N=3 experiments (magnification 40x). [Figure 2D] Figure 1 shows IL-36 receptor expression and activation in microvascular endothelial cells. Western blot showing MAPK and NFκB activation in HRMEC in response to 60 ng / ml (D) IL-36α (E) IL-36β over a 90 minute time course, representative of N=3 experiments. [Figure 2E] Figure 1 shows IL-36 receptor expression and activation in microvascular endothelial cells. Western blot showing MAPK and NFκB activation in HRMEC in response to 60 ng / ml (D) IL-36α (E) IL-36β over a 90 minute time course, representative of N=3 experiments. [Figure 2F]Expression and activation of IL-36 receptor in microvascular endothelial cells. Network analysis of RNA-seq data from HRMEC treated with 100ng / ml IL-36β for 24 hours. The 60 most differentially expressed genes were compared with GO biological processes using ShinyGOv0.61 and pathway enrichment analysis was performed showing the 30 most significant terms. P-value cutoff (FDR) was set at 0.05. [Figure 3A] Representative phase contrast and analyzed network images of untreated, 100ng / ml IL-36α and 100ng / ml IL-36β treated HRMECs 4 hours after plating on Matrigel, showing that IL-36 cytokines affect endothelial cell tube formation and proliferation. Network analysis here was performed using the Angiogenesis Analyzer plugin in ImageJ, representative of N=5 experiments. [Figure 3B] Figure 2. IL-36 cytokine influences endothelial cell tube formation and proliferation. Analysis of tube number, nodule number, and tube length in HRMECs after 4 hours of growth in Matrigel with 100 ng / ml (B) IL-36α (C) IL-36β treatment and 50 ng / ml VEGF as a positive control. Values ​​are representative of N=5 experiments. [Figure 3C] Figure 2. IL-36 cytokine influences endothelial cell tube formation and proliferation. Analysis of tube number, nodule number, and tube length in HRMECs after 4 hours of growth in Matrigel with 100 ng / ml (B) IL-36α (C) IL-36β treatment and 50 ng / ml VEGF as a positive control. Values ​​are representative of N=5 experiments. [Figure 3D] Figure 2 shows that IL-36 cytokine influences endothelial cell tube formation and proliferation. Cell counts of DAPI-stained nuclei in HRMECs treated with 100 ng / ml IL-36α or IL-36β for 24 h, representing N=3 experiments. [Figure 3E]Figure 1 shows that IL-36 cytokines affect endothelial cell tube formation and proliferation. MTS / PMS assay showing increased proliferation with 24-hour treatment of IL-36α or IL-36β (+=50ng / ml,++=100ng / ml), 24-hour treatment with 50ng / ml VEGF was used as positive control, representative of N=3 experiments, statistics performed using one-way ANOVA with multiple comparisons. p<0.05=*, p<0.01=**, p<0.005=***, p<0.001=****. [Figure 4A] Showing that IL-36 cytokine promotes endothelial cell migration, (A,B) are representative images at 10x magnification of HRMEC "wound" healing with (A) IL-36α (B) IL-36β treatment compared to control, where the purple dotted line corresponds to the size of the wound imaged using Image J. [Figure 4B] Showing that IL-36 cytokine promotes endothelial cell migration, (A,B) are representative images at 10x magnification of HRMEC "wound" healing with (A) IL-36α (B) IL-36β treatment compared to control, where the purple dotted line corresponds to the size of the wound imaged using Image J. [Figure 4C] Figure 2 shows that IL-36 cytokine promotes endothelial cell migration. Wound healing rates in HRMEC treated with IL-36α are expressed as wound closure rates, representing N=4 experiments, and two-way ANOVA was performed as the statistical test. [Figure 4D] Figure 2 shows that IL-36 cytokine promotes endothelial cell migration. Comparison of wound healing levels between untreated (unt.) and 100ng / ml IL-36α at 4 and 24 hours after scratching, combined N=4 experiments. Two-way ANOVA with multiple comparisons was performed as the statistical test. [Figure 4E] Figure 2 shows that IL-36 cytokine promotes endothelial cell migration. Wound healing rates in HRMEC treated with IL-36β are expressed as % wound closure, representing N=4 experiments, and two-way ANOVA with multiple comparisons was performed as the statistical test. [Figure 4F]Figure 2 shows that IL-36 cytokine promotes endothelial cell migration. Comparison of wound healing levels between no treatment and 100ng / ml IL-36β at 4 and 24 hours after scratching, N=4 experiments pooled, two-way ANOVA with multiple comparisons was performed as statistical test. [Figure 5A] Figure 1 shows that IL-36 cytokines decrease the permeability of endothelial cell monolayers. Schematic of FITC flux experiments: (1) HRMECs are seeded to form monolayers on semi-permeable transwells, (2) cytokines are treated in the apical and basal chambers, (3) a known concentration of FITC-dextran solution is substituted for the medium in the apical chamber, and (4) the medium from the basal chamber is periodically sampled to measure the FITC flux rate. [Figure 5B] Figure 2 shows that IL-36 cytokines decrease the permeability of endothelial cell monolayers. TEER measurements of HRMEC transwell monolayers after treatment with 100 ng / ml IL-36α or IL-36β for 6 hours. TEER of experimental wells was subtracted from that of blank wells, and blank TEER measurements after treatment were normalized to blank TEER measurements of the same wells before treatment. One-way ANOVA with multiple comparisons was performed as the statistical measure. [Figure 5C] IL-36 cytokine reduces the permeability of endothelial cell monolayers. Flux rate of 4 kDa FITC dextran through HRMEC transwell monolayers pretreated with 100 ng / ml IL-36α cytokine for 6 and 24 hours, with treatment applied to the apical and basal chambers. [Figure 5D] Figure 2 shows that IL-36 cytokine reduces the permeability of endothelial cell monolayers. Permeability of HRMEC treated with 100ng / ml IL-36α was calculated as Papp(cm / s)=(dQ / dT) / (AxC0). T-test was performed as a statistical measure. [Figure 5E]IL-36 cytokine reduces the permeability of endothelial cell monolayers. Flux rate of 4 kDa FITC dextran through HRMEC transwell monolayers pretreated with 100 ng / ml IL-36β cytokine for 6 and 24 hours, with treatment applied to the apical and basal chambers. [Figure 5F] IL-36 cytokine reduces the permeability of endothelial cell monolayers. Permeability of HRMEC treated with 100 ng / ml IL-36β. Calculated as above with T-test performed for statistical determination. [Figure 5G] Figure 2 shows that IL-36 cytokine reduces the permeability of endothelial cell monolayers. Box plot of VE-cadherin2 and cldn5 expression in HRMEC after treatment with 100 ng / ml IL-36β, with gene counts analyzed using iDEP to identify differential expression and highlighted as differentially expressed genes. [Figure 5H] Figure 1 shows that IL-36 cytokine reduces the permeability of endothelial cell monolayers. qPCR of cldn5, tricellulin, and ZO-1 expression performed with RNA extracted from HRMECs treated with 100 ng / ml IL-36β or 10 μM RepSox for 24 h, normalized to untreated controls, and β-actin was used as a housekeeping gene. Here, two-way ANOVA was performed as the statistical test. [Figure 5I] Figure 1 shows that IL-36 cytokines reduce the permeability of endothelial cell monolayers. qPCR for VE-cadherin mRNA expression in HRMEC in response to 100ng / ml IL-36α or IL-36β treatment over a 24 hour time course, normalized to 0 hours with β-actin used as housekeeping gene. One-way ANOVA was used as the statistical measure here and all data represent N=3 experiments, except G, which represents N=2 samples. [Figure 6A]Figure 2 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components of endothelial cells, where (A,B) represent Western blots of VE-cadherin protein expression in response to 24-hour treatment with the indicated doses of (A) IL-36α, (B) IL-36β in HRMEC, representative N=4 experiments. [Figure 6B] Figure 2 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components of endothelial cells, where (A,B) represent Western blots of VE-cadherin protein expression in response to 24-hour treatment with the indicated doses of (A) IL-36α, (B) IL-36β in HRMEC, representative N=4 experiments. [Figure 6C] Figure 2 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components in endothelial cells. Western blot of expression of tight junction proteins ZO-1, tricellin, and occludin upon 24-h treatment with the indicated concentrations of (C) IL-36α and (D) IL-36β in HRMEC. [Figure 6D] Figure 2 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components in endothelial cells. Western blot of expression of tight junction proteins ZO-1, tricellin, and occludin upon 24-h treatment with the indicated concentrations of (C) IL-36α and (D) IL-36β in HRMEC. [Figure 6E] Figure 1. IL-36 cytokine alters protein levels of adhesion and tight junction components in endothelial cells. Flux rate of 70 kDa FITC-dextran through HRMEC transwell monolayers pretreated for 6 h with 100 ng / ml IL-36α and / or 10 μM cycloheximide (CHX), treatments were applied to the apical and basal chambers. [Figure 6F] Figure 1 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components of endothelial cells. Permeability of HRMEC treated with IL-36α and / or 10 μM CHX, calculated as previously described. [Figure 6G] Figure 1 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components of endothelial cells. Flow rate of 70 kDa FITC dextran through HRMEC transwell monolayers pretreated for 6 h with 100 ng / ml IL-36β and / or 10 μM CHX, treatments were applied to the apical and basal chambers, and represents N=3 experiments. [Figure 6H] Figure 1 shows that IL-36 cytokine alters protein levels of adhesion and tight junction components of endothelial cells. Apparent permeability of HRMEC treated with IL-36β and / or CHX was calculated as described above, and T-test was performed as statistical analysis. [Figure 7A] We show that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Dynamic FA profiles acquired 5 days after laser-induced CNV and treatment with IL-36β (0.3 and 3 ng) or vehicle control (0 ng) in C57BL / 6J mice, normalized to signal intensity quantified in the whole image (A), at the site of CNV (B), within blood vessels outside the area affected by CNV (C), or in microvessels (D). [Figure 7B] We show that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Dynamic FA profiles acquired 5 days after laser-induced CNV and treatment with IL-36β (0.3 and 3 ng) or vehicle control (0 ng) in C57BL / 6J mice, normalized to signal intensity quantified in the whole image (A), at the site of CNV (B), within blood vessels outside the area affected by CNV (C), or in microvessels (D). [Figure 7C]We show that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Dynamic FA profiles acquired 5 days after laser-induced CNV and treatment with IL-36β (0.3 and 3 ng) or vehicle control (0 ng) in C57BL / 6J mice, normalized to signal intensity quantified in the whole image (A), at the site of CNV (B), within blood vessels outside the area affected by CNV (C), or in microvessels (D). [Figure 7D] We show that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Dynamic FA profiles acquired 5 days after laser-induced CNV and treatment with IL-36β (0.3 and 3 ng) or vehicle control (0 ng) in C57BL / 6J mice, normalized to signal intensity quantified in the whole image (A), at the site of CNV (B), within blood vessels outside the area affected by CNV (C), or in microvessels (D). [Figure 7E] Figure 1 shows that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Representative FA angiograms at 2.5, 4.5, 6.5, and 8.5 min after fluorescein injection of 0, 0.3, and 3 ng IL-36β with different regions of interest highlighted. [Figure 7F] Figure 1 shows that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Volumetric analysis of CNV lesions based on GS-IB4 isolectin 6 days after laser-induced CNV administration and IL-36β treatment (n=15 for vehicle and 0.3ng IL-36β groups, n=17 for 3ng IL-36β group). Volumetric analysis was performed blinded. Each CNV lesion was assessed before inclusion in the volumetric analysis and all hemorrhages were excluded. Statistics were determined using ANOVA with multiple comparisons. [Figure 7G] Figure 1 shows that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. Representative 3D renderings of GS-IB4 isolectin-based CNV volumes for 0, 0.3, and 3 ng IL-36β. [Figure 7H] Figure 2 shows that IL-36β reduces BRB permeability without affecting neovascular lesion size in vivo. TUNEL staining of paraffin sections taken from C57BL / 6J mice 3 days after intravitreal injection