The atypical chemokine receptor-2 fine-tunes the immune response in herpes stromal keratitis.

Herpes stromal keratitis (HSK) is a blinding corneal disease caused by herpes simplex virus-1 (HSV-1), a common pathogen infecting most of the world's population. Inflammation in HSK is chemokine-dependent, particularly CXCL10 and less so the CC chemokines. The atypical chemokine receptor-2 (ACKR2) is a decoy receptor predominantly for pro-inflammatory CC chemokines, which regulates the inflammatory response by scavenging inflammatory chemokines thereby modulating leukocyte infiltration. Deletion of ACKR2 exacerbates and delays the resolution of the inflammatory response in most models. ACKR2 also regulates lymphangiogenesis and mammary duct development through the recruitment of tissue-remodeling macrophages. Here, we demonstrate a dose-dependent upregulation of ACKR2 during corneal HSV-1 infection. At an HSV inoculum dose of 5.4 x 105 pfu, but not at higher dose, ACKR2 deficient mice showed prolonged clinical signs of HSK, increased infiltration of leukocytes and persistent corneal neovascularization. Viral clearance and T cell activation were similar in ACKR2-/- and wild type mice, despite a transient diminished expression of CD40 and CD86 in dendritic cells. The data suggest that ACKR2 fine-tunes the inflammatory response and the level of neovascularization in the HSK.

The cornea IV immunology, infection, neovascularization, and surgery chapter 1: Corneal immunology.

PURPOSE: of Review: This review offers an informed and up-to-date insight on the immune profile of the cornea and the factors that govern the regulation of such a unique immune environment. SUMMARY: The cornea is a unique tissue that performs the specialized task of allowing light to penetrate for visual interpretation. To accomplish this, the ocular surface requires a distinct immune environment that is achieved through unique structural, cellular and molecular factors. Not only must the cornea be able to fend off invasive infectious agents but also control the inflammatory response as to avoid collateral, and potentially blinding damage; particularly of post-mitotic cells such as the corneal endothelium. To combat infections, both innate and adaptive arms of the inflammatory immune response are at play in the cornea. Dendritic cells play a critical role in coordinating both these responses in order to fend off infections. On the other side of the spectrum, the ocular surface is also endowed with a variety of anatomic and physiologic components that aid in regulating the immune response to prevent excessive, potentially damaging, inflammation. This attenuation of the immune response is termed immune privilege. The balance between pro and anti-inflammatory reactions is key for preservation of the functional integrity of the cornea. RECENT FINDINGS: The understanding of the molecular and cellular factors governing corneal immunology and its response to antigens is a growing field. Dendritic cells in the normal cornea play a crucial role in combating infections and coordinating the inflammatory arms of the immune response, particularly through coordination with T-helper cells. The role of neuropeptides is recently becoming more highlighted with different factors working on both sides of the inflammatory balance.

Immunophysical analysis of corneal neovascularization: mechanistic insights and implications for pharmacotherapy.

The cornea lacks adaptive immune cells and vasculature under healthy conditions, but is populated by both cell types under pathologic conditions and after transplantation. Here we propose an immunophysical approach to describe postoperative neovascularization in corneal grafts. We develop a simple dynamic model that captures not only the well-established interactions between innate immunity and vascular dynamics but also incorporates the contributions of adaptive immunity to vascular growth. We study how these interactions determine dynamic changes and steady states of the system as well as the clinical outcome, i.e. graft survival. The model allows us to systematically explore the impact of pharmacological inhibitors of vascular growth on the function and survival of transplanted corneas and search for the optimal time to initiatetherapy. Predictions from our models will help ongoing efforts to design therapeutic approaches to modulate alloimmunity and suppress allograft rejection.

Combination of dexamethasone and Avastin® by supramolecular hydrogel attenuates the inflammatory corneal neovascularization in rat alkali burn model.

