Graft Site Microenvironment Determines Dendritic Cell Trafficking Through the CCR7-CCL19/21 Axis.

PURPOSE: The graft site microenvironment has a profound effect on alloimmunity and graft survival. We aimed to study the kinetics and phenotype of trafficking antigen-presenting cells (APC) to the draining lymph nodes (DLNs) in a mouse model of corneal transplantation, and to evaluate the homing mechanisms through which graft site inflammation controls APC trafficking. METHODS: Allogeneic donor corneas were transplanted onto inflamed or quiescent graft beds. Host- (YAe+) and donor (CD45.1+ or eGFP+)-derived APCs were analyzed by flow cytometry. Protein and mRNA expression of the CC chemokine receptor (CCR)7 ligands CCL19 and CCL21 were assessed using ELISA and Real-Time qPCR, respectively. Transwell migration assay was performed to assess the effect of DLNs isolated from hosts with inflamed graft beds on mature bone marrow-derived dendritic cells (BMDCs). RESULTS: We found that inflamed graft sites greatly promote the trafficking of both recipient- and graft-derived APCs, in particular mature CCR7+ CD11c+ dendritic cells (DC). CCL19 and CCL21 were expressed at significantly higher levels in the DLNs of recipients with inflamed graft beds. The supernatant of DLNs from recipients with inflamed graft beds induced a marked increase in mature DC migration compared with supernatant from recipients with quiescent graft beds in a transwell assay. This effect was abolished by neutralizing CCL19 or CCL21. These data suggest that graft site inflammation increases the expression of CCR7 ligands in the DLNs, which promote mature DC homing and allorejection. CONCLUSIONS: We conclude that the graft site microenvironment plays a critical role in alloimmunity by determining DC trafficking through the CCR7-CCL19/21 axis.

Chemokine decoy receptor D6 mimicking trap (D6MT) prevents allosensitization and immune rejection in murine corneal allograft model.

Although corneal allotransplantation is performed in the immune-privileged cornea, many grafts are still rejected after transplantation. This study examined the role of chemokine receptor D6 expression in a corneal allograft rejection, investigated the modulation of D6 expression in cells, and determined the effect of D6 on graft survival. Interestingly, D6 was highly expressed in CD45 -: cells and the corneal epithelium of accepted corneal allografts. From the mouse corneal allograft model, TGF-ß was found to play a key role in D6 up-regulation, leading to reduced CCL2, CCL5, and CCL3. To modulate D6 chemokine binding, a D6MT was developed and showed effective chemokine trapping through SPR and FACS assays. By treating corneal allografts with D6MT, the allograft survival rate was improved, and (lymph) angiogenesis was reduced. Direct allosensitization and DC LN homing was drastically reduced in the mouse corneal allograft model. These findings suggest that TGF-ß is a positive regulator of D6 expression, and it is a potential therapeutic target to enhance the survival of corneal allografts.

Effects of topical Janus kinase inhibition on ocular surface inflammation and immunity.

PURPOSE: To determine the effects of topical Janus kinase inhibition on ocular surface inflammation and immunity. METHODS: Ophthalmic 0.003% tofacitinib (CP-690,550) was administered topically to inhibit Janus kinase activation at the ocular surface. Male BALB/c mice 6 to 8 weeks of age were subjected to corneal thermocautery and randomized to receive tofacitinib, vehicle, or no treatment. Corneas were subsequently excised for fluorescence-activated cell sorting and quantitative real-time reverse transcription polymerase chain reaction. Female C57BL/6 mice 6 to 8 weeks of age were exposed to desiccating stress to induce experimental dry eye disease and randomized to receive tofacitinib, tofacitinib and vehicle, vehicle, or no treatment. Corneal fluorescein staining was performed to evaluate clinical disease severity. The corneas and conjunctivae were harvested for immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction. RESULTS: After corneal thermocautery, it was found that tofacitinib treatment decreased the corneal infiltration of CD45+, Gr-1+, and CD11b+ cells on days 1 and 3. Transcripts encoding interleukin (IL)-1ß and IL-6 were significantly decreased by tofacitinib treatment at post-thermocautery day 3. In experimental dry eye disease, tofacitinib treatment twice per day significantly decreased corneal fluorescein staining on days 12 and 15. The corneal infiltration of CD11b+ cells was significantly decreased by tofacitinib treatment twice per day. Tofacitinib treatment twice per day significantly increased the corneal expression of IL-1RA, and significantly decreased the corneal expression of tumor necrosis factor and IL-23. Further, tofacitinib treatment twice per day significantly decreased the conjunctival expression of IL-17A and significantly increased the conjunctival expression of FoxP3. CONCLUSIONS: Topical ophthalmic tofacitinib, a Janus kinase inhibitor, suppressed ocular surface inflammation and immunity in experimental corneal thermocautery and dry eye disease.

