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Accepted Article Preview: Published ahead of advance online publication Mesenchymal stem/stromal cells protect the ocular surface by suppressing inflammation in an experimental dry eye
Min Joung Lee, Ah Young Ko, Jung Hwa Ko, Hyun Ju Lee, Mee Kum Kim, Won Ryang Wee, Sang In Khwarg, and Joo Youn Oh
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Cite this article as: Min Joung Lee, Ah Young Ko, Jung Hwa Ko, Hyun Ju Lee, Mee Kum Kim, Won Ryang Wee, Sang In Khwarg, and Joo Youn Oh, Mesenchymal stem/stromal cells protect the ocular surface by suppressing inflammation in an experimental dry eye, Molecular Therapy accepted article preview online 25 August 2014; doi:10.1038/mt.2014.159
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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 05 March 2014; accepted 18 August 2014; Accepted article preview online 25 August 2014
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Title: Mesenchymal stem/stromal cells protect the ocular surface by suppressing inflammation in an experimental dry eye
Short title: Mesenchymal stem/stromal cells in dry eye syndrome
Authors: Min Joung Lee,1 Ah Young Ko,2 Jung Hwa Ko,2 Hyun Ju Lee,2 Mee Kum Kim,2,3 Won Ryang Wee,2,3 Sang In Khwarg,3 and Joo Youn Oh2,3
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Department of Ophthalmology, Hallym University Sacred Heart Hospital, Anyang,
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Institutions:
Laboratory of Ocular Regenerative Medicine and Immunology, Biomedical
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Korea
Research Institute, Seoul National University Hospital, Seoul, Korea
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Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea
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Correspondence: Joo Youn Oh, MD, PhD. Department of Ophthalmology, Seoul
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National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea. E-mail:
[email protected] Acknowledgement: This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120239).
The authors declare that no competing financial or personal interests exist.
© 2014 The American Society of Gene & Cell Therapy. All rights reserved
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Abstract Dry eye syndrome (DES) is one of the most common ocular diseases affecting nearly 10% of the U.S. 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
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various diseases. Therefore, we here investigated the therapeutic potential of MSCs
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in a murine model of an inflammation-mediated dry eye that was induced by an
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intraorbital injection of concanavalin A. We found that a periorbital administration of
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MSCs reduced the infiltration of CD4 + T cells and the levels of inflammatory cytokines
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in the intraorbital gland and ocular surface. Also, MSCs significantly increased
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aqueous tear production and the number of conjunctival goblet cells. Subsequently,
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corneal epithelial integrity was well-preserved by MSCs. Together, the results
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demonstrate that MSCs protect the ocular surface by suppressing inflammation in
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DES, and suggest that MSCs may offer a therapy for a number of ocular surface
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diseases where inflammation plays a key role.
Key words: Dry eye syndrome, inflammation, mesenchymal stem/stromal cells
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Introduction Dry eye syndrome (DES) is one of the most common ocular disorders. The prevalence of DES ranges from 7% to 33% worldwide,1-8 and studies suggest that approximately 9 million people in the United States suffer from advanced effects of DES that alter the quality of life.8-10 Also, DES results in functional and occupational disability in patients with Sjögren’s syndrome or ocular graft-versus-host disease.10-13
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Unfortunately, most of the treatments to date are based on topical administration of
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tear substitutes, and are only palliative. Thus, efforts are being made to develop novel
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therapies for DES by targeting the underlying causes of the disease.
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The causes of DES are multifactorial. However, inflammation in the ocular surface
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plays a main role in the pathogenesis of DES.14, 15 In fact, an accumulating body of
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evidence supports the notion that DES is a localized autoimmune disease involving
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both innate and adaptive immunity such as CD4 + T cells in the development and
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progression of the disease.14, 15 Accordingly, therapies that inhibit immune response
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may be useful for treating DES. One strategy for modulating excessive immune response is administration of mesenchymal stem/stromal cells (MSCs). MSCs were first found as resident cells forming a niche for hematopoietic cells in the bone marrow of mammals, and have been further explored as reparative cells that limit tissue destruction and enhance repair in various diseases.16 The mechanisms of tissue repair by MSCs are largely attributed to their immune-modulatory effects.17, 18 Therefore, MSCs have been widely tested in clinical trials for a number of immune-mediated diseases with encouraging results.
