Construction Of Immunomodulatory Biomaterial And Its Application In Treg Induction And Immune Homeostasis Regulation

BackgroundThe immune system plays an important role in tissue repair and regeneration.Failure in immune regulation lead to diverse diseases.Regulatory T cells(Treg cells)have been recognized to be critical in preventing excessive inflammatory response and ensuring immune homeostasis through their ability to induce and maintain immune tolerance.Imbalanced immune response caused by abnormal function of Treg cells is closely related to the occurrence and development of various diseases,including allergic asthma,chronic inflammatory diseases such as osteoporosis and enteritis,and various autoimmune diseases.With the in-depth study on Treg cells,researchers attempt to intervene and treat inflammatory diseases,autoimmune disorders,and organ transplantation based on the immunomodulatory effect of Treg cells.At present,many approaches have been discovered to expand Treg cells in vitro and in vivo.Administration of Treg cells was shown to be effective in reversing established arthritis and colitis mice.For autoimmune diseases,antigen-specific Treg cells suppress immune response to specific antigens and show more potent immunosuppressive effects on effector T cells.Therefore,antigen-specific Treg cells represent an optimal approach for treatment of autoimmune diseases.Although antigen-specific Treg cells have shown efficacy in animal models of autoimmunity,dramatic therapeutic effects have not be widely reported in human clinical trials.This may be attributed to the fact that in clinical translation,it is quite difficult to generate immune tolerance in the dysregulated immune system of patients with established autoimmune diseases.It has been suggested that,under inflammatory conditions caused by excessive activated T cells in an established disease state,Treg cells may lose their immunosuppressive function and be converted to T helper 17 cells(Th17).Consequently,patients may show disease relapse.Thus,it is reasonable to remove excessive activated T cells before reestablishment of a balanced immune system.The strategies used thus far to remove excessive activated T cells,such as radiation and antibodies,may also increase the risk of extensive off-target effects on the immune system.Therefore,it is urgent to establish a new strategy of sequential,safe and effective induction of antigen-specific Treg for reestablishing immune homeostasis.With the many advantages of biomaterials,their applications in the field of medicine are more and more extensive.Biomaterials show good plasticity and can achieve precise and sequential multi-functional regulation of cells,tissues,and organs through various modifications.At present,a variety of studies based on biomaterials or using biomaterials as delivery systems to regulate the immune system have been reported,suggesting that biomaterials can be used to regulate the function of T cells,which also represents a promising therapeutic strategy for the treatments of autoimmune diseases.AimTo remove excessive activated T cells and induce Treg cells,we synthesized an immunomodulatory biomaterial.Monocyte chemoattractant protein-1(MCP-1)was encapsulated in the biomaterial to recruit T cells.In addition,Fas ligand(Fas L)was conjugated onto the surface of biomaterial to trigger apoptosis of activated T cells.This design enabled biomaterial to eliminate excessive activated T cells,which triggered professional macrophages to devour the apoptotic T cells and produce high levels of TGF-βthat drive Treg differentiation.Furthermore,the modular design of the biomaterial allowed it to be engineered with autoantigens of experimental autoimmune encephalomyelitis(EAE)or type 1 diabetes(T1D).The sustained release of the autoantigens after induction of T cells apoptosis and TGF-βproduction by macrophages resulted in generation of antigen-specific Tregs in EAE and T1D mice.Methods1.The synthesis of T cell-depleting nanoparticles(TDNs)and their efficacy in vitro.Firstly,TDNs loaded with MCP-1 and Fas L were synthesized based on the structure of MSN.Secondly,splenic T cells were isolated and activated by CD3 and CD28 antibodies.The chemotactic response of activated T cells to the TDNs were observed in a Transwell co-culture system.Finally,the apoptosis of activated T cells induced by TDNs were detected by flow cytometry and Western blot.2 The establishment of mice osteoporosis model and the effect of TDNs on osteoporosis.Female mice were ovariectomized to induce osteoporosis.Bone marrow mesenchymal stem cells(BMMSCs)were isolated,and their surface markers were detected by flow cytometry.BMMSCs were cultured in osteogenic differentiation medium for osteogenic differentiation.The expression of osteogenic related genes and proteins were detected by real time PCR and Western blot,and the calcified nodules were detected by alizarin red staining.Flow cytometry was used to detect the proportion of CD3~+T cells and their apoptotic rate in PBMCs.Micro CT was performed to identify the changes of bone mass in distal femurs.The percentage of T cell subsets was