Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance

BACKGROUNDImmature dendritic cells (imDCs) play an important role in the induction of donor-specific transplant immunotolerance. However, these cells have limitations, such as rapid maturation and a short lifespan in vivo. In previous studies, induced pluripotent stem cells (iPSCs) differentiated into imDCs, and sinomenine (SN) was used to inhibit the maturation of imDCs. AIMTo study the capacity of SN to maintain iPSC-derived imDCs (SN-iPSCs-imDCs) in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance.METHODSIn this study, mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN (iPSCs-imDCs and SN-iPSCs-imDCs). The imDC-related surface markers, endocytotic capacity of fluorescein isothiocyanate-Dextran and apoptosis were analyzed by flow cytometry. The effects of iPSCs-imDCs and SN-iPSCs-imDCs on T-cell stimulatory function, and regulatory T (Treg) cell proliferative function in vitro were analyzed by mixed lymphocyte reaction. Cytokine expression was detected by ELISA. The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting. The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice. Statistical evaluation of graft survival was performed using Kaplan–Meier curves.RESULTSBoth iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained, and their biological characteristics and ability to induce immunotolerance were compared. SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs. Reduced major histocompatibility complex II expression, worse T-cell stimulatory function, higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs (P < 0.05). The levels of interleukin (IL)-2, IL-12, interferon-γ in SN-iPSCs-imDCs were lower than those in iPSCs-imDCs, whereas IL-10 and transforming growth factor-β levels were higher (P < 0.05). The apoptosis rate of these cells was significantly higher (P < 0.05), and the expression levels of cleaved caspase3, Bax and cleaved poly(ADP-ribose) polymerase were higher after treatment with lipopolysaccharides, but Bcl-2 was reduced. In Balb/c mice recipients immunized with iPSCs-imDCs or SN-iPSCs-imDCs 7 d before skin grafting, the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4⁺ T-cell proliferation (P < 0.05) and a higher capacity to induce CD4⁺CD25⁺FoxP3⁺ Treg cell proliferation in the spleen (P < 0.05). The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern. CONCLUSIONThis study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.     

Immunoregulation through IL-10 gene expression and the fate of cytotoxic T lymphocyte-mediated tumor immunotherapy

Gene analysis of tumor associated antigens revealed that tumor antigens are all normal gene product. Inducing tumor reactive cytotoxic T lymphocytes (CT) in the patients is same as inducing autoimmunity in the patients. Immunosuppressive cytokine interleukin-10 (IL-10) plays an important role in maintaining homeostasis or tolerance. To break the tumor tolerance, blocking and IL-10 secretion or intervention in the pathways of IL-10 gene activation is indeed important.     

Human peripheral blood monocyte-derived interleukin-10-induced semi-mature dendritic cells induce anergic CD4(+) and CD8(+) T cells via presentation of the internalized soluble antigen and cross-presentation of the phagocytosed necrotic cellular fragments

We examined the ability of human monocyte-derived interleukin (IL)-10-induced semi-mature dendritic cells (semi-mDCs) that had been pulsed with soluble protein and necrotic cellular fragments to induce an antigen (Ag)-specific anergy in CD4(+) and CD8(+) T cells. IL-10 converted normal immature DCs (iDCs) into semi-mDCs during the maturation. In contrast to normal iDCs and mature DCs, IL-10-induced semi-mDCs as well as IL-10-treated iDCs not only had reduced their allogeneic T cell-stimulatory capacity, but also induced an allogeneic Ag-specific anergy in T cells. Normal mDCs that had been pulsed with tetanus toxin (TT) or allogeneic necrotic cellular fragments caused further activation of TT-specific CD4(+) T cells or allogeneic fibroblast-specific CD8(+) T cells, Ag-pulsed IL-10-induced semi-mDCs induced an anergic state in both cell types. Thus, our results suggest that IL-10-induced semi-mDCs induce an Ag-specific anergy in CD4(+) and CD8(+) T cells via presentation of the internalized protein and cross-presentation of the phagocytosed cellular fragments.     

DCs sensitized with mPD-L1-Ig fusion protein improve the effect of heart transplantation in mice by promoting the generation of T-reg cells

Purpose

To detect the effects of DCs sensitized by mPD-L1-Ig fusion protein in heart transplantation in mice as well as its mechanisms.