Corneal neovascularization (CNV) is one of the leading causes of vision loss and a high-risk factor for transplant rejection. The present study proposed a supramolecular hydrogel comprised of MPEG-PCL micelles and α-cyclodextrin (α-CD) for co-delivery of dexamethasone sodium phosphate (Dexp) and Avastin® (Ava), and further evaluated its therapeutic efficacy in rat alkali burn model. A physical mixing of Dexp/Ava, MPEG-PCL micelles, and α-CD aqueous solution leads to a spontaneous formation of the supramolecular hydrogel via a "host-guest" recognition between MPEG and α-CD. The supramolecular hydrogel provides a relatively quick release of Dexp over Ava during the study of the 5-day in vitro release. The results of in vitro cytotoxicity test and wound healing assay illustrated that the proposed supramolecular hydrogel was non-toxic against L-929 and HCEC cells and did not significantly affect the migration of HCEC cells after 24h incubation. The corneal distribution test suggested that the precorneal duration of Ava was significantly extended by the supramolecular hydrogel with respect to its solution formulation. Moreover, the supramolecular hydrogel showed high ocular biocompatibility and was a non-irritant after topical instillation. Furthermore, the Dexp-Ava hydrogel medication, but not by Ava solution and Ava hydrogel medication, could greatly attenuate the alkali burn-induced corneal inflammation and remarkably suppress the corneal neovascularization via the downregulation of VEGF, CD31, and α-SMA expression in the rat alkali burn model. As a result, the combined Dexp and Ava by supramolecular hydrogel exhibited an advantage over Ava monotherapy approach, which might be a promising alternative therapy for inflammatory CNV.

Frontline Science: Aspirin-triggered resolvin D1 controls herpes simplex virus-induced corneal immunopathology.

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye, caused by HSV-1 infection, and a common cause of vision impairment in humans. The inflammatory lesions in the cornea are primarily caused by neutrophils with the active participation of CD4+ T cells. Therefore, the targeting of these immune cell types and their products represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are lipid mediators derived from docosahexaenoic acid (DHA) and were shown to promote resolution in several inflammatory disease models. In this report, we examined whether AT-RvD1 administration, begun before infection or at a later stage after ocular infection of mice with HSV-1, could control the severity of SK lesions. Treatment with AT-RvD1 significantly diminished the extent of corneal neovascularization and the severity of SK lesions. AT-RvD1-treated mice had fewer numbers of inflammatory cells that included neutrophils as well as Th1 and Th17 cells in the infected cornea. The mechanisms by which AT-RvD1 acts appear to be multiple. These include inhibitory effects on proinflammatory mediators, such as IL-1ß, IL-6, IL-12, CXCL1, MCP-1, MIP-2, vascular endothelial growth factor (VEGF)-A, matrix metalloproteinase 9 (MMP-9), and proinflammatory miRNA, such as miR-155, miR-132, and miR-223, which are involved in SK pathogenesis and corneal neovascularization. In addition, AT-RvD1 attenuated STAT1, which plays an important role in Th1 cell differentiation and IFN-γ expression. These findings demonstrate that AT-RvD1 treatment could represent a useful strategy for the management of virus-induced immunopathological lesions.

Comprehensive Modeling of Corneal Alkali Injury in the Rat Eye.

PURPOSE: To characterize the molecular, clinical, and histopathological profiles in the rat cornea after alkali injury over a 21-day period. METHODS: Alkali injury was induced in one eye of male Lewis rats. Corneal opacity and corneal neovascularization were assessed daily. Real-time qRT-PCR analysis and immunohistochemical staining were conducted to examine inflammation, neovascularization, and fibrosis. RESULTS: We found that within 2 hours of chemical exposure, corneal opacification rapidly developed with an acute increase in various cytokine expressions, while several cytokines demonstrated a secondary peak by day 7. Early neutrophil infiltration peaked at day 1 post-injury while macrophage infiltration peaked at day 7. Throughout the time course of the study, corneal opacity persisted and neovascularization, lymphangiogenesis, and fibrosis progressed. CONCLUSIONS: This study highlights the molecular, clinical, and histopathological changes throughout the progression of alkali injury in the rat cornea. These profiles will assist in the development of new strategies and therapies for ocular alkali injury.

Long-term consequences of topical dexamethasone treatment during acute corneal HSV-1 infection on the immune system.

Herpes simplex virus type 1 (HSV-1) is a leading cause of neurotrophic keratitis (NTK). NTK is characterized by decreased corneal sensation from damage to the corneal sensory fibers. We have reported on the regression of corneal nerves and their function during acute HSV-1 infection. That nerve loss is followed by an aberrant process of nerve regeneration during the latent phase of infection that lacks functional recovery. We recently showed the elicited immune response in the infected cornea, and not viral replication itself, is part of the mechanism responsible for the nerve degeneration process after infection. Specifically, we showed infected corneas topically treated with dexamethasone (DEX) significantly retained both structure and sensitivity of the corneal nerve network in comparison to mice treated with control eye drops, consistent with decreased levels of proinflammatory cytokines and reduced influx of macrophages and CD8+ T cells into the cornea. This study was undertaken to analyze the long-term effect of such a localized, immunosuppressive paradigm (DEX drops on the cornea surface during the first 8 d of HSV-1 infection) on the immune system and on corneal pathology. We found the profound immunosuppressive effect of DEX on lymphoid tissue was sustained in surviving mice for up to 30 d postinfection (p.i.). DEX treatment had prolonged effects, preserving corneal innervation and its function and blunting neovascularization, as analyzed at 30 d p.i. Our data support previously reported observations of an association between the persistent presence of inflammatory components in the latently infected cornea and structural and functional nerve defects in NTK.

