Mee Kum Kim

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

The Anti-inflammatory Effect of Subconjunctival Bevacizumab on Chemically Burned Rat Corneas

Purpose: To investigate the effect of bevacizumab in a model of corneal inflammation. Materials and Methods: Epithelium from the cornea and limbus was completely removed using 100% alcohol in rats. Bevacizumab (1.25 mg/0.05 ml) or normal saline was subconjunctivally injected. One week later, corneas were examined and subjected to hematoxylin-eosin and immunofluorescent staining for VEGF. Expression of IL-2, IFN-γ, IL-6, and TGF-β 1 were analyzed by ELISA. Results: Bevacizumab showed a borderline reduction in corneal neovascularization (11.0 ± 4.8% and 18.0 ± 10.0% in the bevacizumab and control groups, respectively; p = 0.054), while the extent of epithelialization was not affected (5.0 ± 3.4% and 6.1 ± 5.2% in the bevacizumab and control groups, respectively; p = 0.715). The infiltration of inflammatory cells was reduced (99.3 ± 22.3 cells/× 400 in the bevacizumab-injected corneas and 182.3 ± 58.0 cells/× 400 in the controls; p = 0.013). The levels of IL-2, IFN-γ, and IL-6 were decreased in the rats with the bevacizumab injections (44 ± 6 and 79 ± 9 pg/ml for IL-2 in the bevacizumab-injected group and control, respectively, p = 0.025; 45 ± 5 and 67 ± 6 pg/ml for IFN-γ in the bevacizumab-injected group and controls, respectively, p = 0.043; 45 ± 6 and 75 ± 8 pg/ml for IL-6 in the bevacizumab-injected group and controls, respectively, p = 0.030). Conclusions: Bavacizumab showed a reduction in inflammatory cell infiltration and cytokines in chemically burned rat corneas.

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.

Short term effects of topical cyclosporine and viscoelastic on the ocular surfaces in patients with dry eye.

Purpose To compare the short term effects of topical 0.05% cyclosporine (CsA) and a mixture of 0.08% chondroitin sulfate and 0.06% sodium hyaluronate (CS-HA) on dry eye ocular surfaces. Methods 36 patients with moderate to severe dry eye (5 mm/5 min or less with Schirmers test or tear break up time (BUT) less than 6 seconds), were treated with topical application of CS-HA on one eye and CsA on the other 4 times a day for 6-8 weeks. BUT, Schirmers test without anesthesia, and conjunctival impression cytology (CIC; goblet cell density, nucleus to cytoplasmic ratio, and epithelial cell morphology) were evaluated and compared between eyes before and after treatment (repeated measurement of ANOVA). Results After treatment, BUT and tear wettings were significantly prolonged in each group. Topical CsA treated eyes had greater increase in BUT (p=0.026); there was no significant difference in tear wetting (p=0.132). While the 3 parameters of CIC improved in both groups, goblet cell density was significantly higher in eyes treated with CsA (p=0.033). Conclusions While both CS-HA and 0.05% CsA eyedrops improve ocular surfaces, topical CsA may have a better effect on enhancing tear film stability and goblet cell density.

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.

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.

Evaluation of Corneal Biomechanical Properties Following Penetrating Keratoplasty Using the Ocular Response Analyzer