of PBS or 30 ng IL-36β. [Figure 8] Showing that VEGF regulates IL-36R expression signaling, HRMECs were treated with VEGF for the indicated time points and cells were prepared for qPCR, where the expression levels of IL-36R were measured by the ddCT method and data mean ± SD represent n = 3 independent experiments. [Figure 9] Showing that IL-36 cytokine regulates VEGFR2 expression, HRMECs were treated with either IL-36α or IL-36β for the indicated time points and cells were prepared for qPCR, where the expression levels of VEGFR2 were measured by the ddCT method and data mean ± SD represent n = 3 independent experiments. [Figure 10] Figure 1: IL-18 levels in anti-VEGF-treated wet AMD patients increased significantly over 12 weeks. IL-18 levels were measured in aqueous humor of 33 AMD cases (BL, W6, W12) and 13 cataract controls at 6-week intervals. IL-18 levels at baseline and week 6 were significantly lower than cataract controls (A), but there was a significant change in soluble IL-18 from baseline to week 12 and from week 6 to week 12 (B). Data were analyzed using Kruskal-Wallis test and Dunn's multiple comparison test, and all data are presented using mean ± SEM (*p=<0.05). [Figure 11]AMD patients who respond to anti-VEGF therapy show a significant increase in IL-18 in aqueous humor over 12 weeks. IL-18 was measured in samples taken over the course of anti-VEGF treatment, and participants were grouped based on response. In participants who had improved BCVA (A), OCT (B), and objective impression, IL-18 was found to increase significantly between baseline and week 12, and also between weeks 6 and 12 (C). Only patients who did not respond based on OCT measurements showed a significant change in IL-18 levels during treatment. Data are analyzed by two-way ANOVA with Tukey's multiple comparison test. All data are presented using mean ± SEM (*p=<0.05, **p=<0.01, ns=not significant). [Figure 12A] Correlation between overall change in OCT from pre- to post-treatment is shown. Correlation analysis of change in CMT (ΔOCT) and IL-18 levels at baseline (n=11) (A), week 6 (n=11) (B), and week 12 (n=21) (C) in the clinical neovascular cohort. Data were analyzed using graphs showing "r" coefficients and Spearman correlation. [Figure 12B] Correlation between overall change in OCT from pre- to post-treatment is shown. Correlation analysis of change in CMT (ΔOCT) and IL-18 levels at baseline (n=11) (A), week 6 (n=11) (B), and week 12 (n=21) (C) in the clinical neovascular cohort. Data were analyzed using graphs showing "r" coefficients and Spearman correlation. [Figure 12C] Correlation between overall change in OCT from pre- to post-treatment is shown. Correlation analysis of change in CMT (ΔOCT) and IL-18 levels at baseline (n=11) (A), week 6 (n=11) (B), and week 12 (n=21) (C) in the clinical neovascular cohort. Data were analyzed using graphs showing "r" coefficients and Spearman correlation. [Figure 13A]Figure 1 shows a correlation between plasma IL-18 and reduction in retinal edema. Participants were grouped based on functional outcome measures after treatment and found no difference in baseline IL-18 levels between participants who improved or worsened based on BCVA (A). However, patients who improved in OCT measurements after treatment had significantly higher plasma IL-18 at baseline (B). Data were analyzed using Mann-Whitney t-tests and Spearman rank correlation between groups and data are presented using mean ± SEM (*p<0.005, **p=<0.001, ns=not significant). [Figure 13B] Figure 1 shows a correlation between plasma IL-18 and reduction in retinal edema. Participants were grouped based on functional outcome measures after treatment and found no difference in baseline IL-18 levels between participants who improved or worsened based on BCVA (A). However, patients who improved in OCT measurements after treatment had significantly higher plasma IL-18 at baseline (B). Data were analyzed using Mann-Whitney t-tests and Spearman rank correlation between groups and data are presented using mean ± SEM (*p<0.005, **p=<0.001, ns=not significant). [Figure 13C] Figure 1 shows the correlation between plasma IL-18 and reduction in retinal edema. Correlation analysis found no significant association between baseline IL-18 levels and overall change in BCVA (C), but higher baseline IL-18 levels correlated with a greater reduction in retinal thickness during treatment (P=0.01) (D). Data were analyzed using Mann-Whitney t-tests and Spearman rank correlation between groups, and data are presented using mean ± SEM (*p<0.005, **p=<0.001, ns=not significant). [Figure 13D]Figure 1 shows the correlation between plasma IL-18 and reduction in retinal edema. Correlation analysis found no significant association between baseline IL-18 levels and overall change in BCVA (C), but higher baseline IL-18 levels correlated with a greater reduction in retinal thickness during treatment (P=0.01) (D). Data were analyzed using Mann-Whitney t-tests and Spearman rank correlation between groups, and data are presented using mean ± SEM (*p<0.005, **p=<0.001, ns=not significant). [Figure 14A] We show that IL-18 can inhibit VEGF secretion in vivo and in vitro. Three-week-old JR5558 mice (A) were injected (i.p.) with saline (0 μg / kg) or IL-18 (100 μg / kg) for four consecutive days (days 1–4). On day 8, retinas were isolated and VEGF concentrations were measured in protein lysates using the appropriate ELISA. Data are presented as mean ± SEM (n = 4–5 animals per each treatment group) and expressed as pg of VEGF per mg of total retinal protein measured by BCA assay (*p < 0.05 by one-way ANOVA with Tukey post-test compared to saline control). [Figure 14B] We show that IL-18 can inhibit VEGF secretion in vivo and in vitro. Primary human fetal RPE (hfRPE) cells were treated with control medium (0 ng / ml) or a single dose of IL-18 (1000 ng / ml) for 1–72 h. VEGF concentrations in cell-free supernatants were measured at each time point. VEGF secretion rates were calculated from the slope of the linear regression of VEGF concentration (pg / ml) against time (hours) (C). Data are presented as mean ± SEM and represent a single experiment performed in triplicate (*p<0.05, **p<0.001 by Student's t-test compared to control at each time point). [Figure 14C]We show that IL-18 can inhibit VEGF secretion in vivo and in vitro. Primary human fetal RPE (hfRPE) cells were treated with control medium (0 ng / ml) or a single dose of IL-18 (1000 ng / ml) for 1–72 h. VEGF concentrations in cell-free supernatants were measured at each time point. VEGF secretion rates were calculated from the slope of the linear regression of VEGF concentration (pg / ml) against time (hours) (C). Data are presented as mean ± SEM and represent a single experiment performed in triplicate (*p<0.05, **p<0.001 by Student's t-test compared to control at each time point). [Figure 15] Figure 1 shows that IL-18 specifically reduces the expression of VEGFR2 in human retinal endothelial cells (HRMEC). HRMEC were treated with IL-18 (100 ng / ml) for the indicated time points. Cell lysates were prepared for RNA isolation and subjected to qPCR and ddCt methods to assess the relative expression of VEGFR levels using B-actin as a reference control. RT-PCR data are the mean ± SEM of n=3 independent experiments. [Figure 16A] Figure 1 shows HRMEC migration in response to IL-18 and IL-1α stimulation. Confluent HRMEC monolayers were scratched with a pipette tip and treated with IL-18 (50 ng / ml) or IL-1α (10 ng / ml) along with bFGF (50 ng / ml) or VEGF (50 ng / ml) as positive controls. Images were taken immediately after scratching (0 h) and 4, 8, and 24 h after scratching. Images were analyzed using ImageJ imaging software. Data are presented as the mean ± SEM of six separate experiments in triplicate. Data were analyzed using 2-way ANOVA with Dunnett's multiple comparison test (*p<0.05, *p<0.01, ****p<0.0001). [Figure 16B]Figure 1 shows HRMEC migration in response to IL-18 and IL-1α stimulation. Confluent HRMEC monolayers were scratched with a pipette tip and treated with IL-18 (50 ng / ml) or IL-1α (10 ng / ml) along with bFGF (50 ng / ml) or VEGF (50 ng / ml) as positive controls. Images were taken immediately after scratching (0 h) and 4, 8, and 24 h after scratching. Images were analyzed using ImageJ imaging software. Data are presented as the mean ± SEM of six separate experiments in triplicate. Data were analyzed using 2-way ANOVA with Dunnett's multiple comparison test (*p<0.05, *p<0.01, ****p<0.0001). [Figure 17] Figure 1 shows that HRMEC proliferation in response to VEGF is enhanced by IL-18 pretreatment. HRMEC were treated with increasing concentrations of VEGF alone or with 10, 100, or 1000 ng / ml IL-18 pretreatment for 24 hours. Increased VEGF induced increased cell proliferation, which was enhanced after IL-18 treatment. [Figure 18] We show that IL-18 promotes expression of the tight junction protein occludin and inhibits VEGF depletion of occlusion. HRMECS were treated with VEGF, IL-18, or IL-1a for the indicated time points (upper panels), and cells were prepared for RNA isolation and qPCR to measure occlusion expression. HRMECs were treated with VEGF, IL-18, IL-1, or a combination of these cytokines, and cells were prepared for immunoblotting to examine occludin and b-actin expression (lower panels). [Figure 19]We show that IL-18 induces migration of fibroblast-like cells to close wounds created in human retinal pigment epithelial monolayers in vitro. Primary human fetal RPE (hfRPE) cells were treated with control medium (0 ng / ml) or increasing doses of IL-18 (10-1000 ng / ml) immediately after wounding in the center of the well. Microscopic images were acquired at the same location at baseline (0 h) and then every 2 h up to 18 h, and representative images from each treatment group are shown. Cells were stained with DAPI to show nuclei and phalloidin to show F-actin, and brightfield images (phase) are also shown. [Figure 20A] We show that IL-18 activates human choroidal fibroblasts and inhibits VEGF secretion. Human choroidal fibroblasts (HOCF) were treated with increasing doses of IL-18 for 8 hours. Cells were fixed and stained with antibodies targeting the intermediate filaments vimentin (red), Hoechst (blue), and phalloidin (green). [Figure 20B] Figure 1 shows that IL-18 activates human ocular choroidal fibroblasts and inhibits VEGF secretion. HOCF treated with IL-18 (100 ng / ml) for 60 min and immunoblotted for phospho-ERK and phospho-p65, with B-actin and GAPDH as controls. [Figure 20C] We show that IL-18 activates human ocular choroidal fibroblasts and inhibits VEGF secretion. As shown, HOCF were treated with IL-18 (100 ng / ml) for 24 hours. Cellular VEGF expression was analyzed by qPCR. [Figure 20D] We show that IL-18 activates human ocular choroidal fibroblasts and inhibits VEGF secretion.HOCF were treated with 10, 100, or 1000 ng / ml IL-18 for 48 h, and cell death was analyzed by LDH assay. [Figure 21]We show that IL-18 activates bone marrow-derived mesenchymal stem cells. Bone marrow-derived MSCs were treated with 250 ng / ml IL-18 for 8 hours and imaged using confocal microscopy. Cells were stained with phalloidin for F-actin, vimentin, smooth muscle actin (SMA), and collagen 1. [Figure 22] Figure 1 shows increased mesenchymal cell infiltration / activation in animals injected intraperitoneally with IL-18. WT mice underwent laser-induced CNV and were injected IP with IL-18 (100ug / kg). Three days later, eyes were enucleated and flat mounts were stained with F4 / 80, vimentin, and isolectin and imaged by confocal microscopy. [Figure 23] Figure 1. Increased mesenchymal cell infiltration / activation in animals injected intravitreally with IL-18. WT mice underwent laser-induced CNV and were injected intravitreally with IL-18 (0.3 ng / eye). Three days later, eyes were enucleated and flat mounts were stained with F4 / 80, vimentin, and isolectin and imaged by confocal microscopy. [Figure 24] We show that IL-18 reduces the area of ​​fibrovascular scar. WT mice underwent laser-induced CNV and were injected intravitreally with IL-18 (0.3 ng / eye). 14 days later, eyes were enucleated and flat mounts were stained with F4 / 80, vimentin, and isolectin and imaged by confocal microscopy. [Diagram 25] This shows that IL-18 deficiency increases the area of ​​fibrovascular scar. WT and IL-18KO mice were subjected to laser-induced CNV. After 14 days, eyes were enucleated and flat mounts were stained with F4 / 80, vimentin, and isolectin and imaged by confocal microscopy. EXAMPLES

