Ah Young Ko

Intravenous Mesenchymal Stem Cells Prevented Rejection of Allogeneic Corneal Transplants by Aborting the Early Inflammatory Response

Mesenchymal stem/progenitor cells (MSCs) were reported to enhance the survival of cellular and organ transplants. However, their mode of action was not established. We here used a mouse model of corneal allotransplantation and demonstrated that peri-transplant intravenous (i.v.) infusion of human MSCs (hMSCs) decreased the early surgically induced inflammation and reduced the activation of antigen-presenting cells (APCs) in the cornea and draining lymph nodes (DLNs). Subsequently, immune rejection was decreased, and allograft survival was prolonged. Quantitative assays for human GAPDH revealed that <10 hMSCs out of 1 × 10(6) injected cells were recovered in the cornea 10 hours to 28 days after i.v. infusion. Most of hMSCs were trapped in lungs where they were activated to increase expression of the gene for a multifunctional anti-inflammatory protein tumor necrosis factor-α stimulated gene/protein 6 (TSG-6). i.v. hMSCs with a knockdown of TSG-6 did not suppress the early inflammation and failed to prolong the allograft survival. Also, i.v. infusion of recombinant TSG-6 reproduced the effects of hMSCs. Results suggest that hMSCs improve the survival of corneal allografts without engraftment and primarily by secreting TSG-6 that acts by aborting early inflammatory responses. The same mechanism may explain previous reports that MSCs decrease rejection of other organ transplants.

Blockade of CD40–CD154 Costimulatory Pathway Promotes Long‐Term Survival of Full‐Thickness Porcine Corneal Grafts in Nonhuman Primates: Clinically Applicable Xenocorneal Transplantation

The porcine cornea may be a good solution for the shortage of human donor corneas because its size and refractive properties are comparable to those of the human cornea. However, antigenic differences need to be overcome to apply xenocorneal transplantation in actual clinical practice. We aimed to investigate the feasibility of full‐thickness porcine corneas as human corneal substitutes using a CD40–CD154 costimulatory pathway blocking strategy in a clinically applicable pig‐to‐nonhuman primate corneal transplantation model. As a result, the mean survival time of the xenocorneal grafts in recipients who received anti‐CD154 antibody‐based immunosuppressants (POD318 (n = 4); >933, >243, 318 and >192) was significantly longer than that in controls (POD28 (n = 3); 21, 28 and 29; p = 0.010, log‐rank test). Administration of anti‐CD154 antibodies markedly reduced inflammatory cellular infiltrations (predominantly CD8 T cells and macrophages) into the xenocorneal grafts and almost completely blocked xenoantigen‐triggered increases in Th1‐associated cytokines, chemokines and C3a in the aqueous humor. Moreover, systemic expansion of memory T cells was effectively controlled and responses of anti‐Gal/donor pig‐specific antibodies were considerably diminished by programmed injection of anti‐CD154 antibodies. Consequently, porcine corneas might be promising human corneal substitutes when the transplantation is accompanied by potent immunosuppression such as a CD40–CD154 costimulatory pathway blockade.

Mesenchymal Stem/Stromal Cells Protect against Autoimmunity via CCL2-Dependent Recruitment of Myeloid-Derived Suppressor Cells

Exogenously administered mesenchymal stem/stromal cells (MSCs) suppress autoimmunity despite transient engraftment. However, the mechanism is unclear. In this study, we report a novel mechanism by which MSCs modulate the immune system by recruiting myeloid-derived suppressor cells in a mouse model of experimental autoimmune uveitis (EAU). Intravenous infusion of MSCs blocked EAU development and reduced Th1 and Th17 responses. Time course analysis revealed an increase of MHC class IIloLy6G−Ly6ChiCD11b+ cells in draining lymph nodes by MSCs. These Ly6ChiCD11b+ cells suppressed CD4+ cell proliferation and Th1/Th17 differentiation and induced CD4+ cell apoptosis. Adoptive transfer of Ly6ChiCD11b+ cells ameliorated EAU, whereas depletion of Ly6ChiCD11b+ cells abrogated the effects of MSCs. 1.8% of MSCs were present in draining lymph nodes 1 d after infusion, and MSCs with CCL2 knockdown did not increase MHC class IIloLy6G−Ly6ChiCD11b+ cells and failed to attenuate EAU. Therefore, our findings demonstrate that MSCs suppress autoimmunity by recruiting myeloid-derived suppressor cells into sites of inflammation in a CCL2-dependent manner.

Mesenchymal stem/stromal cells protect the ocular surface by suppressing inflammation in an experimental dry eye.

