Yong Woo Ji

Neutralization of Ocular Surface TNF-α Reduces Ocular Surface and Lacrimal Gland Inflammation Induced by In Vivo Dry Eye

PURPOSE The purpose of this study was to investigate the effectiveness of tumor necrosis factor (TNF)-α blocker for treatment of dry eye (DE)-induced inflammation and determine a more effective method to suppress lacrimal gland inflammation. Efficacy of topical versus systemic administration of TNF-α blockers was determined using a murine dry eye (DE) model. METHODS The TNF-α blocker HL036 was developed by modification of the TNF receptor I. Protein purity, binding affinity, and clearance of TNF-α was compared with etanercept. Using DE-induced C57BL/6 mice, corneal erosion and goblet cell counts were measured after subcutaneous or topical treatment with etanercept or HL036. Inflammatory cytokines in cornea and lacrimal glands were determined by quantitative RT-PCR and ELISA. RESULTS HL036 showed TNF-α binding affinity comparable to etanercept, as measured by surface plasmon resonance. HL036 concentration was significantly higher in cornea and anterior segment than etanercept and effectively eliminated TNF-α on ocular surfaces. Etanercept was preferentially concentrated in the posterior segment. Corneal erosion and goblet cell counts were improved only with topically applied etanercept and HL036. Ocular surface IFN-γ, IL-6, and IL-21 were significantly decreased by topical HL036. DE-induced lacrimal gland IFN-γ and IL-6 expression was effectively suppressed by topical etanercept and HL036. CONCLUSIONS Topical TNF-α blockers effectively suppressed lacrimal gland and corneal inflammation by suppressing IFN-γ, IL-21, and IL-6. Differences in TNF-α affinity, clearance, and local concentration of blockers may account for the anti-inflammatory effects in different ocular regions.

Pathogenesis and treatments of TGFBI corneal dystrophies

Transforming growth factor beta-induced (TGFBI) corneal dystrophies are a group of inherited progressive corneal diseases. Accumulation of transforming growth factor beta-induced protein (TGFBIp) is involved in the pathogenesis of TGFBI corneal dystrophies; however, the exact molecular mechanisms are not fully elucidated. In this review article, we summarize the current knowledge of TGFBI corneal dystrophies including clinical manifestations, epidemiology, most common and recently reported associated mutations for each disease, and treatment modalities. We review our current understanding of the molecular mechanisms of granular corneal dystrophy type 2 (GCD2) and studies of other TGFBI corneal dystrophies. In GCD2 corneal fibroblasts, alterations of morphological characteristics of corneal fibroblasts, increased susceptibility to intracellular oxidative stress, dysfunctional and fragmented mitochondria, defective autophagy, and alterations of cell cycle were observed. Other studies of mutated TGFBIp show changes in conformational structure, stability and proteolytic properties in lattice and granular corneal dystrophies. Future research should be directed toward elucidation of the biochemical mechanism of deposit formation, the relationship between the mutated TGFBIp and the other materials in the extracellular matrix, and the development of gene therapy and pharmaceutical agents.

Comparison of surface roughness and bacterial adhesion between cosmetic contact lenses and conventional contact lenses.

Objective: To compare physical characteristics of cosmetic contact lenses (Cos-CLs) and conventional contact lenses (Con-CLs) that might affect susceptibility to bacterial adhesion on the contact lens (CL) surface. Methods: Surface characteristics of Cos-CLs and Con-CLs made from the same material by the same manufacturer were measured by atomic force microscopy (AFM) and scanning electron microscopy. To determine the extent and rate of bacterial adhesion, Cos-CL and Con-CL were immersed in serum-free Roswell Park Memorial Institute media containing Staphylococcus aureus or Pseudomonas aeruginosa. Additionally, the rate of removal of adherent bacteria was evaluated using hand rubbing or immersion in multipurpose disinfecting solutions (MPDS). Results: The mean surface roughness (root mean square and peak-to-valley value) measured by AFM was significantly higher for Cos-CL than for Con-CL. At each time point, significantly more S. aureus and P. aeruginosa adhered to Cos-CL than to Con-CL, which correlated with the surface roughness of CL. In Cos-CL, bacteria were mainly found on the tinted surface rather than on the noncolored or convex areas. Pseudomonas aeruginosa attached earlier than S. aureus to all types of CL. However, P. aeruginosa was more easily removed from the surface of CL than S. aureus by hand rubbing or MPDS soaking. Conclusions: Increased surface roughness is an important physical factor for bacterial adhesion in Cos-CL, which may explain why rates of bacterial keratitis rates are higher in Cos-CL users in CL physical characteristics.