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Here, we investigated the effects of MSCs on the ocular surface in an inflammationmediated dry eye model in mice.
Results Establishment of an inflammation-induced dry eye in mice To create the inflammation-induced dry eye model, we injected 10 or 20 μl
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concanavalin A (ConA; 1, 5, or 10 mg/ml), that is the prototypic T cell mitogen, 19 into
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the intraorbital gland in mice. For control, the same volume of phosphate buffered
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solution (PBS) was injected. One week later, aqueous tear production was measured,
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and the ocular surface was observed for epithelial integrity. Also, intraorbital glands and ocular surface including the cornea and conjunctiva were analyzed by histology
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and assayed for levels of inflammatory cytokines (Figure 1a). We found that 10
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mg/ml ConA induced severe infiltration of CD3 + T cells in the intraorbital gland
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(Figure 1b), and tear production was markedly decreased as measured by a cotton
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thread test (Figure 1c). Also, the levels of IL-2 and IFN-γ that are derived from
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activated T cells20 were significantly increased in the intraorbital gland and ocular surface (Figure 1d-f), whereas the levels of TNF-α, IL-1β, and IL-6 were not affected by ConA (Figure 1f). 20 μl injection of ConA was more effective in inducing inflammation than 10 μl ConA. The integrity of corneal epithelium was significantly disturbed by ConA as indicated by increased corneal dye staining (Figure 1g). Together, the results demonstrate that an intraorbital injection of ConA (20 μl, 10 mg/ml) induced DES in mice by causing inflammation, reducing tear secretion, and disrupting corneal epithelium.
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MSCs increased tear production and suppressed inflammation. To determine whether MSCs have therapeutic effects in an inflammation-mediated dry eye, we administered human or mouse bone marrow-derived MSCs (hMSCs, mMSCs; 1x103 or 1x105 cells/20 μl balanced salt solution; BSS), human dermal fibroblasts (hFbs; 1x105 cells/20 μl BSS), or the same volume of BSS periorbitally after ConA injection into the intraorbital gland (Figure 2a). For negative control, PBS was injected into the intraorbital gland instead of ConA. As expected, aqueous tear
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production was markedly reduced on day 7 by ConA injection (Figure 2b). The tear
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production was significantly increased in the eyes that were treated with hMSCs or
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increasing tear production (Figure 2b).
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mMSCs (Figure 2b). However, the administration of hFbs was not effective in
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The levels of T cell-derived cytokines, IL-2 and IFN-γ20 were significantly reduced in
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the intraorbital gland and ocular surface by hMSCs or mMSCs (Figure 2c, d). Both
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1x103 and 1x105 hMSCs were similarly effective, and the effects of mMSCs were
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similar to those of hMSCs. However, hFbs markedly increased the levels of IL-2 and
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IFN-γ in the intraorbital gland and ocular surface (Figure 2c, d). Corneal staining score, which is indicative of corneal epithelial defects, was significantly lower in the hMSCs- or mMSCs-treated eyes, compared to the BSStreated eyes, suggesting that corneal epithelial integrity was preserved by MSCs (Figure 2e).
MSCs decreased IFN-γ-secreting CD4+ cell infiltration in the intraorbital gland. We further analyzed for the cell population infiltrating the intraorbital gland by ConA.
