Jin Suk Ryu

Intraperitoneal Infusion of Mesenchymal Stem/Stromal Cells Prevents Experimental Autoimmune Uveitis in Mice

Autoimmune uveitis is one of the leading causes of blindness. We here investigated whether intraperitoneal administration of human mesenchymal stem/stromal cells (hMSCs) might prevent development of experimental autoimmune uveitis (EAU) in mice. Time course study showed that the number of IFN-γ- or IL-17-expressing CD4+ T cells was increased in draining lymph nodes (DLNs) on the postimmunization day 7 and decreased thereafter. The retinal structure was severely disrupted on day 21. An intraperitoneal injection of hMSCs at the time of immunization protected the retina from damage and suppressed the levels of proinflammatory cytokines in the eye. Analysis of DLNs on day 7 showed that hMSCs decreased the number of Th1 and Th17 cells. The hMSCs did not reduce the levels of IL-1β, IL-6, IL-12, and IL-23 which are the cytokines that drive Th1/Th17 differentiation. Also, hMSCs did not induce CD4+CD25+Foxp3+ cells. However, hMSCs increased the level of an immunoregulatory cytokine IL-10 and the population of IL-10-expressing B220+CD19+ cells. Together, data demonstrate that hMSCs attenuate EAU by suppressing Th1/Th17 cells and induce IL-10-expressing B220+CD19+ cells. Our results support suggestions that hMSCs may offer a therapy for autoimmune diseases mediated by Th1/Th17 responses.

Topical TSG-6 Administration Protects the Ocular Surface in Two Mouse Models of Inflammation-Related Dry Eye.

PURPOSE To investigate the therapeutic potential of TNF-α stimulated gene/protein (TSG)-6 in two mouse models of inflammation-mediated dry eye syndrome (DES). METHODS We created inflammation-mediated DES in mice by injecting concanavalin A (ConA; 10 mg/mL) into intraorbital and extraorbital lacrimal glands. Recombinant TSG-6 (1 μg in phosphate-buffered solution [PBS]) or the same volume of PBS was administered topically to eyes of the mice four times a day (QID) for 1 week. In parallel experiments, we topically applied TSG-6 (1 μg) or PBS QID to eyes of 12-week-old NOD.B10.H2b mice, a model for primary Sjögrens syndrome. Seven days later, tear production was measured, and the corneal surface was observed for epithelial defects. The number of goblet cells was evaluated in the forniceal conjunctiva. The levels of proinflammatory cytokines were analyzed in the cornea, conjunctiva, and lacrimal glands. Also, in vitro experiments were performed using cultures of corneal epithelial cells (CECs) to test the effects of TSG-6 on cell proliferation and migration. RESULTS Topical TSG-6 administration improved tear production and reduced corneal epithelial defects both in ConA-injected mice and NOD.B10.H2b mice. The conjunctival goblet cell density was higher in TSG-6-treated eyes than in PBS-treated eyes. The expression of proinflammatory cytokines in the cornea, conjunctiva, and intraorbital gland was repressed by TSG-6, while the levels of proinflammatory cytokines in the extraorbital gland were not changed. In vitro experiments revealed that TSG-6 promoted the migration of CECs, but did not affect the proliferation. CONCLUSIONS Topical TSG-6 protected the ocular surface by suppressing inflammation and promoting corneal epithelial wound healing.

Comparison of Topical Application of TSG-6, Cyclosporine, and Prednisolone for Treating Dry Eye.

Purpose: To compare the therapeutic effects of topical tumor necrosis factor (TNF)-&agr;–stimulated gene/protein-6 (TSG-6) with those of cyclosporine and prednisolone eye drops in NOD.B10.H2b mice, a model for inflammation-mediated dry eye. Methods: The 12-week-old NOD.B10.H2b mice were topically administered recombinant TSG-6 (0.1%) 4 times a day, 0.05% cyclosporine (Restasis) twice a day, or 1% prednisolone (Pred Forte) 4 times a day for 1 week. Aqueous tear production was measured by phenol red thread test, and corneal epithelial damage was observed with lissamine green and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Conjunctival goblet cell number was evaluated with periodic acid–Schiff staining. The levels of inflammatory cytokines were analyzed in the ocular surface (cornea and conjunctiva) and intraorbital gland. The dose-dependent effects of topical TSG-6 (0.001, 0.01, and 0.1%) were tested. Results: Tear production and goblet cell density were significantly increased in all groups receiving TSG-6, cyclosporine, and prednisolone. Corneal epithelial staining was markedly reduced by TSG-6 and cyclosporine but not by prednisolone. In prednisolone-treated eyes, corneal epithelial thickness was decreased, and apoptosis of corneal epithelial cells was increased. The levels of interferon gamma and TNF-&agr; in the ocular surface and intraorbital gland were significantly repressed by TSG-6 and cyclosporine, and prednisolone treatment significantly reduced the level of interferon gamma. The effects of TSG-6 on the ocular surface and tear production were dose dependent. Conclusions: Topical TSG-6 was as effective in inflammation-mediated dry eye as cyclosporine eye drops. Topical prednisolone suppressed inflammation but induced apoptosis in the corneal epithelium.