[0107] Materials and Methods Study design The goal of this study was to determine the role of IL-36 in angiogenic processes and vascular permeability in vivo and in vitro. We used FFA to measure permeability in vivo after laser injury and were blinded to treatment conditions during outcome evaluation. As our primary interest was how IL-36 acts in the CNS, specifically the iBRB, where human retinal microvascular endothelial cells are a major component in vivo, we used these cells to model the effects of IL-36 on angiogenic processes and barrier integrity in vivo. The number of experiments, biological replicates, and sample size for each experiment are outlined in the figure legends.

[0108] Retinal flat mount analysis Eyes were enucleated from 12-month-old wild-type C57BL / 6J and IL-36rn- / - mice (received from M. Kopf-Zurich (Tortola L, Rosenwald E, Abel B, Blumberg H, Schafer M, Coyle AJ, Renauld JC, Werner S, Kisielow J & Kopf M (2012) Psoriasiform dermatitis is driven by DC-keratinocyte crosstalk. J Clin Invest 122: 3965-3976) and fixed in 4% PFA for 15 min. After that, the sclera and lens were removed and four radial cuts were made in the eyecups. The eyecups were incubated overnight at 4°C with Griffonia-simplicifolia-isolectin-Alexa488 (ThermoFisher) (1:500) and then mounted. Leica Z-stack images were captured using an SP8 Scanning confocal instrument to image the retinal flat mounts. These images were flattened and analyzed using the REAVER program. Furthermore, the MFI of the flattened Z-stack images was quantified using Image J.

[0109] cell culture Primary human retinal microvascular endothelial cells (HRMECs) (Innoprot) were seeded on tissue flasks coated with fibronectin (Sigma). HRMECs were cultured in endothelial cell basal medium (PromoCell) (hereafter known as growth medium) containing 5% FCS, growth factor supplements and 1% Pen-strep (Sigma). When experimental procedures required the removal of serum from cells, medium without growth factors and containing 0.5% FCS was used. Cultures were incubated at 37°C, 5% CO2, and 95% relative humidity. All cells used for experiments were from passages 4 to 8. For experiments, cells were treated with various concentrations of recombinant human (rh) IL-36α, IL-36β, and VEGF (all from Peprotech).

[0110] Real-time qPCR Total RNA was extracted from HRMECs using the EZNA kit (Omega) and reverse transcribed using M-MLV transcriptase (Promega) according to the manufacturer's protocol. This cDNA, together with the housekeeping gene β-actin, was used as a template for target gene amplification by real-time PCR using SensiFAST SYBR Green (Bioline) to determine the relative abundance of the genes of interest. Relative gene expression was calculated using the 2-ΔΔCT method. Primers used were obtained from Sigma.

[0111] RNA sequencing HRMECs were treated with IL-36β for 24 h and RNA was extracted after treatment as described above. mRNA enrichment was performed on 100 ng of total RNA using the NEBNextPoly(A) mRNA magnetic isolation module. Libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina and sequenced using the NextSeq500 output kit v2.5 (Illumina) with the Illumina NextSeq500 platform. Sequence analysis was performed on MacOS using Python 3.8.5, and all steps were performed in paired-end mode, where applicable. Adapter trimming and quality control were performed using Cutadapt3.1 with a minimum post-trimming length of 21 nucleotides and TruSeq adapter sequences. Alignments were performed with HiSAT2 version 2.2.1 using an index constructed from the Gencode version 37 of the human genome (GRCh38.p13). Aligned reads were sorted and indexed using Samtools version 1.10. For differential gene expression analysis, Wald tests were performed with featureCounts version 2.0.1. For further downstream analysis, PCA plots were generated using the iDEP9.2 online tool, and hierarchical clustering and gene expression pathway analysis were performed. Alignments were visualized using the Integrative Genomics Viewer (IGV).

[0112] immunocytochemistry HRMECs were grown to confluence in chamber slides. They were washed with PBS, fixed with ice-cold methanol, and blocked / permeabilized for 30 min in 5% NGS / 0.05% Triton X-100 prepared in PBS. Primary antibodies (IL-36R Novus Biologicals, VE-cadherin Santa Cruz, IL-1RAcP Invitrogen) were applied overnight and incubated at 40°C. The cells were then washed with PBS before being incubated in secondary antibodies for 2 h at room temperature. Following this incubation, the slides were washed with PBS and stained with Hoechst for 2 min before being washed again, the chambers removed, and a coverslip mounted. The slides were then imaged at 40x using a Zeiss confocal microscope.

[0113] Western blotting Proteins were extracted from cells using RIPA buffer containing protease inhibitors (Sigma). Sample buffer was added to the protein samples and denatured at 100°C for 10 min. Samples were then separated by SDS-PAGE and transferred to PVDF membranes (Sigma). Membranes were blocked with 5% skim milk (Marvel) in TBST. Membranes were incubated overnight at 4°C with primary antibodies diluted in 5% skim milk in TBST. Anti-rabbit or anti-mouse secondary antibodies (Sigma) were applied to the membranes as appropriate and incubated for 1 h at room temperature. Blots were then developed using Western Bright ECL (Advansta). Densitometry of the blots was performed using Image J software.

[0114] Proliferation assay HRMEC were seeded on coverslips in 6-well plates at a density of 5x104 cells / ml and grown overnight in growth medium. They were then washed with PBS, placed in reduced serum medium, and incubated with treatment for 24 hours. Coverslips were then washed with PBS, fixed with ice-cold methanol, and stained with Hoechst (1:10000). Coverslips were then mounted on slides and imaged via fluorescence. N=10 images were taken per slip and averaged to calculate the number of cells per coverslip. For the MTS / PMS assay (Promega), cells were plated on 96-well plates at 5x10 4 Cells were seeded at a density of 1000 cells / ml. They were grown overnight in complete growth medium, washed, and placed in low serum medium with the indicated treatments for 24 h before performing the MTS / PMS assay according to the manufacturer's protocol.

[0115] Tube formation assay Cells were seeded into T75 in full growth medium 2 days prior to the experiment and left to reach approximately 80% confluence. They were placed in reduced serum medium overnight before use in experiments. 96-well plates were coated with 60 μl of Matrigel basement membrane matrix (Corning) and placed in a 37°C incubator for 30 minutes to set according to the manufacturer's instructions. HRMECs were then plated at 8 × 10 4 Cells were seeded onto plates at a density of 1000 cells / ml and treated with cytokines in triplicate as indicated. Plates were placed in an Incucyte and phase contrast images of the wells were taken at 10x magnification at 30 minute intervals. Images were analyzed using the Angiogenesis Analyzer plugin in ImageJ.

[0116] Permeability assay HRMEC were grown and polarized on transwells (Corning) for 5–7 days. Cells were placed in low serum medium and treated with IL-36 cytokine for 6 or 24 h, after which 4 kDa FITC dextran (Sigma) diluted to 1 mg / ml in low serum medium was placed in the apical chamber. Sampling aliquots were periodically removed from the basolateral chamber and placed in a 96-well plate. The basolateral chamber was then replenished with fresh medium. This was repeated at 20 min intervals for 2 h. FITC dextran fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a fluorescence plate reader (BioTek). Relative fluorescence units (RFU) were converted to ng / ml using a FITC dextran standard curve and corrected for background fluorescence and serial dilutions over the course of the experiment. The apparent permeability coefficient (Papp) was calculated as follows:

number

[0117] Transendothelial Electrical Resistance (TEER) Measurement HRMECs were grown in transwells as previously described. They were treated with IL-36 cytokine in both the apical and basal chambers as shown. The TEER of each transwell was measured using an EVOM2 epithelial voltmeter (World Precision Instruments) equipped with an STX2 chopstick electrode. Measurements were taken in ohms, and the initial resistance of the transwell membrane was taken into account by measuring the measurement of a well with culture medium but no cells.

[0118] Inhibition of translation HRMEC were grown in transwells as described above. Upon polarization, HRMEC were treated with 10 μM of the translation inhibitor cycloheximide (Sigma) and / or IL-36 cytokine. 70 kDa FITC dextran (Sigma) diluted to 1 mg / ml in low serum medium was placed in the apical chamber and FITC flux assays were performed as described above.

[0119] Laser-induced CNV and intravitreal injection All relevant national and institutional approvals were obtained before initiating the experiments. CNV was induced in 12-week-old wild-type C57BL / 6J mice using a green 532 nm Iridex Iris laser (532 nm, 140 mW, 100 ms, 50 mm spot size, 3 spots per eye) as described. After laser burn, 3 μl of vehicle or mIL-36β was injected intravitreally at a concentration of 100 ng / ml or 1000 ng / ml. Mice were sacrificed 6 days after laser burn.

[0120] RPE flat mount staining and CNV volume analysis Eyes were enucleated, neural retina was removed, and four radial cuts were made in the eyecup to avoid laser burns. Eyecups were incubated with anti-CD31 antibody (Abcam) overnight at 4°C, followed by goat anti-rat IgG Alexa594 (Abcam) and Griffonia-simplicifolia-isolectin-Alexa488 (Thermo Fisher) (1:400 in phosphate-buffered saline) for 2 h at room temperature before mounting. CNV was assessed by confocal microscopy, and renderings were made from Z-stack images using IMARIS software (Oxford Instruments), and CNV volume was calculated.

[0121] Dynamic fluorescein angiography image acquisition Fluorescein fundus angiography was performed using a Heidelberg Spectralis® HRA+OCT imaging platform (Heidelberg Engineering). After dilating the pupils with 1% (w / v) tropicamide and 2.5% (w / v) phenylephrine, mice were anesthetized using a ketamine / medetomidine mixture and then imaged in FA mode using sodium fluorescein (100 mg / kg, i.p.) as a contrast agent to visualize retinal vessels. A-scan images (field of view 30°, 1536 × 1536 pixels, average of 100 ART frames) were captured at regular intervals (30 s) between 2 and 8.5 min after fluorescein injection using Heidelberg Eye Explorer software (version 1.7.1.0). Regions of interest (CNV lesion site(s), internal vasculature outside the CNV(s), microvessels) were selected and quantified in the registered images using ImageJ. To account for interindividual variability, signal intensity at each time point was normalized to the starting point and expressed as a % value at 2 min.

[0122] Expression and purification of recombinant mouse DEVD-S31-IL-36b and caspase-3 Modified mouse IL-36b containing a caspase-3 processing motif (DEVD) inserted N-terminal to Ser-31 of the known processing site of IL-36b was made by cloning the DEVD-modified mouse IL-36b coding sequence in frame with a polyhistidine tag sequence in the bacterial expression vector pET45b. DEVD-S31-IL-36b was expressed by adding 600 uM IPTG to exponentially growing cultures of endotoxin-free E. coli (ClearColi) bacteria followed by incubation at 37°C for 3 hours. Bacteria were lysed by sonication and the polyhistidine-tagged protein was captured using nickel-NTA agarose (Amintra, Expedeon Ltd) followed by elution in PBS, pH 7.2 in the presence of 100 mM imidazole. Recombinant polyhistidine-tagged caspase-3 was similarly expressed and purified. Bacterial cell lysates and purified proteins were visualized on 12% SDS-PAGE electrophoresis gels stained with Coomassie Brilliant Blue.

[0123] IL-36β in vivo viability assay TUNEL staining was used to determine whether intravitreal injection of mIL-36β causes photoreceptor apoptosis. C57BL / 6J mice were intravitreally injected with vehicle or mIL-36β. Mice were sacrificed 3 days after injection, and eyes were paraffin-embedded and sectioned. After rehydration, TUNEL staining was performed using the in situ cell death detection kit TMR red (Sigma) according to the manufacturer's instructions, and sections were counterstained with DAPI. Sections were imaged by confocal microscopy.

[0124] IL-36 statistical analysis Data are presented as mean plus standard error of the mean or mean minus standard error of the mean. Statistical analysis of FFA permeability profiles was obtained by two-way ANOVA for matched variables. Statistical analysis of liCNV volumes was obtained by one-way ANOVA with multiple comparisons. Statistical comparisons of in vitro experiments were performed comparing treatments with untreated controls using Student's t-test or ANOVA as indicated.

[0125] IL-18 clinical cohort To investigate changes in the intraocular inflammatory profile of AMD patients undergoing anti-VEGF treatment, a hospital cohort of treatment-naive AMD patients was recruited from the Royal Victoria Eye and Ear hospital in Dublin. A total of 46 participants were enrolled in the study, of which 33 were recently diagnosed with neovascular AMD and 13 were "healthy" controls who had undergone cataract surgery. Inclusion criteria for the study relied on newly diagnosed patients with wet AMD requiring treatment with intravitreal anti-VEGF, no previous intraocular treatment in the study eye, no ocular comorbidities, and the patient's ability and willingness to consent to treatment and participation in the study. Written informed consent was obtained at the start of the treatment course.

