Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

By inducing and amplifying dendritic cells(DCs)derived from the bone marrow of asthma murine in vitro,cytokines mRNA were expressed,and the functions of DCS were investigated.Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4,and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates.Large numbers of DCS have been obtained from bone marrow,and they expressed interleukin-13(IL-13),interleukin-9(IL-9),and interleukin-3(IL-3)mRNA.Moreover.the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups(P＜0.05).But,the level of IL-3 mRNA showed no discrepancy between the two groups(P＞0.05).Des are very important in the forming and developing of asthma,which implies that the therapy targeted at DCs will possibly become a new goal.     

OP9-DL1 cell co-culture enhances anti-tumour immunity of mouse bone marrow-derived dendritic cells

DCs (dendritic cells) are the strongest professional APCs (antigen-presenting cells) to initiate immune responses against pathogens, but they are usually incompetent in initiating efficient immune responses in the progress of solid tumours. We have shown that Notch signalling plays a pivotal role in DC-dependent anti-tumour immunity. Compared with the control DCs, OP9-DL1 (Delta-like1) cell co-cultured DCs gained increased tumour suppression activity when inoculated together with tumour cells. This was probably due to the activation of Notch signalling in DCs enhancing their ability to evoke anti-tumour immune responses in solid tumours. Indeed, the OP9-DL1 cell co-cultured DCs expressed higher levels of MHC I, MHC II, CXCR4 (CXC chemokine receptor 4), CCR7 (CC chemokine receptor 7), IL-6 (interleukin 6), IL-12 and TNFα (tumour necrosis factor α), and a lower level of IL-10 than control DCs, resulting in more efficient DC migration and T-cell activation in vivo and in vitro. T-cells stimulated by OP9-DL1 cells co-cultured DCs more efficiently; and were cytotoxic against tumour cells, in contrast with control DCs. These results indicated that up-regulation of Notch signalling in DCs by co-culturing with OP9-DL1 cells enhances DC-dependent anti-tumour immune reactions, making the Notch signalling pathway a target for the establishment of the DC-based anti-tumour immunotherapies.     

Cryptococcus neoformans activates bone marrow-derived conventional dendritic cells rather than plasmacytoid dendritic cells and down-regulates macrophages

Induction of IL-12 and IL-23 is essential for protective immunity against Cryptococcusneoformans. The contribution of dendritic cells vs. macrophages to IL-12/23 production in response to C. neoformans infection is unclear. Activation of conventional bone marrow-derived dendritic cells (BMDC), plasmacytoid BMDC, and bone marrow-derived macrophages (BMMPhi) was assessed by analyzing cytokine responses and the expression of MHC-II, CD86, and CD80 in each cell type. Cryptococcus neoformans induced the release of IL-12/23p40 by BMDC, but not by BMMPhi, in a TLR2- and TLR4-independent but MyD88-dependent manner. Conventional BMDC rather than plasmacytoid BMDC up-regulated MHC-II and CD86, while BMMPhi down-regulated MHC-II and CD86 in response to C. neoformans. The up-regulation of MHC-II and CD86 on BMDC required MyD88. Our data point to conventional DC as critical IL-12/23-producing antigen-presenting cells during cryptococcosis.     

Tumor immunotherapy using bone marrow-derived dendritic cells overexpressing Toll-like receptor adaptors

