The Application Of IL-10 And TGF-?1 Overexpressed MSCs-derived Small Extracellular Vesicles On Experimental Autoimmune Uveitis

Objective:Autoimmune uveitis is one of the leading cause of blindness,characterized by frequency recurrence and difficulty in treating.However,the traditional medical treatment have toxic side effects.The development of new therapies has always been a research hotspot in the field of uveitis.In this study,we observed the therapeutic effects of umbilical cord mesenchymal stem cells(MSC s)-derived small extracellular vesicles overexpressing IL-10 and TGF-?1(sEVs-10,sEVs-T)by using a mice model of experimental autoimmune uveitis(EAU),to provide the theoretical basis for future clinical transformation.Methods:1.Packaging of lentivirus:The lentivirus overexpressing IL-10 and TGF-? and empty vector were packaged with three plasmid system and 293T cells,and were concentrated and purified by ultracentrifugation.2.MSCs culture:MSCs were seperated from human umbilical cord tissue and selected P2 for Lentivirus infection.The expression of the target genes in MSCs was detected by RT-PCR.3.Collection and identification of MSCs-derived sEVs:The supernatant of both uninfected and infected MSCs were collected 48h after lentivirus infection,and separated by ultracentrifugation in order to obtain each group of sEVs(sEVs-N,sEVs-10,sEVs-T,sEVs-V).All groups of sEVs were identified with Nanosight,projection electron microscopy,Western-Blot and ELISA.4.Induction of EAU model:The EAU model was induced in C57BL/6 mice by active immunization with an emulsion containing IRBP651-670,supplemented with Pertussis toxin and tuberculin.5.Tracing experiment:The sEVs were labeled with PKH-26.In vivo tracing:The naive mice were used as control.All of mice were intravenous injected with 100?g sEVs-N on day 11 after immunization and sacrificed after 24h and 48h post injection.Eyeball,spleen,lymph nodes,liver,kidney,and heart tissues were embedded in OCT and frozen sections were prepared.After staining with DAPI,the red fluorescence expression of sEVs was observed by confocal microscope.We also observed the tracing of EAU mice 24h after tail vein injection of sEVs-10,sEVs-T,and sEVs-N.In vitro tracing:Total CD4+T cells were isolated by negative magnetic bead sorting and were co-cultured with each group of sEVs at a concentration of 20 ?g/ml under the stimulation of CD3/CD28 antibody.After 1h,3h and 6h of cultivation,cells were collected for cell smear.After staining with CD4 antibody and DAPI,the expression of red fluorescence was observed using the confocal microscopy.6.Treatment of sEVs:Mice were injected with different doses of sEVs-N(1?g,5?g,10?g)on day 11 post immunization by periocular injection.The control group was injected with PBS.A total of 5 injections were performed once every other day.In the tail vein injection group,sEVs-N of different doses(10?g,50?g)were injected into the tail vein on day 11 after immunization and the control group was also injected with PBS.Comparative experiment:On day 11 post immunization,different groups of mice was injected via tail vein with 50 ?g diverse sEVs respectively(which were named sEVs-N group,sEVs-10 group,sEVs-T group and sEVs-V group).The control group was also injected with PBS.7.Clinical assessment:From the day 9 to day 21 post immunization,the headmounted indirect ophthalmoscope was used every other day to observe the degree of inflammation of the retina in each group of mice,and scored according to Caspi classification.8.Histopathological assessment:Mice were sacrificed on day 18 after immunization and tissue sections were prepared.The histopathological changes of retina were examined by inverted microscope and were scored according to Caspi classification.9.Flow cytometry detection of inflammatory cells:Mice were sacrificed on day 18 after immunization and their eye balls,spleens and lymph nodes were separated to prepare a lymphocyte suspension.After staining with corresponding antibodies,the proportion of Th1 cells,Th17 cells and Treg cells was detected by flow cytometry.10.Test of T cell proliferation in vivo by 5-bromo-2-deoxyuridine(BrdU):Mice were sacrificed 48 hours after treatment with tail vein injection of sEVs.T cells and APCs from spleens and lymph nodes were separated with nylon columns and then were cultured at a 2:1 ratio under different IRBP concentrations(0,1?g/ml,10?g/ml,20?g/ml).After 48 hours of culture,BrdU kit was used to determine the proliferation of T cells.11.Assay of T cell proliferation in vitro by CFDA-SE(CFSE):Total CD4+T cells from spleens of naive mice were isolated by negative magnetic bead sorting and were co-cultured with sEVs at a concentration of 10?g/ml after staining.CD4+T cell proliferation were stimulated in vitro using CD3/CD28 antibody.After 72 hours of incubation,the CFSE fluorescence intensity was measured by flow cytometry.12.T cell differentiation assays in vitro:Naive CD4+T cells from spleens of naive mice were isolated by negative magnetic bead sorting and were co-cultured with sEVs at a concentration of 10 ?g/ml under Thl cell,Th17 cell and Treg cell differentiation conditions,respectively.At the end of culture,the corresponding antibody was stained.Thl,Th17 and Treg cells were analyzed by flow cytometry.13.Proteomics analysis:The