Method

The mPD-L1-IgG1 construct was used to build a yeast expression system, and the fusion protein was expressed by secretion after the transfection of the GS115 yeast strain, purified by affinity chromatography and ion exchange chromatography, and assayed by SDS–PAGE and Western blot. The ability of the fusion protein to bind to the acceptor PD-1 was tested by ELISA, and the ability of the fusion protein to inhibit the function of T cells was tested by mixed lymphocyte reaction (MLR).

Results

We used the new PD-L1-IgG1 fusion protein to sensitize imDCs and maintained the immature state of DCs, so as to induce stable and effective immune tolerance to heart transplantation. After the treatment of DCs by mPD-L1-Ig in vitro, the levels of CD80, CD40 and I-Ab expression on DCs are relatively weaker, the ability of DCs to stimulates the proliferation of allogeneic spleen T cells was significantly decreased (P < 0.01), and the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) secreted by induced allogeneic T cells were significantly decreased (P < 0.01). An in vivo experiment also revealed that DCs sensitized by mPD-L1-IgG1 could prolong the survival time of a transplanted heart to 17.8 ± 1.12 days, and alleviate the pathological change of the cardiac allografts compared with other three groups.

Conclusion

DCs sensitized by the yeast-expressed mPD-L1-Ig fusion protein are shown to alleviate the cardiac allograft rejection in mice.     

双歧杆菌对过敏性哮喘儿童树突状细胞分泌IL-10、IL-12等细胞因子的影响

目的探讨双歧杆菌对过敏性哮喘儿童外周血单核细胞（PBMC）来源的树突状细胞（DC）分泌IL-1β、IL-6、IL-10、IL-12、IL-23和IFN-γ的影响。方法从15例过敏性哮喘儿童和15例非哮喘儿童的外周血单个核细胞诱导生成未成熟DC,加入双歧杆菌后继续培养DC2d,用ELISA方法检测培养上清中IL-1β、IL-6、IL-10、IL-12、IL-23和IFN-γ的水平。结果双歧杆菌能明显刺激哮喘儿童DC分泌IL-12、IFN-γ,IL-1β及IL-6和非哮喘儿童DC分泌IL-12、IL-10、IL-1β及IL-23水平增高。结论双歧杆菌能够刺激过敏性哮喘儿童DC分泌IL-12和IFN-γ,可能改变Th2优势分化,纠正Th1／Th2失衡。同时双歧杆菌还能刺激哮喘儿童DC分泌IL-1p及IL-6增高,达到促进,Th17细胞分化的作用。     

Evidence for interleukin-1-independent stimulation of interleukin-12 and down-regulation by interleukin-10 in Helicobacter pylori-infected murine dendritic cells deficient in the interleukin-1 receptor

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1beta (IL-1beta). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R-/-) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1beta, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R-/- mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa.     

Dendritic cells reduce number and function of CD4+CD25+ cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma

Aim and background: CD4⁺CD25⁺ cells are described as professional regulatory/suppressor T cells that are crucial for the prevention of spontaneous autoimmune diseases. They play an important role in maintaining a balanced peripheral immune system. On the other hand, it has been suggested that regulatory T cells (Treg) suppress antitumor immune responses after tumor-specific vaccinations. Therefore, we determined the percentage of regulatory T cells in cytokine-induced killer (CIK) cells, an effector cell population with high impact for adoptive immunotherapeutic strategies. Results: CIK cells showed strong induction of CD4⁺CD25⁺ cells with high secretion of interleukin 10 (IL-10) after unspecific stimulation of the TCR complex and stimulation with interleukin 2. Depletion of CD25⁺ cells led to an increase in cytotoxic activity and a reduction of IL-10 release. A more pronounced reversal of suppression could be induced by coculture of CIK cells with dendritic cells (DCs). After coculture of CIK cells with DCs, the number of CD4⁺CD25⁺ cells as well as the IL-10 concentration in the supernatant decreased, and the cytotoxic activity against pancreatic carcinoma cells increased. This was shown for cells from healthy donors as well as for cells from patients with pancreatic carcinoma. Conclusion: Our established effector cells possess some regulatory features induced by unspecific TCR-activation that could be prevented by coculture with DCs. CIK cells have desirable properties for immunotherapeutical approaches, especially after coculture with DCs, which could be used additionally for induction of a specific immune response.     

