Jung Hwa Ko

The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury.

To investigate the anti‐inflammatory and anti‐angiogenic effects of mesenchymal stem cells (MSC) in the chemically burned corneas, we mechanically removed the corneal epithelium of rats after 100% alcohol instillation. The rats were then randomized into four groups: fresh media, conditioned media derived from the MSC culture (MSC‐CM), MSC applied topically to the damaged corneas for 2 hours immediately after the injury or MSC‐CM applied either once or 3 times per day for 3 consecutive days. Corneal surface was evaluated every week. After 3 weeks, the corneas were stained with the hematoxylin‐eosin, and the expression of interleukin (IL)‐2, interferon (IFN)‐γ, IL‐6, IL‐10, transforming growth factor (TGF)‐β1, thrombospondin‐1 (TSP‐1), matrix metalloproteinase‐2 (MMP‐2), and vascular endothelial growth factor (VEGF) were analyzed. CD4+ cells were assessed in the corneas. We found that both MSC and three‐time applied MSC‐CM (1) reduced corneal inflammation and neovascularization, (2) decreased IL‐2 and IFN‐γ, although increased IL‐10 and TGF‐β1 as well as IL‐6, (3) reduced the infiltration of CD4+ cells, and (4) upregulated the expression of TSP‐1, although downregulated that of MMP‐2. Interestingly, whereas three‐time application of MSC‐CM was partially effective, transplantation of MSC achieved a better outcome in suppressing corneal inflammation. The results of this study suggest that the anti‐inflammatory and anti‐angiogenic action of MSC in the chemically burned corneas might be mediated in part through paracrine pathways involving soluble factors such as IL‐10, TGF‐β1, IL‐6 and TSP‐1.

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.

Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye

Significance Mesenchymal stem/stromal cells (MSCs) are the focus of intensive efforts directed at developing cell-based therapies in immunologic disorders. However, one of the paradoxical observations made so far is that MSCs engraft transiently in the recipient after exogenous infusion, but achieve long-term therapeutic benefits. Here we demonstrate that MSCs induce the immune tolerance by activating endogenous immune regulatory system of the recipient. Specifically, i.v. administered MSCs induce a population of regulatory monocytes/macrophages in the lung, which are capable of suppressing allo- and autoimmune responses independently of regulatory T cells. The data provide a mechanistic insight into the action of MSCs in immunologic disorders, one of the most frequent indications of diseases for clinical trials using stem cells. Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α–stimulated gene/protein (TSG)-6–dependent manner. As a result, mice were protected against subsequent immune challenge in two models of allo- and autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell–suppressive activities independently of FoxP3+ regulatory T cells. Adoptive transfer of MSC-induced B220+CD11b+ monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II+B220+CD11b+ cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages.

Mesenchymal Stem/Stromal Cells Inhibit the NLRP3 Inflammasome by Decreasing Mitochondrial Reactive Oxygen Species

Mesenchymal stem/stromal cells (MSCs) control excessive inflammatory responses by modulating a variety of immune cells including monocytes/macrophages. However, the mechanisms by which MSCs regulate monocytes/macrophages are unclear. Inflammasomes in macrophages are activated upon cellular “danger” signals and initiate inflammatory responses through the maturation and secretion of proinflammatory cytokines such as interleukin 1β (IL‐1β). Here we demonstrate that human MSCs (hMSCs) negatively regulate NLRP3 inflammasome activation in human or mouse macrophages stimulated with LPS and ATP. Caspase‐1 activation and subsequent IL‐1β release were decreased in macrophages by direct or transwell coculture with hMSCs. Addition of hMSCs to macrophages either at a LPS priming or at a subsequent ATP step similarly inhibited the inflammasome activation. The hMSCs had no effect on NLRP3 and IL‐1β expression at mRNA levels during LPS priming. However, MSCs markedly suppressed the generation of mitochondrial reactive oxygen species (ROS) in macrophages. Further analysis showed that NLRP3‐activated macrophages stimulated hMSCs to increase the expression and secretion of stanniocalcin (STC)‐1, an antiapoptotic protein. Addition of recombinant protein STC‐1 reproduced the effects of hMSCs in inhibiting NLRP3 inflammasome activation and ROS production in macrophages. Conversely, the effects of hMSCs on macrophages were largely abrogated by an small interfering RNA (siRNA) knockdown of STC‐1. Together, our results reveal that hMSCs inhibit NLRP3 inflammasome activation in macrophages primarily by secreting STC‐1 in response to activated macrophages and thus by decreasing mitochondrial ROS. Stem Cells 2014;32:1553–1563