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Hematoxylin-eosin staining showed that the intraorbital gland was severely disrupted with extensive acinar atrophy and periductal infiltration of inflammatory cells on day 7 after ConA injection (Figure 3a). Most of the inflammatory cells infiltrating the intraorbital gland were CD3+ cells (Figure 3a). The treatment with hMSCs or mMSCs markedly decreased the infiltration of CD3 + cells and preserved the structure of the intraorbital gland (Figure 3a). Flow cytometry revealed that the percentage of CD4 + cells or IFN-γ+CD4+ cells was increased in the intraorbital gland after ConA injection, and significantly reduced by treatment with hMSCs (Figure 3b). However, the
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percentage of IFN-γ+CD8+ cells or IL-17+CD4+ cells in the intraorbital gland was not
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increased by ConA and not affected by hMSCs (Supplemental figures 1 and 2).
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Also, the percentage of CD11b +, CD11c+, NK1.1+, or B220+ cells was not altered by
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hMSCs (Supplemental figure 3). In addition, hMSCs did not increase the
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percentage of CD4+Foxp3+ regulatory T cells (Tregs) (Supplemental figure 4).
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MSCs restored goblet cells in the conjunctiva.
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Periodic Acid Schiff (PAS) staining of the conjunctiva showed that the number of
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goblet cells which are responsible for mucous tear production was markedly decreased by ConA injection (Figure 4a). The conjunctival goblet cell counts were significantly higher in the hMSCs- or mMSCs-treated eyes, compared to the BSStreated controls (Figure 4b). The mMSCs were more effective in increasing goblet cells than hMSCs.
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MSCs did not engraft. To evaluate the engraftment of MSCs, we carried out real-time RT PCR assays for human-specific GAPDH (hGAPDH) in the intraorbital gland from mice that received periorbital infusion of 1 x 10 5 hMSCs at day 0 of ConA injection. Results demonstrated that less than 10 hMSCs were present in the intraorbital gland on days 1 and 7 after injection. Therefore, the data indicate that MSCs suppressed immune
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response and improved DES without the long-term engraftment.
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MSCs inhibited T cell proliferation and Th1 differentiation in vitro.
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We next tested whether MSCs might directly inhibit CD4 + cell proliferation and differentiation into IFN-γ-secreting Th1 cells. To evaluate proliferation, we isolated
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CD4+ cells from the peripheral blood of mice, and labeled the cells with 5-
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carboxyfluorescein diacetate succinimide ester (CFSE). The CFSE-labeled CD4+
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cells were stimulated by ConA (5 µg/ml), and co-cultured with hMSCs in transwell for
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5 days. Flow cytometric analysis of CFSE dilution revealed that CD4 + cell proliferation
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was significantly suppressed by co-culture with hMSCs (Figure 5a). To rule out the possibility that hMSCs might inhibit CD4 + cells by directly blocking ConA, we activated CD4+ cells with anti-CD3/CD28, and co-cultured with hMSCs. Similarly, hMSCs suppressed the anti-CD3/CD28-induced proliferation of CD4 + cells (Figure 5b). However, human fibroblasts (hFbs) did not suppress the proliferation of CD4 + cells, indicating that the effects of hMSCs on CD4 + cell suppression were not due to the media exhaustion during co-culture of the cells (Figure 5b). We also evaluated the effects of hMSCs on Th1 differentiation by co-culturing CD4+ cells with hMSCs in the presence of IL-1β and IL-23 for 5 days. Flow cytometry demonstrated that hMSCs
© 2014 The American Society of Gene & Cell Therapy. All rights reserved
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significantly reduced the percentage of IFN-γ+CD4+ cells (Figure 5c).
