| Retinal pigment epithelium cells(RPE)maintain the function and homeostasis of the retina through nutrition,supporting photoreceptor cells,and building barriers.The occurrence of retinal degenerative(RD)diseases is accompanied by apoptosis and dysfunction of RPE cells.Human embryonic stem cells derived RPE(hESC-RPE)transplantation may is a promising clinical strategies to ameliorate the deterioration of this kind of disease.Numbers of animal and clinical experiments have confirmed that hESC-RPE transplantation has obvious short-term effects on RD.Previous studies have reported that more than 95%of transplanted cells mainly play an immunomodulatory role through intercellular substance exchange,autocrine and paracrine.Exosomes,as a kind of nanoscale vesicle,can be produced by almost all living cells,and delivered in the systemic circulation after being secreted out.Exosomes cotain proteins,nucleic acids,lipids and other cargoes,which can change the behavior and state of the target cells by delivering the inner bioactive molecules mentioned.An increasing body of evidence indicates that exosomes play a pivotal role in cell-to-cell communication and material exchange.However,the roles of hESC-RPE-derived exosomes in RD remain not clear.In addition,due to the damage of blood retinal barrier in patients,the transplanted cells will be infiltrated in the inflammatory environment for a long time.After being stimulated by inflammatory factors,the cargoes carried by the exosomes secreted by the transplanted cells will change significantly,and the function of the exosomes will also change.Therefore,exploring the differences in the contents and functions of exosomes secreted by hESC-RPE under different pathophysiological conditions is of great significance for clarifying the effective mechanism of stem cell transplantation and accelerating the clinical transformation of stem cell transplantation.This study hypothesize that exosomes may be one of the key mechanisms for the function after hESC-RPE transplantation.Exosomes secreted by hESC-RPE in different inflammatory microenvironments may have different functions.On the one hand,normal cultured hESC-RPE derived exosomes(Nor-EXO)may alleviate local retinal inflammation by regulating the activation of immune cells in the retina;On the other hand,exosomes secreted by hESC-RPE stimulated by inflammatory factors such as IFN-?(IFN-? EXO)may further enhance the proliferation and activation of immune cells and worsen the RD inflammatory microenvironment.Purposes:To explore the differences in the contents and functions of exosomes secreted by hESC-RPE with or without stimulation of the RD inflammatory microenvironment,and the mechanisms by which exosomes participate in regulating the RD inflammatory microenvironment.Methods:Part 1:Nor-EXO participates in the regulation of RD inflammation microenvironment1.Isolate hESC-RPE-derived exosomes by ultracentrifugation and identify it using Transmission electron microscope(TEM),Western Blot(WB),and Nanosight analysis(NTA).2.Exosomes were transplanted into the subretinal space of RCS rats and PBS was used as the control.The effects of exosomes transplantation on visual function of RCS rats were detected by flash electroretinogram(f-ERG)and grating behavior,and the effects of exosomes transplantation on microglia of RCS rats were detected by immunofluorescence and q-PCR.3.Proteomics was used to characterize the specific protein cargoes carried in the normal cultured hESC-RPE derived exosomes(Nor-EXO),and use multi-databases such as String and GO to find the key biological pathway.4.Co-culture exosomes with retinal explants of RCS rats,and explore the effects of Nor-EXO on pro-inflammatory cytokines and microglia-related cytokines in degenerate retinas through cytokine chips and q-PCR.5.Using the BV2 microglia cell line to clarify the molecular mechanism of Nor-EXO regulating RD.Part 2:IFNy-EXO participates in the regulation of RD inflammation microenvironment1.Pretreated hESC-RPE with IFN-? to mimic the microenvironment of RD in vivo.2.Isolation and purification of normal cultured hESC-RPE-drived exosomes(Nor-EXO)and IFN-? stimulated hESC-RPE-derived exosomes(IFN-? stimulated hESC-RPE derived EXO,IFN-? EXO)to characterize the two different kinds of exosomes by WB,NTA,TEM.3.Two types of exosomes were transplanted into the subretinal space of RCS rats in the early,middle and late stages of RD;changes in visual function of RCS rats after transplantation were detected by flash electroretinogram;flow cytometry and q-PCR were used to detect the effects of different exosomes