Areum Yeo

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.

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.

Langerhans cells prevent subbasal nerve damage and upregulate neurotrophic factors in dry eye disease

The functional role of Langerhans cells (LCs) in ocular surface inflammation and nerve damage in dry eye (DE) disease has yet to be determined. This study was performed to investigate this relationship through both clinical study on DE patients and in vivo mouse models with induced DE disease. In a cross-sectional case-control study (54 eyes of DE patients; 34 eyes of control patients), average cell density, area, and process length of LCs were measured using confocal microscopy. Data were analyzed to determine whether changes in LCs are correlated with subbasal nerve plexus (SNP) parameters (nerve density, beading, and tortuosity). In DE patients, SNP density marginally decreased and nerve beading and tortuosity were significantly increased compared to the control group. The total number of LCs significantly increased in DE patients, and some LCs with elongated processes were found to be attached to nerve fibers. Interestingly, nerve loss and deformation were correlated with inactivation of LCs. In an in vivo experiment to elucidate the role of LCs in ocular surface inflammation and corneal nerve loss, we used a genetically modified mouse model (CD207-DTR) that reduced the population of CD207 (Langerin) expressing cells by injection of diphtheria toxin. In CD207-depleted mice with DE disease (CD207-dDTR+DE), corneal nerves in the central region were significantly decreased, an effect that was not observed in wild-type (WT)+DE mice. In CD207-dDTR+DE mice, infiltration of CD4+, CD19+, CD45+, and CD11b+ cells into the ocular surface was increased, as confirmed by flow cytometry. Increased IL-17 and IFN-γ mRNA levels, and decreased expression of neurotrophic factors and neurotransmitters, were also found in the CD207-dDTR+DE mice. These data support a functional role for LCs in negatively regulating ocular surface inflammation and exhibiting a neuroprotective function in DE disease.

The Effect of TNF-α Blocker HL036337 and Its Best Concentration to Inhibit Dry Eye Inflammation

Purpose Dry eye syndrome is commonly thought of as an inflammatory disease, and we have previously presented data showing the effectiveness of topical TNF-α blocker agents for the treatment of this condition. The purpose of this study was to investigate the effectiveness of the TNF-α blocking agent HL036337 compared to cyclosporine A for the treatment of dry eye induced inflammation in order to establish whether HL036337 represents a more effective method for suppressing inflammation. The efficacy of HL036337 and cyclosporine A was determined using an experimental murine dry eye model. Methods The TNF-α blocker HL036337 is a modified form of TNF receptor I. Using dry eye induced C57BL/6 mice (n = 45), corneal erosion was measured at day 4 and 7 after topical treatment with cyclosporine A or HL036337. To determine the effective treatment dose, 0.25, 0.5, 1, 2.5, and 5 mg/mL of HL036337 were topically administered twice per day to dry eye induced murine corneas for 1 week. Results The optimal concentration of the TNF-α blocker HL036337 for treatment of dry eye induced corneal erosion was determined to be 1 mg/mL. Dry eye induced corneal erosion was improved after 1 week with topically applied cyclosporine A and HL036337 at 1 mg/mL. Conclusions HL036337 administered topically at 1 mg/mL effectively improved corneal erosion induced by dry eye. This finding may also suggest that inhibition of TNF-α can improve dry eye syndrome.

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.

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.

Comparison of Ocular Surface Mucin Expression After Topical Ophthalmic Drug Administration in Dry Eye-Induced Mouse Model

PURPOSE To determine the mucinogenic effect of dry eye (DE) treatment drugs currently in use, we compared the levels of mucin production and inflammatory cytokine expression on the ocular surfaces using a DE-induced mice model. METHODS C57BL/6 mice were separated into 6 groups: a control group, DE-induced mice with the vehicle and treated with cyclosporine A (CsA), rebamipide (Reb), diquafosol tetrasodium (DQS), or prednisolone (Pred). The mRNA expression of MUC 1, 4, 16, 5AC, and proinflammatory cytokines on the corneal epithelia were determined by quantitative real-time polymerase chain reaction. Expression of each MUC was evaluated using flow cytometry and immunohistostaining. Conjunctival goblet cells were analyzed through periodic acid-Schiff (PAS) staining. RESULTS Desiccating stress significantly decreased both mRNA and protein levels of all MUCs in the cornea. CsA mainly enhanced MUC5AC, with an increase in PAS-positive cells, whereas DQS chiefly increased membrane-associated mucins (MM). However, Reb only minimally increased expression of MUC5AC and Pred only increased MUC4. MUC16 did not show any significant change in any group. On the contrary, the mRNA levels of interleukin (IL)-1β, -6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were increased in the DE corneas of the control mice and were reduced by all treatments; in particular, IL-6 was significantly suppressed. CONCLUSION Topical DQS and CsA not only ameliorated ocular surface inflammation under desiccating stress but also upregulated both MM and secretory mucins (SM) and contributed to conjunctival goblet cell recovery, compared to Reb and Pred. Both anti-inflammatory and secretory factors should be considered simultaneously when measuring the treatment effect of DE drugs.