The Evaluation of the Effect of Tolerogenic Probiotics on the Maturation of Healthy Dendritic Cells versus Immature Dendritic Cells.

Background: Dendritic cells, (DCs) as one of the important immune cell populations, are responsible for the initiation, development, and control of acquired immune responses. Myeloid dendritic cells can be used as a vaccine for several autoimmune diseases and cancers. Tolerogenic probiotics with regulatory properties can affect the maturation and development of immature dendritic cells (IDC) into mature DCs with certain immunomodulatory effects. Objective: To assess the immunomodulatory effect of Lactobacillus rhamnosus and Lactobacillus delbrueckii, as two tolerogenic probiotics, in the differentiation and maturation of myeloid dendritic cells. Methods: The IDCs were derived from the healthy donors in GM-CSF and IL 4 medium. Mature DCs (MDC) were produced with L. delbrueckii, L. rhamnosus, and LPS from IDCs. Real-Time PCR and flow cytometry were used to confirm the DC maturation and to determine DC markers as well as IDO, IL10, and IL12 expression levels, respectively. Results: Probiotic-derived DCs showed a significant reduction in the level of HLA-DR (P≤0.05), CD86 (P≤0.05), CD80 (P≤0.001), CD83 (P≤0.001), and CD1a. Also, the expression of IDO (P≤0.001) and IL10 increased while IL12 expression decreased (P≤0.001). Conclusion: Our findings revealed that tolerogenic probiotics could induce regulatory DCs by reducing co-stimulatory molecules along with increasing the expression of IDO and IL10 during the differentiation process. Therefore, the induced regulatory DCs probably can be used in the treatment of various inflammatory diseases.

Monocyte-derived immature dendritic cells negatively regulate hepatic stellate cells in vitro by secreting IL-10.

The development of liver fibrosis is associated with inflammatory responses resulting from chronic liver disease. Immature dendritic cells (imDCs) play an important role in modulating the inflammatory environment of the liver. This study investigated the effects of imDCs on the regulation of hepatic stellate cells (HSCs) during liver fibrosis. We isolated and induced imDCs from monocytes of healthy volunteers, activated LX-2 cells with TGF-ß to establish in vivo liver fibrosis HSCs model, and then set up a cell co-culture system with transwell membranes. imDC surface markers and apoptosis rates of LX-2 cells were detected by flow cytometry. The concentration of IL-10 secreted by imDC was measured through ELISA. The expression of α-SMA in LX-2 after co-culture was examined by qRT­PCR. Proliferation of LX-2 cells were detected by CCK-8. The western blot was used to illustrate the LX-2 activation-related proteins such as Smad3/7 and TGF-ß1. The imDCs co-culture group and the interleukin-10 (IL-10) treatment group had similar results, as they were both able to increase apoptosis, inhibit proliferation, downregulate α-SMA mRNA, and reduce TGF-ß1 and Smad3 protein expression in LX-2 cells. Additionally, the Smad7 protein level was increased after treatment with imDC and IL-10. However, the results in the IL-10 antagonist group showed the opposite trend to that of imDCs and IL-10 groups. Thus, these results suggest that imDC secretion of IL-10 negatively regulates activated LX-2 cells, probably via inhibition of the TGF-ß1/Smad3 pathway and increased expression of Smad7 protein. This may be a potential therapeutic target for liver fibrosis.

Recent advances on the role of various cells-derived exosomes in ocular surface / 中华实验眼科杂志