Myeloid dendritic cells (mDCs) play an important role in the initiation of immune responses to cancer and infectious diseases. Toll-like receptors (TLRs) expressed on mDCs recognize microbial products to elicit signals for mDC maturation, including cytokine production, antigen-presentation and induction of effector cells. TLR agonists work as adjuvants to modulate the function of mDCs. In TLR signaling, MyD88 and TRIF/TICAM-1 are major TLR adaptor molecules, which when overexpressed are able to transduce downstream signals without TLR stimuli. We successfully introduced the adaptors into mouse bone marrow-derived mDCs using lentiviral vectors. Introduction of MyD88 into mDCs in vitro led to the production of IL-6 and IL-12p40 while introduction of TICAM-1 stimulated interferon (IFN)-alpha production. Expression of TICAM-1, but not MyD88, in mDCs slightly induced the co-stimulatory molecule CD86, while significant upregulation of CD86 was observed in response to other TLR stimuli. Both MyD88 and TICAM-1 augmented allogeneic mixed lymphocyte reaction (MLR). Ex vivo mouse spleen cells pre-exposed to tumor antigen exhibited antitumor cytotoxicity when incubated with MyD88- or TICAM-1-expressing mDCs. Using mDC adoptive transfer and a syngeneic mouse tumor implant model, we established an antitumor immunotherapy whereby tumor growth is retarded by adaptor-manipulated mDCs.     

小鼠骨髓源树突状细胞体外诱导培养及初步鉴定

用重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组小鼠白细胞介素4（rmIL-4）体外诱导小鼠骨髓细胞分化为树突状细胞,进行形态学变化观察,分析细胞表面分子,刺激T细胞增殖,探讨小鼠骨髓源树突状细胞（BMDC）体外诱导培养并进行初步鉴定。体外培养9d后BMDC可达80%以上,光镜下可见典型的树突状细胞形态。清楚表达成熟期主要表面标志物,可显著刺激同种异体混合淋巴细胞增殖。获得了较高纯度的BMDC,避免了使用传统磁珠分离方法所带来的成本高,操作复杂,产出率低的弊端,为研究BMDC功能以及运用开展下游实验提供材料。     

Borrelia burgdorferi potently activates bone marrow-derived conventional dendritic cells for production of IL-23 required for IL-17 release by T cells

Lyme borreliosis is characterized by cellular inflammatory responses at multiple body sites. Recently, an association of interleukin-17 (IL-17) and Lyme arthritis was suggested. In this context, it is of special interest that the heterodimeric cytokine IL-23 can act on T cells and initiate the up-regulation of effector cytokines such as IL-17. To determine the role of this specific cytokine cascade for the induction of subsequently induced proinflammatory events we developed an in vitro system to investigate the IL-23-inducing capacity of Borrelia burgdorferi and the potential of the spirochete for inducing the IL-23/IL-17 axis. We used cells derived from mice deficient for IL-23 or IL-12 only or deficient for both IL-12 and IL-23 to define precisely the function of these cytokines. Experiments with bone marrow-derived dendritic cells (BMDC) identified these cells as sources for IL-23 but not for IL-12 after B. burgdorferi exposure. Subsequent investigations with T cell-depleted splenocyte fractions revealed a tight IL-23/IL-17 axis in response to the spirochetes. Monoclonal antibodies that block IL-23 showed further that BMDC-derived IL-23 was required for production of IL-17 in this experimental model. These in vitro data describing a spirochete-induced release of IL-23 may help to define IL-17-dependent inflammatory responses in the disease.     

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN- for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN- for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.     

Pro-inflammatory cytokine expression of spleen dendritic cells in mouse toxoplasmosis

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8α(+)/CD11c(+) splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.     

Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells

A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as “non-dendritic cells” or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these “junk cells” are actually qualified dendritic cells.     

LPS response and endotoxin tolerance in Flt-3L-induced bone marrow-derived dendritic cells