The limitation of IL-10-exposed dendritic cells in the treatment of experimental autoimmune myasthenia gravis and myasthenia gravis

Dendritic cells (DC) are highly specialized antigen presenting cells that play critical roles as instigators and regulators of immune responses including B cell function, antibody synthesis and isotype switch. In this study, we compared immunotherapeutic effect of IL-10-treated DC (IL-10-DC) via both intraperitoneal (i.p.) and subcutaneous (s.c.) delivery in rats with incipient experimental autoimmune myasthenia gravis (EAMG). Spleen DC were isolated from onset of EAMG on day 39 post-immunization, exposed in vitro to IL-10, and then injected into incipient EAMG at dose of 1 x 10(6) cells/rat on day 5 after immunization. Intraperitoneal administration of IL-10-DC suppressed clinical scores, anti-acetylcholine receptors (AChR) antibody secreting cells, antigen-specific IL-10/IFN-gamma production and T cell proliferation compared to control EAMG rats. Importantly, IL-10-DC, if given by s.c. route, failed to ameliorate clinical sign of EAMG. Simultaneously, T cell proliferation, anti-AChR antibody secreting cells and IL-10/IFN-gamma production had no alteration, as compared to control EAMG rats. Both in vitro and in vivo experiments showed that treatment of IL-10 inhibited the migration of DC toward MIP-3beta and lymph node, indicating that in vitro manipulation of DC with IL-10 alters the migration of DC that influences the therapeutic effect in the treatment of autoimmune diseases. In MG patients, neither the improvement of clinical symptom nor the alteration of immunological parameter was observed through s.c. delivery of IL-10-DC, suggesting the limitation of IL-10-DC in the treatment of MG patients.     

Release of iC3b from apoptotic tumor cells induces tolerance by binding to immature dendritic cells in vitro and in vivo

Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Apoptotic cells are known to activate homologous complement and to be opsonized with iC3b. Since maturation of dendritic cells (DC) can be inhibited by binding of iC3b to the complement receptor 3 (CR3, CD11b/CD18) and because immature DC induce tolerance, we investigated the induction of tolerance after pulsing DC with apoptotic cells in the presence or absence of native serum. Apoptosis in pancreatic carcinoma cells was induced either by heat-stress, chemotherapy or anti-Her2 antibody. Monocyte-derived DC were pulsed with apoptotic cells with or without native serum. DC were analyzed for the maturation state by flow cytometry and the cytotoxic activity was determined. Tolerance was prevented by addition of substances such as anti CD11b or N–acetyl-D-Glucosamine (NADG) which block iC3b binding to CR3. Furthermore, binding of iC3b from apoptotic cells to DC was blocked in a syngeneic pancreatic carcinoma mouse model. All of the former strategies for apoptosis induction resulted in iC3b release. Pulsing DC with apoptotic cells in the presence of serum prevents maturation of DC and induces finally tolerance. This tolerance could be prevented almost completely by blocking the interaction of iC3b with the CR3 receptor. This could be shown as well in an immunocompetent mouse model. Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Release of iC3b from apoptotic tumor cells prevents fully maturation of DC and immature DC induce antigen-specific silencing or tolerance. Blocking of iC3b-binding could mostly prevent this effect.     

Modulation of NKT cells and Th1/Th2 imbalance after α-GalCer treatment in progressive load-trained rats

Purpose: The purpose of this study was to determine whether α-galactosylceramide (α-GalCer), a synthetic glycolipid agonist of natural killer T (NKT) cells, can ameliorate exercise-induced immune imbalance. Methods: Eight-week-old female Sprague-Dawley rats were trained with a progressively increasing load for 9 weeks. At 36 h and at 7 d after training, groups of rats were euthanized. The whole blood was used to detect hemoglobin(Hb), plasma was analyzed for hormones testosterone(T) and corticosterone(C), and spleen was harvested for detecting NKT cells and interferon-γ (IFN-γ) and interleukin (IL)-4 producing cells. Results: Two-way analysis of variance (ANOVA) showed significant differences between training and time in Series 1. The results showed, at 36h after training, that the decrease in Hb, T and C concentration reflected overtraining or excessive exercise. At 7 d after training, NKT cell populations decreased, and a T helper 1/T helper 2 (Th1/Th2) lymphocyte imbalance occurred. In Series 2, α-galactosylceramide (α-GalCer), an NKT cell activator was found to enhance NKT cell numbers by 69% and shift the Th1/Th2 lymphocyte imbalance by observably decreasing the frequency of IL-4 secreting cells. Conclusion: These data showed that, in addition to Th1/Th2 self-regulation, α-GalCer played an important modulatory role in the exercise-induced Th1/Th2 lymphocyte imbalance, which may be correlative with NKT immunoregulatory cells.     

Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40hiCD5+ regulatory B cells in vitro and in vivo

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40^hiCD5⁺ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40^hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10^−/−CD5⁺CD19⁺ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40^hiCD5⁺ Breg cells in mice. However, the population of CD40^hiCD5⁺ B cells was minimal in IL-10^−/− mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40^hiCD5⁺ B cells and the autocrine effect of IL-10 is critical to the formation of CD40^hiCD5⁺ Breg cells. [BMB Reports 2015; 48(1): 54-59]     

Leishmania mexicana: participation of NF-kappaB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mφ) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mφ IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-α, and IL-10 and nuclear translocation of NF-κB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-κB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-κB in monocytes with the subsequent decrease in IL-12 production.     

Possible involvement of regulatory T cells in tumor onset and progression in primary breast cancer

Background  The FOXP3 mRNA expression and the other regulatory T cell-related molecules were investigated and compared with clinicopathological parameters in human primary breast cancer. Method  This study included 136 breast cancer patients operated in our department from 2003 to 2006. Total RNA was extracted from frozen normal breast and breast cancer tissues, and the expression of FOXP3, IL-10, TGFβ1 and CCL22 mRNA was evaluated using quantitative real-time RT-PCR. Result   FOXP3, IL-10, TGFβ1 and CCL22 mRNA expressions were significantly higher in cancer tissue than in normal tissue, not only at pT1, 2, and 3 stages but also at the DCIS stage. There were positive correlations between FOXP3 and IL-10, FOXP3 and TGFβ1, as well as FOXP3 and CCL22 mRNA expressions, respectively. FOXP3 and IL-10 mRNA expressions were significantly upregulated in PgR-negative or HER2-positive tumors. Conclusion  These results suggest that regulatory T cells are involved in tumor onset and progression in human primary breast cancer, possibly contributing to poor prognosis of patients with breast cancer.     

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.     

Memory B cells from older people express normal levels of cyclooxygenase-2 and produce higher levels of IL-6 and IL-10 upon in vitro activation

Worldwide the elderly population is increasing. The elderly show deficiencies in immune function. B lymphocytes are essential elements of the immune system responsible for antibody production. This laboratory previously showed that activated human B cells isolated from young adults express cyclooxygenase-2 (Cox-2) and that Cox-2 is essential for optimal antibody responses. Recent data suggests that Cox-2 expression decreases with age in mouse bone tissue. There is no information regarding Cox-2 expression in B cells from older human subjects. We investigated the expression and activity of Cox-2 in naïve and memory B cells from older people. We show that B cells from older subjects show similar Cox-2 protein expression and activity, antibody production and proliferation compared to younger people. However, we found that activated memory B cells from older people produce higher levels of IL-6 and IL-10 compared to young adults. Therefore, the dysregulated cytokine production could contribute to immune senescence in the elderly.     

Association of NKT cells and granulocytes with liver injury after reperfusion of the portal vein

Reperfusion of the liver was conducted by clamping the portal vein for 30 min in mice, followed by unclamping. Unique variation in the number of lymphocytes was induced and liver injury occurred thereafter. The major expander cells in the liver were estimated to be natural killer T cells (i.e., NKT cells), whereas conventional T cells and NK cells increased only slightly or somewhat decreased in number and proportion at that time. Reflecting the expansion of NKT cells in the liver, a Th0-type of cytokine profile was detected in sera, and cytotoxic activity was enhanced in liver lymphocytes. In NKT cell-deficient mice including CD1d (-/-) mice and athymic nude mice, the magnitude of liver injury decreased up to 50% of that of control mice. It was also suspected that accumulating granulocytes which produce superoxides might be associated with liver injury after reperfusion. This might be due to stress-associated production of catecholamines. It is known that granulocytes bear surface adrenergic receptors and that they are activated by sympathetic nerve stimulation after stress. The present results therefore suggest that liver injury after reperfusion may be mainly caused by the activation of NKT cells and granulocytes, possibly by their cytotoxicity and superoxide production, respectively.