Gene Expression In Dendritic Cells Research Articles

The life cycle stages of the opportunistic fungal pathogen Pneumocystis murina provoke a differential immune response in antigen-presenting cells

Abstract Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. The life cycle of Pneumocystis species consists of a trophic stage and an ascus-like cystic stage. Our lab has published data demonstrating that cysts stimulate NF-κB translocation in alveolar macrophages, but the trophic form does not. Here, we demonstrate that infection of wild-type adult mice with a normal mixture of trophic and cystic P. murina life forms elicits a more robust innate immune response in the alveolar spaces and lung parenchyma than infection with the trophic form alone. To determine the pattern recognition receptors that bind trophozoites and cysts, we examined expression of C-type lectin receptors on lung CD11c+ dendritic cells in vivo. Infection with a mixture of trophozoites and cysts resulted in the upregulation of dectin-1 and mannose receptor, while infection with trophozoites alone resulted in upregulation of dectin-2, DEC-205 and mannose receptor. In addition, we demonstrate that bone marrow-derived dendritic cells stimulated with a mixture of trophozoites and cysts produced the proinflammatory cytokines IL-1β and IL-6. In contrast, exposure to trophozoites suppressed β-glucan-induced IL-1β, IL-6, and TNFα production by bone marrow-derived dendritic cells. A targeted gene expression assay is being used to evaluate gene expression in dendritic cells following stimulation with trophozoites alone or a mixture of trophozoites and cysts. We propose that trophozoites suppress β-glucan-signaling, but are sufficient for stimulating protective responses in immunocompetent individuals.

Hypoxia-inducible factor 1 in dendritic cells is crucial for the activation of protective regulatory T cells in murine colitis.

Dendritic cells (DCs) serve as a bridge between innate and adaptive immunity and help to maintain intestinal homeostasis. Inflammatory bowel disease (IBD) is associated with dysregulation of the mucosal immune response. The concomitant hypoxic inflammation in IBD will activate the transcription factor hypoxia-inducible factor-1 (HIF-1) to also drive gene expression in DCs. Recent studies have described a protective role for epithelial HIF-1 in mouse models of IBD. We investigated the role of HIF-1 in DC function in a dextran sodium sulfate (DSS)-induced model of murine colitis. Wild-type and dendritic cell-specific HIF-1α knockout mice were treated with 3% DSS for 7 days. Knockout of HIF-1α in DCs led to a significantly larger loss of body weight in mice with DSS-induced colitis than in control mice. Knockout mice exhibited more severe intestinal inflammation with increased levels of proinflammatory cytokines and enhanced production of mucin. Induction of regulatory T cells (Tregs) was impaired, and the number of forkhead box P3 (Foxp3) Tregs was diminished by dendritic HIF-1α knockout. Our findings demonstrate that in DCs HIF-1α is necessary for the induction of sufficient numbers of Tregs to control intestinal inflammation.

Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human.

C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells (DCs). Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue-specific. Recently, PU.1 has been shown to regulate C1q gene expression in DCs and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR, and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation.

Cutting Edge: MMP-9 inhibits IL-23p19 expression in dendritic cells by targeting membrane stem cell factor affecting lung IL-17 response.

We reported previously that c-kit ligation by membrane-bound stem cell factor (mSCF) boosts IL-6 production in dendritic cells (DCs) and a Th17-immune response. However, Th17 establishment also requires heterodimeric IL-23, but the mechanisms that regulate IL-23 gene expression in DCs are not fully understood. We show that IL-23p19 gene expression in lung DCs is dependent on mSCF, which is regulated by the metalloproteinase MMP-9. Th1-inducing conditions enhanced MMP-9 activity, causing cleavage of mSCF, whereas the opposite was true for Th17-promoting conditions. In MMP-9(-/-) mice, a Th1-inducing condition could maintain mSCF and enhance IL-23p19 in DCs, promoting IL-17-producing CD4(+) T cells in the lung. Conversely, mSCF cleavage from bone marrow DCs in vitro by rMMP-9 led to reduced IL-23p19 expression under Th17-inducing conditions, with dampening of intracellular AKT phosphorylation. Collectively, these results show that the c-kit/mSCF/MMP-9 axis regulates IL-23 gene expression in DCs to control IL-17 production in the lung.