Angiopoietin-2 Blockade Promotes Survival of Corneal Transplants.

Purpose: Corneal transplantation remains the last hope for vision restoration, and lymphangiogenesis (LG) is a primary mediator of transplant rejection. This study was to investigate the specific role of angiopoietin-2 (Ang-2) in transplantation-associated LG and graft rejection. Methods: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice to assess the effects of Ang-2 blockade via neutralizing antibody. Grafts were evaluated in vivo by ophthalmic slit-lamp biomicroscopy and anterior segment optical coherence tomography (OCT) up to 8 weeks after surgery. Additionally, whole-mount corneas were analyzed for lymphatic and blood vessels and macrophages by immunofluorescent microscopy, and draining lymph nodes were assessed for donor-derived cells by flow cytometry. Results: Anti-Ang-2 treatment significantly suppressed LG and graft rejection. In this study, we achieved 75% suppression of LG and 80% graft survival. Our approach also inhibited donor-derived cell trafficking to draining lymph nodes and affected macrophage morphologic phenotypes in the grafted corneas. Additionally, Ang-2 blockade also reduced central corneal thickening, a parameter strongly associated with graft rejection. Conclusions: Ang-2 is critically involved in corneal transplant rejection and anti-Ang-2 treatment significantly improves the outcomes of corneal grafts. Moreover, we have shown that anterior segment OCT offers a new tool to monitor murine corneal grafts in vivo. This study not only reveals new mechanisms for transplant rejection, but also offers a novel strategy to treat it.

Proangiogenic Function of T Cells in Corneal Transplantation.

BACKGROUND: Corneal neovascularization increases the risk of T cell-mediated allograft rejection. Here, we investigate whether T cells promote angiogenesis in transplantation. METHODS: Conventional effector T cells were collected from draining lymph nodes of allogeneic or syngeneic corneal transplanted BALB/c mice. T cells were either cocultured with vascular endothelial cells (VECs) to assess VEC proliferation or used in a mixed lymphocyte reaction assay. Messenger RNA (mRNA) expression of vascular endothelial growth factor (VEGF)-A, -C, and VEGF receptor 2 (VEGF-R2) in VECs was assessed by real-time PCR. VEGF-A protein expression was determined by enzyme-linked immunosorbent assay. Flow cytometry was used to analyze VEGF-R2 expression in corneal CD31 cells, and VEGF-A and IFNγ expression in corneal CD4 T cells. RESULTS: Allogeneic T cells from high-risk (HR) grafted mice induced more VEC proliferation than those from syngeneic transplant recipients (P = 0.03). Vascular endothelial growth factor-A mRNA and protein expression were higher in T cells from draining lymph nodes (P = 0.03 and P = 0.04, respectively) and cornea (protein; P = 0.04) of HR compared with low-risk (LR) grafted hosts. Vascular endothelial growth factor-A, VEGF-C, and VEGF-R2 mRNA expression were increased in VECs when cocultured with T cells from HR transplants compared with LR transplants and naive mice. In addition, IFNγ blockade in T cell/VEC coculture increased VEC proliferation and VEGF-A protein expression, whereas blocking VEGF-A significantly reduced VEC proliferation (P = 0.04). CONCLUSIONS: Allogeneic T cells from corneal transplant hosts promote VEC proliferation, probably via VEGF-A signaling, whereas IFNγ shows an antiangiogenic effect. Our data suggest that T cells are critical mediators of angiogenesis in transplantation.

Integrin Alpha 9 Blockade Suppresses Lymphatic Valve Formation and Promotes Transplant Survival.