[0126] Water sample collection and anti-VEGF treatment Sample collection and processing were performed in a dedicated room under sterile conditions. Three drops of 1% proxymetacaine were administered and allowed to act for 10 minutes. One drop of 1% tropicamide was instilled into the affected eye to dilate the pupil. With the patient in a semi-supine position, disinfection was performed using betadine eye drops on the eye and periorbital area. A disposable spring speculum was placed and 1% proxymetacaine was instilled into the injection site. Aqueous sample collection was performed using a 30-gauge needle attached to a 1-ml syringe, using a temporal approach in the horizontal plane just medial to the limbus. Aqueous humor samples of 0.05 ml to 0.1 ml were collected and immediately transferred to a -80°C freezer for storage. For intravitreal injection of anti-VEGF, the patient was asked to look in the direction opposite to the injection site. A 28-gauge needle was inserted toward the posterior pole of the eye and 125 mg / 0.05 ml of bevacizumab (Avastin®) was injected.

[0127] Best corrected visual acuity (BCVA) Visual acuity was tested in all patients at recruitment, with the patient seated, at a distance of 6 meters, using a Snellen chart via a projector (Nidec). All visual acuities were obtained at the same center by the same staff. Best corrected visual acuity (BCVA) was obtained monocularly, with the patient's most recent correction (spectacles or contact lenses) in place, while the other eye was completely blocked and a pinhole was added. Snellen visual acuity was recorded and later converted to visual acuity score (VAS) (Table 1). Visual acuity was retaken at the 12-week follow-up before the third anti-VEGF treatment.

[0128] [Table 1]

[0129] Ocular Coherence Tomography (OCT) OCT was performed on all eyes at recruitment and at the 12-week follow-up evaluation using a Cirrus HD-OCT5000 (Zeiss). Central macular thickness (CMT) was recorded in micrometers (μm).

[0130] Objective impression The final assessment of response to treatment was made by a consultant ophthalmic surgeon with extensive experience in treating patients with macular disease after observation of the ocular anatomy along with OCT and BCVA measurements, and this assessment was recorded as an objective impression.

[0131] Buffer composition Radioimmunoprecipitation Assay Buffer (RIPA): PBS, 1% (w / v) NP-40, 0.5% (w / v) sodium deoxycholate, 0.1% (w / v) sodium dodecyl sulfate (SDS).Transfer Buffer: 25 mM Tris, 192 mM glycine, 20% (v / v) methanol. Running buffer: 25 mM Tris, 192 mM glycine, 0.1% (w / v) SDS. Tris-buffered saline (TBS): 25 mM Tris, 137 mM NaCl, 2.7 mM KCl (pH ~7.4) TBS-Tween (TBS-T): 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, 0.05% (v / v) Tween™ 20.

[0132] HRMEC cell culture Primary human retinal microvascular endothelial cells (HRMEC) (Angioproteomics) were cultured in endothelial growth medium MV2 kit (Promocell) consisting of basal medium MV2 supplemented with 5% fetal calf serum (FCS; Sigma), 1% penicillin / streptomycin (P / S; Sigma) and a supplement pack containing 5 ng / ml epidermal growth factor (recombinant human), 10 ng / ml basic fibroblast growth factor (recombinant human), 20 ng / ml insulin-like growth factor (long-chain R3 IGF), vascular endothelial growth factor 165 (recombinant human), 1 μg / ml ascorbic acid and 0.2 μg / ml hydrocortisone.

[0133] HRMECs were diluted in Dulbecco's phosphate-buffered saline (DPBS; Sigma) at 1 μg / cm 2The cells were grown on flasks and plates coated with fibronectin (Sigma). Cells were maintained as a monolayer, with medium changed every 2 days and passaged once a week. To passage the cells, the medium was removed and the cells were washed twice with 5 ml of DPBS, the second wash being left for 5 min before removing the cells. After removing the DPBS, the cells were incubated with 2 mL of 0.25% trypsin-EDTA (Sigma) for 30 s and then removed by aspiration. The flask was placed in a 37 °C incubator for 1 min to dissociate the adherent cells. After incubation, the cells were suspended in 4 ml of EGM-MV2 medium. Cells were split 1:4 or diluted with 1–2 × 10 5 Cells were seeded at a density of 1000 cells / ml. Cells were maintained at 37°C and 5% CO2.

[0134] Isolation of primary mouse brain microvascular endothelial cells The following protocol for isolating primary brain microvascular endothelial cells (BMVEC) was adapted from Abbott et al (Abbott, NJ, Hughes, CC, Revest, PA, & Greenwood, J. (1992). Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier. J Cell Sci, 103 ( Pt 1), 23-37) and Assmann et al (Assmann, JC, Muller, K., Wenzel, J., Walther, T., Brands, J., Thornton, P., . . .Schwaninger, M. (2017). Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice. Bio Protoc, 7(10). doi:10.21769 / BioProtoc.2294). The following buffers were prepared prior to isolation:

[0135] Working buffer: Ca2+ / Mg2+ free HBSS, 10mMHEPES, 0.5% (w / v) BSA, 1% P / S. Complete digestion medium: HBSS, 1mg / ml collagenase / dispase, 10mMHEPES, 20u / ml DNase I, 0.147μg / ml TLCK, 1%P / S.

[0136] The following equipment was disinfected with 70% EtOH and exposed to UVC light before separation: 1 pair of large surgical scissors 1 mini dissection scissors 1 Straight tapered forceps 1 curved tapered forceps 1 scalpel with holder 1 Down Stitch Grinder 1 sheet of Whatman filter paper

[0137] Before starting the isolation, culture plates were coated with 100 μg / ml collagen IV (Sigma) and 50 μg / ml fibronectin in PBS and plates were incubated at 37° C. for 2 hours.

[0138] Wild-type (WT) C57BL / 6J mice were sacrificed by CO2 asphyxiation followed by cervical dislocation. The head was sprayed with 70% ethanol and removed with large surgical scissors from behind the ears. The skin was peeled back to expose the skull and excess tissue was removed. Using fine dissection scissors, two horizontal incisions were made along the midline towards the base and front of the skull on both sides, and a vertical cut was made between the eyes. The skull was peeled back using forceps to expose the brain, which was gently lifted and placed in ice-cold working buffer. The olfactory bulbs and cerebellum were removed using a scalpel on a sheet of Whatman® filter paper in a laminar flow hood. The meninges were removed by rotating the brain twice on the filter paper using curved forceps and cut in half along the midline using a scalpel. The brain slices were placed in a Dounce tissue grinder with 5 ml of working buffer and homogenized to a smooth consistency. The homogenate was transferred to a 50 ml tube and the tissue grinder was rinsed with working buffer and added to the homogenate before centrifugation at 600 g for 5 min at 4 °C. The supernatant was removed and the pellet resuspended in 10 ml of 22% (w / v) BSA / PBS and centrifuged at 1000 g for 20 min at 4 °C. This spinning produced a thick myelin plug which was carefully collected and resuspended in 22% BSA to recover more blood vessels. The remaining supernatant was aspirated and the pellet resuspended in complete digestion medium and incubated at 37 °C for 1 h 15 min with shaking every 15 min.

[0139] After digestion, the cell suspension was centrifuged at 600g for 5 min, the supernatant was discarded, and the pellet was resuspended in warm DPBS and finally spun down. During this centrifugation step, the pre-coated culture plates were removed from the incubator, the coating buffer was aspirated, and the wells were washed once with DPBS and air-dried.

[0140] After centrifugation in PBS, vascular fragments were resuspended in selection medium (EGM-MV2 medium plus all supplements and 5 μg / ml puromycin) and then seeded on culture plates. BMVECs are resistant to toxic levels of puromycin due to their high expression of the integral membrane-bound efflux protein P-glycoprotein (Pgp) (Tsai, CE, Daood, MJ, Lane, RH, Hansen, TW, Gruetzmacher, EM, & Watchko, JF (2002). P-glycoprotein expression in mouse brain increases with maturation. Biol Neonate, 81(1), 58-64. doi:10.1159 / 000047185). Cells were first incubated in this medium for 24 h, then the medium was aspirated to remove dead cells and the wells were washed with warm DPBS, followed by a second incubation in puromycin-supplemented EGM-MV2 medium for 24 h. Three days after seeding, the puromycin-supplemented medium was removed and cells were maintained in EGM-MV2 growth medium for 5 days until confluent.

[0141] Isolation and culture of mesenchymal cells from bone marrow Femurs were isolated from mice and using a 20 ml syringe, bones were flushed with DMEM into cell culture dishes. The flush was centrifuged at 1000 rpm for 5 minutes. The supernatant was poured off and resuspended in 1 ml of 15% FBS and aMEM medium and filtered through a 70 um nylon mesh into a 50 ml cap tube. An additional 5 ml of 15% FBS and aMEM was added through the mesh tube into the same 50 ml cap tube and seeded onto TC-coated dishes and cultured in an incubator for 5 days. On day 5, the dishes were washed 3 times with PBS (using PBS without magnesium or calcium), trypsinized, and trypsin was pipetted onto the plates to disrupt the colonies. 10ml of 10% FBS and DMEM was added, the cells were collected in a 50ml cap tube, the process was repeated once more, centrifuged at 1000rpm for 5 minutes, the supernatant was poured off, the cells were resuspended in 1ml of DMEM and 20% FBS, plated into a T75, and fed every 2 days until the cells were confluent, at which point they were split into thirds.

[0142] Cell stimulation Confluent HRMECs were stimulated with 50 or 100 ng / ml recombinant human IL-18 (SB-485232; GSK), 10 ng / ml human IL-1α (R&D Systems), or 50 ng / ml human VEGF. 165 (Peprotech) were diluted in complete growth medium, and only complete growth medium was added to the "untreated" wells. Primary BMVECs were cultured in complete growth medium with 100 ng / ml recombinant mouse IL-18 (SB-528775; GSK), 10 ng / ml mouse IL-1α (Peprotech), or 50 ng / ml mouse VEGF 164 (R&D Systems), and "untreated" wells received growth medium alone.

[0143] MTS cell viability assay Cell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation MTS Assay (Promega) according to the manufacturer's instructions. Cell monolayers were treated for the indicated times, after which treatment supplemented media was removed and incubated with 20 μl of CellTiter 96 AQueous One Solution in 100 μl of complete growth media for 1-4 hours at 37°C and 5% CO2 in a humidified incubator. The absorbance of each well was measured at 490 nm using a SynergyMX plate reader (Biotek, Gen5™).

[0144] Transendothelial Electrical Resistance (TEER) Measurement Transendothelial electrical resistance of endothelial monolayers grown on 0.4 μM transwell inserts (VWR-Corning) was measured using an EVOM2 epithelial volt / ohm meter with an STX2 handheld "chopstick" electrode (World Precision Instruments). STX2 electrodes were sterilized with 70% ethanol for 30 seconds and washed with DPBS to remove excess alcohol. Before the first measurement was taken, the electrodes were calibrated with culture medium. To measure endothelial barrier resistance, the EVOM2 function was set to ohms and a "blank" measurement was taken of a transwell insert without cells in the medium. The electrodes were positioned perpendicular to the transwell, with the long electrode in the external medium contacting the bottom of the outer well and the short electrode contacting the internal medium within the transwell insert. The electrodes were kept constant in this position to achieve consistent readings between wells. When switching between treatment wells, the electrodes were washed and recalibrated to avoid cross contamination. True tissue resistance was calculated using the following formula:

number

[0145] Permeability assay Permeability flux assays were performed on HRMEC and mouse primary BMVEC. For HRMEC, 2 × 10 cells were plated on 6.5 mm Transwell® inserts (Corning) with 0.4 μm pore size precoated with fibronectin. 5 Cells were seeded at a density of 1000 cells / mL. Cells were maintained for a minimum of 5 days before treatment began, with medium changes in the apical and basolateral chambers every 2 days. For primary BMVECs, transwells were coated with collagen IV and fibronectin as described. After isolation, 200 μl of vessel fragments were seeded onto the transwells in EGM-MV2 growth medium supplemented with puromycin. After 24 hours, this medium was replaced with fresh EGM-MV2 and puromycin, and maintained in EGM-MV2 alone for another 24 hours. Cells were left for a minimum of 5 days to reach confluence before treatment began.

[0146] Experimental wells were treated with either IL-18 (100 ng / ml), IL-1α (10 ng / ml) or VEGF (50 ng / ml) for 24 hours prior to the assay, or for 48 hours in pre-treated wells. Prior to starting the assay, fresh EGM-MV2 medium was placed in the basolateral chamber and the apical chamber medium was replaced with 1 mg / ml FITC-isothiocyanate dextran (4 or 70 kDa) (Sigma) diluted in EGM-MV2 medium. Immediately after this, as the baseline 0 time point, 100 μl aliquots were taken from the basolateral chambers of treated and control wells and transferred to a 96-well plate (Corning), wells were replenished with 100 μl fresh EGM-MV2 medium and further samples were taken at 20 min intervals for 2 hours.

