Investigation Of The Role Of Mesenchymal Stem Cells In Experimental Autoimmune Uveoretinitis

ObjectiveUveitis is the most common type of refractory autoimmune diseases of the eye. MSCs can exert profound immunosuppression both in vitro and in vivo by inhibiting the proliferation and function of a number of immune cell types. In the present study, we plan to investigate the effect of MSCs on EAU development and clarify its mechanism, to provide new ideas and alternative approaches to clinical treatment of uveitis.Methods1. The enriched MSCs isolated from Lewis or Wistar rats were incubated until they covered about 90% of the bottom of the culture bottle. The phenotype of cultured cells was characterized by flow cytometry and inducing differentiation in vitro.2. To induce EAU, the antigen IRBP was emulsified 1:1 (vol/vol) with Complete Freund's adjuvant (CFA), containing Mycobacterium tuberculosis H37RA, emulsified completely and injected subcutaneously into two hind footpads. The immunization was followed by intraperitoneal administration of pertussis toxin within 24 hours. Animals were randomly divided into experimental group and control. To study the preventive effect of MSCs treatment simultaneously with immunization, or therapeutic effect of delayed administration of MSCs on established EAU, the immunized rats were treated intravenously with syngeneic or allogeneic MSCs diluted in PBS or with an equal volume of PBS in control groups, for 3 consecutive days, at different stages of EAU, starting from day 0, day 9, day 12 or day 16 after administration of IRBP. The clinical situations of EAU were examined with a slit lamp regularly and histopathological changes were examined by staining with hematoxylin and eosin 20 days after immunization. The clinical signs of inflammation and histopathological severity were scored according to Caspi.3. Mononuclear cells (MNCs) were obtained from the spleen and the lymph nodes of various groups and incubated in the presence of IRBP peptide for 3 days. T-cell proliferation was studied thereafter by a standard 3H-thymidine incorporation assay.4. The levels of Thl and Th2 cytokine production were measured in supernatants derived from 72 hour cultures of MNCs stimulated by IRBP under various conditions, using a commercially available ELISA kit according to the manufacturer's instructions.5. At the end of the experiment, we collected peripheral blood from IRBP-immunized rats that were treated or untreated with MSCs. Cells were then counted and examined by flow cytometry to evaluate the expression of CD4 and CD25.6. Total RNA was isolated from spleen and lymph nodes and Real-time polymerase chain reaction (PCR) was performed to measure FoxP3 Gene expression levels.Results1. MSCs were successfully separated and identified.2. Established rat model of EAU.3. In group treatment simultaneous with immunization,9 days after immunization and 12 days after immunization, MSC infusion significantly reduced disease severity (P< 0.05). MSC treatment simultaneously with immunization showed amazing preventive effect, while MSC injection from day 16, upon disease stabilization showed no significant clinical improvement (P>0.05). Consistent with the clinical effect, histological examination of the retinal sections showed significant less retinal architecture damage in MSC treatment groups except for the last group (P< 0.05).4. MSCs significantly inhibited proliferation of pathogenic T cells in vitro and prevented T cells response on in vivo injection (P< 0.05).5. MSCs treatment significantly reduced Thl and elevated Th2 cytokine secretions in EAU rats and shifted the immune balance from Thl to Th2 dominance.6. MSCs treatment significantly increased the proportion of Tregs, and real-time quantitative PCR results showed that the expression of FOXP3-mRNA in MSCs-treated group were significantly higher, which indicated that MSCs play a role by up-regulating Tregs.ConclusionTaken together, these results suggest that MSCs can effectively prevent and ameliorate EAU. Their action is apparently through the inhibition of pathogenic T-cell responses, modulation of the balance of Thl/Th2 and increasing the proportion of regulatory T cells, which confirms the therapeutic plasticity of MSCs on immunological diseases due to their capacity for modulating systemic autoimmunity.