Packed Red Blood Cells Modulate Expression Of Dendritic Cell Signal Transduction and Antigen Presentation Pathways

IntroductionClinical studies suggest packed red blood cell (PRBC) transfusion modulates recipients' immune responses, resulting in increased rates of infection, morbidity and mortality. In addition, there is evidence that increased length of storage of PRBC before transfusion is associated with worse outcomes. The mechanisms driving immunosuppression and transfusion-related poor patient outcomes remain largely undefined. As dendritic cells (DC) play a critical role in initiation and regulation of the immune response, this study investigated transfusion-mediated modulation of expression of DC genes involved in inflammation, antigen presentation and signal transduction. MethodsMonocyte-derived DC (MoDC) were differentiated following exposure to PRBC (at either D2, D14, D28 or D42 of routine storage) in an in vitro transfusion model. Gene expression profiling of MoDC generated “post-transfusion” was assessed at each time point using quantitative real time PCR using ΔΔCT method (relative to GAPDH and matched “no transfusion control”) and data analysed using ANOVA (95% CI). Expression array (Applied Biosystems) consisted of 28 genes broadly categorised into 1) chemokines, cytokines and receptors, 2) antigen presentation, 3) signal transduction. ResultsPRBC transfusion significantly modulated expression of genes associated with signal transduction (CD86, CD44, TAP2, all p<0.05) and antigen presentation (NFκB2, RELA, RELB, all p<0.05). Modulation of gene expression was associated with the storage age of the PRBC to which the “recipient” was exposed. Networks of gene interactions were generated (Ingenuity Systems) to illustrate potential mechanisms of immune deregulation that are associated with transfusion-related DC modulation. ConclusionsThis study reports, for the first time, transfusion-driven modulation of DC gene expression that was associated with the storage age of PRBC transfused. These results provide a basis for the early assessment of immune function that we plan to translate into clinical studies to evaluate the efficacy of diagnostic tools to predict poor patient outcomes. Disclosures:No relevant conflicts of interest to declare.

Dendritic cell reprogramming by the hypoxic environment

Myeloid dendritic cells (DCs) are professional antigen-presenting cells central to the orchestration of innate and acquired immunity and the maintenance of self-tolerance. The local microenvironment contributes to the regulation of DC development and functions, and deregulated DC responses may result in amplification of inflammation, loss of tolerance, or establishment of immune escape mechanisms. DC generation from monocytic precursors recruited at sites of inflammation, tissue damage, or neoplasia occurs under condition of low partial oxygen pressure (pO2, hypoxia). We reviewed the literature addressing the phenotypic and functional changes triggered by hypoxia in monocyte-derived immature (i) and mature (m) DCs. The discussion will revolve around in vitro studies of gene expression profile, which give a comprehensive representation of the complexity of response of these cells to low pO2. The gene expression pattern of hypoxic DC will be discussed to address the question of the relationship with a specific maturation stage. We will summarize data relative to the regulation of the chemotactic network, which points to a role for hypoxia in promoting a migratory phenotype in iDCs and a highly proinflammatory state in mDCs. Current knowledge of the strict regulatory control exerted by hypoxia on the expression of immune-related cell surface receptors will also be addressed, with a particular focus on a newly identified marker of hypoxic DCs endowed with proinflammatory properties. Furthermore, we discuss the literature on the transcription mechanisms underlying hypoxia-regulated gene expression in DCs, which support a major role for the HIF/HRE pathway. Finally, recent advances shedding light on the in vivo influence of the local hypoxic microenvironment on DCs infiltrating the inflamed joints of juvenile idiopathic arthritis patients are outlined.