[0147] The amount of FITC-dextran that crossed the endothelial monolayer and reached the basolateral chamber was measured using a spectrofluorometer (Optima Scientific) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Relative fluorescence units were converted to ng / ml using a FITC-dextran standard curve (ranging from 1 mg / ml to 0.001 μg / ml) and expressed as the apparent permeability coefficient (P app ) was calculated as follows:

number

[0148] Wound healing (scratch) assay Cells were seeded in 12-well plates and grown until confluence was reached. 24 h prior to scratching, culture medium was replaced with 0.5% serum medium without supplements for 24 h to induce cell quiescence. Using a p200 pipette tip, the cell monolayer was scratched once along the center of the well, washed once with warmed DPBS to remove debris, and then replaced with 0.5% serum-supplemented medium with experimental treatment. Images were acquired using an OlympusIX51 inverted microscope with Cell^A imaging software (Olympus). Images were taken at baseline immediately after scratching and at appropriate time points up to 24 h (HRMEC) or 48 h (BMVEC). The area within the scratch was quantified using ImageJ software, and each time point was normalized to baseline and expressed as percentage closure.

[0149] Protein isolation from cells Cell culture supernatant was aspirated and wells were washed once with ice-cold DPBS. Cells were lysed on ice in RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS) containing a 1:100 dilution of protease and phosphatase inhibitor cocktail (Sigma). Wells were scraped with a p200 pipette tip and lysates were transferred to Eppendorf tubes, centrifuged at 14,000g for 15 min to remove cell debris and immediately stored at -20°C.

[0150] Measurement of protein concentration Protein was quantified using the Pierce™ Bicinchoninic Acid Assay Kit (BCA Assay) (Thermo Fisher Scientific) according to the manufacturer's instructions. Protein standards were made by serially diluting bovine serum albumin (BSA) ranging from 0 to 2,000 μg / ml. Samples were added in duplicate to wells of a 96-well plate. Working BCA reagent was prepared by mixing 50 parts BCA Reagent A to 1 part BCA Reagent B. This working reagent was added to the wells and the plate was incubated at 37°C for 30 min. Absorbance was measured at 562 nm on a Synergy™ Mx microplate reader (BioTek).

[0151] Preparation of SDS-PAGE gel The separating and stacking gels for SDS-PAGE were hand-made. Gels were made and run using a mini-PROTEAN® tetra hand-casting system (Bio-Rad). A casting plate consisting of a short plate and a 1.5 mm spacer plate was assembled and held in place in the casting frame by clamps. The separating gel was poured onto the plate leaving enough space for the stacking gel, equivalent to the length of the comb plus 1 cm. An overlay solution of water-saturated n-butanol was applied to prevent drying as the gel cured. After polymerization, the n-butanol was removed and the top of the separating gel was washed thoroughly with distilled water before adding the stacking gel and comb.

[0152] Once set, the gel was removed from the casting frame, the comb removed, and the gel inserted into the vertical electrophoresis module with the short plate facing the inner chamber. The module was placed into the gel tank and the inner and outer buffer chambers filled with 1X running buffer. The gel was loaded with protein samples and standards and initially run at 100 volts to allow the samples to stack, then increased to 150 volts to allow separation in the resolving gel.

[0153] Sample preparation 5 μl of 5X sample buffer (250 nM Tris pH 6.8, 10% w / v SDS, 50% glycerol, 0.5% bromophenol blue, 500 mM DTT) was added to 20 μl of protein sample, boiled at 95° C. for 5 minutes, and cooled. Equal amounts of sample were loaded into each well along with 8 μl of PageRuler™ Plus press-tend ladder (Thermo-Fisher).

[0154] Protein transfer Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) using a mini Trans-Blot® cell system (Bio-Rad). After electrophoresis, the short casting plate was carefully separated from the gel and the stacking gel was discarded. PVDF activated with 100% methanol was placed on top of the gel, and the gel / PVDF was sandwiched between filter paper soaked in 1X transfer buffer and placed in a transfer cassette. To prevent overheating of the buffer, a cooling unit was added to the gel tank, and the tank was filled with 1X transfer buffer and operated at 100 volts for 1 hour.

[0155] Immunoblotting After transfer, the membrane was blocked by incubating in 5% nonfat dry milk (Marvel) in TBS-T with shaking for 1 h at room temperature. The membrane was incubated overnight at 4°C with gentle agitation with primary antibody diluted in 5% Marvel or BSA, depending on the antibody specifications. Primary antibody was retrieved and stored at -20°C. The membrane was washed three times for 5 min with TBS-T, and the membrane was incubated with secondary antibody diluted in 5% Marvel for 1 h at room temperature. The secondary antibody was removed and discarded, and excess secondary antibody was removed from the membrane by washing three times for 5 min with TBS-T.

[0156] The membrane was removed from TBS-T and dried for 20 seconds before marking the protein standards using a WesternBright™ ChemiPen™ (Advansta). Proteins were detected using in-house enhanced chemiluminescence (ECL) reagents or the WesternBright™ ECL HRP Substrate Kit (Advansta). In both cases, equal volumes of substrates A and B were mixed and pipetted onto the membrane immediately after marking the protein standards. The membrane was sandwiched between acetate sheets in a film cassette, and a sheet of X-ray film (FujiFilm) was placed on top of the membrane in a darkroom and sealed into the cassette. Exposure times varied for each antibody. After exposure, the X-ray film was removed and immersed in a bath of developer until protein signals were observed. The film was washed in a water bath and then placed in a fixer bath and incubated for 30 seconds with gentle agitation. Excess fixer was washed off and the film was left to dry. Alternatively, after incubation with ECL reagent, membranes were placed in a LAS-3000Imager (Fujifilm) and the focus and exposure time were controlled using the LAS-3000 software. Densitometry of images was performed using ImageJ software to calculate band intensities for each treatment sample and these were normalized to the band intensity of the loading control protein β-actin.

[0157] Plasma collection Whole blood was collected into EDTA tubes and centrifuged at 10,000 g for 5 min. Plasma was carefully transferred into 1 ml aliquots and stored at -80°C until evaluation.

[0158] IL-18 ELISA Plasma levels of IL-18 were measured by sandwich ELISA. 96-well plates were coated with 100 μl of 0.5 μg / ml anti-human IL-18 capture antibody (R&D Systems) diluted in 50 mM sodium carbonate-bicarbonate buffer (pH 9.4) (Thermo Scientific) and incubated overnight at 4° C. The plates were washed with TBS containing 0.1% TweenX3 and blocked with SuperBlock™ TBS for 1 h at room temperature. The plates were washed three times and 50 μl of plasma was added to the plates in triplicate. Samples were incubated for 2 h at room temperature with shaking. The plates were incubated with 100 μl of 100 ng / ml biotinylated anti-human IL-18 detection antibody (R&D Systems) diluted in reagent diluent and incubated for 1 h at room temperature with shaking. Excess antibody was washed three times in TBS-T and the plate was incubated with streptavidin-HRP reporter tag (Thermo Scientific) for 20 minutes with shaking. From this step onwards, the plate was protected from light. After washing the plate three times, 3,3',5,5-tetramethylbenzidine (TMB) (Sigma) was added and left at room temperature, where the solution was oxidized by HRP to form a blue precipitate. The reaction was stopped using a stop solution of 1:12 parts HCl to water. Absorbance was read at 450 nm on a Synergy™ Mx microplate reader (BioTek).

[0159] IL-18 binding protein (IL-18BP) ELISA The concentration of IL-18BP in plasma was measured using the DuoSet® ELISA kit (R&D Systems). The capture antibody was diluted in DPBS to a working concentration of 2 μg / ml and a clean ELISA plate (Greiner Bio-One) was coated with 50 μl of this solution, sealed and incubated overnight at room temperature. The plate was washed three times with PBS-T (0.05%) wash buffer and 200 μl of reagent diluent (1% BSA in PBS) was added to block the wells. The plate was blocked for 1 hour at room temperature with gentle agitation before being washed as before and 50 μl of plasma was added. Plasma samples were added in triplicate and incubated for 2 hours at room temperature with shaking. After removing the plasma and washing the plate, the detection antibody was added, diluted in reagent diluent to a working concentration of 25 ng / ml and incubated for 1 hour at room temperature with shaking. After this, the plate was washed and the streptavidin-HRP provided in the kit was diluted 1:20 in reagent diluent and 50 μl was added to each well. From this step onwards, the plate was protected from light. The Strep-HRP solution was washed off and 50 μl of TMB solution was added and incubated until a blue colour was observed. After a sufficient time had passed, an equal volume of STOP solution was added to each well to stop the reaction and the absorbance was read at 450 nm.

[0160] Free IL-18 calculation The concentration of free IL-18, which is the proportion of cytokine not bound to the inhibitor IL-18BP, was calculated using the law of mass action (Migliorini et al., 2010). Following measurement of total IL-18 and IL-18BP concentrations by ELISA, the molecular weights of IL-18 (18.4 kDa) and IL-18BP (17.6 kDa), the stoichiometry of the IL-18 / IL-18BP complex was 1:1 and the dissociation constant (K) of 0.4 nM were determined. d ) was used to apply the calculations.

[0161] Law of mass action:

number

number

[0162] cDNA synthesis All RNA samples were diluted to 50ng / μl before cDNA synthesis. RNA was reverse transcribed using 0.25μl M-MLV reverse transcriptase (10 units / μl) (Promega) in a master mix consisting of 2μl 5X M-MLV buffer (Promega), 2μl dNTPs (10mM) (New England Biolabs), 0.5μl random hexamers (2μM) (IDT), and 0.25μl RNaseout (40 units / μl) (Invitrogen). The same amount of RNA as the master mix was added to an RNAase / DNAse-free PCR tube and incubated at 20°C for 10 minutes, 42°C for 40 minutes, and 95°C for 3 minutes in a Veriti96-well thermo cycler (Applied Biosystems). After synthesis, cDNA was diluted 2-fold (25ng) or 1-fold (5ng) in RNAse-free H20 and stored at 4°C.

[0163] Real-time quantitative PCR (qPCR) All RT-PCR assays were performed using SensiFAST SYBR Hi-ROX Kit (Bioline) with primers designed using Primer-BLAST (NCBI). For each target, a master mix consisting of 4 μl SYBR Green mix, 0.5 μl forward primer (4 μM) and 0.5 μl reverse primer (4 μM), and 3 μl RNAse-free H20 was prepared and thoroughly mixed by vortexing. 8 μl of master mix and 2 μl of cDNA were added to each well of a 0.1 mL MicroAMP® Fast 96-well reaction plate (Applied Biosystems), sealed with optical adhesive film (Applied Biosystems), and spun down for 30 seconds in a centrifuge equipped with a microplate rotor (Eppendorf). Plates were run using a QuantStudio 3 Real-Time PCR System (Applied Biosystems) or a StepOnePlus Real-Time PCR System (Applied Biosystems) with run conditions set as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 1 min. A melt curve stage of 95°C for 1 s, 60°C for 20 s, 95°C for 1 s was added to assess primer quality. Data were analyzed using QuantStudio™ Design & Analysis software (Applied Biosystems) for comparative C. t (ΔΔC t ) method, in which test samples were normalized to an endogenous control (β-actin), and then the ΔCt of the test samples was normalized to the sample control (untreated sample), and then 2 -ΔΔCt was calculated to determine the relative fold change of the test sample versus the sample control.

[0164] IL-18 statistical analysis Analysis of aqueous cytokines was performed using the nonparametric Kruskal-Wallis test with Dunn's multiple comparisons for comparing three or more independent groups. Mann-Whitney t-test was used for two independent groups. Correlation analysis between cytokines and ΔBCVA or ΔOCT was performed using Spearman's rank correlation. Analysis of changes in cytokine levels between baseline and week 12 between improved or worsened groups was performed using two-way ANOVA with matched samples between time points, with Sidak's multiple comparisons test between improved / worsened groups and Tukey's multiple comparisons test between time points.

[0165] The invention will now be described in more detail by reference to the following non-limiting examples.

[0166] Example 1 Loss of IL-36 receptor antagonist leads to microvascular remodeling in the CNS To investigate the effects of uninhibited IL-36 signaling on microvascular integrity in the central nervous system (CNS) in vivo, we examined structural changes in the retinal vascular network using 12-month-old mice lacking an IL-36 receptor antagonist (IL-36rn- / -). The retina is part of the CNS, and retinal blood vessels are functionally similar to cerebral blood vessels. Using isolectin staining to highlight the vascular structure of retinal flat mounts, we revealed that IL-36rn- / - mice had altered retinal vasculature compared to age-matched wild-type controls, and we observed that IL-36rn- / - mice had a higher density of blood vessels (Figure 1A).

