The Biological Characteristics Of Recombinant Glucose-regulated Protein78-induced Murine DC

[Objective]The78kD glucose-regulated protein (Grp78) is an endoplasmic reticulum chaperone that belongs to the heat-shock protein (HSP) family. Recent reports have suggested that Grp78is highly expressed in a variety of tumor tissues. Dendritic cells (DCs) are potent professional antigen-presenting cells (APCs).They play a pivotal role in the induction of immunity or tolerance depending on their activation and maturation status, phenotype, and source of origin. Immature DCs (immDC) are an example of tolDCs, but they are not stable and can become immunogenic by undergoing final maturation in response to proinflammatory cytokines and/or pathogen-derived molecules. ImmDC may therefore be unsafe to be used as a therapeutic tool. However, stable tolDC can be generated in vitro by, for instance, genetic engineering or pharmacological modulation of DC. TolDC shows great therapeutic potential in inhibiting destructive immune responses in mouse models of bone marrow or organ transplantation and autoimmunity. Therefore, we set up a hypothesis to test if Grp78could induce tolerance DC with the functional stability. In this study, mouse recombinant Grp78which was prepared by our lab before was supplemented into the BMDC culture system. Then the differentiation and maturation of CD1lc+cells were detected. These tolerogenic DCs were further identified for their phenotype, cytokine secretion profile and the ability to induce differentiation of Foxp3+Treg. This study could lay foundation for the role of Grp78in immune regulation.[Methods] 1.Preparation of recombinant Grp78Mouse recombinant Grp78was prepared by our lab previously.Briefly, Escherichia coli expression strain BL21-(DE3) containing the recombinant PGEX-4T-3-BiP plasmid was grown at37℃in Luria-Bertani (LB) medium containing Ampicillin (100mg/ml). Isopro-pyl-D-thiogalactopyranoside (0.8mM) was added to the medium to induce expression of the recombinant protein. The culture was incubated for a further6hr at37℃. Cells were pelleted by centrifugation. GST Spin Purification Kit was used for purification of the recombinant bacterial proteins.2.Cell cultureBone marrow-derived dendritic cells (BM-DCs) were isolated from BALB/c mouse and cultured in RPMI1640complete medium supplemented with10%(v/v) fetal bovine serum,1mM sodium pyruvate,50μM2-mercaptoethanol,10ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF), and10ng/ml recombinant murine interleukin (IL)-4in the presence or absence of Grp78(10μg/ml) for7days. Lipopolysaccharide (LPS,500ng/ml) was supplemented at the final12hr of seven-days-culture of dendritic cells. DC phenotypic characteristics were determined by flow cytometry.3.Flow cytometryThe following antibodies were used for cell surface marker analysis:CD11c-PE-cy7, CD40-APC, CD83-APC, CD80-PE-cy5, MHC-II-FITC, CD3-PE.Cells were incubated at4℃for30min, centrifuged, and resuspended in FACS buffer. For intracellular staining, Via-probe was added prior to fixation and permeabilization. Data were collected on a BD PharMingen and analyzed by FlowJo software.4.DC cytokine production and migrationSupernatants were harvested16h after activation of DC with LPS and then stored at-20℃. Cytokines (TGF-β,INF-γ, IL-10, TNF-α, HMGB1, NO) were quantified using sandwich ELISA. 5.Relative quantitative real-time PCRThe total RNA was isolated from the BMDCs using the Iso-plus reagent according to the manufacturer’s protocol. The complementary DNA was synthesized using moloney murine leukemia virus reverse transcriptase (M-MLV RT), Taq polymerase,1μg of total RNA, and10pmol of the oligo(dT)15primer. The real-time PCR reactions were performed in duplicate using a Light Cycler480PCR system (Roche, IN, USA) following the manufacturer’s instructions. The relative quantification was performed using the levels of GAPDH, which was used as the endogenous house keeping control.6.BMMC-T cell coculturesSplenocytes and lymphocytes from spleen were cocultured in48-well plates with0.3mg/mL anti-CD3molecular complex (BD) stimulation of T lymphocytes. Supernatants were harvested after4or6days. Proliferation was assessed by incorporation of CFSE. Expression of CD4, CD25, and Foxp3was assessed by flow cytometry as described above.7.Animal modelThe CD11c+cells loaded by NIT cells were infused to Balb/c mice by tail vein, once a week, three times.10days after last infusion, mice were killed. Lymphocytes from spleen, lymph nodes, blood were analyzed for the characteristic of murine T cells.