Fms-like tyrosine kinase-3 ligand (Flt-3L) stimulates the differentiation of bone marrow cells into dendritic cells (DCs) and was used as an adjuvant therapy in the experimental model of burn wound sepsis. In this study, we describe the phenotypical characteristics of an Flt-3L-dependent DC culture (FLDC) system following LPS stimulation, which induces an inflammatory response, and after a second LPS stimulation, which induces tolerance. Priming of FLDCs with LPS via TLR4 has been shown to induce the activation of all three mitogen-activated protein kinase (MAPK) families and enhance NF-κB complex translocation into the nucleus. Stimulated FLDCs express all maturation markers and exhibit an increase in IL-12p40 production and to a lesser extent, IL-10 production. In contrast, LPS stimulation of tolerized FLDCs was not associated with TLR4 up-regulation and led to MAPK inhibition. The decrease in p38 and JNK activation was correlated with an impairment of IL-12p40 production. Endotoxin tolerance in FLDCs was associated with enhanced ERK1/2 activation, an increase in MKP-1 phosphatase expression, a decrease in NF-κB translocation to the nucleus and an increase in IL-10 production. Overall, DCs generated from bone marrow with Flt-3 ligand have similar characteristics to DC subtypes found in the steady state in vivo, which can acquire endotoxin tolerance in some circumstances.     

TLR7刺激对系统性红斑狼疮易感型小鼠树突状细胞免疫功能的影响

目的研究TLR7通路对于系统性红斑狼疮易感Fas-/-MRLlpr/lpr小鼠树突状细胞免疫功能的影响。方法 从5周龄MRLlpr/lpr小鼠骨髓提取DC体外培养,以TLR7激活剂咪喹莫特处理48h,流式细胞仪检测细胞表面分子CD80、MHCⅡ表达。ELISA法检测细胞培养上清中细胞因子IFN-γ、TNF-α、IL-10。结果咪喹莫特处理后,树突状细胞表面抗原提呈相关分子MHCⅡ和CD80分子均降低,分泌的IFN-γ、TNF-α和IL-10均明显增加。结论 TLR7通路调节系统性红斑狼疮易感小鼠MRLlpr/lpr小鼠DC抗原呈递能力,具有影响免疫应答作用。     

An engineered novel lentivector specifically transducing dendritic cells and eliciting robust HBV-specific CTL response by upregulating autophagy in T cells

Dendritic cells (DCs) play a predominant role in initiating cell immune responses. Here we generated a DC-targeting lentiviral vector (LVDC-UbHBcAg-LIGHT) and evaluated its capacity to elicit HBV-specific cytotoxic T lymphocyte (CTL) responses. DC-SIGN-mediated specific transduction using this construct was confirmed in DC-SIGN-expressing 293T cells and ex vivo-cultured bone marrow cells. LVDC-UbHBcAg-LIGHT-loaded DCs were highly effective in inducing HBV-specific CTLs. Mechanistic studies demonstrated autophagy blocking led to a significant increase in apoptosis and obvious inhibition of CD8 + T cells entry into S-phase, correspondingly attenuated LVDC-UbHBcAg-LIGHT-loaded DC-induced T cell responses. This observation was supported by accumulation of pro-apoptotic proteins and the main negative cell cycle regulator-CDKN1B that otherwise would be degraded in activated T cells where autophagy preferentially occured. Our findings revealed an important role of autophagy in the activation of T cells and suggested LVDC-UbHBcAg-LIGHT may potentially be used as a therapeutic strategy to combat persistent HBV infection with higher security.     

Impact of histamine on dendritic cell functions

A rapidly growing body of evidence highlighted that histamine, a small biogenic amine, is implicated in the regulation of DC (dendritic cell) functions. It is well established that DCs represent the most potent antigen-presenting cells of the body, linking innate and acquired immunity and regulating the outcome of immune responses. Signals, associated with ongoing inflammation and uptake of foreign antigens, promote maturation of DCs and activation of T-cell responses in secondary lymphatic organs. These bone marrow-derived cells patrol continuously all over the body. During their persistent migration, several mediators may influence the behaviour and functions of DCs. Histamine, produced by mast cells, basophils or DCs themselves, may have an important role in the life cycle of DCs. From the differentiation, through their never-ending circulation, until the induction of T-cell response, histamine is present and influences the life cycle of DCs. Here, we summarize recent progress in histamine research with respect to DC functions. We also point out some controversial aspects of histamine action on DCs.     