[0167] To quantify whether these observed differences were significant, REAVER analysis was used to measure total vessel length, number of branch points, and vascular tortuosity in representative flattened Z-stack images of wild-type and IL-36rn− / − mice (Figure 1B).

[0168] To confirm vascular density, we also measured the maximum fluorescence intensity (MFI) of these representative images. IL-36rn- / - mice exhibited significantly increased retinal vessel length, increased incidence of vascular branch points, and increased overall vascular density compared to wild-type animals (Figure 1C-E).

[0169] There was no evidence of increased tortuosity in IL-36rn mice (Figure 1F). These data suggest that excessive IL-36 signaling can cause structural remodeling of the retinal microvasculature.

[0170] Example 2 CNS microvascular endothelial cells are directly activated by IL-36 cytokines Vascular network analysis of IL-36rn- / - mice demonstrated that IL-36 signaling influenced microvascular geometry, although IL-36 may act directly or indirectly on endothelial cells, suggesting a proangiogenic role for IL-36 via indirect mechanisms in non-CNS-related diseases.

[0171] We utilized the publicly available Endothelial Translatome Database (http: / / www.rehmanlab.org / ribo) to determine whether endothelial cells of the CNS actively express IL-36R (ilr1l2) and family cytokine receptors. This resource provides information on genes undergoing active translation under baseline conditions and following in vivo inflammatory (LPS) challenge.

[0172] We found that brain endothelial cells basally and actively express IL-36R (Il1rl2), IL-18R (il18r1), IL-18R accessory protein (il18rap), IL-1R accessory protein (Il1rap), and IL-1R (il1r1), and that expression of IL-36R, IL-18Rap, and IL-1R increased during inflammation ( Figure 2 A).

[0173] Next, expression levels in primary human retinal microvascular endothelial cells (HRMEC) were examined. Real-time qPCR was performed on RNA isolated from confluent HRMEC monolayers. Expression levels of IL-18R, IL-18RAP, IL-1RAP and IL-36R were compared using β-actin as a housekeeping gene and presented relative to IL-18R (as the least abundant receptor). Both IL-36R mRNA and the accessory protein IL-1RAcP required for IL-36 signaling are endogenously expressed at significant and comparable levels in HRMEC (Figure 2B).

[0174] Next, the localization of IL-36R was analyzed in HRMECs by confocal microscopy. HRMECs were grown to confluence and stained for IL-36R and the endothelial-specific plasma membrane protein VE-cadherin. IL-36R was found at the plasma membrane and colocalized with the endothelial adherens junction component VE-cadherin (Figure 2C, white arrows).

[0175] It was also found in the nucleus, indicating that, like several other IL-1 family receptors, IL-36R contains a nuclear localization signal (Figure 2C, open red arrows). Indeed, analysis of the amino acid sequence of the IL-36R protein using cNLS Mapper revealed two putative bipartite nuclear localization signals from residues 501 and 506, with high NLS scores of 6.6 and 5.9, respectively.

[0176] We next investigated whether IL-36R could signal in HRMEC. IL-36R, like other IL-1R family members, activates the transcription factor NFκB and mitogen-activated protein kinase (MAPK) p38 in many cell types. HRMEC were cultured to confluence and treated with recombinant human IL-36α or IL-36β for 90 min. IL-36α and IL-36β treatment phosphorylated both NFκB p65 (Ser536) and MAPK p38 (Thr180 / Tyr182) within 5 min of cytokine stimulation (Fig. 2D and E), confirming that the IL-36 signaling pathway was functional in HRMEC.

[0177] To identify the functional impact of IL-36 modulation on endothelial cells in an unbiased manner, we performed RNA sequencing on HRMECs treated with IL-36β for 24 h and conducted network analysis of the data using iDEP and ShinyGO (Figure 2F). Using a hierarchical clustering tree to visualize the relationships between enriched GO terms, we identified that the top pathways affected by IL-36 were “angiogenesis” and “vascular morphogenesis”, in addition to “angiogenesis” and its related pathways, particularly “cell proliferation”, “cell migration”, and “cell adhesion”, among others.

[0178] Example 3 IL-36 cytokines directly promote microvascular remodeling in vitro The ability to form blood vessels or tubes is essential for the process of vascular remodeling and reorganization, and newly formed blood vessels can mature or regress. The increase in branching points in IL36rn mice and the network analysis of gene expression changes downstream of IL-36 stimulation of HRMECs suggested a role for IL-36 cytokines in microvascular remodeling. To investigate the effect of IL-36 cytokines on this process in HRMECs that have the ability to form mature tubes and visible networks 4 hours after seeding, we utilized a Matrigel-dependent tube formation assay. Figure 3A shows the network complexity of IL-36 cytokine treatment.

[0179] IL-36α demonstrated the potential ability to increase tube length when compared to unstimulated controls, but did not increase network complexity as indicated by the number of branch points (Figure 3B).

[0180] IL-36β significantly increased tube length compared to unstimulated controls (left panel) and could also increase network complexity, as shown by a significant increase in branch points (middle panel) and tube number (right panel) (Figure 3C). In all cases, VEGF was used as a control.

[0181] Angiogenesis and vascular remodeling are stages in the multistep process of angiogenesis, which also involves endothelial cell proliferation and migration. To determine whether IL-36 treatment directly affected HRMEC proliferation, cells were treated with IL-36α or IL-36β, and both cytokines led to an increase in cell number after 24 h (Figure 3D).

[0182] Furthermore, we performed an MTS / PMS assay, which again showed a significant increase in cell proliferation in response to exposure to IL-36α or IL-36β ( Fig. 3E ), although neither cytokine was as effective as VEGF alone.

[0183] Example 4 IL-36 cytokines directly induce endothelial cell migration We next assessed whether IL-36 cytokines could promote endothelial cell migration. HRMECs were grown to confluence before the monolayer was "scratched" to create a wound. Cells were then treated with IL-36α or IL-36β, and images of wound closure were acquired periodically to assay cell migration into the wound (Figure 4A-F). IL-36α or IL-36β treatment applied at the time of scratch significantly increased the rate of wound closure and cell migration (Figure 4A-F), demonstrating that IL-36 cytokines can drive endothelial cell migration in vitro.

[0184] Example 5 IL-36 cytokines directly enhance monolayer integrity and reduce vascular permeability One of the most important characteristics of endothelial cells is their ability to form and maintain a stable vascular network. Loss of barrier properties in established vascular networks can lead to disease, and similarly, pathologies associated with neovascularization are often due to neovessels failing to maintain proper barrier function. Because the CNS is an immune-privileged tissue, the inner blood-retinal barrier (iBRB), similar to the BBB, can tightly control the influx and outflow of substances into and out of the retina.

[0185] These data indicate that IL-36 cytokines have the properties to drive angiogenic processes in vitro that are normally accompanied by a loss of barrier function. To investigate whether IL-36 cytokines directly affect endothelial monolayer permeability, transendothelial electrical resistance (TEER) and FITC-dextran transendothelial flux assays were performed on HRMECs treated with IL-36α or IL-36β. The assays were performed when the cells reached confluence (Figure 5A).

[0186] TEER measurements were taken before the start of the flux assay and, when compared to baseline values, showed that treatment with either IL-36α or IL-36β significantly increased TEER compared to untreated controls, indicating that resistance across the HRMEC monolayer barrier is improved by the IL-36 cytokine (Figure 5B).

[0187] After exposure to IL-36α for 6 or 24 h, the velocity of FITC-dextran migrating through the HRMEC monolayer was significantly reduced ( Fig. 5C ).

[0188] Accordingly, IL-36α induced a significant decrease in apparent permeability (Papp), indicating a direct IL-36α-dependent increase in endothelial monolayer tightness (FIG. 5D).

[0189] In the case of IL-36β, the decrease in FITC-dextran flux rate was significant at 24 h (Fig. 5E), and Papp was also significantly decreased at 24 h after treatment (Fig. 5F).

[0190] Taken together, these data suggest that IL-36 cytokines do not induce permeability but rather actually enhance HRMEC barrier function in vitro.

[0191] Example 6 IL-36 cytokines increase the expression of components of tight junctions and cadherin junctions Retinal vascular integrity and the control of low-flow fluid-phase transcytosis are largely determined by the endothelial cell barrier complex. This complex is composed of tight junctions, gap junctions, and adherens junctions, which regulate the influx and outflow of molecules from the circulation to the tissue and vice versa. VE-cadherin is a key regulator of vascular permeability and a component of adherens junctions. On the other hand, tight junctions (TJs) are composed of proteins such as ZO-1, tricellulin, occludin, and claudin-5.

[0192] Interestingly, analysis of the RNA-seq dataset showed that both protocadherin VE-cadherin 2 (24-h adjusted p=0.04) and claudin 5 (24-h adjusted p=0.001) were two genes highly induced by IL-36β (both in the top 25 most variable genes) (Figure 5G). Furthermore, ShinyGO network analysis pointed to cell-cell adhesion as a pathway significantly regulated by IL-36β (Figure 2F).

[0193] Recently, it was found that the TGFβR inhibitor RepSox enhances barrier integrity through upregulation of the TJ protein claudin-5. For comparison, we compared gene expression of the TJ proteins claudin-5, tricellulin, and ZO-1 in response to either IL-36β or RepSox (Figure 5H). We found that IL-36β induced claudin-5 and tricellulin to levels comparable to RepSox, whereas ZO-1 transcripts were unaffected by either stimulus. We next analyzed VE-cadherin transcripts and observed that IL-36 cytokines could similarly induce VE-cadherin gene expression in a time-dependent manner (Figure 5I).

[0194] Example 7 Inhibition of protein translation leads to loss of IL-36-mediated barrier integrity We next examined whether the increased transcripts translated into enhanced protein expression levels of VE-cadherin (Figures 6A and B) and the TJ proteins ZO-1, tricellulin, and occludin (Figures 6C and D) in response to IL-36α (Figures 6A and C) or IL-36β (Figures 6B and D) treatment. Increased expression of VE-cadherin was observed 24 hours after IL-36 cytokine treatment (Figures 6A and B). Enhanced TJ expression, accompanied by a dose-dependent increase in the expression of ZO-1, tricellulin, and occludin, was also observed 24 hours after IL-36 cytokine treatment (Figures 6C and D).

[0195] Taken together, these results suggest that the enhanced barrier function observed in vitro after IL-36 cytokine stimulation may be due in part to upregulation of multiple barrier components by IL-36 cytokines. To test this, we investigated whether inhibition of protein translation results in a loss of IL-36-dependent barrier enhancement. HRMECs grown on transwells were incubated with the translation inhibitor cycloheximide (CHX) and stimulated with IL-36α or IL-36β prior to flux assays. Inhibition of protein translation results in an absolute loss of IL-36α- and IL-36β-dependent barrier enhancement (Figure 6E-H). This strongly suggests that IL-36-dependent gene expression and translation of junctional proteins are the primary mechanisms by which IL-36 improves monolayer integrity.

[0196] Example 8 IL-36β enhances barrier function of the CNS microvasculature under inflammatory conditions The barrier-enhancing properties of the IL-36 cytokine in vitro were surprising, and we wanted to investigate whether this function was also reflected in the in vivo environment.The retina is supplied with nutrients by two vascular beds: the inner retinal vasculature that contributes to the iBRB, and the palisading choroidal vasculature of the outer retina.

[0197] To investigate the effect of IL-36 on vascular integrity of the CNS in vivo, IL-36β was administered at two doses (0.3 ng / eye or 3 ng / eye) directly into the vitreous immediately after laser-induced injury in both eyes of C57BL / 6J mice. Laser-induced injury promotes the growth of pathological new blood vessels from the choroidal vessels of the outer retina, called choroidal neovascularization (CNV). These early CNVs have weak barrier properties and are fluorescein permeable, allowing the acquisition of dynamic fundus fluorescein angiography (FFA) imaging in vivo. FFA was performed 5 days after IL-36β administration (Figure 7A-D, E are representative images).