PURPOSE: The lymphatic pathway mediates transplant rejection. We recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis, and these valves express integrin alpha 9 (Itga-9) and play a critical role in directing lymph flow. In this study, we used an allogeneic corneal transplantation model to investigate whether Itga-9 blockade could suppress valvulogenesis after transplantation, and how this effect would influence the outcomes of the transplants. METHODS: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice. The recipients were randomized to receive subconjunctival injections of either Itga-9 blocking antibody or isotype control twice a week for 8 weeks. Corneal grafts were assessed in vivo by ophthalmic slit-lamp biomicroscopy and analyzed using Kaplan-Meier survival curves. Additionally, whole-mount full-thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves. RESULTS: Anti-Itga-9 treatment suppressed lymphatic valvulogenesis after transplantation. Our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea. More importantly, Itga-9 blockade led to a significant promotion of graft survival. CONCLUSIONS: Lymphatic valvulogenesis is critically involved in transplant rejection. Itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival.

Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts.

BACKGROUND: Angiogenin (ANG), a component of tears, is involved in the innate immune system and is related with inflammatory disease. We investigated whether ANG has an immune modulatory function in human corneal fibroblasts (HCFs). METHODS: HCFs were cultured from excised corneal tissues. The gene or protein expression levels of interleukin (IL)-1beta (ß), IL-4, IL-6, IL-8, IL-10, complements, toll-like receptor (TLR)4, myeloid differentiation primary response gene (MYD)88, TANK-binding kinase (TBK)1, IkappaB kinase-epsilon (IKK-ε) and nuclear factor-kappaB (NF-κB) were analyzed with or without ANG treatment in tumor necrosis factor-alpha (TNF-α)- or lipopolysaccharide (LPS)-induced inflammatory HCFs by real-time polymerase chain reaction (PCR), Western blotting and immunocytochemistry. Inflammatory cytokine profiles with or without ANG were evaluated through immunodot blot analysis in inflammatory HCFs. Corneal neovascularization and opacity in a rat model of corneal alkali burn were evaluated after application of ANG eye drops. RESULTS: ANG decreased the mRNA levels of IL-1ß, IL-6, IL-8, TNF-α receptor (TNFR)1, 2, TLR4, MYD88, and complement components except for C1r and C1s and elevated the mRNA expression of IL-4 and IL-10. Increased signal intensity of IL-6, IL-8 and monocyte chemotactic protein (MCP)-1 and MCP-2 induced by TNF-α or LPS was weakened by ANG treatment. ANG reduced the protein levels of IKK-ε by either TNF-α and LPS, and decreased TBK1 production induced by TNF-α, but not induced by LPS. The expression of NF-κB in the nuclei was decreased after ANG treatment. ANG application lowered corneal neovascularization and opacity in rats compared to controls. CONCLUSION: These results demonstrate that ANG reduces the inflammatory response induced by TNF-α or LPS in HCFs through common suppression of IKK-ε-mediated activation of NF-κB. This may support the targeting of immune-mediated corneal inflammation by using ANG.

Inhibition of RelA expression via RNA interference induces immune tolerance in a rat keratoplasty model.

RelA, the most important regulator of NF-kB activity, and its mechanisms in keratoplasty immune rejection have not been fully investigated. In the present study, lentivirus-mediated silencing of RelA expression in a bone marrow-derived dendritic cell (BMDC) model was tested. The BMDCs were transfected with RelA-shRNA to induce an immature, maturation-resistant and tolerogenic phenotype, while not significantly changing IFN-γ, IL-10 and IL-17 expression. A fully allogeneic rat cornea transplant model was established for in vivo studies. The allograft mean survival time (MST) of lv-shRelA-DC injection groups were significantly longer than the untreated BMDC group and control group. The corneal opacity and neovascularization scale of the lv-shRelA-DC injection groups were slight compared to pair control others. Postoperative flow cytometric analysis revealed that the percentage of Treg positive cells was dramatically increased in animals that received an lv-shRelA-DC injection. ELISA and qRT-PCR analyses of serum showed that IFN-γ and IL-17 expression were suppressed by lv-shRelA-DC treatement. In vivo experiments demonstrated that IL-10 induced immunosuppression was partly attributed to injection of lv-shRelA-DC throughout the experiment, differing from the general anti-inflammatory factors. Luciferase and Chromatin IP evaluation showed that RelA knockdown in BMDCs significantly reduces DNA binding to IFN-γ, IL-10 and the IL-17 promoter and inhibited of transcriptional activity. Taken together, this study illustrates a significant role of RelA in mediating the corneal neovascularization by affecting IL-17 expression. Our comprehensive analysis shows that the significant role of RelA provides a novel and feasible therapeutic approach for the prevention of corneal allograft rejection.

