Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL-12 and NF-kappa B. In this paper, we demonstrate that transfection of DC with IL-10-specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL-10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL-10 by siRNA was accompanied by increased CD40 expression and IL-12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL-10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL-10 siRNA-treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL-10 siRNA-transfected DC enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL-10 siRNA-transfected antigen-presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.     

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca(2+) release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter-receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte-derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down-modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN-gamma > CD40 cross-linking). Although weak, IFN-gamma consistently induces DC maturation. De novo-synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF-kappa B activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti-CD38 mAb induces up-regulation of CD83 expression and IL-12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL-12 secretion and MDDC-induced allogeneic T cell proliferation.     

CD40L-expressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.     

p38MAPK通路调控树突状细胞表达PD-L1的实验研究

目的探讨脂多糖刺激单核细胞源树突状细胞表达程序性死亡因子配体1(PD-L1)与p38MAPK信号通路的关系。方法体外培养树突状细胞,用p38抑制剂SB203580阻断p38MAPK通路后再用LPS刺激树突状细胞。光学显微镜观察各组细胞的形态变化;流式细胞术测定CD86和PD-L1表达的平均荧光强度;Western blot测定PD-L1蛋白表达。结果光镜下观察LPS刺激组细胞先经历梭型贴壁后逐渐恢复圆形,树突较多;SB203580和LPS共同刺激组细胞树突退化,未刺激组细胞成团悬浮,树突较多。SB203580和LPS共同刺激组细胞CD86、PD-L1较LPS刺激组的明显降低(P<0.05或P<0.01),与未刺激组的相比CD86差异无统计学意义,但PD-L1降低(P<0.05)。SB203580和LPS共同刺激组细胞PD-L1的蛋白表达量较其它两组的均明显减少(P<0.01或P<0.01)。结论 LPS通过p38MAPK信号通路调控树突状细胞表达PD-L1。     

IL—12以及IL—12P40对子宫内异症患者NK细胞功能的免疫调节

目的通过观察正常人及子宫内膜异位症(EM)患者外周血及腹腔液中IL-12、IL-12P40含量变化,分析其分子诱导NK细胞对子宫内膜细胞的细胞毒效应的影响,探讨IL-12、IL-12P40分子与EM患者NK细胞功能低下的相关性.方法ELISA酶联免疫检测IL-12、IL-12P40含量以及MTT释放法检测NK细胞毒活性.结果①EM患者腹腔上清液可使正常人外周血NK细胞杀伤活性(16.80%±3.6%)明显下降(8.37%±4.5% )(P＜0.05).② EM患者IL-12含量血清为66.38±12.6 pg/ml,低于对照组84.97±13.7 pg/ml(P＜0.05);腹腔液中为77.76±14.6 pg/ml,低于对照组106.92±10.7 pg/ml(P＜0.05).③EM患者IL-12P40含量血清为35.64±10.6 pg/ml, 与对照组无明显差异;腹腔液含量为79.76±12.6 pg/ml, 高于对照组40.54±10.0 pg/ml(P＜0.05), 并与疾病的程度呈负相关.④IL-12能增强NK细胞对子宫内膜细胞的杀伤,并呈剂量依赖.在10 ng/ml浓度诱导后杀伤率为31.90%,高于对照组的16.80%.⑤IL-12P40能拮抗IL-12对NK细胞活性的诱导,当加入IL-12P40后,使IL-12诱导的活性(29.3%)下降为19.36%.结论子宫内膜异位症患者腹腔液中IL-12P40含量的增高可能与NK细胞功能下降有关.     

p38 MAPK signaling mediates retinoic acid-induced CD103 expression in human dendritic cells

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin α_E), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.     

