Reza Dana

Dry Eye Disease: An Immune-Mediated Ocular Surface Disorder

Dry eye disease is a multifactorial disorder of the tears and ocular surface characterized by symptoms of dryness and irritation. Although the pathogenesis of dry eye disease is not fully understood, it is recognized that inflammation has a prominent role in the development and propagation of this debilitating condition. Factors that adversely affect tear film stability and osmolarity can induce ocular surface damage and initiate an inflammatory cascade that generates innate and adaptive immune responses. These immunoinflammatory responses lead to further ocular surface damage and the development of a self-perpetuating inflammatory cycle. Herein, we review the fundamental links between inflammation and dry eye disease and discuss the clinical implications of inflammation in disease management.

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.

Topical Bevacizumab in the Treatment of Corneal Neovascularization Results of a Prospective, Open-Label, Noncomparative Study

OBJECTIVE To study the safety and efficacy of topical bevacizumab in the treatment of corneal neovascularization (NV). DESIGN In a prospective, open-label, noncomparative study, 10 eyes from 10 patients with stable corneal NV were treated with topical bevacizumab, 1.0%, for 3 weeks and followed up for up to 24 weeks. MAIN OUTCOME MEASURES The primary safety variables were the occurrence of ocular and systemic adverse events throughout the course of the study. The primary efficacy variables were neovascular area, the area of the corneal vessels themselves; vessel caliber, the mean diameter of the corneal vessels; and invasion area, the fraction of the total corneal area covered by the vessels. RESULTS From baseline visit to the last follow-up visit, mean reductions were 47.1% (standard deviation [SD], 36.7%) for neovascular area, 54.1% (SD, 28.1%) for vessel caliber, and 12.2% (SD, 42.0%) for invasion area. The decreases in neovascular area and vessel caliber were statistically significant (P= .001 and P< .001, respectively). However, changes in invasion area did not achieve statistical significance (P= .19). Visual acuity and central corneal thickness showed no significant changes. Topical bevacizumab was well tolerated with no adverse events. CONCLUSIONS Short-term topical bevacizumab therapy reduces the severity of corneal NV without local or systemic adverse effects. APPLICATION TO CLINICAL PRACTICE Topical bevacizumab provides an alternative therapy in the treatment of stable corneal NV. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00559936.

Corneal sensation and subbasal nerve alterations in patients with herpes simplex keratitis: an in vivo confocal microscopy study.

PURPOSE To study and correlate corneal sensation in patients with herpes simplex keratitis (HSK) with density and morphologic features of subbasal corneal nerves by in vivo confocal microscopy (IVCM). DESIGN Prospective, cross-sectional, controlled, single-center study. PARTICIPANTS Thirty-one eyes with the diagnosis of acute (n = 7) or chronic (n = 24) HSK and their contralateral clinically unaffected eyes were studied and compared with normal controls (n = 15). METHODS In vivo confocal microscopy (Confoscan 4; Nidek Technologies, Gamagori, Japan) and corneal esthesiometry (Cochet-Bonnet; Luneau Ophthalmlogie, Chartres, France) of the central cornea were performed bilaterally in all patients and controls. Patients were grouped into normal (> 5.5 cm), mild (> 2.5-5.5 cm), and severe (≤ 2.5 cm) loss of sensation. MAIN OUTCOME MEASURES Changes in corneal nerve density, total nerve number, main nerve trunks, branching, and tortuosity were evaluated after IVCM and were correlated to corneal sensation, disease duration, and number of recurrences. RESULTS Herpes simplex keratitis eyes, as compared with controls, demonstrated significant (P < 0.001) decrease in mean nerve density (448.9 ± 409.3 vs. 2258.4 ± 989.0 μm/frame), total nerve number (5.2 ± 4.5 vs. 13.1 ± 3.8), main nerve trunks (2.3 ± 1.6 vs. 4.7 ± 1.2), and nerve branches (3.2 ± 4.3 vs. 9.8 ± 3.3). In contralateral unaffected eyes, mean nerve density (992.7 ± 465.0 μm/frame), total nerve number (7.8 ± 3.3), and branches (4.5 ± 2.3) were decreased significantly as compared with controls (P < 0.002). Reduced nerve density, total nerve count, and main trunks in HSK eyes were correlated significantly with corneal sensation across all subgroups (P < 0.001). Nerve density decreased within days of infection and was correlated to frequency of episodes in patients with HSK (P < 0.02). CONCLUSIONS In vivo confocal microscopy revealed that the loss of corneal sensation in HSK correlates strongly with profound diminishment of the subbasal nerve plexus after herpes simplex virus infection. Surprisingly, the contralateral, clinically unaffected eyes also demonstrated a diminishment of the subbasal nerve plexus as compared with normal subjects, revealing bilateral nerve alteration in an apparently unilateral disease.

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.

Contribution of Macrophages to Angiogenesis Induced by Vascular Endothelial Growth Factor Receptor-3-Specific Ligands

Vascular endothelial growth factor receptor (VEGFR)-2 is a major stimulator of hemangiogenesis (HA), whereas VEGFR-3 stimulates lymphangiogenesis (LA). Contrary to this understanding, we demonstrate that implantation of pellets containing VEGFR-3-specific ligands (VEGF-C156S and recombinant murine VEGF-D) into the corneal stroma induce not only LA but also robust HA characterized by blood vessels that are positive for VEGFR-3 expression. The implantation of pellets containing VEGFR-3-specific ligands also leads to the recruitment of VEGF-A-secreting macrophages. Depletion of these infiltrating macrophages using clodronate-liposome administration shows a significant reduction in HA as well as LA. Blockade of either VEGFR-2 or VEGFR-3 signaling reduces both HA and LA; however, the percent reduction of HA is greater in the VEGFR-2 blockade group. In addition, in the VEGFR-3 blockade group, the percent reduction of HA is significantly greater with VEGFR-3-specific ligands than that by VEGF-A or VEGF-C. Collectively, our data suggest that VEGFR-3-specific signaling can induce new blood vessels, to which macrophages contribute a major role, and signify its potential as an additional therapeutic target to the existing VEGF-A/VEGFR-2 signaling-based antiangiogenesis strategies.

Ocular Graft-versus-Host Disease: A Review

Graft-versus-host disease (GVHD) is a common cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Systemic findings involving the skin, gastrointestinal tract, and liver often overshadow the other manifestations of GVHD. Ocular surface disease remains the most common cause of long-term morbidity in GVHD. Herein, the etiology, pathophysiology, clinical manifestations, and treatment of acute and chronic systemic GVHD are reviewed, with a focus on ocular GVHD.