Daniel R. Saban

Autoimmunity in Dry Eye Is Due to Resistance of Th17 to Treg Suppression

Dry eye disease (DED), an inflammatory autoimmune disorder affecting the ocular surface, degrades visual performance and the quality of life of >10 million people in the United States alone. The primary limitation in the effective treatment of DED is an incomplete understanding of its specific cellular and molecular pathogenic elements. Using a validated mouse model of DED, herein we functionally characterize the different T cell subsets, including regulatory T cells (Tregs) and pathogenic effector T cells, and determine their contribution to the pathogenesis of DED. Our data demonstrate the presence of dysfunctional Tregs and the resistance of pathogenic T cells, particularly Th17 cells, to Treg suppression in DED. In addition, we clearly show that in vivo blockade of IL-17 significantly reduces the severity and progression of disease, which is paralleled by a reduction in the expansion of Th17 cells and restoration of Treg function. Our findings elucidate involvement of a previously unknown pathogenic T cell subset (Th17) in DED that is associated specifically with Treg dysfunction and disease pathogenesis and suggest a new target for dry eye therapy.

Levels of Foxp3 in Regulatory T Cells Reflect Their Functional Status in Transplantation

Foxp3 expressing CD4+CD25+ regulatory T cells (Tregs) have been shown to prevent allograft rejection in clinical and animal models of transplantation. However, the role of Foxp3 in regulating Treg function, and the kinetics and mechanism of action of Tregs in inducing allograft tolerance in transplantation, are still not fully understood. Thus, we investigated the kinetics and function of Tregs in a mouse model of orthotopic corneal transplantation, the most common form of tissue grafting worldwide. In this study, using in vitro functional assays and in vivo Treg adoptive transfer assays, we show that far more relevant than Treg frequency is their level of Foxp3 expression, which is directly associated with the potential of Tregs to prevent allograft rejection by producing regulatory cytokines and suppressing effector T cell activation. In addition, our data clearly demonstrate that Tregs primarily suppress the induction of alloimmunity in regional draining lymph nodes rather than suppressing the effector phase of the immune response in the periphery. These findings provide new insights on Treg dynamics in transplantation, which are crucial for designing therapeutic strategies to modulate Treg function and to optimize Treg-based cell therapies for clinical translation.

Anti-angiogenesis Effect of the Novel Anti-inflammatory and Pro-resolving Lipid Mediators

PURPOSE Resolvins and lipoxins are lipid mediators generated from essential polyunsaturated fatty acids that are the first dual anti-inflammatory and pro-resolving signals identified in the resolution phase of inflammation. Here the authors investigated the potential of aspirin-triggered lipoxin (LX) A4 analog (ATLa), resolving (Rv) D1, and RvE1, in regulating angiogenesis in a murine model. METHODS ATLa and RvE1 receptor expression was tested in different corneal cell populations by RT-PCR. Corneal neovascularization (CNV) was induced by suture or micropellet (IL-1 beta, VEGF-A) placement. Mice were then treated with ATLa, RvD1, RvE1, or vehicle, subconjunctivally at 48-hour intervals. Infiltration of neutrophils and macrophages was quantified after immunofluorescence staining. The mRNA expression levels of inflammatory cytokines, VEGFs, and VEGFRs were analyzed by real-time PCR. CNV was evaluated intravitally and morphometrically. RESULTS The receptors for LXA4, ALX/Fpr-rs-2 and for RvE1, ChemR23 were each expressed by epithelium, stromal keratocytes, and infiltrated CD11b(+) cells in corneas. Compared to the vehicle-treated eye, ATLa-, RvD1-, and RvE1-treated eyes had reduced numbers of infiltrating neutrophils and macrophages and reduced mRNA expression levels of TNF-alpha, IL-1 alpha, IL-1 beta, VEGF-A, VEGF-C, and VEGFR2. Animals treated with these mediators had significantly suppressed suture-induced or IL-1 beta-induced hemangiogenesis (HA) but not lymphangiogenesis. Interestingly, only the application of ATLa significantly suppressed VEGF-A-induced HA. CONCLUSIONS ATLa, RvE1, and RvD1 all reduce inflammatory corneal HA by early regulation of resolution mechanisms in innate immune responses. In addition, ATLa directly inhibits VEGF-A-mediated angiogenesis and is the most potent inhibitor of NV among this new genus of dual anti-inflammatory and pro-resolving lipid mediators.