sEVs-N and sEVs-10 samples were digested with enzymes to obtain peptides,and then were detected using the SWATH method.The obtained differential proteins were analyzed by bioinformatics.Results:1.The high titers of lentivirus overexpressing IL-10,TGF-? and vector lentivirus were successfully packaged.2.The umbilical cord MSCs overexpressing IL-10 and TGF-? were constructed.Each group of sEVs was successfully isolated and identified.3.Mice models of EAU were successfully induced.4.Tracer results:After tail vein injection,sEVs were mainly distributed in the lymph nodes,spleen,and liver in EAU mice.Only a small amount of expression can be observed in the kidney,heart and retina,which decayed with time.The sEVs of each group could reach the retinal tissue after being injected intravenously.Although sEVs were concentrated in the lymph nodes,spleen,and liver in naive mice.There was less distribution in the lymph nodes and spleen than EAU mice at the same injection dose.No sEVs was observed in the retina while the expression in the liver was increased.The results of cell tracing showed that after being incubated for 1h,a large amount of red fluorescence could be observed in T cells in each group.No obvious difference was observed among the groups of 1h,3h and 6h.5.Clinical observation:After periocular injection of sEVs-N,gradient results showed that there was no statistically significant difference in the clinical scores between the treatment group and the PBS group(P>0.05).After tail vein injection of sEVsN,the clinical score of the group treated with 50?g sEVs-N was significantly lower than that of the PBS group(P<0.05).The results of the comparative experiment showed that the clinical score of the sEVs-10 group was significantly lower than that of the other groups(P<0.05);the difference between the sEVs-T group and the PBS group,sEVs-N and sEVs-V group was not statistically significant(P>0.05).6.Histopathological observation:The histopathological score of sEVs-10 group was significantly lower than that of other groups(P<0.05);the histopathological score of sEVs-N group and sEVs-V group was significantly lower than that of sEVs-T group and PBS group(P<0.05).There was no significant difference in the histopathological score of sEVs-T compared with PBS group(P>0.05).7.Flow cytometry results:The proportion of Thl and Th17 cells in the eyeballs of the sEVs-10 group decreased significantly(P<0.05),while the proportion of Treg cells in the spleen and lymph nodes increased significantly(P<0.05).The sEVs-N and sEVs-V groups also obtained similar results in contrast to the PBS and sEVs-T groups.There was no statistically significant difference between the sEVs-T group and the PBS group(P>0.05).8.T cell proliferation assays in vivo:Compared to other groups,the sEVs-10 group can significantly inhibit the proliferation of T cells at the concentration of 20?g/ml IRBP in vivo.The sEVs-N group and the sEVs-V group can also significantly inhibit the proliferation of T cells in vivo in contrast to the PBS group.There was no statistically significant difference between the sEVs-T group and the PBS group(P>0.05).9.T cell proliferation assays in vitro:sEVs-N can inhibit CD3/CD28 antibody mediated T cell proliferation in a concentration-dependent manner.Each group of sEVs at the concentration of 10 ?g/ml could inhibit the CD3/CD28 antibodymediated T cells proliferation in vitro.Among them,sEVs-T had the strongest effect,sEVs-10 followed,and sEVs-N and sEVs-V are the weakest.10.T cell differentiation assays in vitro:Each sEVs group can significantly inhibit Th1 cell differentiation,of which sEVs-T has the strongest effect,followed by sEVs-10,sEVs-N and sEVs-V have the weakest ability.In terms of Th17 differentiation,each sEVs group could significantly inhibit Th17 cell differentiation.There was no statistical difference between the sEVs-10 group and the sEVs-N group,and the sEVs-T inhibition ability was significantly weaker than that of other sEVs groups.In terms of Tregs differentiation,sEVs-N and sEVs-V had no effect on the differentiation of Naive T cells into Tregs.A small increase in the proportion of Treg cells could be observed in the sEVs-10 group and the sEVs-T group.11.Proteomics:sEVs-10 and sEVs-N samples detected a total of 1616 proteins,including 209 differential proteins.Among them,there were 125 kinds of upregulated proteins,including KRT16,etc.,and 84 kinds of down-regulated proteins,including HSP90AA1,etc.Conclusions:1.Intravenous injection of MSCs-derived sEVs can significantly ameliorate EAU.MSCs-derived sEVs overexpressing IL-10 have a stronger immunosuppressive effect on EAU disease progression in mice.2.Overexpression of IL-10 can enhance the inhibitory ability of MSCs-derived sEVs on T cell proliferation both in vitro and in vivo,Thl cell differentiation in vitro,and can also promote Treg cell differentiation in vitro,thereby exerting a stronger immunosuppressive effect.However,overexpression of TGF-? has pleiotropic effects which can promote Th17 differentiation and proliferation of effector T cells.Further research is still needed.3.Lentivirus-mediated overexpression of IL-10 by MSCs has a huge impact on the protein expression in sEVs.Differential proteins are also widely involved in autoimmune-related biological reaction.