Exosomes are membranous vesicles secreted by cells and can be widely involved in intercellular communication, anti-inflammation, immunoregulation, etc.Mesenchymal stem cell (MSC), regulatory T cell (Treg), immature dendritic cell (imDC) and myeloid-derived suppressor cell (MDSC) are the main ocular surface-related exosomes origins.Exosomes derived from different cells play their roles by delivering different biological molecules to recipient cells.Exosomes derived from MSC play a positive role in ocular surface inflammation and immune-related diseases by inhibiting T cell proliferation, transforming macrophage phenotype, regulating T helper (Th) cell differentiation and up-regulating Treg expression, reduce neovascularization and inflammation, and foster a microenvironment to promote corneal wound healing at the same time.Exosomes derived from Treg contain inducible NO synthase and microRNA (miRNA) including miR-503, miR-330 and miR-9, which can interfere with cell cycle progression, induce apoptosis, induce the differentiation of other T cells into Treg phenotype, inhibit T cell allograft rejection to induce immune tolerance.Exosomes derived from imDC inhibit corneal allograft rejection by delivering miR-682.MDSC-derived exosomes promote Treg expansion in vivo and in vitro, inhibit the proliferation and cytotoxicity of activated T cells, and express miR-29a-3p and miR-93-5p, which can inhibit the differentiation of Th1 and Th17 cells.Given the anti-inflammatory and immunosuppressive effects of exosomes, this paper reviewed the studies on ocular surface inflammation and immune-related diseases such as corneal injury, mucopolysaccharide storage disease, dry eye, Sj?gren syndrome and ocular graft-versus-host disease.

Histone deacetylase inhibitors suppress immature dendritic cell's migration by regulating CC chemokine receptor 1 expression.

The modulation of immature dendritic cells (iDCs), which involves processes such as phagocytosis, migration, and maturation, is considered a beneficial research theme. Once activated by an antigen, iDCs turn to mature DCs (mDCs) and migrate towards secondary lymphoid organs, and initiate the progress of cellular immunity. Histone deacetylase inhibitors (HDACis) are also thought to be a major modulator of cellular immunity. Herein, we demonstrate that HDACis (trichostatin-A (TSA), sodium butylate (SB), scriptaid (ST)) play a central regulatory role in the migratory activity of iDCs. In our results, TSA, SB and ST showed the potent inhibitory effect on the migration of iDCs stimulated by MIP-1α. The inhibitory activities of HDACis were found to be caused by reduction of CCR1 expression on the cell surface, and by the inhibition of phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and c-Jun N-terminal kinase (JNK).

Isolation,induced culture and identification of immature dendritic cells in vitro / 中国免疫学杂志

Objective:To establish a fast method for the generation of immature dendritic cells(DCs) from human peripheral blood mononuclear cells(hPBMCs) in vitro.Methods: High purity human CD14+ monocytes were collected using density Ficoll gradient centrifugation and MACS beads sorting system.iDCs(Immature DC) were induced after cultured with rhGM-CSF and rhIL-4 on the fourth day.Fluorescence activated cell sorting(FACS) was used to identify cell surface markers(CCR5) and capabilities of antigen uptake of iDCs on the fourth day.Ordinary optical microscope,scanning electron microscopy and transmission electron microscopy were used to observe surface and internal structure of iDCs on the fourth day of culture conditions.Results: FACS result shows that the purity of CD14+ monocytes collected from hPBMCs were more than 94%.The antigen uptake capability and CD195 of iDCs was detected on the fourth day of cultured conditions.Typical surface and internal structure characteristics of iDCs were observed.Conclusion: Rapid induction culture is an effective method for obtaining a large number of iDC with typical characteristics in vitro,and can be used for further experimental study.

Immature dendritic cell exosomes suppress experimental autoimmune myasthenia gravis.

Immature dendritic cell-derived exosomes (iMDEX) display a certain degree of immunosuppressive activity in autoimmune diseases. However, the role of iMDEX in experimental autoimmune myasthenia gravis (EAMG) is still unclear. Therefore, we tested the effects of mouse bone marrow (BM)-derived iMDEX on tolerance induction in a mouse model of EAMG. In this study, we found that the CELLine culture system produced more exosomes, the morphology and phenotype of these exosomes were found to be identical when compared with traditional cell culture. And, iMDEX(1000) ameliorated the progression of EAMG by reducing AChR-reactive lymphocyte proliferation, AChR antibody levels and pro-inflammatory cytokine levels.

