Person:

Dana, Reza

Recent experimental and clinical data suggest that there is a link between dry eye disease (DED) and T cell-mediated immunity. However, whether these immune responses are a consequence or cause of ocular surface inflammation remains to be determined. Thus far, only models of acute DED have been used to derive experimental data. This is in contrast to clinical DED which usually presents as a chronic disease. In the present study, using a murine model of chronic DED, it was established that the chronic phase of the disease is accompanied by Th17 responses at the ocular surface, and that a significant memory T cell population can be recovered from chronic DED. This memory response is predominantly mediated by Th17 cells. Moreover, adoptive transfer of this memory T cell population was shown to induce more severe and rapidly progressing DED than did the adoptive transfer of its effector or naïve counterparts. Not only do these results clearly demonstrate that effector memory Th17 cells are primarily responsible for maintaining the chronic and relapsing course of DED, but they also highlight a potentially novel therapeutic strategy for targeting memory immune responses in patients with DED.

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. Here 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.

Certain components of a graft that provoke alloimmunity may not be vital for graft function or critical as targets of rejection. Corneal transplantation is an example of this since graft epithelium plays a role in allosensitization, while corneal graft endothelium—which shares the same alloantigens—is the critical target in allorejection. In this study, we found that exploiting this biology by replacing donor epithelium of an allograft with an allodisparate 3rd-party epithelium yields a marked enhancement in transplant survival. Such “chimeric” allografts consisted of a C3H/He (H-2k) corneal epithelium over a C57BL/6 (H-2b) epithelial-denuded cornea (or v.v.) and orthotopically placed on BALB/c (H-2d) hosts. Conventional corneal allografts (C3H/He, or C57BL/6) or isografts (BALB/c) were also transplanted on BALB/c hosts. Alloreactive T cell frequencies (CD4+ IFN-gamma+) primed to graft endothelium were strongly diminished in chimeric relative to conventionally allografted hosts. This was corroborated by decreased T cell infiltration (p=0.03) and a marked enhancement of allograft survival (p=0.001). Our results represent the first successful demonstration of chimeric tissue, epithelial-denuded allograft plus 3rd-party allodisparate epithelium, in the promotion of allograft survival. Moreover, chimeric grafting can be readily performed clinically, whereby corneal allograft rejection remains a significant problem particularly in inflamed graft beds.

Purpose

In the present study, the authors developed novel models to stimulate blood vessel formation (hemangiogenesis) versus lymphatic vessel formation (lymphangiogenesis) in the cornea.

Methods

Micropellets loaded with high-dose (80 ng) or low-dose (12.5 ng) basic fibroblast growth factor (bFGF) were placed in BALB/c corneas. Angiogenic responses were analyzed by immunohistochemistry to quantify blood neovessels (BVs) and lymphatic neovessels (LVs) to 3 weeks after implantation. Areas covered by BV and LV were calculated and expressed as a percentage of the total corneal area (percentage BV and percentage LV). Hemangiogenesis (HA) and lymphangiogenesis (LA) were also assessed after antibody blockade of VEGFR-2 or VEGFR-3

Results

Although high-dose bFGF stimulation induced a more potent angiogenic response, the relative LV (RLV = percentage LV/percentage BV × 100) was nearly identical with high- and low-doses of bFGF. Delayed LA responses induced 3 weeks after implantation of high-dose bFGF resulted in a lymphatic vessel-dominant phenotype. Interestingly, the blockade of VEGFR-2 significantly suppressed BV and LV. However, the blockade of VEGFR-3 inhibited only LV (P = 0.0002) without concurrent inhibition of BV (P = 0.79), thereby resulting in a blood vessel-dominant phenotype

Conclusions

An HA-dominant corneal phenotype can be obtained in BALB/c mice 2 weeks after implantation of an 80-ng bFGF micropellet with VEGFR-3 blockade. Alternatively, an LA-dominant corneal phenotype can be obtained 3 weeks after implantation of an 80-ng bFGF micropellet without supplementary modulating agents. These models will be useful in evaluating the differential contribution of BV and LV to a variety of corneal abnormalities, including transplant rejection, wound healing and microbial keratitis.