on the proportion of immune cells and the expression of related proinflammatory cytokines in vivo.4.The proteome was used to analyze the changes of protein expression profile between the exosomes derived from hESC-RPE with IFN-? stimulated or not,enrich immune-related pathways and predict the differences function.5.Co-culture hESC-RPE with two different types of exosomes in vitro,then characterize the effects of different exosomes on the immunogenicity,activity and function of hESC-RPE,and the chemotaxis of immune cells by flow cytometry and q-PCR.6.The proportion,proliferation and granzyme B of natural killer cells and CD 8+cytotoxic T cells in peripheral blood mononuclear cells of patients with RD were characterized by carboxyfluorescein diacetate succinimide,solid-phase enzyme-linked immunospot assay and flow cytometry.Results:Part 1:Nor-EXO participates in the regulation of RD inflammation microenvironment1.Acquire exosomes by ultracentrifugation the cell culture fluid of normal cultured hESC-RPE,it showed a classic double-concave saucer structure under TEM,the average diameter detected by NTA was 119.7nm,western blotting showed that exosomal markers such as CD63?CD81?TSG101 can be detected on the surface of exosomes we acquired,it is proved that ultracentrifugation can obtain relatively pure exosomes.2.Compared with the PBS transplanted group,after Nor-EXO was transplanted into the subretinal space of RCS rats,the visual function of RCS rats was preserved,b-wave amplitude(p<0.05)and visual acuity(p<0.05)significantly increased,suggesting that Nor-EXO transplantation delays visual function loss in RCS rats.3.Immunofluorescence showed that the morphology of microglia in the retina of Nor-EXO transplanted RCS rats was mostly in the resting state "branched",while the microglia cells in the retina of PBS transplantation group was mostly exhibited an amoebic-like activated state;and the levels of microglial-related proinflammatory cytokines such as IFN-y and CCL-2 in the retina of the Nor-EXO transplanted group were significantly lower(p<0.05),indicating that Nor-EXO transplantation alleviate the microglial-mediated inflammatory response in the retina of RCS rats.4.The analysis of proteomic data shows that the proteins carried by Nor-EXO are mainly enriched on the pathway of Negative regulation of acute inflammatory response(q<0.05).5.Cytokine microarray showed that co-culture with Nor-EXO significantly reduced the expression levels of IL-1?,IL-1?,sICAM-1 and other proinflammatory cytokines in RCS rat retinal explants(p<0.05);the results of q-PCR indicated that Nor-EXO significantly decreased the expression of activated microglial marker CD68 at the transcription level(P<0.05);It is suggested that Nor-EXO reduce the level of pro-inflammatory cytokines in retinal explants of RCS rats by down regulating the quantity of activated microglia.Part 2:IFNy-EXO participates in the regulation of RD inflammation microenvironment1.IFN-y stimulated hESC-RPE for 24 hours,then flow cytometry showed that the expression level of HLA antigen on the surface of hESC-RPE increased,it is suggested that the high immunogenicity hESC-RPE model was successfully constructed in vitro.2.Acquire exosomes by ultracentrifugation the cell culture fluid of IFN-?stimulated hESC-RPE,it showed a classic double-concave saucer structure under TEM,the average diameter detected by NTA was 123.4 nm,western blotting showed that exosomal markers such as CD63?CD81.TSG101 can be detected on the surface of exosomes we acquired.It indicated that there is no significant difference between Nor-EXO and IFN-? EXO in morphology and particle size.3.Nor-EXO or IFN-? EXO were transplanted into the subretinal space of RCS rats in the early,middle and late stages of RD.Flash electroretinogram showed that the transplantation of IFN-? EXO at three time points resulted in the rapid loss of b wave amplitude of f-ERG in RCS rats(P<0.05),while Nor-EXO significantly preserve b wave amplitude at the early and middle stages of RD(P<0.05),suggesting that Nor-EXO transplantation could protect the residual visual acuity of RCS rats,but its protective effect weakened with the deepening of degeneration;IFN-? EXO transplantation leads to further loss of visual function in RCS rats.4.Flow cytometry showed that IFN-? EXO transplantation significantly increased the proportion of NK cells and CD8+T cells in degenerative retinas of RCS rats(p<0.01),while Nor-EXO decreased the proportion of NK cells(p<0.05),which has no significant effect on CD8+T cells;q-PCR results suggest that IFN-? EXO dramatically