Inhibition of tryptophan metabolism did not negate the effect of hMSCs Previous studies have shown that hMSCs inhibit T cell proliferation by secreting indoleamine 2,3-dioxygenase (IDO) that catabolizes tryptophan required for T cell proliferation.21-23 Based on these reports, we tested whether IDO secretion by hMSCs might mediate the inhibitory effects of hMSCs on CD4+ cell proliferation. However, the
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addition of an IDO inhibitor 1-methyl-D-tryptophan (1-MT) did not ablate the effects of
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hMSCs in suppressing ConA-induced CD4+ cell proliferation in vitro or in increasing
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tear production or goblet cell counts in ConA-induced dry eye in mice (Supplemental
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figure 5). The data, therefore, indicate that the immune-modulatory effects of MSCs
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in the Con A-mediated dry eye might be independent of IDO.`
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Discussion
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Data demonstrate that a local infusion of MSCs protected the ocular surface in an
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inflammation-mediated dry eye. The beneficial effects of MSCs were largely mediated by suppression of inflammation and by restoration of tear production and conjunctival goblet cells. Especially, MSCs decreased the number of IFN-γ-secreting CD4+ cells in vivo, and suppressed CD4+ cell proliferation and IFN-γ+CD4+ cell differentiation in vitro. Given that CD4+ cells play an important role in the ocular surface inflammation including simple DES and devastating ocular GVHD,15, 20 our results suggest that MSCs may be a useful therapy for a number of ocular surface diseases which are accompanied by inflammation.
© 2014 The American Society of Gene & Cell Therapy. All rights reserved
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In the present study, we injected ConA into the intraorbital gland to create an inflammation-mediated dry eye in mice. ConA is a prototypic T cell mitogen that expands and activates T cells.19 Therefore, the model made it possible to follow the inflammatory response of the intraorbital gland and ocular surface and the immunemodulating effects of MSCs on the ocular inflammation. As a result, we observed that an intraorbital injection of ConA induced CD4+ T cell-mediated inflammation in the intraorbital gland and ocular surface, and thereby decreased tear production, reduced conjunctival goblet cells, and disrupted corneal epithelial integrity, all of which are
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characteristic findings of DES. The periorbital administration of MSCs suppressed
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activation of CD4+ T cells, and enhanced tear production and ocular surface integrity.
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However, the causes of DES are diverse, and DES can be induced by different
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pathophysiologic mechanisms as well as T cell-mediated inflammation.1 Thus, further
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studies using other animal models that mimic different mechanisms of DES 24 would
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help elucidate the effects of MSCs in dry eye.
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Although our data convincingly indicate that MSCs suppressed T cell-mediated
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inflammation in the ocular surface and prevented the progression of DES, the
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mechanism is not yet clear. In this study, the effects of hMSCs were similar to mMSCs. Of note was the finding that hFbs did not improve DES, and rather elicited strong immune response in the intraorbital gland and the ocular surface. These findings indicate that the therapeutic effects of hMSCs on inflammatory DES are not the result of xenogeneic reaction of human cells injected in mice, but the unique effects that MSCs possess. We did not detect MSCs in the intraorbital gland on days 1 and 7 after injection into mice. This finding is consistent with previous reports that MSCs rapidly disappear in vivo after injection, and repair tissues without the long-term engraftment and through
© 2014 The American Society of Gene & Cell Therapy. All rights reserved
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suppression of early immune response. As for the mechanism of T cell suppression, several reports suggested that hMSCs inhibit T cells by inducing Tregs.25-28 We did not observe an increase in Tregs by hMSCs in this study. However, it is possible that MSCs might recruit other immunoregulatory cells than Tregs.29 Other studies suggested that inhibition of tryptophan metabolism by IDO is responsible for T cell suppression by hMSCs. 21-23 However, 1-MT, an IDO inhibitor, was not effective in reversing the effects of hMSCs
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in vivo and in vitro in our study. Moreover, some of the immune-modulatory effects of
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MSCs are species-specific. For instance, inducible nitric oxide synthase (iNOS) is
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known to mediate the immune-suppressive activity of mMSCs,30 whereas IDO is
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involved in the immunosuppressive activity of hMSCs. 21-23 Hence, although we
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observed similar beneficial effects of hMSCs and mMSCs on DES, the mechanism(s) might be different between hMSCs and mMSCs. Further studies would be necessary
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to find out the immune-modulatory mechanisms of MSCs in DES. In conclusion, we here demonstrate that a periorbital infusion of human or mouse
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bone marrow-derived MSCs protected the ocular surface by suppressing
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inflammation in an experimental dry eye which was mediated by CD4 + T cellmediated response. Our study has an implication for further exploration of MSCs as a novel therapy for patients with severe DES and other inflammatory ocular diseases.