Impaired maturation and function of dendritic cells by mycobacteria through IL-1beta

Dendritic cells (DC) are pivotal for initiation and regulation of innate and adaptive immune responses evoked by vaccination and natural infection. After infection, mycobacterial pathogens first encounter monocytes, which produce pro-inflammatory cytokines, including IL-1beta, TNF-alpha and IL-6. The role of these cytokines in DC maturation remains incompletely understood. Here, we show that maturation of DC from monocytes was impaired by pretreatment of monocytes with low doses of IL-1beta. Under these conditions, Mycobacterium leprae-infected DC failed to stimulate antigen-specific T cell responses. Expression of CD86 and CD83 and production of IL-12 in response to lipopolysaccharide and peptidoglycan were diminished. In contrast, these DC functions were not impaired by pretreatment with TNF-alpha, IL-6 or IL-10. When monocytes were infected with M. bovis Bacillus Calmette-Guérin, and subsequently differentiated to DC, the activity of these DC was suppressed as well. Thus, IL-1beta acts at early stages of differentiation of DC and impairs biological functions of DC at later stages. Therefore, production of IL-1beta by mycobacteria-infected antigen-presenting cells counteracts effective stimulation of innate and adaptive immune responses.     

Elevated serum interleukin (IL)-12p40 levels in common variable immunodeficiency disease and decreased peripheral blood dendritic cells: analysis of IL-12p40 and interferon-gamma gene

Common variable immunodeficiency disease (CVID) is a heterogeneous syndrome characterized by low immunoglobulin serum levels and recurrent bacterial infections. Several studies suggest that CVID patients have a polarized immune response towards a T helper type 1 phenotype (TH1). However, the factors causing the TH1 polarization remain to be determined in this disease. In the present study, serum interleukin (IL)-12, interferon (IFN)-gamma levels and the IL-12p40 and IFN-gamma gene were studied in CVID patients. Furthermore, we evaluate dendritic cells (DCs) compartment, myeloid dendritic cells (mDCs) and plasmocytoid dendritic cells (pDCs), which help to differentiate naive T cells preferentially into TH1 and TH2, respectively. The serum IL-12p40 subunit levels were increased significantly in CVID patients compared to healthy controls. We examined whether these elevated serum IL-12p40 levels are associated with IFN-gamma or IL-12p40 gene polymorphisms, or with new mutations in the IL-12p40 promoter gene. In our hands, no new mutations were found and gene polymorphisms frequencies in CVID patients were similar to the control population. In conclusion, the elevated serum levels of IL-12p40 found in our CVID patients were not related to these genetic variations. The DC compartment analysis did not show an imbalance between pDCs and mDCs, but revealed the presence of low numbers and percentage of both DC populations in CVID.     

Essential roles of IL-12 and dendritic cells but not IL-23 and macrophages in lupus-like diseases initiated by cell surface HSP gp96

Proinflammatory cytokine IL-23 but not IL-12 is critical for the pathogenesis of organ-specific autoimmune diseases including experimental autoimmune encephalitis and collagen-induced arthritis. The contribution by IL-23 in systemic autoimmune diseases such as lupus is undefined. We addressed this question in a murine lupus-like disease model, initiated by enforced cell-surface expression of an ER HSP gp96 in C57BL/6 background. We found a significant increase of p40 in the sera in these mice that preceded the onset of diseases. However, autoimmunity was abrogated in transgenic mice expressing membrane-bound gp96 reconstituted with p35-/- BM, but not with p19-/- BM. Moreover, we found that dendritic cells (DC) but not macrophages were the main producers of p40. To dissect the roles of DC further, we depleted DC using a diphtheria toxin-based inducible DC depletion system. We demonstrated that the integrity of DC was essential for autoimmunity. Our results thus revealed that IL-12 and DC are critical for the pathogenesis of lupus-like disease precipitated by cell surface gp96. This study further highlighted the significant biological differences between IL-12 and IL-23.     

Combined use of toll-like receptor agonists and prostaglandin E(2) in the FastDC model: rapid generation of human monocyte-derived dendritic cells capable of migration and IL-12p70 production

Phenotypical maturation, IL-12p70 production and migration upon chemokine receptor CCR7 ligation are currently proposed as requirements for the use of human monocyte-derived dendritic cells (DC) in antitumoral vaccination. We have previously described a short-term protocol for DC generation from monocytes including stimulation with TNF-alpha, IL-1beta and PGE(2) (FastDC). These "conventional" FastDC are mature, migrate in response to CCR7 ligation and effectively stimulate autologeous T cells in vitro, but are deficient in IL-12p70 production. Here, conventional FastDC were compared to FastDC activated with different TLR ligands. High levels of IL-12p70 were induced by combined activation of FastDC with TLR4 and TLR7/8 ligands. IL-12 secretion could be maximized by additional T cell-derived stimulation. However, TLR-stimulated FastDC failed to migrate upon CCR7 ligation, independent of additional activation with CD40 ligand and IFN-gamma. The presence of PGE(2) during TLR ligation fully restored migratory capacity of FastDC, but left IL-12p70 production and activation of tumor antigen-specific cytotoxic T cells unaffected, challenging previous findings obtained with standard 7-day monocyte-derived DC. The FastDC model thus not only represents an effective tool for antitumoral vaccination, but may also provide novel insights into human DC biology.     