Dry Eye-Induced CCR7+CD11b+ Cell Lymph Node Homing Is Induced by COX-2 Activities

PURPOSE We aimed to determine the role of CCR7+CD11b+ cell lymph node (LN) homing and T-cell differentiation in dry eye (DE)-induced immunopathogenesis and investigate the therapeutic effects of cyclooxygenase-2 (COX-2) and prostaglandin E2/eicosanoid-prostanoid (PGE2/EP) inhibitors against DE. METHODS Six-week-old female C57BL/6 mice were housed in a controlled-environment chamber and administered topical selective COX-2 inhibitors or EP2 antagonists. Expression of major histocompatibility complex (MHC)-IIhigh, CD11b+, CCR7+, IFN-γ+, IL-17+, and CD4+ in the corneas and draining LNs was evaluated using flow cytometry. Mixed lymphocyte reactions (MLRs) with carboxyfluorescein diacetate succinimidyl ester labeling and intracellular cytokine staining were used to verify DE-induced corneal dendritic cell function. mRNA expression of COX-2, EPs, and proinflammatory cytokines in ocular surface was evaluated using quantitative RT-PCR and immunohistochemical staining. RESULTS Dry eye significantly increased MHC-IIhighCD11b+ and CCR7+CD11b+ cells in the cornea and LNs, and MLR revealed CCR7+CD11b+ cells from DE corneas stimulated IL-17+CD4+ cell proliferation. mRNA levels of COX-2, EP2, IFN-γ, TNF-α, IL-6, and IL-17 were significantly higher in DE ocular surface but were suppressed by topical COX-2 inhibitors and EP2-specific blockers. Immunohistochemical staining showed COX-2 and matrix metalloproteinase expression in DE corneal epithelia that was diminished by both topical treatments. Furthermore, both topical treatments significantly reduced frequencies of MHC-IIhigh, CD11b+, and CCR7+CD11b+ cells in the corneas and LNs, but also IL-17+CD4+ cells in LNs. CONCLUSIONS Topical COX-2/EP2 treatment reduces CCR7+CD11b+ cells on the ocular surface with inhibition of cellular LN homing and suppresses Th17 immune response, suggesting the COX-2/PGE2/EP axis contributes to immuno-inflammatory pathogenesis on the ocular surface and may be a novel therapeutic target in DE.

Automated Measurement of Tear Film Dynamics and Lipid Layer Thickness for Assessment of Non-Sjögren Dry Eye Syndrome With Meibomian Gland Dysfunction.

Purpose: To investigate automated values from an advanced corneal topographer with a built-in real keratometer, color camera, and ocular surface interferometer for the evaluation of non-Sjögren dry eye syndrome (NSDES) with meibomian gland dysfunction (MGD). Methods: Sixty-four patients (64 eyes) diagnosed with NSDES with MGD were enrolled. All eyes were evaluated using the Ocular Surface Disease Index (OSDI), fluorescence staining score, tear film breakup time (TBUT), Schirmer test, and MGD grade. Noninvasive Keratograph average tear film breakup time (NIKBUTav), tear meniscus height (TMHk), meibomian gland (MG) dropout grade, and lipid layer thickness (LLT) using interferometry were measured. Results: Among automated indexes, NIKBUTav (mean 7.68 ± 4.07 s) and the MG dropout grade (mean 1.0 ± 0.5) significantly correlated with the OSDI (mean 40.6 ± 22.9) (r = −0.337, P = 0.006; and r = 0.201, P = 0.023, respectively), as did all conventional indicators, except the Schirmer score (mean 9.1 ± 5.9 mm). TMHk (mean 0.21 ± 0.18 mm) had significant correlation with the Schirmer score, the staining score (mean 1.2 ± 0.7), TBUT (mean 3.8 ± 1.8 s), and NIKBUTav (r = 0.298, P = 0.007; r = −0.268, P = 0.016; r = 0.459, P < 0.001; and r = 0.439, P < 0.001, respectively), but not any MGD indicator, even the MG dropout grade. NIKBUTav showed significant correlations with all clinical parameters and other automated values, except the Schirmer score and LLT (mean 83.94 ± 20.82 nm) (all ≥ 0.25 and P < 0.01). The MG dropout grade highly correlated with all indexes except TMHk (all ≥ 0.25 and P < 0.05). LLT was significantly associated with TBUT, MGD grade (mean 2.0 ± 0.7), and MG dropout grade (r = 0.219, P = 0.047; r = −0.221, P = 0.039; and r = 0.433, P < 0.001, respectively), although it was not related to patient symptoms. Conclusions: Automated noninvasive measurements using an advanced corneal topographer and LLT measured with an ocular surface interferometer can be alternatives to conventional methods to evaluate tear conditions on the ocular surface; the former device can provide information about conformational MG changes in NSDES with MGD.

Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

Purpose By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs). Methods C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45+ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition. Results DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45+ cell infiltration in LGs. Likewise, CD45+ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice. Conclusions Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.

Gr-1intCD11b+ myeloid-derived suppressor cells accumulate in corneal allograft and improve corneal allograft survival

We identified the characteristics of myeloid‐derived suppressor cells (MDSCs) and investigated their mechanism of induction and their functional role in allograft rejection using a murine corneal allograft model. In mice, MDSCs coexpress CD11b and myeloid differentiation antigen Gr‐1. Gr‐1+CD11b+ cells infiltrated allografted corneas between 4 d and 4 wk after surgery; however, the frequencies of Gr‐1+CD11b+ cells were not different between accepted and rejected allografts or in peripheral blood or BM. Of interest, Gr‐1intCD11b+ cells, but not Gr‐1hiCD11b+ cells, infiltrated the accepted graft early after surgery and expressed high levels of immunosuppressive cytokines, including IL‐10, TGF‐β, and TNF‐related apoptosis‐inducing ligand. This population remained until 4 wk after surgery. In vitro, only high dose (>100 ng/ml) of IFN‐γ plus GM‐CSF could induce immunosuppressive cytokine expression in Gr‐1intCD11b+ cells. Furthermore, adoptive transfer of Gr‐1intCD11b+ cells reduced T cell infiltration, which improved graft survival. In conclusion, high‐dose IFN‐γ in allograft areas is essential for development of Gr‐1intCD11b+ MDSCs in corneal allografts, and subtle environmental changes in the early period of the allograft can result in a large difference in graft survival.

Meibomian gland dysfunction associated with periocular radiotherapy

Purpose: To investigate the influence of periocular radiotherapy on meibomian glands. Methods: We evaluated 28 patients (40 eyes) who received radiotherapy (RT group) for conjunctival or orbital lymphoma and 30 age-matched control subjects (60 eyes). Subjects underwent slit-lamp examination of the eyelids, Schirmer test, meibography, and evaluation of tear film breakup time (TBUT), Ocular Surface Disease Index (OSDI) scores, meibomian glands evaluation (meiboscore, meibum expressibility, and lid margin abnormality scores), and tear film lipid layer thickness using an ocular surface interferometer. These parameters were compared between subjects in the RT and control groups. Results: Meiboscores as well as meibum expressibility and OSDI scores in the RT group were significantly higher compared with those in the control group (1.6 ± 0.9 vs. 0.4 ± 0.6, 1.6 ± 1.0 vs. 0.2 ± 0.4, and 48.1 ± 21.4 vs. 6.2 ± 4.4, respectively, P < 0.001, all), whereas the Schirmer value (9.2 ± 5.1 vs. 12.3 ± 5.2, P = 0.004), TBUT (4.2 ± 2.5 vs. 6.4 ± 2.6, P = 0.001), and lipid layer thickness (61.0 ± 29.3 vs. 85.2 ± 20.0, P < 0.001) in the RT group were lower compared with those in the control group. The percentage of meibomian gland dropout was significantly correlated with age (P = 0.025) and total radiation dose (P = 0.012), regardless of the target location of irradiation. Even low-dose irradiated eyes (<30 Gy) exhibited significantly higher meiboscores (P < 0.001) and shorter TBUT (P = 0.005) compared with control eyes. Conclusions: Eyes that received periocular radiotherapy exhibited relatively high tear film instability induced by meibomian gland dysfunction, contributing to the high severity of dry eye symptoms.