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells

We investigated chondrogenesis of cell-mediated sox9 gene therapy as a new treatment regimen for cartilage regeneration. pIRES2-EGFP vector containing a full-length mouse sox9 cDNA was transfected into bone marrow-derived mesenchymal stem cells (MSCs) by lipofection and chondrogenic differentiation of these cells was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich micromass aggregate with EGFP expressing MSCs was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice to analyze in vivo chondrogenesis. A massive tissue formation in about 2mm diameter was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained. These results indicated that cell-mediated sox9 gene therapy could be a novel strategy for hyaline cartilage damage.     

一种拮抗HIV-1并靶向DC的融合基因的设计及表达鉴定

艾滋病病毒 (Human immunodeficiency virus,HIV) 通过与靶细胞膜的融合感染宿主细胞,研究表明阻断HIV与受体靶分子的结合可以阻止HIV进入宿主细胞,抑制HIV病毒的感染。设计合成了一个包含CD4和CCR5与HIV-1结合的主要功能结构区,及Flt3-L和Mip-3α分子的融合基因,构建了2个融合基因的真核表达载体pABK-CKR5-CD4/Flt3L-Mip3α (pABK-HIV-MF) 和pABK-CKR5-CD4 (pABK-HIV-MT),在人胚肾293细胞中进行了表达。RT-PCR、细胞免疫荧光技术、ELISA和Western blotting检测结果表明融合基因在真核细胞中获得了正确的表达,这为进一步研究其对于HIV-1的拮抗并靶向树突状细胞 (DC) 清除研究奠定了基础。     

Retracted: The role of HOTAIR-induced downregulation of microRNA-126 and interleukin-13 in the development of bronchial hyperresponsiveness in neonates

Long noncoding RNAs, including HOTAIR, are involved in the pathogenesis of a wide range of diseases. This study aimed to explore the mechanism underlying the involvement of HOTAIR in neonatal bronchial hyperresponsiveness (BHR). A total of 105 newborns were recruited in this study to collect their peripheral blood mononuclear cell and serum samples, which were then divided into different genotype groups based on the genotypes of rs4759314, rs874945, and rs7958904. The real-time polymerase chain reaction, western blot analysis, computational analyses, and luciferase assays were performed to establish the regulatory relationships between the HOTAIR, microRNA-126 (miR-126), and interleukin-13 (IL-13). The level of HOTAIR, miR-126, and IL-13 among rs4759314 AA, AG, and GG groups, as well as among rs874945 GG, AG, and AA groups was similar. However, the level of HOTAIR was increased in the rs7958904 GG group, accompanied by a decreased level of miR-126 and IL-13. In addition, the level of airway responsiveness was comparable among rs4759314 AA, AG, and GG groups, as well as among rs874945 GG, AG, and AA groups. However, the airway responsiveness in the groups rs7958904 CG and CC was much stronger than that of the GG group. We also demonstrated that, by directly binding to miR-126, HOTAIR reduced the expression of miR-126, which in turn decreased the expression of IL-13. In summary, we demonstrated the role of HOTAIR-induced downregulation of miR-126 and IL-13 in the development of BHR in neonates.     

microRNA modified tumor-derived exosomes as novel tools for maturation of dendritic cells