IL-10 Indirectly Regulates Corneal Lymphangiogenesis and Resolution of Inflammation via Macrophages.

The role of IL-10, a primarily anti-inflammatory cytokine, in the regulation of inflammatory lymphangiogenesis is undetermined. Herein, we show that IL-10 modulates corneal lymphangiogenesis and resolution of inflammation. IL-10 was not expressed in healthy corneas but was up-regulated in inflamed corneas by infiltrating macrophages. Macrophages up-regulated the expression of prolymphangiogenic vascular endothelial growth factor-C upon stimulation with IL-10. Consistently, corneal inflammation resulted in reduced expression of vascular endothelial growth factor-C and decreased corneal lymphangiogenesis in IL-10-deficient mice (IL-10(-/-)). The effect of IL-10 on lymphangiogenesis was indirect via macrophages, because IL-10 did not directly affect lymphatic endothelial cells. The expression of proinflammatory cytokines and the numbers of infiltrating macrophages increased and remained elevated in inflamed corneas of IL-10(-/-) mice, indicating that IL-10 deficiency led to more severe and prolonged inflammation. The corneal phenotype of IL-10 deficient mice was mimicked in mice with conditional deletion of Stat3 in myeloid cells (lysozyme M Cre mice Stat3(fl/fl) mice), corroborating the critical role of macrophages in the regulation of lymphangiogenesis. Furthermore, local treatment with IL-10 promoted lymphangiogenesis and faster egress of macrophages from inflamed corneas. Taken together, we demonstrate that IL-10 indirectly regulates inflammatory corneal lymphangiogenesis via macrophages. Reduced lymphangiogenesis in IL-10(-/-) and lysozyme M Cre Stat3(fl/fl) mice is associated with more severe inflammatory responses, whereas IL-10 treatment results in faster resolution of inflammation. IL-10 might be used therapeutically to terminate pathological inflammation.

Soluble vascular endothelial growth factor receptor-3 suppresses allosensitization and promotes corneal allograft survival.

PURPOSE: To investigate the effect of VEGF-C and VEGF-D blockade via soluble VEGFR-3 (sVEGFR-3) on T cell allosensitization, corneal neovascularization, and transplant survival. METHODS: Corneal intrastromal suture placement and allogeneic transplantation were performed on BALB/c mice to evaluate the effect of sVEGFR-3 on corneal neovascularization. Soluble VEGFR-3 trap was injected intraperitoneally to block VEGF-C/D (every other day starting the day of surgery). Immunohistochemical staining of corneal whole mounts was performed using anti-CD31 (PECAM-1) and anti-LYVE-1 antibodies to quantify the levels of hem- and lymphangiogenesis, respectively. Mixed lymphocyte reaction (MLR) was performed to assess indirect and direct host T cell allosensitization and the frequencies of IFN-γ-producing T cells in the draining lymph nodes were assessed using flow cytometry. Graft opacity and survival was evaluated by slit-lamp biomicroscopy. RESULTS: Treatment with sVEGFR-3 resulted in a significant blockade of lymphangiogenesis 2 weeks post-transplantation and significantly prolonged corneal allograft survival compared to the control group at 8 weeks post-transplantation (87.5 % vs. 50 %), and this was associated with significant reduction in the frequencies of allosensitized T cells and decreased frequencies of IFN-γ-producing CD4 T cells. CONCLUSIONS: Soluble VEGFR-3 suppresses corneal lymphangiogenesis and allograft rejection and may offer a viable therapeutic modality for corneal neovascularization and corneal transplantation.

Inhibitory effects of topical cyclosporine A 0.05% on immune-mediated corneal neovascularization in rabbits.

BACKGROUND: We aimed to study the inhibitory effects of topical cyclosporine A (CsA) 0.05% on immune-mediated corneal neovascularization, and to compare its efficacy with those of dexamethasone 0.1% and bevacizumab 0.5%. METHODS: Immune-mediated corneal neovascularization was created in 36 right eyes of 36 rabbits. The rabbits were then randomized into four groups. Group I received CsA 0.05%, Group II received dexamethasone 0.1%, Group III received bevacizumab 0.5%, and Group IV received isotonic saline twice a day for 14 days. The corneal surface covered with neovascular vessels was measured on the photographs. The rabbits were then sacrificed and the corneas excised. Paraffin-embedded sections were stained with hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. RESULTS: The means of percent area of corneal neovascularization in Group I, II, III, and IV were 24.4%, 5.9%, 37.1%, and 44.1%, respectively. The inhibitory effect of CsA 0.05% was found to be better than the effect found in the bevacizumab 0.5% and control groups (p = 0.03 and p = 0.02, respectively). CsA 0.05% was found to have significantly lesser inhibitory effects on corneal neovascularization than dexamethasone 0.1% (p < 0.001). Apoptotic cell density was higher in Group III and Group IV than in Group I and Group II. There was no difference between Group I and Group II in terms of apoptotic cell density (p = 0.7). CONCLUSIONS: Topical CsA 0.05% was shown to have an inhibitory effect on immune-mediated corneal neovascularization in rabbits.