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes

By engaging CD36 on murine macrophages, thrombospondin-1 prevents excessive macrophage VEGF-C production and corneal neovascularization.

Characterization of Effector T Cells in Dry Eye Disease

PURPOSE Dry eye disease (DED) is associated with ocular surface inflammation that is thought to be mediated primarily by CD4 T cells. The purpose of this study was to investigate whether this T cell-mediated immune response is generated in the lymphoid compartment and to characterize the functional phenotype of the T cells activated in DED. METHODS DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled environment chamber and to systemic scopolamine. T cells from regional draining lymph nodes (LNs) of DED mice and normally sighted mice were analyzed for surface activation markers (CD69 and CD154), chemokine and cytokine receptors, and proliferation potential. RESULTS Draining LNs of DED mice showed increased frequencies of CD69- and CD154-expressing T cells with higher proliferative capacity. In addition, these LN T cells primarily showed a helper T-cell (Th)1 phenotype, expressing significantly higher levels of IFN-gamma and IL-12Rbeta2 but not IL-4R. Similarly, the LNs of DED mice showed significantly increased frequencies of T cells expressing CXCR3 and CCR5, but not CCR4, suggesting a bias toward a Th1 phenotype. CONCLUSIONS These data demonstrate that a Th1-type immune response is induced in the regional LNs of DED mice. The identification of specific cytokine/chemokine receptors overexpressed by these T cells may signify potential novel targets/strategies for the treatment of DED.

Dependence of Corneal Stem/Progenitor Cells on Ocular Surface Innervation

PURPOSE Neurotrophic keratopathy (NK) is a corneal degeneration associated with corneal nerve dysfunction. It can cause corneal epithelial defects, stromal thinning, and perforation. However, it is not clear if and to which extent epithelial stem cells are affected in NK. The purpose of this study was to identify the relationship between corneolimbal epithelial progenitor/stem cells and sensory nerves using a denervated mouse model of NK. METHODS NK was induced in mice by electrocoagulation of the ophthalmic branch of the trigeminal nerve. The absence of corneal nerves was confirmed with β-III tubulin immunostaining and blink reflex test after 7 days. ATP-binding cassette subfamily G member 2 (ABCG2), p63, and hairy enhancer of split 1 (Hes1) were chosen as corneolimbal stem/progenitor cell markers and assessed in denervated mice versus controls by immunofluorescent microscopy and real-time PCR. In addition, corneolimbal stem/progenitor cells were detected as side population cells using flow cytometry, and colony-forming efficiency assay was performed to assess their function. RESULTS ABCG2, p63, and Hes1 immunostaining were significantly decreased in denervated eyes after 7 days. Similarly, the expression levels of ABCG2, p63, K15, Hes1, and N-cadherin transcripts were also significantly decreased in denervated eyes. Stem/progenitor cells measured as side population from NK mice were decreased by approximately 75% compared with normals. In addition, the authors found a significant (P = 0.038) reduction in colony-forming efficiency of stem/progenitor cells harvested from denervated eyes. CONCLUSIONS Corneolimbal stem/progenitor cells are significantly reduced after depletion of sensory nerves. The data suggest a critical role of innervation in maintaining stem cells and/or the stem cell niche.

Corneal Penetration of Topical and Subconjunctival Bevacizumab

PURPOSE To investigate the ability of bevacizumab to penetrate the cornea after topical application or subconjunctival injection. METHODS Bevacizumab 1% was topically applied three times a day to the corneas of mice (BALB/c) with intact corneas (n = 14), and with corneal neovascularization (n = 14). Animals were euthanized at 1, 6, 12, and 24 hours, and 2, 4, and 7 days for immunohistochemical analyses. Donkey anti-human IgG labeled with Cy3 was used for bevacizumab immunoreactivity detection. Additionally, one-time topical bevacizumab 1% was tested in corneas with denuded epithelium (n = 16). In another group (n = 16), a single dose of 0.5 mg bevacizumab was injected subconjunctivally. Animals were euthanized at 1, 6, and 24 hours, and 2, 4, 7, 14, and 21 days for immunohistochemical studies. RESULTS Bevacizumab was barely detected beyond the very superficial layer of the corneal epithelium in mice with intact corneas even after 7 days of topical administration. Application of bevacizumab in mice with corneal neovascularization; however, showed variable penetration into the corneal stroma. Experimentation with single application of topical bevacizumab in corneas with denuded epithelium or subconjunctivally injected bevacizumab showed intense staining for bevacizumab. CONCLUSIONS Topically applied bevacizumab has limited capacity to penetrate the corneas with intact epithelium. However, bevacizumab can penetrate the neovascularized cornea after topical application. This study demonstrates that subconjunctivally injected bevacizumab in eyes with an intact cornea penetrates well into the corneal stroma.