Tumor-derived exosomes (TEX) are known by their immune suppression effects as well as initiation mediators in cancer progression and metastasis. Meanwhile, they are appropriate sources to induce immunity against tumor cells, as consist of tumor specific and associated antigens. The aim of the current study is modifying TEX with microRNA miR-155, miR-142, and let-7i, to enhance their immune stimulation ability and induce potent dendritic cells (DC). For this, exosomes were isolated from mouse mammalian breast cancer cell line; 4T1, and subjected to miR-155, miR-142, and let-7i by electroporation. Immature DCs were generated from mouse bone marrow in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). To mature DCs, lipopolysaccharide (LPS), TEX, and modified TEX were used. The expression level of miRNAs and their target genes (IL-6, IL-17, IL-1b, TGFβ, SOCS1, KLRK1, IFNγ, and TLR4) was determined. TEX were nanovesicles with spheroid morphology which expressed CD81, CD63, and TSG101, as exosome markers, at protein level. MHCII, CD80, and CD40 as maturation markers were assessed by flow cytometry. Overexpression of miRNAs were confirmed in exosomes and mDCs. Up and downregulation of target genes confirmed the gene network in DC maturation. We found that Let-7i could efficiently induce the DC maturation, as well as miR-142 and miR-155 have enhancing effects. These findings reveal that the modified TEX would be a hopeful cell-free vaccine for the cancer treatment.     

Leptospira interrogans is recognized through DC-SIGN and induces maturation and cytokine production by human dendritic cells

Leptospirosis is a global zoonotic disease, caused by pathogenic Leptospira species including Leptospira interrogans, that causes public health and livestock problems. Pathogenesis, immune response and cellular receptors for Leptospira are not well understood. Interaction of dendritic cells (DCs) with L. interrogans serovar Autumnalis L-643 and BL-6 isolated from leptospirosis patients, and both virulent and avirulent serovar Pyrogenes 2317 strains isolated from animal were investigated. Carbohydrate analysis using lectins showed that all of these leptospires contained high mannose components as a common backbone and DC-SIGN was involved in leptospires' attachment. Interaction of the L. interrogans strains with DCs induced maturation, but had different effects on IL-10, IL-12p70 and tumor necrosis factor (TNF)-alpha production. Both virulent and avirulent Pyrogenes 2317 and Autumnalis BL-6 but not L-643 strains induced IL-12p70 and TNF-alpha production, but minimal IL-10 secretion. These data demonstrated that L. interrogans binds DC-SIGN and induces DCs maturation and cytokine production, which should provide new insights into cellular immune processes during leptospirosis.     

Effect of patulin on the interdigitating dendritic cells (IDCs) of rat thymus

Patulin is a common fungal contaminant of ripe apples used for the production of apple juice concentrates and it is also present in other fruits, vegetables and food products. Patulin is a secondary metabolite produced by species of the genera Penicillium, Aspergillus and Byssochlamys. Patulin has been reported to be mutagenic, carcinogenic and teratogenic. Antigen-presenting cells (APCs) are of prime importance in the innate immune response; they capture antigen in tissues and then migrate to the lymphoid organs to present the antigen to T lymphocytes. Thus, they are crucial for the initiation of immunity. Interdigitating dendritic cells (IDCs) are a subset of APCs that are present at the lymphatic organs. In the thymus, they act in positive and negative selection during T cell development. In the present study, patulin was administered orally to growing male rats aged 5-6 weeks. A dose of 0.1 mg kg(-1) bw day(-1) was given to rats for a period of 60 or 90 days daily. The effect of patulin on the IDCs of thymus was investigated by transmission electron microscopy (TEM), and the results were evaluated in terms of cell destruction. In the rats of the control group, it was observed that the IDCs had an indented nucleus, a clear cytoplasm and numerous membrane extensions. In the cytoplasm, a well-developed golgi complex, mitochondria, granular endoplasmic reticulum and a small number of lysosomal structures were observed. At day 60 of patulin-treated rat groups (P-60), loss of cristae in mitochondria and chromatin margination and lysis in the nucleus were found. It was observed that the IDCs had a perinuclear area of cytoplasm surrounded by a peripheral electron-lucent zone. In the cytoplasm of the 90-day patulin-treated rat group (P-90), a peripheral electron-lucent zone was also found, similar to the P-60 group. Additionally increase in vesicular and lysosomal structures, increase in apoptotic bodies and condensation of chromatin in the nucleus were noted. It was observed that patulin leads to apoptotic body formation and cell apoptosis in the IDCs of rat thymus especially in the P-90-treated groups.