Purpose To evaluate corneal biomechanical properties in eyes that had previously undergone penetrating keratoplasty (PK) using the ocular response analyzer (ORA). Methods We recruited 26 patients who had received unilateral PK. Corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated intraocular pressure (IOPg), and cornea-compensated intraocular pressure (IOPcc) were measured with the ORA and were compared to the measurements from the contralateral eyes that did not undergo PK. Results The CH was 8.95±2.59 mmHg in eyes that underwent PK and 9.78±1.45 mmHg in the contralateral eyes that did not undergo PK (p=0.077). The CRF was 10.26±2.64 mmHg in post-PK eyes and 9.75±1.45 mmHg in the contralateral eyes (p=0.509), and the CH-CRF was significantly smaller in post-PK eyes (-1.31±2.32 mmHg in post-PK eyes vs. 0.03±0.88 mmHg in fellow eyes, p=0.016). The IOPg and IOPcc were significantly higher in the PK group than they were in the control group. The IOPccs were 20.81±7.81 mmHg and 16.27±2.49 mmHg in post-PK and control eyes, respectively (p=0.011); and the IOPgs were 19.22±7.34 mmHg and 15.07±3.03 mmHg in post-PK and control eyes, respectively (p=0.019). The IOPcc-gs were 1.59±2.81 mmHg and 1.21±1.30 mmHg in post-PK and control eyes, respectively (p=0.412), and the central corneal thickness (CCT)s were 489.11±90.60 µm and 556.24±42.84 µm in post-PK and control eyes, respectively (p=0.068). Conclusions Following PK, CH tended to decrease while CRF tended to increase, significantly decreasing CH-CRF. A significantly higher intraocular pressure and a thinner CCT following PK may have contributed to the observed changes in these corneal biomechanical parameters.

Long-term outcomes of first-line treatment with doxycycline in patients with previously untreated ocular adnexal marginal zone B cell lymphoma.

Ocular adnexal lymphoma (OAL) has been associated with Chlamydophila psittaci infection, for which doxycycline has been suggested as a treatment option. We conducted this study to evaluate the long-term results of first-line doxycycline treatment in patients with OAL. Ninety patients with histologically confirmed OAL with marginal zone B cell lymphoma were enrolled. Each patient received one or two cycles of doxycycline (100xa0mg bid) for 3xa0weeks. After a median follow-up period of 40.5xa0months (8–85), the 5-year progression-free survival (PFS) rate was 60.9xa0%. All patients were alive at the last follow-up date. Thirty-one patients (34xa0%) showed local treatment failure without systemic spread. However, PFS rate in these patients was 100xa0% after salvage chemotherapy and/or radiotherapy. PFS was independently predicted in multivariate analysis by the tumor-node-metastasis (TNM) staging (hazard ratio [HR], 4.35; 95xa0% confidence interval [CI], 2.03–9.32; Pu2009<u20090.001) and number of cycles of doxycycline (HR, 0.31; 95xa0% CI, 0.14–0.69; Pu2009=u20090.004). No serious adverse event was reported during doxycycline therapy. In conclusion, first-line doxycycline therapy was effective and safe. Patients who failed to respond to doxycycline therapy were successfully salvaged with chemotherapy and/or radiotherapy without compromising long-term outcomes. Patients with T1N0M0 disease could be considered good candidates for first-line doxycycline.

Clinical comparison of ciliary sulcus and pars plana locations for posterior chamber intraocular lens transscleral fixation

PURPOSE: To compare the clinical outcomes of transscleral fixation of a posterior chamber intraocular lens (PC IOL) in the ciliary sulcus or pars plana. SETTING: Department of Ophthalmology, Seoul National University College of Medicine, Seoul, South Korea. DESIGN: Comparative case series. METHODS: This retrospective chart review comprised eyes having ciliary sulcus or pars plana fixation of a 3‐piece foldable acrylic PC IOL between January 2003 and August 2010. The postoperative corrected distance visual acuity (CDVA), efficacy index, safety index, endothelial cell count (ECC), and complication rates in the 2 groups were compared. RESULTS: The ciliary sulcus group comprised 38 eyes and the pars plana group, 56 eyes. There was no significant between‐group difference in the postoperative CDVA, efficacy index, safety index, or ECC. The mean spherical equivalent difference was larger in the ciliary sulcus group. Intraocular lens dislocation and pupillary capture of the IOL optic occurred more frequently in the ciliary sulcus group (P=.001 and P=.041, respectively). However, retinal detachment, IOL decentration or tilt, cystoid macular edema, secondary glaucoma, and vitreous hemorrhage did not differ significantly between the 2 groups. CONCLUSION: The pars plana location for PC IOL transscleral fixation was as safe and effective as the ciliary sulcus location. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.