Expression of the chemokine decoy receptor D6 mediates dendritic cell function and promotes corneal allograft rejection.

PURPOSE: To identify the role of chemokine receptor 6 (D6) expression by dendritic cells (DCs) and its role in corneal transplant immunity. METHODS: Flow cytometry analysis was used to assess the expression level of the D6 chemokine receptor in different leukocyte populations and DC maturation following lipopolysaccharides (LPS) stimulation of bone marrow-derived DCs isolated from wild-type (WT) or D6(-/-) mice (C57BL/6 background). Mixed-lymphocyte reactions and delayed-type hypersensitivity assays were performed with bone marrow-derived DCs from WT or D6(-/-) mice to evaluate T-cell alloreactivity. Adoptive transfer experiments with T cells from WT or D6(-/-) hosts with BALB/c corneal allografts were performed. Graft opacity was assessed over an 8-week period, and graft survival was plotted using Kaplan-Meier survival curves. RESULTS: Expression of the D6 chemokine receptor was significantly higher in DCs compared to other leukocyte subpopulations, including neutrophils, lymphocytes, and monocytes/macrophages. LPS challenge of D6(-/-) bone marrow-derived DCs elicited significantly lower levels of major histocompatibility complex II and costimulatory molecules (CD40, CD80, and CD86) compared to WT bone marrow-derived DCs, indicating the role of the D6 chemokine receptor in DC maturation. Further, DCs isolated from D6(-/-) mice induced less T-cell proliferation (p≤0.001) and interferon-gamma production in T cells of draining lymph nodes compared to WT mice following corneal transplantation (p≤0.001). Moreover, adoptively transferred T cells from D6(-/-) corneal transplanted mice to WT mice led to impaired graft rejection, compared to the hosts that received T cells from the WT transplanted mice. CONCLUSIONS: We demonstrated D6 chemokine receptor expression by DCs and identified its critical function in multiple aspects of DC biology, including maturation and consequent elicitation of alloreactive T-cell responses that are responsible for corneal allograft rejection.

The CCR6/CCL20 axis mediates Th17 cell migration to the ocular surface in dry eye disease.

PURPOSE: Th17 cells are believed to be the primary effector cells in the pathogenesis of dry eye disease (DED). However, the mechanisms by which Th17 cells migrate from the lymphoid tissues to the ocular surface are unknown. The purpose of this study was to investigate the role of the C-C chemokine receptor 6/C-C chemokine ligand 20 (CCR6/CCL20) chemokine axis in mediating Th17 cell migration in DED. METHODS: DED was induced by housing C57BL/6 mice in a low-humidity environment supplemented with scopolamine treatment. Th17 cell expression of CCR6 was evaluated using flow cytometry and ocular surface expression of CCL20 was measured using PCR and ELISA assays. CCL20 neutralizing antibody was administered subconjunctivally to DED mice and disease severity, including the frequency of conjunctival Th17 cells, was evaluated. RESULTS: CCR6 is preferentially expressed by Th17 cells in both normal and DED mice and DED significantly upregulates ocular surface expression of CCL20. Disruption of CCR6/CCL20 binding with CCL20 neutralizing antibody decreases T-cell migration in vitro and reduces Th17 cell infiltration of the conjunctiva when administered in vivo, significantly improving clinical signs of DED. These changes were accompanied by a decrease in ocular surface inflammatory cytokine levels and corneal CD11b+ cell frequencies. Treatment also significantly reduced the generation of Th17 cells. CONCLUSIONS: Local neutralization of CCL20 decreases Th17 cell infiltration of the ocular surface in DED, leading to improvement in clinical signs of disease. This suggests that CCR6/CCL20 interactions direct Th17 cell migration in DED and that disruption of this axis may be a novel therapeutic approach to this condition.