detected by flow cytometry,and cytokines in serum were detected by ELISA.Finally,we evaluated the biological safety of TDNs by biochemical index detection and histological staining.3.The synthesis of IHMs and its efficacy in EAE mice.Firstly,Modular IHMs microparticles loaded with MOG35-55 were synthesized(IHMMOG).Microparticles were firstly characterized by scanning electron microscopy,dynamic light scattering and energy dispersive X-ray spectroscopy.Secondly,the EAE mice model was established and the therapeutic effect of IHMMOG on EAE was observed.The proportion and apoptotic rate of CD3~+T cells in PBMCs were detected by flow cytometry.The histopathological changes of spinal cord were evaluated by H&E staining and Luxol fast blue(LFB)staining.Finally,the induction of antigen-specific Treg cells in spinal cord was detected by tetramer method.4.The synthesis of IHMGAD and its efficacy in mice with type 1 diabetes.Firstly,IHMGAD microparticles loaded with the autoantigen of type 1 diabetes(GAD524-543)were synthesized and were injected in NOD/Shi Lt J mice.Blood glucose level was monitored by blood glucose meter.Secondly,H&E staining was performed to observe the degree of inflammatory infiltration and the number of islets in pancreas.Then,the ratio of Th1 cells and Treg cells in pancreas was detected by flow cytometry.Finally,CD4~+and CD4~+CD25~-T cell subsets in spleen were sorted,and the response of lymphocytes to specific antigen stimulation was detected.Results1.TDNs induced the apoptosis of activated T cells in vitro.Firstly,we confirmed that MCP-1 was successfully encapsulated in the mesoporous and Fas L was conjugated on the surface of MSN.Secondly,T cell morphology changed significantly,and the expression of Fas and CD25(a marker of T cell activation)increased dramatically after T cells were activated.Flow cytometry results showed that the apoptotic rate of activated T cells increased.In addition,the expression of Bax and Cleaved caspase-3 increased while that of Bcl-2 decreased after TDNs treatment.2.TDNs administration ameliorated the osteopenia phenotype in ovariectomy mice by inducing Treg cells.Firstly,a mouse model of osteoporosis induced by estrogen deficiency was successfully established,which showed a significant decrease in bone mass of femur.After TDNs treatment,the proportion of CD3~+T cells in PBMCs decreased significantly,accompanied by the increase of apoptotic rate.It was further observed that the proportion of Treg cells in spleen was increased while the proportion of Th17 cells was decreased.Meanwhile,the bone mass of femur was significantly rescued,and the osteogenic ability of BMMSCs was also increased significantly.3.IHMMOG induced antigen-specific Treg to treat EAE mice.Firstly,the mice EAE model was established successfully.After injection of IHMMOG,the apoptotic rate of T cells in PBMCs increased significantly.In addition,significant improvements in mean clinical scores were observed.The infiltration of inflammatory cells in spinal cord was alleviated,and the loss of myelin was significantly reduced.More importantly,the proportion of MOG35-55-specific Treg in spinal cord increased evidently.These data indicated that the antigen-specific Treg cells generated by MDMOG require MCP-1-mediated T cell recruitment,Fas L-induced T cell apoptosis and the release of TGF-β.4.IHMGAD induced antigen-specific immune tolerance to treat type 1 diabetes.First,we showed that the blood glucose level of NOD/Shi Lt J mice was effectively controlled after IHMGAD injection.Histological results showed that the inflammatory infiltration of islets was significantly improved after IHMGAD treatment.In addition,the proportion of Treg cells in pancreatic lymph nodes increased,while the proportion of Th1 cells decreased.Moreover,we demonstrated that splenic lymphocytes also acquired antigen-specific immune tolerance to GAD524-543.These data indicated that the antigen-specific immune tolerance generated by MDGAD require MCP-1-mediated T cell recruitment,Fas L-induced T cell apoptosis and the release of TGF-β.Conclusion1.TNDs show immunomodulatory function and can induce apoptosis of activated T cells in vitro.2.TDNs reestablish immune homeostasis and treat osteoporosis by inducing Treg cells.We demonstrated that TDNs enables rapid release of MCP-1 to recruit activated T cells and then induces their apoptosis through the conjugated Fas L.This delicate design enabled TDNs to eliminate excessive activated T cells.Apoptotic signals recognized by macrophages promote the induction of Treg cells and help ameliorate osteoporosis.3.IHMs with modular design induce antigen-specific Treg cells by loading different autoantigens to treat EAE and type 1 diabetes.IHMMOG and IHMGAD can induce T cell apoptosis and promote the secretion of TGF-βin vivo.The release of autoantigen loaded in IHMs at a low density promotes the production of antigen-specific Treg cells,and finally ameliorate EAE and type 1 diabetes in NOD/Shi Lt J mice.We have successfully constructed a modular biomaterial with immunomodulatory function,which can be applied for the treatment of a variety of autoimmune disorders.