[0198] IL-36β was chosen over IL-36α because it tends to broadly induce the strongest response in in vitro experiments. A significant enhancement of overall barrier function was observed with 3 ng of IL-36β (Figure 7A). This effect was particularly pronounced at the CNV site (Figure 7B). However, in addition, IL-36β-dependent enhancement of barrier function was also detected in the inner retinal vasculature, including both the superficial and deep vascular plexus outside the area affected by laser injury (Figure 7C), and even in the microvessels of the deep vascular plexus (Figure 7D).

[0199] These data identify IL-36β as a potential vascular stabilizing factor that improves barrier integrity at sites of direct injury and microvascular inflammation.

[0200] Example 9 IL-36β has therapeutic potential to stabilize pathological vascular permeability These data supported the function of IL-36 in enhancing barrier properties and pointed to the potential of IL-36β as a therapeutic regulator of microvascular leakage. However, given that there were data demonstrating that IL-36 signaling could induce angiogenic steps in vitro, and evidence of baseline vascular remodeling in vivo, it was necessary to examine the impact of IL-36β on angiogenesis in vivo in a pathological setting. To do this, the volume of choroidal neovascular lesions was assessed by isolectin (Ib4) staining. Volumetric analysis shows that IL-36β does not increase neovascular lesion size, and no significant changes in lesion volume are observed at either dose of IL-36β (0.3 ng / eye or 3 ng / eye) (representative images in Figure 7F,G).

[0201] These data indicate that IL-36 does not exacerbate pathological angiogenesis. In other non-CNS disease models, IL-36 has been shown to upregulate IL-1a / β expression in immune cells. IL-1a / β can lead to pathological infiltration of immune cells, swelling, and neuronal cell death. To examine whether IL-36β similarly induces neuronal cell death, we injected IL-36β at doses ranging up to 30 ng per eye and performed terminal deoxynucleotidyl transferase dUTP nick-end labeling on retinal cross sections to detect DNA fragmentation. Figure 7H shows that, in contrast to IL-1, IL-36β does not induce cell death or cause structural abnormalities in neural retinal tissue, even at concentrations 10-fold higher than the dose required to reduce microvascular permeability.

[0202] Example 10 Reciprocal regulation of IL-36 and VEGF signaling HRMEC can respond to both VEGF and IL-36 signaling. These data indicate that their regulation is reciprocal, as VEGF treatment reduces IL-36R expression (Figure 8), whereas both IL-36α and IL-36β cytokines can reduce VEGFR2 expression (Figure 9).

[0203] Overall, this IL-36 dataset indicates that IL-36 treatment of angiogenic diseases may not only be effective alone, but also function as an adjunct to anti-VEGF therapy, since (1) it reduces VEGFR2 expression, thereby inhibiting residual VEGF signaling, and (2) it is expected to promote VEGF-independent compensatory endothelial cell proliferation, in parallel with enhancing cell-cell junctions at contact, stabilizing vascular lesions, and thus resulting in smaller stable neovascular membranes. Thus, the combination of anti-VEGF and IL-36 is therapeutic.

[0204] Example 11 Anti-VEGF treatment significantly increased aqueous IL-18 levels over 12 weeks in hospitalized patients with wet AMD We then measured the concentration of endogenous IL-18 in the aqueous humor of the neovascular clinical cohort and compared it to cataract controls. Changes in IL-18 concentrations were followed over the first 3-month course of bevacizumab in the hospital cohort, and analyzed changes in IL-18 concentrations in both responders and non-responders as determined by best corrected visual acuity (BCVA) and optical coherence tomography (OCT) measurements to determine whether baseline plasma or ocular IL-18 concentrations correlated with overall improvement in these measures after treatment. A cohort of untreated men and women newly diagnosed with CNV secondary to AMD from RVEEH and the Beacon Clinic were consented. Prior to administration of the first dose of bevacizumab in the untreated clinical cohort, blood and water samples were collected to measure baseline levels of IL-18. IL-18 levels were then grouped based on participants' response to treatment as determined by BCVA and OCT measurements.

[0205] Aqueous IL-18 concentrations were assessed three times over a 12-week period in 33 participants. One baseline sample was taken immediately prior to intravitreal bevacizumab injection and two more samples taken at 6-week intervals. We found that there was a significant increase in aqueous IL-18 levels between baseline measurements and week 12 (p=0.01), and the progressive increase in IL-18 levels was also significant from week 6 to week 12 (p=0.02). These data indicate that IL-18 levels increase gradually over time with anti-VEGF treatment for wet AMD (Figure 10).

[0206] Example 12 Patients with AMD who respond to anti-VEGF therapy have a significant increase in aqueous humor IL-18 over a 12-week period Aqueous samples were classified into AMD cases that improved or worsened during the treatment period based on the patient's response to bevacizumab. No significant differences in overall levels were found between responders and non-responders. However, one notable difference observed between patients who responded to treatment and those who did not was that aqueous IL-18 concentrations increased significantly from baseline to week 12 in patients who improved based on all measures: BCVA (p=0.01) (Figure 11A), OCT (p=0.01) (Figure 11B), and objective impression (p=0.003) (Figure 11C). Improvers measured by objective impression also showed a significant increase in IL-18 between weeks 6 and 12 (p=0.02). No significant increase in IL-18 levels was found between baseline and week 12 for BCVA and objective impression in patients who did not respond to treatment, but a significant difference was found between IL-18 levels measured by OCT from week 6 to week 12 in non-responders ( P = 0.04), indicating a delayed or suboptimal response to anti-VEGF treatment.

[0207] Example 13 Increased aqueous IL-18 levels correlate with reduced macular edema The correlation between the global change in OCT over the treatment period and the log-transformed IL-18 levels at the three sampling stages was evaluated. Global changes in OCT yielded negative results, indicating a reduction in central macular thickness (CMT) in improved patients. A moderate inverse correlation was found between IL-18 and ΔOCT at week 6 (Spearman r = -0.40) (Figure 12), with higher levels of IL-18 associated with a greater reduction in retinal thickness, and this trend continued at week 12, albeit weaker (Spearman r = -0.25).

[0208] Example 14 Plasma IL-18 correlates with reduced retinal edema Based on BCVA measurements, there was no significant difference in baseline plasma IL-18 concentrations between responders and non-responders (Figure 13A), and there was a weak positive correlation between baseline plasma IL-18 concentrations and BCVA measurements (Figure 13D). In contrast, it is very clear that patients who responded to anti-VEGF treatment and had a decrease in retinal thickness / edema after the course of treatment, as measured by OCT, had significantly higher plasma IL-18 levels at baseline compared to patients who did not respond to treatment (Figure 13B). Furthermore, IL-18 levels correlated strongly with the overall change in OCT over the course of treatment (Figure 13E), with higher baseline plasma IL-18 correlated with a greater decrease in retinal thickness (Spearman r=-0.47, *p=0.01).

[0209] Example 15 IL-18 can inhibit VEGF secretion in vivo and in vitro In the retinas of young animals (3 weeks old), VEGF levels were significantly decreased after treatment with a lower dose (100 μg / kg) of IL-18 (Figure 14A). The RPE performs many important support functions to maintain retinal homeostasis, one of which is the production of growth factors, including VEGF. In vitro, the levels of secreted VEGF were found to increase linearly over time, suggesting that VEGF is constitutively produced by the RPE. Supplementation of the cell culture medium with IL-18 (1000 ng / ml) reduced VEGF concentrations at (almost) all time points examined (1-72 h) (Figure 14B) and also significantly slowed the overall rate of VEGF secretion (Figure 14C).

[0210] Example 16 IL-18 specifically reduces VEGF2 expression in human retinal endothelial cells (HRMEC) HRMEC respond to VEGF through signaling from a variety of VEGF receptors. The primary one thought to be involved in pathogenic angiogenesis is VEGFR2. IL-18 specifically reduces VEGFR2 on these retinal endothelial cells (Figure 15).

[0211] Example 17 IL-18 stimulates increased HRMEC migration across the wound Cell migration is a key component in the formation of new blood vessels. During angiogenesis, in response to changes in the surrounding environment, endothelial "tip" cells utilize migration to initiate the sprouting of new blood vessels. In wound healing, this movement of endothelial cells is required to repair and maintain vascular integrity. Using the scratch assay as a model of wound healing, we assessed endothelial cell migration in response to IL-18 and IL-1α. We found that IL-18 significantly increased the rate of wound closure when compared to untreated controls and was comparable to VEGF-induced wound closure at 24 hours (Figure 16A). Interestingly, we found that VEGF increased the rate of closure more than IL-18 at 4 and 8 hours after scratching, although not significantly. IL-18-treated cells 8 hours after scratching were comparable to untreated controls (NT vs. IL-18; x - diff =-0.99, p=0.99), whereas the mean difference between VEGF and control wells was 1.0 with FGF-treated cells in between (NT vs. FGF; - diff =-4.366, p=0.79) Significant difference (NT vs. VEGF; x - diff =-9.35, p=0.08). However, by 24 hours, the rate of closure in IL-18-treated cells was significantly increased (x - diff =-12.58, p=0.002), which was comparable to VEGF (NT vs VEGF; x - diff =-12.25p=0.01), both outperformed FGF (NT vs FGF; x - diff =-28.4, p=0.0001) (Figure 16B). These data suggest that the effects of VEGF on endothelial cell migration are strongest during the first few hours after exposure, whereas IL-18 signaling may be delayed in comparison but is ultimately able to induce a strong pro-migratory response.

[0212] Example 18 IL-18 enhances VEGF-induced proliferation in HRMECs Using the MTS assay, HRMEC were treated with increasing doses of VEGF. This corresponded to increased proliferation up to 5 ng / ml, with minimal benefit of the higher dose (Panel 1). This VEGF-induced proliferation was increased after a 24-hour pretreatment with increasing doses of IL-18. Note that IL-18 alone can also enhance HRMEC proliferation (Figure 17).

[0213] Example 19 IL-18 promotes expression of the tight junction protein occludin and inhibits its depletion by VEGF HRMEC cells were treated with IL-18 and the expression of the tight junction protein occludin was examined (Figure 18). IL-18 increased the expression of this tight junction protein, whereas in comparison, both VEGF and IL-1a decreased the expression of occludin. IL-18 can rescue the stabilizing inhibition of occludin expression by VEGF, whereas IL-1a does not rescue the VEGF-mediated inhibition of occludin. These data indicate that IL-18 can stabilize junctions between endothelial cells and enhance barrier integrity.

[0214] The above data indicate that IL-18 reduces edema by (1) decreasing available VEGF and the ability of endothelial cells to respond to it, and (2) promoting endothelial cell migration through upregulation of occludin, stabilizing cell-cell junctions at contact, improving vascular stability, and inhibiting permeability of vascular junctions.

[0215] Example 20 IL-18 induces migration of fibroblast-like cells to close wounds in human retinal pigment epithelial monolayers in vitro The decrease in vascular permeability may be due to the retinal pigment epithelium (RPE) enveloping the neovascular lesion and blocking leakage. To assess whether IL-18 influences this process, a series of scratch assays were performed in cultures of hfRPE cells (Figure 19). Although some pigmented RPE were observed to migrate into the wound, the most striking observation was the appearance of fibroblast-like cells migrating across the wound in response to IL-18 treatment. Because hfRPE cells are primary cultures isolated from primary tissue, it is likely that contaminating fibroblast-like cells remained in the cultures throughout the isolation process.

[0216] Example 21 IL-18 activates human choroidal fibroblasts. Given the observation of fibroblast-like cells in the hfRPE scratch assay, the ability of IL-18 to activate human ocular choroidal fibroblasts (HOCFs) was evaluated. Figure 20 shows that IL-18 activates HOCFs, triggering a morphological change in which they adopt a more stellate, active phenotype in which the intermediate filament vimentin migrates from a perinuclear position to extend deeper toward the cell membrane. IL-18 also significantly reduces baseline secretion of VEGF from HOCFs. LDH assays show that IL-18 does not cause HOCF death and may enhance HOCF viability.

[0217] Example 22 IL-18 activates the mesenchymal constriction phase of the wound healing process Mesenchymal contraction is a key step in the wound healing process that involves the interaction of the cytoskeletal filaments vimentin and smooth muscle actin (SMA). Mesenchymal stem cells (MSCs) were isolated from mouse bone marrow and treated with IL-18 to evaluate the effect of IL-18 on these filaments (Figure 21). IL-18 had a strong effect on the appearance of vimentin and SMA in MSCs, indicating that IL-18 can promote the mesenchymal contraction phase of wound healing. IL-18 also increased collagen in MSCs, indicating that IL-18 can promote the production of extracellular matrix for cell migration.