Effect of desiccating environmental stress versus systemic muscarinic AChR blockade on dry eye immunopathogenesis.

PURPOSE: A majority of experimental data on dry eye disease (DED) immunopathogenesis have been derived from a murine model of DED that combines desiccating environmental stress with systemic muscarinic acetylcholine receptor (mAChR) inhibition. However, to our knowledge the effects of pharmacologic mAChR blockade on the pathogenesis of experimental DED have not been evaluated systemically. The purpose of our study was to investigate the differential effects of desiccating environmental stress and mAChR inhibition on the pathogenesis of DED. METHODS: DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled-environment chamber or to systemic scopolamine, or by performing extraorbital lacrimal gland excision. Clinical disease was assessed using corneal fluorescein staining (CFS) and the cotton thread test (CTT). Corneal CD11b(+) and conjunctival CD3(+) T-cell infiltration were evaluated by flow cytometry. T-cells from draining cervical lymph nodes (CLN) and distant inguinal lymph nodes (ILN) were analyzed for Th1, Th2, Th17, and Treg responses by flow cytometry and ELISA. RESULTS: Desiccating environmental stress and systemic mAChR blockade induced similar clinical signs of DED. However, desiccating environmental stress imparted higher conjunctival CD3(+) T-cell infiltration, and greater Th17-cell activity and Treg dysfunction than mAChR blockade, while mAChR blockade decreased tear secretion to a greater extent than desiccating environmental stress. Systemic mAChR blockade attenuated Th17 activity and enhanced Th2 and Treg responses without affecting Th1 activity. CONCLUSIONS: In vivo inhibition of mAChRs variably affects CD4(+) T-cell subsets, and desiccating environmental stress and systemic mAChR blockade induce DED through different primary pathogenic mechanisms.

Nerves and neovessels inhibit each other in the cornea.

PURPOSE: To evaluate the regulatory cross-talk of the vascular and neural networks in the cornea. METHODS: b-FGF micropellets (80 ng) were implanted in the temporal side of the cornea of healthy C57Bl/6 mice. On day 7, blood vessels (hemangiogenesis) and nerves were observed by immunofluorescence staining of corneal flat mounts. The next group of mice underwent either trigeminal stereotactic electrolysis (TSE), or sham operation, to ablate the ophthalmic branch of the trigeminal nerve. Blood vessel growth was detected by immunohistochemistry for PECAM-1 (CD31) following surgery. In another set of mice following TSE or sham operation, corneas were harvested for ELISA (VEGFR3 and pigment epithelium-derived factor [PEDF]) and for quantitative RT-PCR (VEGFR3, PEDF, and CD45). PEDF, VEGFR3, beta-3 tubulin, CD45, CD11b, and F4/80 expression in the cornea were evaluated using immunostaining. RESULTS: No nerves were detected in the areas subject to corneal neovascularization, whereas they persisted in the areas that were neovessel-free. Conversely, 7 days after denervation, significant angiogenesis was detected in the cornea, and this was associated with a significant decrease in VEGFR3 (57.5% reduction, P = 0.001) and PEDF protein expression (64% reduction, P < 0.001). Immunostaining also showed reduced expression of VEGFR3 in the corneal epithelial layer. Finally, an inflammatory cell infiltrate, including macrophages, was observed. CONCLUSION: Our data suggest that sensory nerves and neovessels inhibit each other in the cornea. When vessel growth is stimulated, nerves disappear and, conversely, denervation induces angiogenesis. This phenomenon, here described in the eye, may have far-reaching implications in understanding angiogenesis.

Safety and efficacy of the multitargeted receptor kinase inhibitor pazopanib in the treatment of corneal neovascularization.