Delayed Onset of Lattice Corneal Dystrophy Type IIIA Due to a Novel T621P Mutation in TGFBI

To the Editor: We recently identified a family with lattice corneal dystrophy type IIIA (LCD IIIA) associated with a novel threonine-to-proline (T621P) mutation in the transforming growth factor-beta induced (TGFBI) gene. The 50-year-old female proband (II-2) visited our clinic because of decreased visual acuity. Slit-lamp examination disclosed thick, branching, refractile, ropy lattice lines located in both stromal corneas, which extended to the limbus radially (Figures AA-AC, available in the online version of this article). The right cornea showed a higher number of lattice lines than the left cornea. Molecular analysis involving bidirectional complete sequencing demonstrated a novel T621P mutation in exon 14 of TGFBI. After we identified this new mutation, we performed genetic and clinical examinations on 10 other family members (Severance Hospital Institutional Review Board [No. 4-2014-1046]). The corneas of elderly affected members (I-1, II-1, and II-2) showed the LCD IIIA phenotype with asymmetry between both corneas, as reported for LCD IIIA. Three family members in their mid-twenties (III-1, III2, and III-4) had clear corneas despite having the mutated TGFBI gene (Figure AD). This suggests that the family had a late-onset form of LCD IIIA. The 26-year-old son of the proband (III-3), who had undergone LASIK 5 years earlier at a different facility, maintains clear corneas and showed no mutation in the recent genetic test. He was aware that his grandmother and mother had visual problems but was not aware of the genetic basis of the condition. Therefore, he did not notify the surgeon of any visual problems of the family before the LASIK procedure because he was not asked about them. The relatively late onset of LCD IIIA is favorable for young affected individuals in that visual acuity is well preserved until advanced age. However, the III-3 family member, who is the only one having the normal gene among the four genetically examined cousins, highlights the worrying possibility of young adults harboring the mutation but having clear corneas and undergoing LASIK.

Corneal lymphangiogenesis facilitates ocular surface inflammation and cell trafficking in dry eye disease

PURPOSE While the normal cornea has limited innervation by the lymphatic system, chronic immune-inflammatory disorders such as dry eye (DE) can induce lymphangiogenesis in the ocular surface. Using a conditional knock-down murine model, Lyve-1Cre;VEGFR2flox mice, this study investigated the role of lymphangiogenesis in the pathophysiology of DE. METHODS DE was induced in both wild type (WT) B6 and Lyve-1Cre;VEGFR2flox mice. Tissue immunostaining and volumetric gross measurements were used to assess changes in the ocular surface, skin, and lymph nodes (LNs). The expression of lymphangiogenic factors (TNF-α, IL-6/-8/-12/-17, VEGF-C/-D, IFN-γ, VEGFR-2/-3, Lyve-1, and podoplanin) and the frequency of immune cells (CD4, CD11b, and CD207) on the ocular surface and lacrimal glands were quantified by real-time polymerase chain reaction and flow cytometry. RESULTS Compared to WT mice, there were fewer lymphatic vessels and a reduction in lymphangiogenic markers in the ocular surface and skin of Lyve-1Cre;VEGFR2flox mice. After DE induction, mRNA levels of TNF-α, IL-8, and IFN-γ were significantly reduced in Lyve-1Cre;VEGFR2flox mice compared to WT mice (p < .01). Surprisingly, the LNs from Lyve-1Cre;VEGFR2flox mice with DE were significantly smaller and populated by fewer dendritic cells and effector T cells than those from WT mice (p < .001). Furthermore, immunostaining showed corneal nerves in the DE-induced Lyve-1Cre;VEGFR2flox mice were notably intact like in the naïve condition. CONCLUSIONS Inhibition of lymphangiogenesis in the cornea effectively attenuates not only the inflammatory response including trafficking of immune cells but also preserves corneal nerves under desiccating stress. Corneal lymphangiogenesis might be a contributing factor in deterioration on the ocular surface homeostasis.