The Protective Effect Of Exosomes From TGF-beta1 Gene Modified Bone Marrow-derived Immature DC In Murine Inflammatory Bowel Disease And The Underlying Mechanisms

Autoimmune disease is a kind of disease that is caused by body immune system responsing to autoantigen.Inflammatory bowel disease(IBD) including ulcerative colitis(UC) and Crohn' s disease(CD) is a kind of non-specific inflammatory bowel disease that is mainly caused by abnormal immune response of immune system to normal intestinal flora.In western countries,incidence of IBD is 4-6 out of one hundred thousand.In our country,because of the change of dietetic structure,incidence of IBD increased every year.Reported by domestic literature,cases in this five years is eight times more than the corresponding period in 90' s of last century.The main symptom of IBD is diarrhea,grume sanguinopurulent stool and bellyache. Furthermore,lasting for a long time,the risk of cancer mutation increases,so IBD threatens the people' s health of our country badly.In clinal,allopathy is the main therapeutic means of IBD and there still has not an effective procedure of treatment,so the demand of exploring a new way to cure IBD thoroughly is urgent.Transforming Growth Factor-beta 1(TGF-beta1) including 112 amino acid is a dimer protein with molecular weight of 25KD.It is strictly conservative in each species.For example,human,rat and mouse have totally identical sequence.Almost all cells have receptors of TGF-beta1 and its influence of growth,differentiation in cells is very extensive.In T cells,TGF-beta1 can inhibit their proliferation,inhibit the differentiation of Th1 and Th2,inhibit the differentiation of CTL,induce the generation of regulatory T cells(Treg);in B cells,TGF-beta1 can inhibit their proliferation,induce IgA class switch,it also can induce the apoptosis of immature and resting B cells;in NK cells,TGF-beta1 can inhibit the secretion of IFN-γand the cytolytic function of NK cells.In dendritic cells(DC),TGF-beta1 can inhibit their maturation and antigen presenting;furthermore,TGF-beta1 can inhibit the function of active macrophages(MOs) and granulocytes.DC are the most potent professional antigen presenting cells(APC) that are able to modulate T cell immunity in either a positive or negative manner,depending upon their lineage and state of maturation.There are several subpopulations of DC including myeloid DC,plasmacytoid DC,and Langerhans cells that play different roles in the regulation of the immune responses.In addition to their ability to stimulate immunity, these different DC populations,under certain conditions,are involved in T cell immunosuppression and/or induction of central and peripheral tolerance.For example, IL-4 or Fas L gene modified DC can inhibit delayed-type hypersensitivity(DTH) and collagen-induced arthritis(CIA).Exosomes are small vesicles released by various live cells,50-100nm,that have structure of lipid bi-layer not derived from cells membrane.In vivo,exosomes play bi-directional regulatory role in immune system.In murine models,exosomes derived from tumor share the tumor antigen can present the antigen to DC,which activate the immunity of anti-tumor of DC and then eradicate established tumor.Exosomes from APC carry MHC-Ⅰ,MHC-Ⅱand T cell costimulatory molecules on their surface, suggesting that they could play important roles in immune regulation.In murine models,exosomes from vIL-10,Fas L or IL-4 gene modified DC can inhibit DTH and CIA.Furthermore,adoptive transfer experiment show that gene modified exosomes can induce the generation of regulatory APC and T cells and then exert the function of systemic immune inhibition.Bone marrow derived immature DC(MD-imDC) because of their immature stage in differentiation,expressing low level MCHC and costimulatory molecules,so MD-imDC derived exosomes can inhibit the activation of immune cells and induce immunological tolerance.In murine cervical heterotopic heart transplantation model, TGF-beta1 gene modified MD-imDC can exert function of systemic immune inhibition, prolonging the survival of allograft.Treg play a very important role in immune balance in intestine.TGF-beta1 can induce the generation of Treg.Under the condition of losing TGF-beta signal,mice show more severe IBD development,so maintenance of TGF-beta signal is very important in regulating immune homeostasis in the intestine. The aim of this project is to explore if TGF-beta1-exo can play the function of systemic immune inhibition and rebuild immune balance in intestine.In this project dextran sodium sulphate(DSS) was used to induce murine IBD model.Based on this model,TGF-beta1 gene modified DC derived exosomes(TGF-beta1-exo) were pre-injected i.p.and the protective effect of TGF-beta1-exo during