Impact of the hypomethylating agent 5-azacytidine on dendritic cells function

Recent evidence suggested that 5-azacytidine (5-aza) can impact important immune functions via epigenetic modifications, making it an attractive candidate for pharmacologic manipulation of the immune system. The aim of this work was to study the effects of 5-aza on human dendritic cells (DC) generated from peripheral blood monocytes, and to test the type of immune response induced in patients treated with 5-aza. On the phenotypic level, CD40 and CD86 expression was significantly increased on mature DC exposed to 5-aza (5-aza-DC), compared with control untreated DC. Mature control DC and mature 5-aza-DC secreted comparable amounts of interleukin (IL)-6, IL-12p70, IL-23, and tumor necrosis factor-α. However, mature 5-aza-DC secreted significantly lower levels of IL-10 and IL-27 compared to mature control DC (p = 0.04 and p = 0.005, respectively). In the peripheral blood of 14 patients (7 males and 7 females; age range, 53-81 years) with advanced myeloid malignancies (8 acute myeloid leukemia and 6 myelodysplastic syndrome) treated with 5-aza, there was a significant decrease of IL-4-secreting CD4(+) T cells (p = 0.001), and a significant increase of IL-17A- and IL-21-secreting CD4(+) T cells (p = 0.003 and p = 0.01, respectively, compared to 5 healthy donors) suggesting a Th17 response pattern in the blood of patients receiving 5-aza. In all, these data suggest potentially novel mechanisms of action of epigenetic therapies, such as 5-aza, which may have broader implications for immunotherapeutic strategies.

17β‐estradiol induces CD40 expression in dendritic cells via MAPK signaling pathways in a minichromosome maintenance protein 6–dependent manner

The human immune system exhibits sexual dimorphism in autoimmune diseases such as systemic lupus erythematosus (SLE). Female sex hormones, including 17β-estradiol, are strongly implicated in the gender bias in SLE. CD40 is a costimulatory molecule and plays a crucial role in modulating the immune response of effector cells. We have previously shown that 17β-estradiol up-regulated CD40 expression and altered minichromosome maintenance protein 6 (MCM6) gene expression in dendritic cells (DCs). The mechanism of the correlation between CD40 and MCM6 in the presence of 17β-estradiol remains unknown. This study was undertaken to elucidate this mechanism and to explain the role of MCM6 in the gender bias in SLE. Bone marrow-derived DCs transfected with small interfering RNA (siRNA) for MCM6 were treated with 17β-estradiol in the absence or presence of CpG. The expression levels of costimulatory molecules, activity of MAPKs, and levels of MCM6 protein were measured. Moreover, the functions of DCs, including proliferation, apoptosis, endocytosis, and cytokine production, were analyzed. In addition, levels of messenger RNA for MCM6 were detected in DCs purified from SLE patients. Regardless of the presence or absence of CpG, 17β-estradiol induced CD40 expression via the activation of p38 and JNK, but not ERK. The activation of p38 and JNK enhanced MCM6 expression, which then induced CD40 expression. Suppression of MCM6 in DCs abolished the up-regulation of 17β-estradiol-induced CD40 expression. Importantly, MCM6 expression was significantly increased in SLE patients compared with healthy controls. Our findings indicate that 17β-estradiol induces CD40 expression in DCs via p38 and JNK MAPKs in an MCM6-dependent manner. MCM6 may be a critical mediator of sex-based differences in autoimmune disease.

