The Investigation Of Effects Of Exosomes Derived From Mesenchymal Stem Cell On Experimental Autoimmune Uveoretinitis

ObjectiveUveitis is one of the common cause of visual disability.Experimental autoimmune uveoretinitis(EAU)is a useful tool for studying the pathogenesis and developing new therapies for human uveitis.Our previous studies demonstrated that both syngeneic and allogeneic mesenchymal stem cells(MSCs)can ameliorate IRBP-induced EAU in rats.Exosomes are small membranous vesicles that are secreted by most cell types,encapsulating abundant proteins and RNAs that can mediate MSC function.It has been proved that exosomes derived from MSCs can down-regulate immune response or induce immune tolerance in some autoimmune reponses.This study evaluated the efficiency of locally administration of exosomes derived from MSCs on EAU in Lewis rats and investigated the mechanism,so as to optimize the strategy of MSC-based therapy and provide a novel biologic for clinical translation in treating uveitis.Methods1.Culture of MSCs:MSCs were seperated from human umbilical cord,cultured and amplified in vitro.The phenotypes of cultured cells were characterized by flow cytometry.2.Isolation of exosomes from MSCs:culture medium containing exosomes depleted serumfrom p3-p5 MSCs were collected,and exosomes were isolated by multiple centrifugations.The concentration was tested by BCA kits.The exosomes preparation was 0.22μm filtered and stored in-20℃freezer until use.3.Induction of EAU model:The interphotoreceptor retinoid-binding protein(IRBP)R16 segment was emulsified 1:1(vol/vol)with Complete Freund’s adjuvant(CFA),containing Mycobacterium tuberculosis H37RA(2.5g/l).The antigen was injected subcutaneously into one hind footpad of Lewis rats.4.Exosomes treatment:36 EAU rats were randomly divided into the exosomes treatment group and the PBS control group.The EAU rats were treated by periocular injection of different doses of exosomes starting on the onset day or with an equal volume of PBS for consecutive 7 days.5.Clinical observation:Clinical signs of inflammation were examined by slit-lamp from the day 6 after immunization and scored according to Caspi criteria.6.Histopathological observation:On the day 15 and 21 after immunization,histopathological changes of retina were examined by hematoxylin and eosin staining.The inflammatory responses of retinas were scored according to Caspi criteria.7.Electrophysiology(ERG):ERG was performed on day 12,15 and 21 of the procedure.The amplitudes of the a-wave and b-wave were measured.8.Analysis of T cell subsets and inflammatory cells by flow cytometry:On the day 15after immunization,monocytes from the cervical draining lymph nodes and eyes were separated and stained by mouse anti-rat m Abs against CD4,IL-17,IFN-γ,Foxp3,CD68,Gr-1 and CD161.The proportions of T cell subsets and inflammatory cells were analyzed by flow cytometry.9.Assay of T cell proliferation by 5-bromo-2-deoxyuridine(Brdu):On the day 12after immunization,spleen T lymphocytes were seperated and incubated with exosomes and different doses of specific and non-specific antigen in vitro for 48hours,and labeled with Brdu.The Brdu incorporation was assessed at 450nm using an enzyme-linked immunosorbent assay reader.10.Chemotaxis Assay:On the day 12 after immunization,spleen monocytes were separated.The chemoattractive effect of exosome was examined by an in vitro chemoattractant assay.The migration of monocytes to the lower chamber containing CCL21 and exosomes was detected after 4 hour incubation.Results1.The MSCs were successfully proliferated and identified.2.Rat models of EAU were successfully established.3.Clinical observation:Sub-tenon injection of exosomes significantly reduced intraocular inflammation as evaluated by slit-lamp biomicroscopy.(P<0.05)4.Consistent with the clinical evaluation,exosomes treatment greatly reduced the inflammatory cell infiltration and retinal damage on day 15 and 21.(P<0.05)5.Electrophysiology(ERG):The amplitudes for a-waves and b-waves were significantly larger in treatment group than those in control goup on day 12,day 15and day 21.(P<0.05)6.Analysis of T cell subsets and inflammatory cells by flow cytometry:Exosomes treatment down regulated the proportions of CD4~+IFN-γ~+,CD4~+IL-17~+,Gr-1~+,CD161~+and CD68~+cells in eyes(P<0.05),while was of no effect on the cells in lymph nodes.7.Assay of spleen T lymphocyte proliferation by Brdu:No inhibitory effect was observed in T cell proliferation by h UC-MSC derived exosomes under R16 or Con A stimulation.8.Chemotaxis Assay:Chemoattractive effects of CCL-21 on CD4~+T cells and inflammatory cells were suppressed by h UC-MSC exosomes.ConclusionExosomes derived from h UC-MSCs can effectively ameliorate EAU by inhibiting the migration of inflammatory cells.