Prednisolone induces apoptosis in corneal epithelial cells through the intrinsic pathway

Glucocorticoid eye drops are one of the most widely used medications in ophthalmology. However, little is known about the effects of glucocorticoids on corneal epithelial cells that are directly exposed to topically-administered glucocorticoids. Here we investigated the effects of prednisolone, a synthetic glucocorticoid analogue frequently used in the clinic, on corneal epithelial cells. Results showed that prednisolone decreased survival of corneal epithelial cells by inhibiting proliferation and inducing apoptosis in a dose-dependent manner. The levels of mitochondrial reactive oxygen species (mtROS), cleaved caspase-3, and -9 were increased by prednisolone. The effects of prednisolone on apoptosis and mtROS were blocked 1) by the glucocorticoid receptor (GR) antagonist RU-38486, 2) in cells with GR siRNA knockdown, and 3) by treatment with N-acetylcysteine. Transcript levels of pro-inflammatory cytokines were increased in corneal epithelial cells upon hyperosmolar stress, but repressed by prednisolone. In NOD.B10.H2b mice, topical administration of 1% prednisolone increased apoptotic cells in the corneal epithelium. Together, data indicate that prednisolone induces apoptosis in corneal epithelial cells through GR and the intrinsic pathway involving mtROS, caspase-9, and -3. The pro-apoptotic effects of glucocorticoids along with their anti-inflammatory effects should be considered when glucocorticoid eye drops are used in patients with ocular surface disease.

Prospective Clinical Trial of Corneal Reconstruction With Biomaterial-free Cultured Oral Mucosal Epithelial Cell Sheets

Purpose: To investigate the efficacy and safety of transplantation with biomaterial-free cultured oral mucosal epithelial cell sheets (COMECs) for ocular reconstruction in subjects with total limbal stem cell deficiency. Methods: A prospective clinical trial (NCT02149732) was conducted in 8 subjects with total limbal stem cell deficiency after approval from the institutional review board of Seoul National University Hospital (H-0707-043-213) and the Ministry of Food and Drug Safety of Korea. COMECs were prepared in a culture system without the use of any temperature-sensitive polymers or carriers. The COMECs were transplanted without suture fixation. Four subjects underwent penetrating keratoplasty after stabilization of the COMEC transplant. Stable epithelialization, changes in visual acuity, and postoperative complications were evaluated for 6 months. Corneal cytokeratins (K) of 4 subjects who underwent penetrating keratoplasty were stained with an immunofluorescent agent. Results: The ocular surface was successfully reconstructed in 6 eyes. Complete stable epithelialization was achieved within a mean of 53.6 days. Visual improvement (≥2 lines) was achieved in 62.5% of the eyes. K12 (corneal phenotype), K4, and K13 (mucosal phenotype) were well expressed in grafts after keratoplasty, whereas K1, K8, and K19 were barely expressed. No ocular infections, local tumor formation, or remarkable systemic complications were observed. Ocular reconstruction using COMECs failed in 2 eyes, which had full symblepharon in 4 quadrants. Conclusions: Transplanting biomaterial-free COMECs seems to be an efficient and safe procedure to reconstruct the ocular surface in patients who are completely limbal stem cell deficient without a full symblepharon.

Mesenchymal stromal cells promote B-cell lymphoma in lacrimal glands by inducing immunosuppressive microenvironment

Mesenchymal stromal cells (MSCs) have therapeutic potential for various diseases because of their anti-inflammatory and immunosuppressive properties. However, the immunosuppressive microenvironment allows tumor cells to evade immune surveillance, whereas maintenance of inflammation is required for tumor development and progression. Hence, MSCs may promote or suppress tumors in a context-dependent manner. We here investigated the effects of bone marrow-derived MSCs in a murine model of lacrimal gland B-cell lymphoma. Co-injection of MSCs with B lymphoma cells enhanced tumor growth in lacrimal glands without long-term engraftment. Of note, MSCs induced greater infiltration of immune and immune-regulatory cells near tumor: CD4+ cells, CD11b+ cells, CD4+Foxp3+ regulatory T cells and CD11b+Ly6C+Ly6G− myeloid-derived suppressor cells. Concurrently, there was up-regulation of immune-related molecules including TNF-α, IL-1β, TGF-β1, and arginase in glands treated with MSCs. Apoptosis in tumor was less severe in mice treated with MSCs compared to those without MSCs; however, MSCs did not directly inhibit apoptosis of B lymphoma cells in an in vitro co-culture. Together, data demonstrate that MSCs create immunosuppressive milieu by recruiting regulatory immune cells and promote B-cell lymphoma growth in lacrimal glands.

Transcription Profiling of NOD-like Receptors in the Human Cornea with Disease.