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Materials and Methods Animals The experimental protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital Biomedical Research Institute (IACUC No. 12-0360). Eight-week-old female BALB/c mice were purchased from Orient Bio Inc. (Seongnam, Korea), and maintained in a specific pathogen-free environment with continuously available water and food. Animals were treated in
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strict accordance with the ARVO statement for the use of animals in ophthalmic and
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vision research.
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Preparation of cells
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The hMSCs were obtained from the Center for the Preparation and Distribution of
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Adult Stem Cells (http://medicine.tamhsc.edu/irm/msc-distribution.html) that supplies
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standardized preparations of MSCs enriched for early progenitor cells to over 300
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laboratories under the auspices of an NIH/NCRR grant (P40 RR 17447-06). Animal
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experiments were performed with passage two hMSCs from one donor. The cells consistently differentiated into three lineages in culture, were negative for hematopoietic markers (CD34, CD36, CD117, and CD45), and were positive for mesenchymal markers CD29 (95%), CD44 (>93%), CD49c (99%), CD49f (>70%), CD59 (>99%), CD90 (>99%), CD105 (>99%), and CD166 (>99%). The hMSCs were cultured in complete culture medium (CCM) containing 17 % fetal bovine serum (FBS; Gibco®/Life Technologies, Grand Island, NY), 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen TM/Life Technologies, Carlsbad, CA) until 70 % confluence was reached, and harvested with 0.25% trypsin/1 mM EDTA at
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37°C for 2 min for injection. After washing, the cells were resuspended in BSS (BioWhittaker, Walkersville, MD) for injection. The hFbs were primary cultured fibroblasts from human skin, and obtained from CELLnTEC (Bern, Switzerland). The cells were cultured and prepared for injection in the same manner as hMSCs. The mMSCs were prepared from the marrow of long bones of hindlimbs of eightweek-old female BALB/c mice using the previously described method 31 with
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modification. Briefly, the bone marrow extracts were obtained by flushing the cut ends
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of the bones, and cell suspension was filtered through a 70 μm strainer to remove
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bone spicules. After centrifugation, the cells were resuspended in CCM, and
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distributed into culture plates at density of 1 x 10 6 cell/cm2. The plates were cultured
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undisturbed at 37°C with 5% CO2 in a humidified chamber (normoxia condition) for 3
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days. After 3 days, the nonadherent cells were removed by gentle swirling, and
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adherent cells were replaced with CCM. After additional 4 days of culture, the plates
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were washed with serum-free medium, and harvested with 0.25% trypsin/1 mM EDTA
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at 37°C for 2 min before injection.
Animal models and treatment To create dry eye, mice were anesthetized with intraperitoneal injection of zolazepam-tiletamine (Zoletil®, Virbac, Carros, France), and 10 or 20 μl ConA (Sigma-Aldrich, Saint Louis, MO), that was diluted in PBS at concentrations of 1, 5, and 10 mg/ml, was injected into the intraorbital gland through transconjunctival approach using a Hamilton syringe with a 33 gauge needle (Hamilton, Reno, NV) under an operating microscope (Carl Zeiss, Jena, Germany). The same volume of
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PBS was injected and served as negative control. Immediately after ConA injection, hMSCs (1x10 3 or 1x105 cells/20 μl BSS), mMSCs (1x105 cells/20 μl BSS), or the same volume of BSS were injected into the periorbital space using 30 gauge needle-syringe (Becton Dickinson & Co., Franklin Lakes, NJ). In some experiments, 1-MT (10 mg; Sigma-Aldrich) was injected periorbitally along with hMSCs. The experiments were repeated in three independent sets, and each set
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included at least 5 mice per group.
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Measurement of tear production
Aqueous tear production was measured with standardized phenol red-impregnated
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cotton threads (FCI Ophthalmics, Pembrooke, MA). The threads were applied to the
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ocular surface in the lateral canthus for 60 sec, and wetting of the thread was
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Corneal dye staining
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measured in millimeters.