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.

Stanniocalcin-1 Protects Retinal Ganglion Cells by Inhibiting Apoptosis and Oxidative Damage

Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.

Identification of alpha-Gal and non-Gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry.

Purpose: To investigate the expression of α-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry. Methods: The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immunochemical staining was added to confirm the non-Gal antigen expression in pig corneal cells. Results: Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of α-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed. Conclusions: As well as α-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the α-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.

Investigating the relationship between serum interleukin-17 levels and systemic immune-mediated disease in patients with dry eye syndrome.

Purpose To investigate the association between dry eye syndrome (DE) and serum levels of interleukin (IL)-17 in patients with systemic immune-mediated diseases. Methods IL-17 and IL-23 levels were measured in the sera of patients whose tear production was <5 mm on the Schirmer test. Subjects included patients with chronic graft-versus-host disease (GVHD), rheumatoid arthritis (RA), Sjogrens syndrome (SS), systemic lupus erythematosus (SLE), and no systemic disease. Corneal/conjunctival fluorescein staining was scored and the correlation between the score and the IL-17 level was evaluated. Results A strong correlation existed between IL-17 level and the type of systemic disease. IL-17 was significantly elevated in patients with chronic GVHD compared to those with RA and SS. IL-17 was not detectable in patients with SLE or in those without systemic disease. IL-23 was not detected in any of the subjects. IL-17 was significantly increased in patients with high fluorescein staining scores. Conclusions Our data suggest that IL-17 is involved in the pathogenesis of DE in patients with systemic immune-mediated diseases.

Neonatal systemic inflammation in rats alters retinal vessel development and simulates pathologic features of retinopathy of prematurity

BackgroundAlteration of retinal angiogenesis during development leads to retinopathy of prematurity (ROP) in preterm infants, which is a leading cause of visual impairment in children. A number of clinical studies have reported higher rates of ROP in infants who had perinatal infections or inflammation, suggesting that exposure of the developing retina to inflammation may disturb retinal vessel development. Thus, we investigated the effects of systemic inflammation on retinal vessel development and retinal inflammation in neonatal rats.MethodsTo induce systemic inflammation, we intraperitoneally injected 100 μl lipopolysaccharide (LPS, 0.25 mg/ml) or the same volume of normal saline in rat pups on postnatal days 1, 3, and 5. The retinas were extracted on postnatal days 7 and 14, and subjected to assays for retinal vessels, inflammatory cells and molecules, and apoptosis.ResultsWe found that intraperitoneal injection of LPS impaired retinal vessel development by decreasing vessel extension, reducing capillary density, and inducing localized overgrowth of abnormal retinal vessels and dilated peripheral vascular ridge, all of which are characteristic findings of ROP. Also, a large number of CD11c+ inflammatory cells and astrocytes were localized in the lesion of abnormal vessels. Further analysis revealed that the number of major histocompatibility complex (MHC) class IIloCD68loCD11bloCD11chi cells in the retina was higher in LPS-treated rats compared to controls. Similarly, the levels of TNF-α, IL-1β, and IL-12a were increased in LPS-treated retina. Also, apoptosis was increased in the inner retinal layer where retinal vessels are located.ConclusionsOur data demonstrate that systemic LPS-induced inflammation elicits retinal inflammation and impairs retinal angiogenesis in neonatal rats, implicating perinatal inflammation in the pathogenesis of ROP.