Pattern Recognition Scavenger Receptor CD204 Attenuates Toll-like Receptor 4-induced NF-κB Activation by Directly Inhibiting Ubiquitination of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6

The collaboration and cross-talk between different classes of innate pattern recognition receptors are crucial for a well coordinated inflammatory response and host defense. Here we report a previously unrecognized role of scavenger receptor A (SRA; also known as CD204) as a signaling regulator in the context of Toll-like receptor 4 (TLR4) activation. We show that SRA/CD204 deficiency leads to greater sensitivity to LPS-induced endotoxic shock. SRA/CD204 down-regulates inflammatory gene expression in dendritic cells by suppressing TLR4-induced activation of the transcription factor NF-κB. For the first time, we demonstrate that SRA/CD204 executes its regulatory functions by directly interacting with the TRAF-C domain of TNF receptor-associated factor 6 (TRAF6), resulting in inhibition of TRAF6 dimerization and ubiquitination. The attenuation of NF-κB activity by SRA/CD204 is independent of its ligand-binding domain, indicating that the signaling-regulatory feature of SRA/CD204 can be uncoupled from its conventional endocytic functions. Collectively, we have identified the molecular linkage between SRA/CD204 and the TLR4 signaling pathways, and our results reveal a novel mechanism by which a non-TLR pattern recognition receptor restricts TLR4 activation and consequent inflammatory response.

Expression of Circulatory Dendritic Cells and Regulatory T-Cells in Patients With Different Subsets of Coronary Artery Disease

Dendritic cells (DCs), regulators of innate and adaptive immunity, may play an important role in atherosclerosis. DC invasion was found in early atherosclerotic lesions. We aimed to characterize circulating DC gene expression in patients with different subsets of coronary artery disease (CAD). Peripheral blood mononuclear cells were quantified using real-time polymerase chain reaction and fluorescence activated cell sorting in patients with acute coronary syndrome (ST-elevation myocardial infarction [STEMI], n = 35; non-ST-elevation myocardial infarction [NSTEMI], n = 30) and stable CAD (6 months after stent implantation without progression, n = 15) compared with control subjects (n = 15). DCs and T-cells (TCs) were characterized using specific primers for CD1a (immature), CD86 (mature), CD123 (plasmacytoid), BDCA1 (myeloid), CD178 (activated TCs), and FOXP3 (regulatory TCs). To evaluate whether serum of patients with STEMI induces DC differentiation, incubation of patient serum was performed. CD86 was upregulated and CD1a downregulated in all patients with CAD (P < 0.05). Patients with STEMI and NSTEMI showed a downregulation of CD1a compared with patients with stable CAD (P ≤ 0.01). In contrast, stable patients with CAD had elevated CD178 levels compared with patients with STEMI and NSTEMI (P ≤ 0.04). In patients with STEMI, FOXP3 was downregulated compared with control subjects (P < 0.0001). Incubation of STEMI serum induced an upregulation of CD1a and CD86 in a human DC cell line. Coincubation with a blocking antibody for heat shock protein 60 inhibited this upregulation. DCs are differentially regulated in patients with different subsets of CAD. Mature DCs are upregulated and immature DCs are downregulated in patients with CAD. Patients with STEMI show a significant downregulation of regulatory TCs. Circulating shock protein 60 induces DC differentiation in patients with STEMI.

Immunoproteasomes regulate gene expression in dendritic cells (100.36)

Abstract A constitutive form of the proteasome (CP) is expressed in all eukaryotic cells and is involved in many cellular processes. Immune and cytokine-activated cells also express a second type of proteasome named the immunoproteasome (IP). Since its discovery, it has been thought that the main role of IPs is to produce MHC I-associated peptides. However, an increasing number of studies report peptide-independent effects of IPs. In line with this, we recently showed using microarrays that knocking-out two IP-subunits (LMP7 and MECL1) in bone marrow-derived dendritic cells alters the expression of 171 genes mostly associated with immune functions, with enriched clusters on chromosomes. We now show by qPCR experiments that the disruption of both LMP7 and MECL1 is necessary to alter gene expression. Bioinformatic predictions of transcription factor binding sites suggest that this effect of IPs hinges mainly on transcriptional regulation. Comparing the abundance of unspliced and mature mRNAs in WT and IP knock-out cells shows that IPs also influence the maturation of certain transcripts. However, IPs do not affect mRNA stability overtime when blocking mRNA synthesis. The control of gene expression by IPs might explain aberrant T and B cell phenotypes previously described in IP-subunit knock-out mice. A better comprehension of how IPs regulate gene expression could help us understand the selective pressure that allowed the conservation of more than one type of proteasome in vertebrates.