Osteopontin has a crucial role in osteoclast-like multinucleated giant cell formation.

The osteoclast (OC) is a major player in the pathogenic bone destruction of inflammatory bone diseases such as rheumatoid arthritis and Langerhans cell histiocytosis. Recently, it was shown that immature dendritic cells (iDC) fuse faster and more efficiently than monocytes in forming OC-like multinucleated giant cells (MGCs), and that osteopontin (OPN) is involved in the pathogenesis of inflammatory bone diseases. In this study, we hypothesized that OPN is a key factor for generation of OC-like MGCs from iDCs. We used an in vitro culture system to differentiate iDCs, derived from monocytes obtained from the blood of healthy donors, into OC-like MGCs. We evaluated OPN levels and expression of OPN receptors during the course of differentiation. OPN has an arginine-glycine-aspartic acid (RGD) motif, and protease cleavage reveals a SVVYGLR motif. The concentrations of both full-length and cleaved forms of OPN increased during the course of OC-like MGC formation. Expression of OPN RGD- and SVVYGLR-recognizing receptors also increased at later stages. We analyzed whether blocking OPN binding to its receptors affected OC-like MGC formation. Monocytes treated with OPN siRNA were able to differentiate into iDCs effectively; however, differentiation of these iDCs into OC-like MGCs was significantly reduced. The formation of OC-like MGCs was not significantly reduced by RGD synthetic peptide. By contrast, SVVYGLR synthetic peptide caused a significant reduction. These data suggest that the cleaved form of OPN plays a critical role in driving iDC differentiation into OC-like MGCs in the early phase of differentiation, in an autocrine and/or paracrine fashion.

Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes.

The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes.

Mechanism of immune tolerance induced by donor derived immature dendritic cells in rat high-risk corneal transplantation.

AIM: To study the role of immature dendritic cells (imDCs) on immune tolerance in rat penetrating keratoplasty (PKP) in high-risk eyes and to investigate the mechanism of immune hyporesponsiveness induced by donor-derived imDCs. METHODS: Seventy-five SD rats (recipient) and 39 Wistar rats (donor) were randomly divided into 3 groups: control, imDC and mature dendritic cell (mDC) group respectively. Using a model of orthotopic corneal transplantation in which allografts were placed in neovascularized high-risk eyes of recipient rat. Corneal neovascularization was induced by alkaline burn in the central cornea of recipient rat. Recipients in imDC group or mDC group were injected donor bone marrow-derived imDCs or mDCs of 1×10(6) respectively 1 week before corneal transplantation via tail vein. Control rat received the same volume of PBS. In each group, 16 recipients were kept for determination of survival time and other 9 recipients were executed on day 3, 7 and 14 after transplantation. Cornea was harvested for hematoxylin-eosin staining and acute rejection evaluation, Western blot was used to detect the expression level of Foxp3. RESULTS: The mean survival time of imDC group was significantly longer than that of control and mDC groups (all P<0.05). The expression level of Foxp3 on CD4(+)CD25(+)T cells of imDC group (2.24±0.18) was significantly higher than that in the control (1.68±0.09) and mDC groups (1.46±0.13) (all P<0.05). CONCLUSION: Donor-derived imDC is an effective treatment in inducing immune hyporesponsiveness in rat PKP. The mechanism of immune tolerance induced by imDC might be inhibit T lymphocytes responsiveness by regulatory T cells.