PURPOSE: To evaluate the safety and efficacy of topical pazopanib in the treatment of corneal neovascularization (CNV). METHODS: Twenty eyes of 20 patients with stable CNV were enrolled in a prospective, open label, noncomparative study and treated with topical pazopanib 0.5% for 3 weeks, and followed for 12 weeks. The primary endpoint was to determine the tolerability and safety of topical pazopanib in the treatment of CNV defined by the occurrence of ocular and systemic adverse events during the study. The secondary endpoint was to evaluate the effect of topical pazopanib on the reduction of (1) neovascular area (NA), defined as the area of the corneal vessels themselves, (2) invasion area (IA), defined as the fraction of the total cornea into which the vessels extend, (3) vessel length (VL), defined as the mean measurement of the extent of vessels from end to end, and (4) vessel caliber (VC), defined as the mean diameter of the corneal vessels. RESULTS: There were no severe adverse events following the use of topical pazopanib. Compared with the baseline visit, NA and VL showed a statistically significant decrease at week 3 (P = 0.02 and 0.01, respectively); and NA, IA, and VL statistically significantly decreased at week 12 (P = 0.03, 0.04, and <0.01, respectively). Visual acuity maintained without changes after the 12 week follow-up. CONCLUSIONS: This preliminary study suggests that topical treatment with pazopanib 0.5% is safe, well tolerated, and may have a role as an alternative for the treatment of CNV (ClinicalTrials.gov number, NCT01257750).

PDE4 inhibition suppresses IL-17-associated immunity in dry eye disease.

PURPOSE: To determine the effect of phosphodiesterase type-4 (PDE4) inhibition on IL-17-associated immunity in experimental dry eye disease (DED). METHODS: Murine DED was induced, after which a PDE4 inhibitor (cilomilast), dexamethasone, cyclosporine, or a relevant vehicle was administered topically. Real-time PCR, immunohistochemical staining, and flow cytometry were employed to evaluate the immuno-inflammatory parameters of DED with a focus on IL-17-associated immunity. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease progression. RESULTS: DED induction increased proinflammatory cytokine expression, pathogenic immune cell infiltration, and CFS scores. Cilomilast significantly decreased the expression of TNF-α in the cornea (P ≤ 0.05) and IL-1α, IL-1ß, and TNF-α in the conjunctiva (P ≤ 0.05) as compared with vehicle control. Cilomilast treatment markedly decreased the presence of CD11b+ antigen-presenting cells in the central and peripheral cornea (P ≤ 0.05), and led to decreased conjunctival expression of cytokines IL-6, IL-23, and IL-17 (P ≤ 0.05). Moreover, cilomilast decreased the expression of IL-17 and IL-23 in the draining lymph nodes (P ≤ 0.05). Topical cilomilast was significantly more effective than vehicle at reducing CFS scores (P ≤ 0.05). The therapeutic efficacy of cilomilast was comparable or superior to that of dexamethasone and cyclosporine in all tested measures. CONCLUSIONS: Topical cilomilast suppresses the generation of IL-17-associated immunity in experimental DED.

b-FGF induces corneal blood and lymphatic vessel growth in a spatially distinct pattern.

PURPOSE: To study the spatial variances in ligand expression and angiogenic effect in response to the inflammatory response induced by basic fibroblast growth factor (b-FGF). METHODS: b-FGF micropellets (80 ng) were implanted in the temporal side of the cornea of Balb/c mice. On days 1, 3, and 7, blood (heme-) and lymphangiogenesis were observed by immunofluorescence staining of corneal flat mounts with LYVE-1 and CD31 to identify lymphatic and blood vessels, respectively. A second group of corneas were harvested for quantitative real-time polymerase chain reaction. Each cornea was divided into 2 different areas: (1) pre-pellet area and (2) opposite-pellet area. Expression of vascular endothelial growth factor (VEGF) ligands was evaluated using real-time polymerase chain reaction in each respective zone. RESULTS: Blood vessels grew into the cornea from the pre-pellet area, whereas corneal lymphatic vessels grew from the opposite-pellet area toward the center of the cornea. VEGF-A was upregulated in the pre-pellet, whereas VEGF-D expression was mostly observed in the opposite-pellet area. VEGF-C level increased simultaneously in both areas. CONCLUSIONS: A single inducing factor, that is, b-FGF, may simultaneously provoke hemangiogenesis and lymphangiogenesis in different locations of the cornea through differential expression of VEGF ligands. This distinctive spatial pattern should be considered while evaluating the corneal predilection for inflammation beyond that which is directly visible by slit lamp examination.

Vascular endothelial growth factor-C promotes alloimmunity by amplifying antigen-presenting cell maturation and lymphangiogenesis.