Genome‐wide analysis of gene expression in dendritic cells from rat regenerating liver after partial hepatectomy

Dendritic cells (DCs) play a pivotal role in orchestrating immune response occurring in liver regeneration (LR). However, there are few details about relationship between DCs and LR at the molecular level. In this study, we firstly obtained high-purity DCs by the combination of Percoll density gradient centrifugation and immunomagentic bead sorting, then measured genome-wide gene expression in DCs from rat regenerating liver after partial hepatectomy (PH) using rat genome 230 2·0 microarray composed of 25,020 genes, and verified the reliability of microarray data with RT-PCR method. Among 25,020 genes present on the array, 1621 known genes and 1307 unknown genes, totally 2928 genes, were identified to be LR-related. H-clustering analysis showed that 2928 genes were grouped into three expression patterns: up-regulation, down-regulation and complex changes in expression. And 1621 known genes were functionally grouped into at least 23 biological categories. When expression patterns were combined with gene functions, as a whole, the genes involved in immune/defence response, inflammatory response and secretion of active substance in DCs were highly enriched in down-regulated pattern. DCs exhibited the reduced immune/defence and inflammatory response, and the suppressed secretion capacity of active substances after PH. A further analysis of genes expressed in the phase-dependent manner during LR suggested the rapid induction of genes encoding some transcription factors and cytokines in DCs at immediate-early phase, the unobvious enhancement of metabolic process, immunity and inflammation at proliferation phase, while the impairment of detoxification, immunity and inflammation at terminal phase.

Vitamin A Upregulates Matrix Metalloproteinase-9 Activity by Murine Myeloid Dendritic Cells through a Nonclassical Transcriptional Mechanism , ,

Myeloid dendritic cells (DC) are specialized antigen-presenting immune cells. Upon activation in peripheral tissues, DC migrate to lymph nodes to activate T lymphocytes. Matrix metalloproteinase (MMP)-9 is a gelatinase essential for DC migration. We have previously shown that all-trans retinoic acid (atRA), a bioactive metabolite of vitamin A, significantly augmented DC MMP-9 mRNA and protein production. We investigated the mechanisms by which atRA increased MMP-9 activity in vitro. Mouse myeloid DC cultured with atRA demonstrated increased gelatinase activity compared with cells cultured with retinoic acid receptor (RAR)-α antagonist. Adding MMP-9 inhibitor significantly blocked DC gelatinase activity and increased adherence of DC in a dose-dependent manner. AtRA-induced Mmp-9 gene expression in DC was blocked by transcriptional inhibition. Because the Mmp-9 promoter contains no canonical retinoic acid response element (RARE), we performed additional studies to determine how atRA regulated DC Mmp-9 transcription. Electrophoretic mobility shift assays for the consensus Sp1, activating protein-1, and nuclear factor-κB binding sites located in the Mmp-9 promoter did not indicate greater nuclear protein binding in response to atRA. Chromatin immunoprecipitation assays indicated RARα and histone acetyltransferase p300 recruitment to, and acetylation of, histone H3 at the Mmp-9 promoter was greater after atRA treatment. These data suggest that atRA regulated DC adhesion in vitro partly through MMP-9 gelatinase activity. Mmp-9 expression was enhanced through a transcriptional mechanism involving greater RARα promoter binding, recruitment of p300, and subsequent histone H3 acetylation, despite the absence of a consensus RARE.