Mechanism of Immune Hyporesponsiveness Induced by Recipient-Derived Immature Dendritic Cells in Rat Liver Transplantation / 中国普外基础与临床杂志

Objective To study the mechanism of immune hyporesponsiveness of allograft rejection induced by transfusion nonpufsed allopeptide syngeneic immature dendritic cell(imDC) generated from recipient bone marrow progenitors and to explore a possible strategy for liver allograft protection in clinic.Methods Forty experimental rats were randomly divided into 4 group: control group,cyclosporine A(CsA) group,mature DC(mDC) group and imDC group.In control group,Wistar rats only received liver transplantation.In CsA group,Wistar rats underwent liver transplantation plus CsA treatment(10 mg/(kg?d)).In mDC group,recipient-derived mDC 1?106 were infused intravenously through the penile vein to Wistar rats.In imDC group,ImDC with the dose of 1?106 were injected into Wistar rats via the dorsum vein of penile.In each group,five recipients were killed on the 10th day after transplantation,the other five recipients were left to observe survival time.The levels of ALT,AST,TBIL,IL-2,IFN-?,IL-4 and IL-10 were detected.The acute rejection and the expression of FasL/Fas in the grafts were detected by HE and immunohistochemical staining.Western blot was used to detect Scurfin protein expression of CD4+ CD25+ T cells.Results The median survival time of the liver allografts in CsA group and imDC group were significantly longer than that in control group and mDC group(P

Immature dendritic cells alone can induce mixed allogeneic chimerism / 中国免疫学杂志

100 d) than those injected BMCs at the fifth day (

The possible mechanisms of establishing allogeneic chimerism and prolonging allograft survival by immature dendritic cells / 中国免疫学杂志

Objective:To explore the possibly mechanisms of inducing allogeneic chimerism and prolonging allografts survival in recipients by immature dendritic cells (imDCs) Methods:Bone marrow cells (BMCs) derived from donor (C57BL/6) were used to generate imDCs The splenocytes of recipients were pretreated by inactivated imDCs in vitro, or the recipients (Balb/C) were injected of inactivated imDCs via vein in vivo Then collected the splenocytes mixed with the inactivated splenocytes from donor to detect the responsiveness Mixed lymphocyte reaction was also used to evaluate the reactivity of the chimerism mice to the donor splenocytes At the same time the diversion of Th1/Th2 paradigm was studied by semi quantitative RT PCR Results:The splenocytes conditioned with imDCs pretreatment expressed hypo responsiveness to the donor stimulation, and the immunized mice also proliferated less degree compared with the naive mice The hyporeactivity was evidently seen within 72 hours after stimulation by donor splenocytes There was significant difference between them The chimerism mice showed unresponsiveness to donor antigens, while reactivity to the third party antigens was retained The result of RT PCR suggested, to some extent, there was a diversion of Th1/Th2 paradigm in the establishment of chimerism in the model Conclusion:The putative mechanism of immature dendritic cells inducing the generation of allogeneic chimerism may based on the hypo responsiveness produced by imDCs, and there may also exist some kinds of diversion of Th1/Th2 paradigm

A SCANNING ELECTRON MICROSCOPE STUDY ON THE IMMATURE DENDRITIC CELLS BEFORE AND AFTER THE ELECTRANSFECTION OF HUMAN HEPATIC CANCER CELL RNA / 解剖学报

Objective To observe the morphologic changes of immature dendritic cells(imDCs) before and after the electransfection of human hepatic cancer cell RNA. Methods Monocytes were purified from human peripheral blood,and induced into imDCs.Then human hepatic cancer cell RNA was electransfected into monocyte-derived imDCs.ImDCs were identified by the immunocytochemical method with 7 specific antibodies before and after electransfection.These dendritic cells were observed by scanning electron microscopy. Results After electransfection of human hepatic cancer cell RNA there were few changes of molecule expressions in imDCs.ImDCs were in round,oval and irregular shapes before electransfection.Their sizes were not identical but all bigger than monocytes.There were many protrusions in different shapes which looked like dendrite,or/and bubble,veil cloud on the surface of these imDCs.Although there were some cell fusions and cell deaths after electransfection,most imDCs recovered from the damage.Electransfecting human hepatic cancer cell RNA into imDCs would make pores on cell membrane.Conclusions The pores on cell membrane make it possible that the exogenous material enters imDCs.This study can prove the possibility of electransfecting human hepatic cancer cell RNA into imDCs to make cancer vaccine,which provides a new way for tumor biologic therapy.