up-regulates the expression levels of chemokines such as CCL-2,CCL-5,CXCL9 in the retina of RCS rats(p<0.05),In addition,Nor-EXO can significantly reduce the expression of CCL-2(p<0.05),and has almost no effect on its chemokines.It is suggested that IFN-? EXO recruits NK cells and CD8+T cells,which leads to enhanced retinal inflammation response in RCS rats and changes its retinal inflammation microenvironment.5.By analyzing the proteomic data of Nor-EXO and IFN-? EXO,a total of 428 differentially expressed proteins were detected,and pathways such as Antigen processing and presesntation via MHC Class I,celluar response to IFN-??Cytokine signaling in immune response and Leukocyte transdothelial migration(q<0.05).It is suggested that IFN-? EXO causes the recruitment and activation of NK cells and CD8+T cells by enhancing the presentation of MHC antigens,leading to increased release of pro-inflammatory cytokines and enhanced inflammatory response in the retina.6.After co-culturing two different types of exosomes with hESC-RPE for 48 h,flow cytometry revealed that IFN-?EXO significantly increased the expression level of HLA antigen on the surface of hESC-RPE(p<0.01);flow cytometry and q-PCR suggested IFN-y EXO promotes hESC-RPE apoptosis(p<0.01),decreases the expression of functional genes such as RPE65,PEDF,bestrophin(p<0.05),and increases the transcription of chemokines such as CCL5,CXCL-9 Level expression(p<0.05),while no significant effect on hESC-RPE co cultured with Nor-EXO.It is suggested that IFN-?EXO up-regulates the expression of HLA antigen on the surface of hESC-RPE,induces hESC-RPE apoptosis,down-regulates the expression level of functional genes and up-regulates chemotaxis of immune effector cells.7.After co-cultured the two types of exosomes with PBMCs from patients with RD for 48 h,flow cytometry revealed that IFN-? EXO significantly increased the proportion of NK and CD8+T cells in PBMCs of patients with RD(p<0.01)and enhanced proliferation of NK cells(p<0.05);ELISPOT results showed that IFN-?EXO dramatically promoted the activation of NK cells and CD8+T cells and the secretion of granzyme B(p<0.01),while Nor-EXO down regulation the secretion of Granzyme B(p<0.05).The results mentioned above strongly prove that,in contrast to Nor-EXO's ability to alleviate inflammation,IFN-? EXO activates two types of immune cells and induces the secretion of immune effectors,thereby worsening the inflammatory microenvironment of transplanted cells.ConclusionIn this study,two kinds of exosomes were transplanted into the subretinal cavity of RCS rat,which is a classical model of RD,to explore the role of exosomes in the inflammatory microenvironment of RD and its regulatory mechanism.1.In this study,it was the first time to observe that Nor-EXO transplantation can alleviate the activation of microglial cells and inhibit the secretion of proinflammatory cytokines to save visual function in RCS rats.IFN-? EXO transplantation significantly increased the proportion of NK cells and CD8+T in the retina which leads to rapid loss of visual function in RCS rats.2.Functional clustering of differentially expressed proteins between Nor-EXO and IFN-? EXO suggested that IFN-? EXO up-regulated the secretion of pro-inflammation cytokines and enhanced antigen presentation while Nor-EXO negative regulated inflammatory response.3.In this study,retinal explant culture was used to confirm that nor-exo significantly reduced the expression level of CCL-2 in the denatured retina of RCS rats,and that nor-exo reduced the expression level of related cytokines by inhibiting the number of activated M1 microglia.It is clear that the inhibition of norexo on microglia mediated inflammation is an important mechanism to limit the further deterioration of the inflammatory microenvironment of the degenerative retina.4.In this study,it was observed that IFN-? exo co cultured with hesc-rpe significantly increased the expression of HLA antigen on the cell surface,worsened the survival of hesc-rpe and down regulation the expression of function related genes,and enhanced the chemotaxis of immune cells.5.In this study,we creatively use ELISPOT to detect the effect of two types of exosomes on RD patients' PBMCs,it was the first time to discover that contrary to the inhibitory effect of Nor-EXO,IFN-? EXO significantly increase the proportion.proliferation and activation of NK cells and CD8+T cells in RD patirnts' PBMCs,and further worsen the RD microenvironment.It Provides strong evidence for findings from animal and cell models. |
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