Prostaglandin D2 inhibits the production of interleukin-12 in murine dendritic cells through multiple signaling pathways

Prostaglandin (PG) D(2), and its metabolites, are known to be important mediators during acute and chronic inflammation. However, their functions during the early phases of the immune response are poorly documented. In the present study, we show that PGD(2 )inhibits, in a dose-dependent manner, the CD40- and LPS-induced secretion of the Th1-driving factor IL-12 by murine splenic dendritic cells (DC), the most potent antigen-presenting cells. The inhibition of IL-12 production is mediated only in part by the cell surface G alpha s protein-coupled D prostanoid receptor (termed DP1) but not by the G alpha i protein-coupled DP receptor, DP2. We show that recruitment of DP1 in DC results in the activation of a cyclic AMP/protein kinase A pathway that is partially responsible for the inhibition of IL-12 production. We also suggest that the DP1-independent effects exerted by PGD(2) on IL-12 production may be due to the action of ist PGJ(2), but not PGF(2)alpha, metabolites. Electrophoretic mobility shift assays demonstrated that PGD(2) affects NF-kappa B activation through (the) DP1-independent pathway(s). Together these data suggest that PGD(2), by interacting with DP1 and by binding to other target cellular proteins, may regulate immune responses by affecting IL-12 production in DC.     

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.     

Triptolide inhibits IL-12 production and T cell-stimulatory capacity of dendritic cells

Extracts of Tripterygium wilfordii Hook F.(TWHF ) are effective in traditional Chinesemedicine for treatment of autoimmune diseasessuch as rheumatoid arthritis, systemic lupus ery thematosus, nephritis and asthma [1, 2]. Trip tolide, a diterpenoid triepoxide, is derived fromTWHF and is responsible for most of the immuno suppressive and anti inflammatory effects ofTWHF. Triptolide has been shown to be effectivein the treatment of autoimmune diseases, …     

人IL-12的克隆、表达及生物活性的鉴定

目的：克隆中国人ＩＬ－２ｐ４０基因,构建ＩＬ－１２的真核基因表达载体。方法：采用ＲＴ－ＰＣＲ从北京地区人脐血树突状细胞（ＤＣ）中克隆ＩＬ－１２ｐ４０ｃＤＮＡ基因,并进行序列分析。利用ｐｃＤＮＡ３．１和ｐＬＸＰＸＳＮ构建ＩＬ－１２表达载体,转染人肝癌细胞后对其进行生物学和免疫学分析。结果：北京地区人ＤＣ中ＩＬ－１２ｐ４０ｃＤＮＡ基因２１０位密码子有独特的结构,为ＧＣＣ（Ａｌａ）。并且２３９和２９１位     

IL-2Rbeta links IL-2R signaling with Foxp3 expression

Immunological tolerance to self antigens is a tightly regulated process. Recent work has demonstrated that the forkhead family member Foxp3 is a critical element in the differentiation and function of mouse CD4(+)CD25(+) regulatory T cells (Treg). Recent work has suggested an important role for IL-2 in the development and maintenance of Treg. To directly assess the effect of IL-2 signaling on Treg development and function, we analyzed mice that were genetically deficient in components of the IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Ralpha) displayed a slight decrease in Treg within the thymus, while peripheral numbers are unchanged. In contrast, we found that mice deficient in CD122 (IL-2Rbeta) had a profound reduction in both thymic and peripheral Treg, coinciding with more rapid development of a fatal lymphoproliferative disease. Expression of a Foxp3 transgene restored Treg and protected against the onset of autoimmunity. Thus, a signal mediated by IL-2Rbeta is essential for the development and homeostasis of Foxp3(+) Treg in vivo.     

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-gamma production by allergen and PPD specific T cells

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-gamma. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-gamma production. Synthesis of IFN-gamma can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-gamma and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-gamma were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-gamma levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.