IBD development was evaluated.PARTⅠ.The isolation and identification of exosomes from TGF-beta1 gene modified bone marrow-derived immature DC and their function in vitro.DCs,the highly effective specialized antigen-presenting cells,play a pivotal role in antigen presentation in the proper context of the required antigenic and costimulatory molecules to induce T cell-mediated immune responses.The functions are highly correlated with their differentiation status.Mature DCs highly express major histocompatibility complex class-Ⅱ(MHC-Ⅱ) molecules and co-stimulatory molecules, such as CD80/86,CD40,providing two signals to activate T lymphocytes to initiate immune responses,while immature DCs which are deficient in these molecules show tolerogenic.So,heterogeneous properties of DCs either tolerogenic or immunogenic determine the outcome of immune responses.The tolerogenic properties of DCs can be amplified by delivery of genes encoding immunosuppressive molecules that are not or seldom produced by DCs.The genes which are the most feasible to induce peripheral tolerance include vIL-10,TGF-beta,CTLA4-Ig,inducible NOS,FasL,and so on. Manipulation of these agents has been shown to render DC tolerogenic characteristics.Cultured in rmGM-CSF only,we obtained DCs with steady immature phenotype and the purity is ideal averaging between 60-70%.Identified day 7 DCs by FACS,we found DCs expressed CDllc the marker molecule of murine DCs and low level of MHC-Ⅱ,CD40,CD86.To best improve the infection efficiency of Ad TGF-beta1 to large amount DCs, we purified adenoviruses by CsCl density gradient centrifugation and then infected DCs by MOI50.During the 2h' serum-free infection,we put the mixture of DCs and adenoviruses onto rotatable bed with 37℃and keep the bed rotating which can make the cell and viruses contact completely.By this way,we improved the infection efficiency greatly and based on this,we characterized the immunophenotype of DCs by FACS to define the influence of TGF-beta1 gene modification on DCs maturation.We found the control DCs exhibited features of immature DCs.The infection of Ad Lac Z did not change the features obviously.The expression of MHC-Ⅱ,CD86,CD40 were all inhibited on TGF-beta1 gene modified DCs and the most obvious inhibition was observed in CD80.IL-12 is one of primary cytokines secreted from DC,which can drive T cells toward help T cell 1(Th1) and their secretion of IL-2 and IFN-γthat result in strong immunological response.The IL-12 level in culture supernatant of mature DC was significantly higher than that of immature DC.After stimulation by LPS,accelerated the maturation of DC.The concentration of IL-12 from the supernatant of TGF-beta1 gene modified DC was lower than the control group,while LPS stimulation widened the differences.In vitro each groups DC was inactivated and set up in 120 hours primary MLR cultures with naive BALB/c T cells.DC transduced with TGF-beta1 gene,consistent with their surface phenotype and capability of secreting of IL-12,induced a comparatively low level of allogeneic T cell proliferation.DC transduced with Lac Z gene show slightly stronger ability to stimulate allogeneic T cell proliferation than control DC.We transfected TGF-beta1 gene into DC with ideal efficiency and then obtained TGF-beta1-DC with steady immature phenotype.Furthermore,we validated their inhibitory function to T cells ensuring their immunological tolerant capability.Based on this,we isolated exosomes from supernatant of TGF-beta1 gene modified DC. Supernatant from 1~*10~6DC can produce 1μg exosomes in first 24 hours.After that, alonging with the apoptosis of DC,the production of exosomes also decreased.In view of the maturation of DC with time,we collected the exosomes from first 48h DC.We visualized the exosomes with high purity under the EM,those particles with the sizes at 50-100nm,with clear bi-lipid membrane.After adsorbed onto latex,phenotype of exosomes was detected by FACS.Results showed that TGF-beta1-exo expressed low MHC-Ⅱmolecules,CD80 and undetectable CD86,CD40,which suggested they might bear the function of immune regulation.CD11c the marker of murine DC also could be detected on exosomes which ensured us their genesis of DC.Tsg101 and Hsp70 the characteristic protein of exosomes also could be detected in exosomes by WB.Tsg101 is a marker of endosome and related to the formation of exosomes.Compared with Lac Z-exo and Control-exo,TGF-beta1-exo beared lower Hsp70 which is know to convert T cell tolerance to autoimmunity in vivo.This further suggests the immune regulatory function of exosomes.Compared with Control-exo and Lac Z-exo,only TGF-beta1-exo expressed TGF-beta1.This result showed that TGF-beta1 could be sorted to exosomes through unknown