Dry eye syndrome (DES) is one of the most common ocular diseases affecting nearly 10% of the US population. Most of the currently available treatments are palliative, and few therapeutic agents target biological pathway of DES. Although DES is a multifactorial disease, it is well-known that inflammation in the ocular surface plays an important role in the pathogenesis of DES. Mesenchymal stem/stromal cells (MSCs) have been shown to repair tissues by modulating excessive immune responses in various diseases. Therefore, we here investigated the therapeutic potential of MSCs in a murine model of an inflammation-mediated dry eye that was induced by an intraorbital injection of concanavalin A. We found that a periorbital administration of MSCs reduced the infiltration of CD4(+) T cells and the levels of inflammatory cytokines in the intraorbital gland and ocular surface. Also, MSCs significantly increased aqueous tear production and the number of conjunctival goblet cells. Subsequently, corneal epithelial integrity was well-preserved by MSCs. Together, the results demonstrate that MSCs protect the ocular surface by suppressing inflammation in DES, and suggest that MSCs may offer a therapy for a number of ocular surface diseases where inflammation plays a key role.

Cross-reactivity between decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants

The purpose of this study is to investigate cross‐reactivity between hypertonic saline‐treated decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants. Five Chinese rhesus macaques, which had undergone anterior partial thickness corneal transplantation using hypertonic saline‐treated decellularized porcine corneal lamellae in preceding experiments, were used as recipients for subsequent full‐thickness corneal allografts. To determine whether sensitization of recipients to xenoantigens leads to cross‐reactivity against alloantigens, we compared; (i) allogeneic one‐way mixed lymphocyte reaction (MLR) of peripheral blood mononuclear cells (PBMCs) from xeno‐sensitized recipients with that of PBMCs from naïve rhesus macaques, and (ii) amounts of IgG antibodies that bound to the PBMCs of a rhesus panel (five monkeys) before and after xeno‐sensitization. Graft survival and immunologic profiles including memory T‐cell subsets and donor rhesus‐specific antibodies were also evaluated. No hyperacute or acute rejection was observed within a month of subsequent allotransplantation in any recipient. Alloreactivity by MLR was not different between xeno‐sensitized rhesus recipients and naïve rhesus monkeys. Panel‐reactive IgG antibodies were unchanged after xeno‐sensitization, and no change in donor rhesus‐specific antibodies was observed in any recipient. No significant changes in memory T‐cell subsets were observed during the early post‐operative period in any recipient. Decellularized porcine corneal lamellae may not increase cross‐reactivity to alloantigens, and thus, porcine corneal lamellae may be used as a bridge to subsequent corneal allografting.

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.

Intravenous Infusion of Mesenchymal Stem/Stromal Cells Decreased CCR7+ Antigen Presenting Cells in Mice with Corneal Allotransplantation

Abstract Purpose: To investigate the effects of intravenous (IV) infusion of human mesenchymal stem/stromal cells (hMSCs) on activation and migration of CCR7+ antigen presenting cells (APCs) in allogeneic corneal transplantation. Materials and Methods: We first analyzed the cellular and molecular profiles of draining lymph nodes (DLNs) in early and late phases after syngeneic or allogeneic corneal transplantation in mice, and then investigated the effects of hMSCs on APCs expressing CCR7, a key molecule implicated in APC migration to DLNs. Results: After early transplantation, the numbers of MHC class II+CD11b+CD11c−, MHC class II+CD11b−CD11c+, and MHC II+CD11b+CD11c+ cells as well as the levels of APC-derived cytokines (IL-12a and IL-12b) driving the Th1 response were increased in both syngeneic and allogeneic transplants indicating activation of APCs. In late phase, the numbers of CD3+CD4+CD8− and CD3+CD4−CD8+ cells and the levels of T cell-derived cytokines were increased in allogeneic transplants, but not in syngeneic transplants indicating immune rejection. The peri-transplant infusion of IV hMSCs significantly reduced the numbers of CCR7+CD11b+ or CCR7+CD11c+ cells in DLNs and the cornea in the early phase. Also, the expression of CCR7 and its ligands, CCL19, CCL21, and CXC3R as well as IL-12 were markedly decreased by hMSCs in the cornea and DLNs. Conclusions: IV hMSCs reduced the activation and migration of CCR7+ APCs in the cornea and DLNs in allogeneic corneal transplantation.

