| ObjectiveThe immunoregulatory effects of MSCs have become a hot research topic in recent years and MSCs have been used in the therapy of immune disease. Uveitis is the most common type of autoimmune diseases of the eye. In the present study, we plan to investigate the effect of MSCs on EAU development and the dynamic changes of Thl/Th2/Thl7/Treg lymphocytes and their cytokine production, to provide foundation for the application of MSCs in clinical uveitis treatment.Methods1. MSCs were harvested from the bone marrows of Wistar rats by adherent culture. The phenotype of cultured MSCs was characterized by flow cytometry.2. Male Lewis rats were randomly divided into normal group, MSCs-treatment group and control group. To induce EAU, the antigen interphotoreceptor retinoid binding protein (IRBP) R16peptide emulsified completely with Complete Freund’s adjuvant (CFA) was injected subcutaneously into hind footpad, the immunized rats were treated intravenously with MSCs simultaneously or on day12after immunization for consecutive3days. In control group, the immunized rats were treated intravenously with an equal PBS.3. Starting from day4after immunization, the inflammation situations of EAU were examined with a slit lamp regularly and scored accoding to Caspi.4. The rats were executed on day6.9,12,15.20after immunization respectively and the eyes were collected. Histopathological changes were examined by staining with hematoxylin and eosin and scored accoding to Caspi.5. The rats were executed on day6,9,12,15,20after immunization respectively. Mononuclear cells (MNCs) were obtained from the spleen and the lymph nodes of various groups and incubated for72hours, the dynamic levels of Th1/Th2/Th17/Treg and IL-27cytokine production in supernatants were measured with ELISA kits.6. MNCs were examined by flow cytometry to evaluated the proportion of Th17and Tregs to CD4+T cells and the dynamic changes were analyzed.7. MNCs were obtained from EAU rats on day12after immmuization and cocultured with MSCs in96-well plates for72hours. The dynamic levels of Th1/Th2/Th17/Treg and IL-27cytokine production in supernatants were measured with ELISA kits.8. Aqueous humor was harvested on day6,9,12,15,20after immunization respectively, the dynamic levels of Th1/Th2/Th17/Treg and IL-27cytokine production were measured by ELISA kits.Results1. MSCs were successfully separated and identified.2. Established rat model of EAU.3. In control group, most rats began to show early signs of uveitis on day6, severe illness developed on day12, then recovered gradually. MSCs infusion strikingly reduced disease severity, reaching statistical significance when compared with control rats (P<0.05).4. In control group, little inflammatory cells infiltrated the retina on day6, large amounts of inflammatory cells infiltrated the retina and the organizational structure of retina was almost destroyed on day12, the following condition subsided. Histological examination of the retinal showed significant less retinal architecture damage in MSCs treatment group (P<0.05).5. Cytokine productions of MNCs:In control group, the changes of Th1/Th17cytokines production presented the same trend with the development of EAU and the productions of cytokines were significantly positively correlated with histopathological scores, MSCs significantly reduced the productions of Th1and Thl7cytokines in comparison with control group (P<0.05). The changes of Th2, Treg and IL-27cytokines production presented the contrary trend with the development of EAU and the cytokines productions were significantly negatively correlated with histopathological scores, MSCs significantly elevated the productions of Th2, Treg and IL-27cytokines in comparison with control group (P<0.05).6. Flow cytometry:In control group, the changes of proportions of Th17cells to CD4+T cells presented the same trend with the development of EAU and the proportions of Th17cells were significantly positively correlated with histopathological scores, MSCs significantly decreased the proportion of Th17in comparison with control group (P<0.05). The changes of proportions of Treg cells to CD4+T cells presented the contrary trend with the development of EAU and the proportions of Treg cells were significantly negatively correlated with histopathological scores. MSCs significantly upregulated the proportion of Treg in comparison with control group (P<0.05).7. MSCs cocultured with the MNCs in vitro:MSCs significantly decreased the productions of IL-2, IFN-γ, IL-6and IL-17, increased the productions of TGF-β, IL-4, IL-10and IL-27(P<0.05).8. Cytokines in aqueous humor:In control group, the changes of the productions of IFN-γ in aqueous humor presented the same trend with the development of EAU and the productions of IFN-γ were significantly positively correlated with histopathological scores, MSCs significantly reduced the production of IFN-γ in comparison with control group (P<0.05). The changes of the productions of IL-10presented the contrary trend with the development of EAU and the productions of IL-10were significantly negatively correlated with histopathological scores, MSCs significantly elevated the production of IL-10in comparison with control group (P<0.05).Conclusions1. Th1, Th2, Th17and Treg cells were all engaged in the development of EAU. Th1/Th17effector responses were considered as responsible for the pathology in EAU. Th2and Tregs responses were considered as protective in EAU.2. MSCs can effectively prevent and ameliorate EAU.3. MSCs inhibited Th1and Th17cells’ differentiation and the productions of their hallmark cytokines IFN-γ and IL-17through decreasing the productions of IL-2and IL-6, promoted Th2and Treg cells differentiation and the productions of their hallmark cytokines IL-10through increasing the productions of IL-4and TGF-β, which confirmed the immunoregulatory plasticity on EAU.4. IL-27was considered as protective in EAU, which implied the therapeutic plasticity of MSCs on EAU due to the increased expression level of IL-27. |
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