PURPOSE: To investigate the role of anti-vascular endothelial growth factor (VEGF)-C therapy in corneal graft survival and concomitant suppression of hem- and lymph-angiogenesis. METHODS: Corneal suture model in BALB/c mice was placed and immunohistochemical staining was performed with CD31/PECAM-1 and LYVE-1 to quantify the level of blood and lymphatic vessels. Corneal transplants were done in BALB/c mice from C57BL/6 mice donors; grafts were subsequently scored for opacity. VEGF-C was blocked in the angiogenesis and transplant model using neutralizing monoclonal anti-VEGF-C (VGX-100) by intraperitoneal injection. To determine the function of VEGF-C in maturation of antigen-presenting cells (APCs), bone marrow-derived dendritic cells were generated and matured in the presence or absence of VEGF-C. RESULTS: VEGF-C expression was demonstrated to be markedly upregulated in corneal graft rejection. VEGF-C blockade, through administration of a VEGF-C blocking monoclonal antibody, suppresses corneal angiogenic responses, inhibits trafficking and maturation of APCs, and significantly improves allotransplant survival. CONCLUSIONS: These data suggest VEGF-C as a potentially important target in corneal transplant pharmacotherapy and immunobiology.

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.

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes.

Lymphangiogenesis plays an important role in tumor metastasis and transplant outcome. Here, we show that thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein and naturally occurring inhibitor of angiogenesis inhibits lymphangiogenesis in mice. Compared with wild-type mice, 6-mo-old TSP-1-deficient mice develop increased spontaneous corneal lymphangiogenesis. Similarly, in a model of inflammation-induced corneal neovascularization, young TSP-1-deficient mice develop exacerbated lymphangiogenesis, which can be reversed by topical application of recombinant human TSP-1. Such increased corneal lymphangiogenesis is also detected in mice lacking CD36, a receptor for TSP-1. In these mice, repopulation of corneal macrophages with predominantly WT mice via bone marrow reconstitution ameliorates their prolymphangiogenic phenotype. In vitro, exposure of WT macrophages to TSP-1 suppresses expression of lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and VEGF-D, but not of a primarily hemangiogenic factor VEGF-A. Inhibition of VEGF-C is not detected in the absence or blockade of CD36. These findings suggest that TSP-1, by ligating CD36 on monocytic cells, acts as an endogenous inhibitor of lymphangiogenesis.

Interferon-γ-secreting NK cells promote induction of dry eye disease.

NK cells have been increasingly reported to be an important effector in autoimmune diseases. However, nothing is known in this regard in DED, the most common eye pathology, which is characterized by sustained inflammation on the ocular surface. In the present study, we have examined the profile of NK cells on the ocular surface as well as in the draining lymphoid tissues during the development of this disease. Our data demonstrate activated NK cells during the disease-induction phase. Moreover, in vivo depletion of NK cells in mice results in reduced disease severity and diminished proinflammatory cytokines. Furthermore, we show that NK cells are also able to modulate the maturation of APCs, which is correlated with IFN-γ from NK cells. Together, our findings provide new in vivo evidence that IFN-γ-secreting NK cells can promote induction of DED via direct target tissue damage and indirect influence on the priming phase of an adaptive immune response in secondary lymphoid tissue.

Effect of topical azithromycin on corneal innate immune responses.

PURPOSE: To determine the effect of azithromycin (AZM) in a murine model of corneal inflammation. METHODS: The effect of topical AZM was studied in murine corneal inflammation. Corneal inflammation was induced by thermal cautery in BALB/c mice. Leukocyte infiltration at different time points was analyzed by flow cytometry. At set time points, real-time polymerase chain reaction was performed to quantify the expression of different inflammatory cytokine transcript in the cornea. Corneal samples were analyzed immunohistochemically for the expression of intercellular adhesion molecule-1 (ICAM-1). Corneal neovascularization (CNV) was induced by micropellet (VEGF-A) placement. Mice were then treated topically with either AZM or vehicle. CNV was evaluated morphometrically. RESULTS: Eyes receiving AZM showed a significant decrease in corneal infiltration compared with the vehicle-treated group. AZM also significantly decreased messenger RNA expression levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and ICAM-1 in the cornea. There was no significant difference in CNV between the AZM- and vehicle-treated groups. CONCLUSIONS: After an inflammatory insult, topical AZM significantly reduced leukocyte infiltration into the cornea. This was further supported by an associated decrease in expression of IL-1ß, TNF-α, and ICAM-1 in the cornea, indicating AZM may have a potential anti-inflammatory effect on corneal inflammation.

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.