TSG-6 Protects Corneal Endothelium From Transcorneal Cryoinjury in Rabbits

PURPOSE To investigate the effect of an anti-inflammatory protein, TNF-α stimulated gene/protein (TSG)-6 and an antiapoptotic protein, stanniocalcin (STC)-1 on corneal endothelium in rabbits with transcorneal cryoinjury. METHODS Transcorneal freezing (-80°C) was applied to rabbit corneas for 30 seconds. Immediately post injury, either TSG-6 (10 μg/100 μL), STC-1 (10 μg/100 μL), or the same volume of balanced salt solution (BSS) was injected into the anterior chamber. Each eye was examined for corneal opacity, corneal thickness, endothelial cell density, and endothelial hexagonality every 2 to 6 hours for 48 hours post injury. The concentrations of myeloperoxidase (MPO) and IL-1β were measured in the aqueous humor every 6 hours. At 48 hours post injury, each cornea was assayed for TNF-α, IL-1β, IL-6, and MPO, and histologically evaluated with alizarin red-trypan blue staining, hematoxylin-eosin staining, and immunostaining for neutrophils. RESULTS Tumor necrosis factor-α stimulated gene/protein-6 significantly decreased the development of corneal opacity and edema after cryoinjury compared with STC-1 or BSS. The corneal endothelial cell density and hexagonality were markedly preserved by TSG-6. The mRNA levels of TNF-α, IL-1β, and IL-6 in the cornea and the protein levels of MPO and IL-1β in the aqueous humor and cornea were significantly lower in TSG-6-treated eyes than BSS-treated controls. Similarly, the expression of fibroblast growth factor-2 was reduced by TSG-6 treatment. Histologic evaluation demonstrated that neutrophil infiltration of the cornea was decreased in TSG-6-treated eyes. CONCLUSIONS Tumor necrosis factor-α stimulated gene/protein-6 protected corneal endothelial cells from transcorneal cryoinjury through suppression of inflammation.

Analysis of macrophage phenotype in rejected corneal allografts.

PURPOSE We investigated the phenotype of macrophages infiltrating rejected corneal allografts. METHODS We performed allogeneic or syngeneic corneal transplantation in mice, and humanely killed animals at day 28 during allograft rejection when 60% of corneal allografts were rejected. We divided allografts into two groups: grafts with rejection as rejectors and grafts without rejection as nonrejectors, and analyzed for macrophage infiltration and their phenotype using immunohistochemistry. In addition, we investigated the time course of proinflammatory cytokines and chemokines by analyzing corneal grafts at days 7, 28, and 42 using real-time RT-PCR. Also, we assayed human corneal allografts with chronic graft failure. RESULTS We found that a large number of CD11b(+), F4/80(+), or inducible nitrous oxide synthase cells (iNOS(+)) infiltrated corneal allografts during rejection in mice, while the cells were found rarely in syngeneic or allogeneic grafts that were not rejected. There were rare CD11c(+) cells in rejectors and nonrejectors. Many mannose receptor cells (MRC(+)) were present in nonrejectors, but not in rejectors. The levels of Th1 cytokines, IFN-γ, and IL-2 were highly increased in rejectors at day 28, indicating immune rejection. Also, the levels of IL-12a, IL-1β, TNF-α, CCL3, and iNOS that are produced by activated macrophages were markedly increased in rejectors at day 28, compared to syngeneic grafts or nonrejectors. Similarly, human corneal allografts with chronic graft failure had higher levels of IL-12a, IL-1β, CCL3, and iNOS than controls. CONCLUSIONS Increased numbers of macrophages in rejected corneal allografts implicate that these cells might contribute to the immunopathogenesis of corneal graft rejection.

Effect of Electrocauterization on the Inflammation of the Conjunctiva in Experimental Animal Model

Purpose Recently, conjunctivochalasis repair surgery using electrocauterization has been gaining popularity. However, patients with electrocauterized conjunctivoplasty tend to complain of more postoperative pain than patients undergoing simple excision with suturing. Therefore, we investigated the effects of electrocauterization on inflammation of the conjunctiva using an experimental animal model and compared these with the effects of simple excision with suturing. Methods Ten New Zealand white rabbits underwent cauterization in the right eyes and excision and suturing in the left eyes. For each eye, we excised or electrocauterized the inferior bulbar conjunctiva, 1 mm in width and 6 mm in length, 2 mm from the limbus. A fine-needle electrode was inserted subconjunctivally, and electrocauterization was performed. In the contralateral eye, the corresponding area was excised and re-approximated with 10-0 nylon sutures. Sutures were removed after 14 days. Tissue samples were obtained at 21 days post-procedure, and inflammatory cells were counted in five randomly selected fields (×200) on hematoxylin-eosin stained slides. Tumor necrosis factor (TNF)-α and interleukin (IL)-1β concentrations in tears were measured using enzyme linked immunosorbent assays. Results All cauterized eyes demonstrated smooth surface healing without scarring after 5 days, whereas sutured eyes presented with mild edema with some scarring until the suture was removed. The number of inflammatory cells was significantly greater in sutured eyes compared with cauterized eyes (p = 0.035, Mann-Whitney U-test) at 21 days post-procedure. Tear TNF-α and IL-1β concentrations at 21 days were similar in both groups. Conclusions Electrocauterization for conjunctivoplasty seems to be advantageous in terms of inflammation compared with simple suturing and excision.