[Results]1.Impact on differentiation of BMMC induced by Grp78Compared with the control group, Grp78significantly inhibited the differentiation of CDllc+cells BMDC (P=0.003), and with the Grp78concentration increased, CDllc+cells was decreased in a dose-dependent manner, while adding LPS had no significant effect for BMDC differentiate into CD11c+cells. Further analyzed cell surface marker CD11b and Grl by flow cytometry, showed Grp78group, and Grp78-LPS group the percentage of myeloid suppressor cells formed increases, and as the concentration of Grp78increased, MDSC ratio gradually increased, suggesting MDSC may involvement negatively regulate effects of Grp78.2. Expression of surface molecules I-A/E of murine bone marrow-derived LPS-DC, Grp78-DC, Grp78-LPS-DC.The results show Grp78-DC, Grp78-LPS-DC surface expression of I-A/E expression decreased significantly than LPS-DC (P<0.01).3.Expression of surface molecules of murine bone marrow-derived LPS-DC, Grp78-DC, Grp78-LPS-DC.Compared with LPS stimulation group, Grp78significantly reduced DC activation marker CD83expression on DC (P=0.011), as well as costimulatory molecules CD40, CD86expression. Expression of co-inhibitory molecule B7-H3and B7-H4were upregulated.4.Expression of cytokines of different groups of mice BMMCThe BMMC induced by Grp78reduced of secretion of NO, but the secretion of HMGB1, TGF-β have no difference.5. Expression of cytokines TNF-α, TGF-β, IL-6, IL-10, IL-4, IL-12of the different groups of mice BMMCThe BMMC induced by Grp78increased secretion of IL-10,decreased secretion of IFN-γ, TNF-α, have no effect of secretion of TGF-β; at the mRNA level, BMMC reduced by Grp78reduced expression of inflammatory cytokine TNF-a.6. Glucose uptake ability of LPS-DC, Grp78-DC, Grp78-LPS-DCGlucose uptake ability of Grp78-DC, Grp78-LPS-DC is significantly higher than LPS-DC, suggesting that CDllc+cells induced by Grp78were maintained strong ability of dextran uptake.7.The characteristic of murine splenic T cells induced by BMMCSplenocytes and lymphocytes from spleen were cocultured, BMMC induced by Grp78can significantly induce CD4+CD25+Foxp3+T cells with generated higher rate (P=0.009).8. The characteristics of T cells from mice which were infused of different groups of mice bone marrow-derived DC in vivo The CD11c+cells loaded by NIT cells were infused to Balb/c mice by tail vein, once a week, three times.10days after last infusion, mice were killed. Lymphocytes from spleen, lymph nodes, blood were analyzed for the characteristic of murine T cells.The results showed that, CD4+Tcells has no significant difference in the lymph nodes of mice, but CD4+CD25+Foxp3+T cells after Grp78-DC infusion significantly increased(P<0.05), and each group of mice spleen CD4+CD25+Foxp3+Tcells and the percentage of CD4+IL-17+T,CD4+IL-4+T, CD4+IL-10+T and CD4+IFN-y+cells had no significantly difference.[Conclusion]This paper first discusses the impact of Grp78on mouse bone marrow-derived DC differentiation and maturation, and to explore its function compare with the GM-CSF group, LPS group.The results showed that:①Grp78significantly inhibited the differentiation of bone marrow-derived mononuclear cells to DC, and increased the percentage of MDSC, suggesting MDSC may involvement negatively regulate effects of Grp78;②CD11c+cells induced by Grp78were maintained strong ability of dextran uptake, but down of the expression of cell surface IA/E molecules and costimulatory molecules and upregulated inhibitory molecules, reduced secretion of inflammatory cytokines, increased secretion of IL-10; suggesting CD11c+cells induced by Grp78have tolerated DC phenotype;③Co-cultured with fellow murine splenic T cells, BMMC induced by Grp78has induced significantly higher of Treg;④The vivo experiments show that, after Grp78-DC infusion, the mouse lymph node CD4+CD25+Foxp3+Treg cells was significantly higher than other DC group, these results suggest that Grp78-DC and Grp78-LPS-DC has a characteristics of tolerance DC.