ABSTRACT Purpose: To investigate the expression of nucleotide-binding oligomerization domain-like receptors (NLRs) in human corneas with disease and corneal cells. Methods: The expression of NOD1, NOD2, NLRP1, and NLRP3 was analyzed using real-time RT-PCR in (1) corneas with active infection, history of herpetic stromal keratitis (HSK), chronic allograft rejection, and limbal stem cell deficiency (LSCD), and (2) human corneal cells after lipopolysaccharide (LPS) stimulation. Healthy corneas and cells without LPS served as controls. Results: The mRNA levels of NOD2 and NLRP3 were increased in corneas with infection and HSK. Conversely, the levels of NOD1, NOD2, NLRP1, and NLRP3 transcripts were decreased in corneas with LSCD. In corneas with rejection, the expression of NOD1 and NLRP1 was downregulated. Corneal endothelial cells upregulated the expression of NOD2 and NLRP3 upon LPS. Conclusions: The changes in the NLR expression may reflect different susceptibility to infectious and non-infectious injuries in corneas with various diseases.

Distribution of Interleukin-22–secreting Immune Cells in Conjunctival Associated Lymphoid Tissue

Purpose Interleukin (IL)-22 is a cytokine involved in epithelial cell regeneration. Currently, no research studies have analyzed the distribution of the three distinct IL-22–secreting cell populations in human or mouse conjunctiva. This study investigated the distribution of the three main populations of IL-22–secreting immune cells, αβ Th cells, γδ T cells, or innate cells (innate lymphoid cells [ILCs] or natural killer cells), in conjunctival associated lymphoid tissues (CALTs) in human and mouse models. Methods We collected discarded cadaveric bulbar conjunctival tissue specimens after preservation of the corneo-limbal tissue for keratoplasty from four enucleated eyes of the domestic donor. The bulbar conjunctiva tissue, including the cornea from normal (n = 27) or abraded (n = 4) B6 mice, were excised and pooled in RPMI 1640 media. After the lymphoid cells were gated in forward and side scattering, the αβ Th cells, γδ T cells, or innate lymphoid cells were positively or negatively gated using anti-CD3, anti-γδ TCR, and anti–IL-22 antibodies, with a FACSCanto flow cytometer. Results In normal human conjunctiva, the percentage and number of cells were highest in αβ Th cells, followed by γδ T cells and CD3− γδ TCR − IL-22+ innate cells (presumed ILCs, pILCs) (Kruskal-Wallis test, p = 0.012). In normal mice keratoconjunctiva, the percentage and total number were highest in γδ T cells, followed by αβ Th cells and pILCs (Kruskal-Wallis test, p = 0.0004); in corneal abraded mice, the population of αβ Th cells and pILCs tended to increase. Conclusions This study suggests that three distinctive populations of IL-22–secreting immune cells are present in CALTs of both humans and mice, and the proportions of IL-22+αβ Th cells, γδ T cells, and pILCs in CALTs in humans might be differently distributed from those in normal mice.

Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking

Purpose To investigate whether the protection of corneal limbus from riboflavin exposure during collagen cross-linking (CXL) prevents limbal epithelial stem cell (LESC) loss. Methods Ten New Zealand white rabbits received an epithelium-off CXL using an accelerated protocol. Seven days before procedure, 5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected. During procedure, riboflavin was applied to the corneal surface within a 9 mm diameter retention ring in 5 rabbits, thereby preventing the limbus from riboflavin exposure. In other 5 rabbits, riboflavin was instilled every 2 min, allowing the spillover to the limbus. One day after UVA irradiation, corneas were subjected to histological and molecular assays. Results There were no differences in corneal thickness and epithelial healing between the groups. The numbers of BrdU-labelled and p63+ limbal epithelial cells were markedly reduced in the group without a ring, but significantly increased when a ring was used. Robust expression of CK3/12 was observed in the limbal epithelium in the group with a ring. The mRNA levels of ABCG2, FGF2, IL-1β, and IL-6 were significantly increased in the corneas with a ring. Conclusions Protection of limbus from riboflavin during CXL was effective in preserving LESCs. However, inflammation was increased in the cornea treated with riboflavin using a ring.

Corneal Toxicity of Topical Tacrolimus Ointment in Mice with Corneal Epithelial Injury

PURPOSE To investigate the effect of tacrolimus ointment on corneal epithelium. METHODS Eight-week-old male BALB/c mice were divided into 3 groups. In group 1, no injury was made. In group 2, the central 2-mm corneal epithelium was scraped off. In group 3, the whole corneal and limbal epithelium was removed after absolute ethanol application. In each group, the corneas were observed and treated daily with 0.03% or 0.1% tacrolimus ointment (Protopic®). The viability of cultivated human corneal epithelial cells was also examined after 24-h incubation with various concentrations of tacrolimus. RESULTS Tacrolimus ointment significantly delayed the epithelial healing in corneas with epithelial injuries (groups 2 and 3) in dose- and frequency-dependent manners, whereas it did not have any effects on uninjured corneas (group 1). The proportion of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells and mRNA levels of inflammatory cytokines were higher in tacrolimus-treated corneas, compared with controls. Tacrolimus did not directly reduce the viability of corneal epithelial cells in culture. CONCLUSIONS Topical application of tacrolimus ointment onto the ocular surface induced toxicity in the cornea with epithelial defects through the impairment of epithelial healing and induction of apoptosis.