Dry eye disease (DED), an inflammatory autoimmune disorder affecting the ocular surface, degrades visual performance and the quality of life of more than 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 which is associated specifically with Treg dysfunction and disease pathogenesis, and suggest a new target for dry eye therapy.

Purpose

To develop and compare effective strategies for depleting graft-derived passenger leukocytes which include antigen-presenting cells from corneal buttons, and to assess the effectiveness of this strategy in promoting graft survival using a high-risk (HR) model of corneal transplantation.

Methods

Corneal buttons harvested from C57BL/6 mice were used in three ex vivo strategies of passenger leukocyte depletion. Two strategies involved storage in Optisol-GS medium at different temperatures for prolonged periods. A third strategy utilized complement-dependent cytotoxicity (CDC) by treating the buttons with anti-CD45 mAb plus complement. Whole-mount corneal buttons or cells from enzyme digested corneas were analyzed using confocal microscopy or flow cytometry, respectively, for the pan-leukocyte surface marker CD45. HR host beds were created and used to evaluate the efficacy of passenger leukocyte depletion on transplant survival.

Results

Passenger leukocyte numbers in the buttons were significantly reduced by all three treatments. CDC was the most efficient strategy for passenger leukocyte depletion with 39% reduction (P < 0.00005) of CD45+ cells, and negligible damage to the endothelial layer, achievable within 24 h. However, passenger leukocyte depletion failed to improve HR graft longevity.

Conclusions

Anti-CD45 antibody plus complement-mediated targeting of donor tissue is the most efficient way to deplete corneal passenger leukocytes and can considerably reduce the time required for cell depletion. However, depletion of graft passenger leukocytes does not have a significant effect on promoting graft survival even in the HR setting.

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-1β, 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-α, IL-1α, IL-1β, VEGF-A, VEGF-C, and VEGFR2. Animals treated with these mediators had significantly suppressed suture-induced or IL-1β–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.

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.

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-1β (IL-1β), 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-1β, TNF-α, and ICAM-1 in the cornea, indicating AZM may have a potential anti-inflammatory effect on corneal inflammation.

Purpose.

In addition to Langerhans cells (LCs), other dendritic cells (CD11c+) have recently been shown to express Langerin (c-type lectin). In skin, (non-LC) Langerin+ dendritic cells initiate adaptive immunity. However, whether such dendritic cells (DC) reside in the cornea, an immune-privileged tissue, is unknown.

Methods.

Normal C57BL/6 corneas were harvested for qRT-PCR analyses of Langerin expression in the epithelium versus stroma. Immunohistochemistry for Langerin was also performed. Single-cell preparations of epithelium versus stroma were FACS analyzed for CD11c, CD11b, and CD103 expression. Fluorescence microscopy of corneas from muLangerin-eGFP mice (in which all CD11c+ Langerin+ cells express eGFP), huLangerin-DTA mice (only LCs are constitutively deleted), and huLangerin-Cre eYFP-flox (only LCs express eYFP) was performed.

Results.

qRT-PCR, immunohistochemistry, and FACS analysis identified CD11c+ Langerin+ cells in the epithelium and stroma. Similarly, corneas of muLangerin-eGFP mice contained eGFP+ cells in the epithelium and stroma. However, FACS analysis indicated phenotypically differing CD11c+ Langerin+ populations in the epithelium (CD11blowCD103low) versus stroma (CD11b+CD103low). Additionally, corneas from huLangerin-DTA mice were devoid of Langerin+ cells in the epithelium but were detectable in the stroma. In corneas from huLangerin-Cre eYFP-flox, eYFP+ cells were detectable in the epithelium but not in the stroma.

Conclusions.

The normal corneal epithelium is endowed with CD11c+ Langerin+ cells that are LCs, whereas the stroma is endowed with a separate population of (non-LC) Langerin+ DCs. These findings should henceforth facilitate the examination of Langerin-expressing DC subsets in the immunopathogeneses of conditions such as keratoconjunctivitis sicca, allergic keratoconjunctivitis, and corneal allograft rejection.