[0218] Taken together, the above in vitro data indicate that IL-18 promotes the mesenchymal cell migration phase and the mesenchymal cell contraction phase of wound healing, accelerating the pace of healing and resulting in smaller lesions and smaller scars.

[0219] Example 23 Increased mesenchymal cell infiltration / activation in IL-18-injected animals We verified the in vitro findings and observed whether IL-18 could promote migration and activation of mesenchymal cell infiltration in vivo. Wild type mice were injected intraperitoneally with IL-18 (Figure 22) and subjected to laser-induced injury to produce choroidal neovascular lesions (CNV). Three days later, eyes were enucleated, prepared for flat mounting, and stained with F4 / 80 to assess infiltrating mononuclear cells and vimentin to assess infiltrating mesenchymal cells. It was evident that IL-18 increased vimentin and these vimentin positive cells appeared to form cages encapsulating endothelial cells (isolectin +ve) and mononuclear cells around the lesion. We repeated this experiment, this time injecting IL-18 directly into the eye by intravitreal injection on the same day that the mice received laser-induced injury (Figure 23). A similar vimentin staining pattern to that observed with systemic injection was observed, with IL-18 driving vimentin-positive cells to encapsulate neovascular lesions.

[0220] Example 24 IL-18 reduces the area of ​​fibrovascular scar Vimentin is secreted into the extracellular matrix where it contributes to the initial scar tissue. When vimentin staining patterns were evaluated 14 days after laser injury, we found less vimentin positive staining in WT mice treated with IL-18 compared to vehicle controls (Figure 24). Conversely, in IL-18-deficient mice (IL-18KO), the area of ​​vimentin staining was larger compared to that of WT mice (Figure 25).

[0221] Taken together, these in vitro and in vivo data suggest that IL-18 (1) promotes compensatory proliferation of multiple cell types, including endothelial cells, fibroblasts, macrophages, and mesenchymal stem / stromal cells, and (2) accelerates the rate of mesenchymal activation and contraction, thereby promoting healing of neovascular lesions, reducing edema, and reducing scar areas. VEGF inhibition alone prevents vascular leakage, but blocks endothelial cell proliferation and maturation, impeding the healing process and keeping neovascular lesions in an early pathological immature state, i.e., lesions that cannot heal. IL-18 is useful as an adjunct to anti-VEGF therapy because it (1) reduces the expression of VEGFR2, thereby inhibiting residual VEGF signaling, (2) promotes VEGF-independent compensatory endothelial cell proliferation in parallel with strengthening cell-cell junctions upon contact, thereby stabilizing vascular lesions and resulting in smaller, more stable neovascular membranes, and (3) accelerates the rate of mesenchymal activation and contraction, leading to faster healing of neovascular lesions, reduced edema, and reduced scar area. Thus, the combination of anti-VEGF and IL-18 is therapeutically effective.

[0222] Anti-VEGF treatment removes VEGF from the retina but does not induce a healing response. These data indicate that IL-36 is well suited to reduce the permeability of neovascular lesions, whereas IL-18 is well suited to enhance mesenchymal activation and contraction. The combination of anti-VEGF (any type) treatment with IL-18 and IL-36 should potently inhibit the permeability of neovascular lesions and promote wound healing responses.

[0223] It is well established that vascular barrier disruption and excessive plasma leakage contribute to a variety of disease processes, particularly when affecting neuropathy and retinal diseases. Regulation of vascular permeability and angiogenesis occurs through a variety of direct and indirect mechanisms, and various members of the IL-1 family of interleukins can mediate aspects of these processes. Indeed, proteomic profiling has revealed considerable overlap in the pathways and biological functions regulated by IL-1β and VEGF in activated endothelial cells. Although VEGF is both a driver of permeability and a proangiogenic agent, the most immediate and potent effect of anti-VEGF treatment as a therapeutic modality is thought to be the regulation of vascular permeability. One of the more established areas of IL-36 research concerns the role of IL-36 in psoriasis, where there is evidence that IL-36 may contribute to the persistent angiogenesis of psoriatic plaques. Here, the aim was to evaluate whether IL-36 can regulate microvascular angiogenesis and / or vascular permeability associated with the CNS.

[0224] Notably, IL-36 significantly enhanced endothelial barrier function. This protection from vascular permeability was observed not only in areas of injury-induced neovascularization but also in microvessels of the iBRB distal to the laser injury. Immune dysregulation and inflammatory processes have been associated with neovascularization both clinically and experimentally, and the vascular permeability observed in the inner retinal vessels is likely the result of laser-induced local inflammation. Neovascularization and neovascular permeability observed in response to laser injury are highly dependent on VEGF upregulation. These data therefore indicate that IL-36β may enhance vascular barrier integrity and protect against both VEGF-induced and inflammation-induced vascular permeability. This is opposite to, and not common to, the established role of IL-1 in promoting vascular permeability. Although barrier disruption is associated with many retinal and neuropathies, few barrier-strengthening agents have been discovered that specifically enhance barrier integrity and increase resistance to vascular leakage. Angiopoietin 1 (Ang1) and TGFβR inhibitors (RepSox) are two such factors, both of which enhance barrier integrity through induction of tight junction protein expression, and both of which are under intense investigation for their therapeutic potential. Similar to the mechanisms underlying RepSox and Ang1, these data indicate that IL-36 cytokines also enhance barrier function, at least in part due to IL-36-dependent transcriptional regulation of tight junction proteins and VE-cadherin, which are directly involved in maintaining vascular integrity. When protein translation was inhibited by cycloheximide, IL-36 treatment lost its ability to enhance barrier function.

[0225] IL-36 appears to be more similar to Ang1. Although many molecules are known to regulate either angiogenesis or permeability, the overlap between molecules that can regulate both is highly selective. Both VEGF and angiopoietin are potent proangiogenic factors that function together in the early stages of vascular development, with VEGF acting early in angiogenesis and Ang1 acting later in vascular remodeling, maturation, and stabilization. However, VEGF causes increased vascular permeability, whereas Ang1 stabilizes blood vessels and protects them from VEGF-induced plasma leakage. Furthermore, although long-term expression of Ang1 generates a large number of large blood vessels, acute administration of Ang1 in vivo does not increase neovascular lesion size when administered to adult mice in multiple disease models; instead, Ang1 induces a highly ordered, hierarchical stable, and leakage-resistant vascular structure. This study similarly found that long-term unregulated IL-36 signaling modeled using il36rn- / - mice demonstrates the role of IL-36 in vascular modeling. IL-36 was also found to be a pro-angiogenic factor promoting proliferation, migration, and tube formation of primary endothelial cells in vitro. However, it is noteworthy that despite these clear pro-angiogenic characteristics of IL-36, introduction of recombinant IL-36β into adult mice did not increase neovascular lesion volume in vivo in a pathological setting (a mouse model of laser-induced CNV) as would be expected for a pro-angiogenic cytokine.

[0226] The biggest difference between normal and new pathological vessels is that new vessels lack tight junction proteins, which do not prevent plasma and serous fluid in neovascular lesions from leaking into the surrounding tissue. IL-36 appears to upregulate the angiogenic process, promoting the tightening of endothelial cell junctions, as well as leading to vascular remodeling and maturation, resulting in a stable vascular network that does not cause edema. These data demonstrating this are supported by network analysis of RNA sequencing data. In this context, there is room to consider the IL-36 cytokine as a wound healing molecule, which in the context of the retina may promote regulated vascular healing. Taken together, these data show that IL-36 is a cytokine with barrier-strengthening properties that are useful in reducing microvascular leakage in diseases that reduce microvascular leakage due to inflammation and elevated VEGF.

[0227] The evidence indicates that IL-36 is highly expressed in primary human retinal endothelial cells (HRECs) and acts to reduce levels of VEGFR2, but not VEGFR1, which inhibits the pathological effects of VEGF signaling. Furthermore, IL-36 is believed to rescue VEGF-induced permeability of HRECs cultured on Transwell filters and prevent migration of Evans Blue BSA across polarized HREC monolayers. Furthermore, IL-18, expressed throughout ocular tissues, has potent wound-healing properties and can induce rapid wound closure while enhancing the integrity of tight junction components of retinal and choroidal endothelial cells.

[0228] IL-36 cytokines were first identified in 1999 after searching DNA databases for IL-1 homologs. Since then, it has been established that there are three distinct but highly homologous signaling molecules in the IL-36 subfamily: IL36α, IL36β, and IL36γ. To date, the IL-36 signaling cascade has been most extensively studied in psoriasis, where activation of IL-36R promotes keratinocyte inflammation in both plaque and pustular phenotypes of the disease. Early studies suggested that, at least in psoriasis, IL-36R signaling may act indirectly as a proangiogenic mediator via macrophage activation, but its role in regulating vascular permeability remains unclear.

[0229] In this study, we used the mouse retinal vasculature as an accessible in vivo model amenable to manipulation to study the direct effects of IL-36β cytokine on angiogenesis and permeability in the adult mouse CNS. This model has the advantage of allowing the in vivo acquisition of fluorescein angiography images, allowing accurate measurement of vascular permeability in real time at the site of injury and in uninjured vessels. In stark contrast to IL-1, we found that IL-36β acts as a potent inhibitor of vascular permeability and upregulates the translation of key components of the adherens layer and tight junctions.

[0230] Notably, IL-36 can directly promote angiogenic steps involved in the wound healing process in vitro, but importantly, IL-36R signaling demonstrates that the proangiogenic features promoted by IL-36 in vitro are uncoupled from its ability to enhance vascular integrity and reduce vascular permeability in vivo in an acute setting, thus potentially providing a therapeutic application for reducing microvascular leakage in vivo. Furthermore, introduction of recombinant IL-36β by intravitreal injection is well tolerated and non-toxic to the surrounding neural tissue. These data expand the function of the IL-36 cytokine to include its unusual action of enhancing vascular remodeling and endothelial barrier integrity.

Claims

1. A pharmaceutical composition for use in the treatment or prevention of an eye disease, the pharmaceutical composition comprising IL-36.

2. The pharmaceutical composition of claim 1, further comprising IL-18.

3. The pharmaceutical composition of claim 1 , wherein the treatment comprises administering the pharmaceutical composition to a subject in need thereof.

4. 4. The pharmaceutical composition of claim 3, wherein the treatment further comprises administering an anti-VEGF agent to the subject in need thereof.

5. The pharmaceutical composition of claim 4 , wherein the anti-VEGF agent and the pharmaceutical composition are administered simultaneously.

6. The pharmaceutical composition of claim 4 , wherein the anti-VEGF agent and the pharmaceutical composition are administered sequentially.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the use is in the treatment of wound healing.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the treatment comprises administration of cells expressing IL-36 and / or IL-18.

9. 7. The pharmaceutical composition according to claim 1, wherein the eye disease is selected from any one or more of age-related macular degeneration (AMD), nascent geographic atrophy, incomplete retinal pigment epithelium and outer retinal atrophy (iRORA), wet AMD, geographic atrophy, dry AMD, macular edema, retinopathy, diabetic retinopathy, and glaucoma.

10. 7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the anti-VEGF agent is selected from an anti-VEGF antibody or a fragment thereof, an anti-VEGF polyclonal antibody or a fragment thereof, an anti-VEGF monoclonal antibody or a fragment thereof, a VEGF inhibitor, a small molecule VEGF inhibitor, a tyrosine kinase inhibitor, a small molecule tyrosine kinase inhibitor, a fusion protein comprising VEGF or a fragment thereof, a recombinant fusion protein comprising VEGF or a fragment thereof, and any combination thereof.

11. The pharmaceutical composition according to any one of claims 4 to 6, wherein the anti-VEGF agent is selected from aflibercept, axitinib, bevacizumab, cabozantinib, lapatinib, lenvatinib, pazopanib, ponatinib, ramucirumab, ranibizumab, regorafenib, sorafenib, sunitinib, vandetanib, and combinations thereof.

12. 7. The pharmaceutical composition of any of claims 1 to 6, wherein said treatment comprises administration by a route selected from topical, parenteral, intraarterial, intravenous, intraocular, intravitreal, and each combination thereof.

13. The pharmaceutical composition according to any one of claims 1 to 6, wherein the treatment comprises the administration of a unit dose of at least 0.3 ng of IL-36 and / or IL-18.

14. The pharmaceutical composition according to any one of claims 1 to 6, wherein the IL-36 is selected from IL36α, IL36β, and IL36γ.

15. Use of IL-36 in the manufacture of a medicament for the treatment or prevention of an eye disease.

16. The use of claim 15, wherein the medicament further comprises IL-18.

Images