Effects of topical and subconjunctival bevacizumab in high-risk corneal transplant survival.

PURPOSE To investigate whether corneal graft survival could be improved by topical or subconjunctival bevacizumab in a murine model of vascularized high-risk corneal transplantation. METHODS Before corneal transplantation, intrastromal sutures were placed for 2 weeks in the corneas of BALB/c mice, inducing intense angiogenesis. Allogeneic corneal transplantation was performed using C57BL/6 donor mice. Topical bevacizumab (2.5%) was delivered 3 times a day for 3 weeks in one treatment group, and 0.02 mL (0.5 mg) bevacizumab was injected subconjunctivally at days 0, 4, 8, and 15 after transplantation in the other treatment group. The control group received no treatment. Grafts were examined twice a week for 8 weeks by slit-lamp microscopy and were photographed once a week by slit-lamp digital camera and scored for opacity. For assessment of corneal neovascularization (NV), a quantitative method was used to measure three primary metrics including neovascular area, vessel caliber, and neovessel invasion area. RESULTS Both topical and subconjunctival bevacizumab treatment reduced neovascular area and vessel caliber; however, the regression of corneal NV was more profound when treated subconjunctivally. The mean percentage reduction of neovascular area was 55% (P < 0.05) by week 8 in the subconjunctival treatment group and 33% (P = 0.15) in the topical group. Only subconjunctival bevacizumab treatment resulted in significant regression of neovessel invasion area (P < 0.05). All corneal transplants in both the control and the topical groups were rejected by 4 weeks after transplantation. However, in the subconjunctival treatment group, 33% of corneal grafts survived (P < 0.01). CONCLUSIONS Subconjunctival bevacizumab may offer an adjunctive measure to conventional therapies in preventing graft rejection in high-risk corneal transplantation.

Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model

Aims/hypothesisWe sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells.MethodsWild-type, gp130−/− and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp+ chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood.ResultsBM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b+/CD45hi/ CCR2+/Ly6Chi inflammatory monocytes. Diabetic gp130−/− mice were protected from development of diabetes-induced changes in their HSCs.Conclusions/interpretationThe BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

Suppression of Inflammatory Corneal Lymphangiogenesis by Application of Topical Corticosteroids

OBJECTIVES To analyze whether topical application of corticosteroids inhibits inflammatory corneal lymphangiogenesis and to study the potential underlying antilymphangiogenic mechanisms. METHODS Inflammatory corneal neovascularization was induced by suture placement, and the corneas were then treated with topical fluorometholone, prednisolone acetate, or dexamethasone sodium phosphate. After 1 week, the corneas were stained with lymphatic vessel endothelial hyaluronan receptor 1 for detection of pathological corneal lymphangiogenesis. The effect of these corticosteroids on macrophage recruitment was assessed via fluorescence-activated cell sorting analysis. The effect of these corticosteroids on proinflammatory cytokine expression by peritoneal exudate cells was tested via real-time polymerase chain reaction. Furthermore, the effect of steroid treatment on the proliferation of lymphatic endothelial cells was assessed via enzyme-linked immunosorbent assay. RESULTS Treatment with corticosteroids resulted in a significant reduction of inflammatory corneal lymphangiogenesis. The antilymphangiogenic effect of fluorometholone was significantly weaker than that of prednisolone and dexamethasone. Corneal macrophage recruitment was also significantly inhibited by the application of topical steroids. Treatment of peritoneal exudate cells with corticosteroids led to a significant downregulation of the RNA expression levels of tumor necrosis factor and interleukin 1β. Additionally, proliferation of lymphatic endothelial cells was also inhibited. CONCLUSIONS Corticosteroids are strong inhibitors of inflammatory corneal lymphangiogenesis, with significant differences between various corticosteroids in terms of their antilymphangiogenic potency. The main mechanism of the antilymphangiogenic effect seems to be through the suppression of macrophage infiltration, proinflammatory cytokine expression, and direct inhibition of proliferation of lymphatic endothelial cells. CLINICAL RELEVANCE Steroids block corneal lymphangiogenesis, the main risk factor for immune rejections after corneal transplantation. The different antilymphangiogenic potency of these drugs should be taken into account when using steroids in clinical practice (eg, after keratoplasty).