To evaluate the corneal epithelial integrity, corneal staining was performed by administering one drop of 3% Lissamine™ Green B (Sigma-Aldrich) to the inferior lateral conjunctival sac. The corneal surface was observed, and photographed with a digital camera fitted with a macro lens (Canon Inc., Melville, NY). The total area of fluorescein staining of the cornea was scored in a blinded manner as follows: score 0 for no punctuate staining; score 1 when one fourth or less was stained; score 2 when one half or less was stained; and score 3 when one half or more was stained. The average corneal staining score was calculated for each mouse.
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Histopathology The whole eyeball and intraorbital gland were excised, and fixed in formalin. The samples were sliced in 4 μm sections and subjected to hematoxylin-eosin, PAS, or CD3 immunohistochemical staining. To count goblet cells, four different sections through superior and inferior conjunctival fornices were selected in the PAS-stained slides, and the average counts of PAS-stained cells were calculated under a
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microscope using x 10 object. For CD3 immunohistochemical staining, a rabbit anti -
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mouse CD3 (ab5690, Abcam, Cambridge, MA) was used as a primary antibody.
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Real-time RT-PCR
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For RNA extraction, the intraorbital gland or ocular surface including the cornea and
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conjunctiva was cut into small pieces with microscissors, lysed in RNA isolation
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reagent (RNA Bee, Tel-Test, Friendswood, TX), and homogenized using a sonicator
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(Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL). Total RNA was
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extracted using RNeasy Mini kit (Qiagen, Valencia, CA) and used to synthesize double-stranded cDNA by reverse transcription (SuperScript III; InvitrogenTM/Life Technologies). Real-time amplification was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA): IL-2 (TaqMan Gene Expression Assays ID, Mm00434256_m1), IFN-γ (TaqMan Gene Expression Assays ID, Mm01168134_m1), TNF-α (TaqMan Gene Expression Assays ID, Mm00443260_g1), IL-1β (TaqMan Gene Expression Assays ID, Mm00434228_m1), and IL-6 (TaqMan Gene Expression Assays ID, Mm00446190_m1). An 18s rRNA (TaqMan Gene Expression Assays ID, Hs03003631_g1) was used for normalization of gene
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expression. For all the PCR probe sets, TaqMan Gene Expression Assay kits were purchased from Applied Biosystems. The assays were performed in dual technical replicates for each biological sample.
Standard curve for human GAPDH A standard curve was generated by adding serial dilutions of hMSCs to mouse tissue as previously described.32 Briefly, 0, 10, 100, 1,000, 10,000, or 100,000 hMSCs were
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added to each mouse intraorbital gland, respectively. Following RNA extraction, cDNA was generated using 1 µg total RNA, and real-time amplification was
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performed using hGAPDH (TaqMan® Gene Expression Assays ID, Hs99999905_05).
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The values were normalized to total eukaryotic 18s rRNA . The standard curve was made based on hGAPDH expression from a known number of hMSCs added to one
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Flow cytometry
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mouse intraorbital gland.
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Intraorbital gland was placed and minced between the frosted ends of two glass slides in RPMI media (WelGENE, Inc., Daegu, Korea) containing 10% FBS and 1% penicillin–streptomycin. Cell suspensions were collected, and incubated for 30 min at 4°C with fluorescein-conjugated anti-mouse antibodies: CD4, CD8, CD11b, CD11c, CD25, B220, NK1.1, IFN-γ, Foxp3 (eBioscience, San Diego, CA), and IL-17A (BD Pharmingen™, San Diego, CA). For intracellular staining, the cells were stimulated for 4 h with 50 ng/ml phorbol myristate acetate and 1 μg/ml ionomycin in the presence of GolgiPlug (BD Pharmingen™). The cells were assayed for fluorescence using a FACSCanto flow cytometer (BD BioSciences, Mountain View, CA). Data were
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analyzed using Flowjo program (Tree Star, Inc., Ashland, OR).