Pathogenesis of herpes stromal keratitis--a focus on corneal neovascularization.

The cornea is a complex sensory organ that must maintain its transparency for optimal vision. Infections such as with herpes simplex virus can result in blinding immunoinflammatory reactions referred to as herpes stromal keratitis (HSK). In this review we discuss the pathogenesis of HSK referring to work mainly done using animal model systems. We briefly discuss the role of multiple cell types and soluble mediators but focus on the critical role of corneal vascularization (CV) in contributing to corneal damage. We describe how VEGF and other angiogenic molecules are induced following infection and discuss the many ways by which CV can be controlled. Speculations are made regarding future approaches that could improve the management of HSK.

Effects of subconjunctival ranibizumab in a presensitized rat model of corneal graft.

We evaluated whether corneal graft survival in presensitized corneal transplantation was affected by subconjunctival ranibizumab in a rat model. The effect of ranibizumab in the presensitized corneal transplantation has not been previously reported, although anti-VEGF was attempted on a non-presensitized model in other studies. Corneas were transplanted from Brown Norway to Spraque Dawley rats. The recipient rats were randomly assigned to three groups: Group 1, skin autograft and subconjunctival injection of PBS; Group 2, skin allograft and injection of PBS; and Group 3, skin allograft and injection of ranibizumab (vascular endothelial growth factor antibody). A skin graft was performed 2 weeks before corneal transplantation. On days 3, 7, 11, and 14 after transplantation, the grafts were scored. The number of corneas with graft rejection on day 14 was significantly higher in Group 2 than in Group 1 or 3 (6/15 [40.0%] in Group 1, 13/15 [86.7%] in Group 2, and 4/15 [26.7%] in Group 3). The mean clinical scores for edema, opacity, and new vessels in Group 3 were significantly lower than those in Group 2, while the edema score in Group 1 was significantly lower than that in Group 2 on day 14. Before corneal allotransplantation, presensitization by skin grafting accelerated the graft rejection process. In a short-term presensitized rat model of keratoplasty, application of subconjunctival ranibizumab prevented graft rejection.

Interleukin-17 enhanced immunoinflammatory lesions in a mouse model of recurrent herpetic keratitis.

Interleukin-17 (IL-17), mainly produced by activated (memory) T cells, has been found in the corneas from herpetic stromal keratitis (HSK) patients. To better understand the role of IL-17 and to optimize fidelity to human recurrent HSK, in this study, we utilized a mouse model of recurrent HSK, examined the expression of IL-17 and Th17 cells, and determine the alterability of virus-induced corneal inflammation after anti-IL-17 antibody treatment during murine recurrent HSK. We found that Th17 cells were obviously up-regulated in both cornea and DLNs of recurrent mice. Peak IL-17 protein present in recurrent cornea in conjunction with peak opacity mediated by CD4(+) T cells. Systemic administration of anti-IL-17 antibody resulted in a diminished severity of corneal opacity, neovascularization, and CD4(+) T cells infiltration compared to control. Anti-IL-17 treatment down-regulated the mRNA and protein levels of TNF-α expression in recurrent corneas, and decreased HSV-specific DTH responses. Our results indicate that elevated IL-17 expression may be involved in the development of recurrent HSK. The likely mechanisms of action for IL-17 are through up-regulating TNF-α expression and promoting HSV-specific DTH responses. Thus, IL-17 might constitute a useful target for therapeutic intervention in recurrent HSK.

Minocycline inhibits alkali burn-induced corneal neovascularization in mice.

The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV). A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS) or minocycline twice a day (60 mg/kg or 30 mg/kg) for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs). The mRNA expression levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs), interleukin-1α, 1ß, 6 (IL-1α, IL-1ß, IL-6) were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1ß and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1ß, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1ß, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d.) can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.

Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.