mechanisms.TGF-beta1 was further quantified by ELISA assay and the amount of TGF-beta1 in four cycles of freeze/thaw exosomes and intact exosomes was 15pg/μg and 8pg/μg.This result suggested that there were both secreting and membrane-associated TGF-beta1 in exosomes.In vitro each group exosomes 120 hours primary MLR,TGF-beta1-exo induced a comparatively low level of allogeneic T cell proliferation.While Contro-exo and Lac Z-exo also show slight inhibitory effect.Except for inhibiting T cells proliferation,we also found TGF-beta1-exo could inhibit the activation of lymphocytes induced by Con A.That result made us much more ensure the inhibitory function of TGF-beta1-exo.In conclusion,we isolated exosomes from TGF-beta1 gene modified DC successfully and characterized them by EM,FACS and WB.Furthermore,we testified their immune inhibitory function in vitro.All those results set up solid foundation for our next therapeutic experiments in vivo.PARTⅡ.The protective effect of exosomes from TGF-beta1 gene modified bone marrow-derived immature DC in murine inflammatory bowel disease and the underlying mechanisms.We then examined the in vivo effects of TGF-beta1-exo on the development of IBD using a C57BL/6 murine IBD model induced by DSS.IBD was induced by giving 1.5%DSS for continuous 9 days.Two days before DSS drinking,C57BL/6 were injected i.p.with Control-exo,Lac Z-ex and TGF-beta1-exo at the dose of 10μg/per mouse and on day 3,day 5 after DSS drinking mice were received another injection of each group exosomes i.p.at the same dose.There also had one group with drinking water as normal control and the other drinking DSS only with no treatment as positive control.Compared to control mice,which maintained their weight,the DSS-induced mice(no treatment) suffered extensive weight loss—statistically significant from day 5 after induction,reaching 16%by day 9.Treatment of Contro-exo or Lac Z-exo did not prevent the DSS-induced weight loss and similarly to the group of untreated mice,they all reached 15%by day 9.In contrast,the weight loss of the DSS-induced mice treated with TGF-beta1-exo was significantly lower decreasing by only 5%from their original weight by day 9.The daily monitored disease activity index(DAI,combined score of body weight,bleeding and stool consistency),as well as the intestinal bleeding were also reduced by treatment of TGF-beta1-exo but not by treatment of Contro-exo or Lac Z-exo.Histological assessment of colonic damage in Control-exo,Lac Z-exo or untreated mice,9 days after DSS induction,revealed extensive severe nearly diffuse inflammation involving the mucosa,submucosa and in some cases extending through all intestinal layers(transmural inflammation).This was associated with a pronounced disruption of the normal architecture and crypt loss.In colons of DSS-induced mice treated with TGF-beta1-exo less damage and more conserved glandular structure were revealed,although widespread leukocyte infiltrations were found.To look into the mechanisms hidden behind the results we had obtained,we isolated lymphocytes from spleens and mesentery lymph nodes(mLNs) of inflammatory site of each group mice and analyzed for relative Foxp3~+ Treg cell nembers.Compared to other group mice,lymphocytes from spleens of TGF-beta1-exo treated mice contained the similar relative Foxp3~+ Treg cell nembers.While among the lymphocytes from mLNs of inflammatory site,the relative Foxp3~+ Treg cell nembers increased statistically.To further analyze the cause of that difference,we labeled exosomes with CFSE and then coincubate CFSE labeled exosomes with lymphocytes from spleens or mLNs of IBD or control mice.We found that lymphocytes from mLNs of IBD mice could internalized more exosomes than control mice but that difference could not be observed between lymphocytes from spleens of IBD and control mice.In conclusion,the development of IBD induced by DSS of TGF-beta1-exo treated mice was much slower than that of other groups.Unlike the slightly inhibitory effect observed in MLR in vitro,Control-exo and Lac Z-exo could not show any protective effect in the development of IBD induced by DSS.Compared to other groups,the relative Foxp3~+ Treg cell nembers increased statistically among the lymphocytes from mLNs of inflammatory site of TGF-beta1-exo treated mice,which may be one of the mechanisms of TGF-beta1-exo can show the protective effect in the development of IBD induced by DSS.Lymphocytes from inflammatory site could internalize more TGF-beta1-exo that resulted in the high intensity of TGF-beta1 in inflammatory site probably contribute one of the cause of the results that the relative Foxp3~+ Treg cell nembers increased statistically among the lymphocytes from mLNs of inflammatory site but not spleens.