In vitro T cell proliferation and Th1 differentiation assays Mouse CD4+ T cells were purified from the peripheral blood of BALB/c mice using CD4 MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions, and cultured in RPMI 1640 medium (WelGENE, Inc.) containing 10% FBS (Gibco®/Life Technologies) and 1% penicillin/streptomycin (PS).
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We found that 86.4% of CD4+ T cells survive in RPMI 1640 medium containing 10%
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FBS and 1% PS after 5 days of culture. For co-culture studies, we also used RPMI
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1640 medium supplemented with 10% FBS and 1% PS, because the survival of
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MSCs in this medium was similar to the survival of MSCs in CCM, while the survival
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of CD4+ T cells in CCM markedly decreased to 57% after 5 days of culture. For
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proliferation assay, the cells were labeled with 1 µl of 5 mM CFSE (InvitrogenTM/Life
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Technologies) for 10 min at 37°C in the dark. The CFSE-labeled CD4+ cells were co-
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cultured with hMSCs or hFbs (CELLnTEC) in transwell at a ratio of 10:1 (CD4 + cells
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to hMSCs or hFbs) in RPMI 1640 medium supplemented with 10% FBS and 1% PS,
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and stimulated with 5 µg/ml ConA or 2.5µg/ml anti-CD3/anti-CD28 monoclonal antibodies (eBioscience) for 5 days. In some experiments, 1-MT (500 μM or 1 mM; Sigma-Aldrich) was added to the culture with hMSCs. The CFSE fluorescence was assessed using FACSCanto flow cytometer (BD BioSciences). The experiments were performed three times independently. For differentiation assay, CD4 + cells were co-cultured with hMSCs in transwell at a ratio of 10:1 (CD4+ cells to MSCs) in the presence of 25 ng/ml IL-1β and 25 ng/ml IL23 (R&D systems, Minneapolis, MN) for 5 days. CD4 + cells were then stimulated for 4
© 2014 The American Society of Gene & Cell Therapy. All rights reserved
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h at 37°C with 50 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin in the presence of GolgiPlug (BD Pharmingen™), and incubated with the fluorescenceconjugated anti-mouse IFN-γ antibody (eBioscience) after fixation and permeabilization. The cells were analyzed by FACSCanto flow cytometer (BD BioSciences) and Flowjo program (Tree Star, Inc.). All the experiments were performed six times independently. Statistical analysis
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Values were compared between the groups using the one-way ANOVA or two-tailed
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Student’s t test (Prism®, GraphPad Software, Inc., La Jolla, CA), and shown as the
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mean value ± SEM or SD. Differences were considered significant at p < 0.05.
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Acknowledgement
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This study was supported by a grant of the Korean Health Technology R&D Project,
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Ministry of Health & Welfare, Republic of Korea (A120239).
There is no conflict of interest to declare.
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Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6.
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Figure legends Figure 1. Establishment of inflammation-induced dry eye in mice. (a) Concanavalin A (ConA) was injected into an intraorbital space in mice. Phosphate buffered solution (PBS) was injected as vehicle control. One week later, the tissues were subjected to assays. (b) Immunostaining showed extensive infiltration of CD3 + T cells in the intraorbital gland. (c) Aqueous tear production as measured by phenol red
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thread test was significantly reduced by ConA injection. (d, e) Real-time RT-PCR
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assay showed that either 10 or 20 μl injection of ConA (10 mg/ml) significantly
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increased the levels of IL-2 and IFN-γ transcripts which are the cytokines derived
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from activated T cells. (f) In the ocular surface including the cornea and conjunctiva,
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the levels of IL-2 and IFN-γ transcripts were increased by ConA injection (10 mg/ml,
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20 μl), while the levels of TNF-α, IL-1β, and IL-6 were not affected. (g) The corneal
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dye staining indicated the development of corneal epithelial erosion in ConA-injected
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eyes. n=10 to 20 in each group. The data are presented as the mean +/± SEM. * p