Retraction

While bivalent antibodies can block ligand-receptor interactions, IgM pentamers efficiently cross-link cell surface targets and evoke physiological responses. We have described one such interaction between an IgM antibody (Ab) and the B7-DC costimulatory molecule expressed by dendritic cells that induces strong antitumor immunity and modulates pathogenic responses associated with allergic asthma. Progressive changes in gene expression in dendritic cells activated by an IgM B7-DC cross-linking Ab resulted in the increased expression in 350 genes and decreased expression of more than 200 genes over the course of 24 h following Ab treatment. In particular, up-regulation of the caspase inhibitor FLIP and the chemokine receptor CCR7, and the down-regulation of the CXCR4 receptor provide a mechanistic basis of Ab-induced survival and enhanced migration into draining lymph nodes. Increased expression of both cell surface and secreted molecules known to be mediators of the immunomodulatory properties of dendritic cells was detected at both the levels of RNA and protein expression. This analysis documents the ability of IgM Ab to activate a gene expression cascade leading to important biological changes in cellular function and provides mechanistic insight into the potent immunomodulatory properties attributed to this Ab.

Suppressive effects of transcription factor GATA-1 on cell type-specific gene expression in dendritic cells

To evaluate the effects of the transcription factor GATA-1 on determining cell fate between dendritic cell (DC) and mast cell (MC) lineages, GATA-1 was exogenously expressed in bone marrow-derived (BM) DCs. Exogenous expression of GATA-1 by a retrovirus in BMDCs inhibited expression of CD11c, CD80, CD86, and major histocompatibility complex class II with suppression of antigen-presenting ability and morphological changes toward granulocyte-like cells. Transcription of MC proteases and c-kit was markedly upregulated by GATA-1. Expression of IRF-4 and -8 was markedly suppressed, whereas PU.1 mRNA level was not affected by GATA-1. Chromatin immunoprecipitation assay showed that recruitment of PU.1 on the IRF-8 promoter was reduced in GATA-1-expressing DCs. These results indicate that GATA-1 suppresses PU.1 function but not PU.1 transcription. Thus, GATA-1 appears to determine cell fate by regulating several cell-specific transcription factors.

Dexamethasone synergistically increases secretion of the anti-inflammatory cytokine interleukin(IL)-10 in murine dendritic cells (DCs) upon viral infection: Potential implication to stress-mediated modification of susceptibility to viral infection. (94.4)

Abstract Glucocorticoids (GC) are end-products of the HPA axis, which play central roles in stress response, including pathogen infection, and are widely used as therapeutic agents for inflammatory diseases. It is known that stressed condition altered the susceptibility to viral infection. Since it is not known how GC influence the function of DCs upon viral infection, we infected murine DC with Newcastle disease virus (NDV) in the presence of glucocorticoid receptor (GR) ligand dexamethasone (Dex), and examined mRNA expression of 84 genes, which play various roles in the TLR signaling cascade. We found that NDV infection potently induced mRNA expression of 24 genes. Among them, 7 genes (interferon (IFN)-γ, interleukin (IL)-6, IL-10, lymphotoxin A, C-type lectin domain family 4, nuclear factor of κ light chain gene enhancer in B cell inhibitor β and prostaglandin-endoperoxidase synthase 2) were further up- or down-regulated by Dex pre-treatment. Particularly on IL-10, Dex synergistically up-regulated NDV-induced mRNA by 7-fold. The Dex effect was mediated by GR, as inhibitor RU 486 abolished the effect. In contrast to IFN-γ and IL-6, which are Th1 cytokines and response at early time point (6 h), kinetics of IL-10 mRNA showed the highest synergistic effect of NDV and Dex at a later time point (24 h). ELISA results showed consistent results with mRNA expression. Taken together, our study indicates that GC modulate host immunity, altering TLR-related gene expression in DCs.

The RNA binding protein tristetraprolin influences the activation state of murine dendritic cells

Dendritic cells (DCs) serve to maintain peripheral tolerance under steady state conditions. Upon triggering by activation signals they initiate strong immune responses. The activation of DCs is accompanied by a rapid upregulation of proinflammatory cytokines, which were shown in other cell types to be regulated by mechanisms at the transcriptional and posttranscriptional level. Tristetraprolin (TTP), an important RNA binding protein, is involved in the regulation of mRNA stability of such cytokines. In this study we analyzed the significance of TTP for mouse DCs, which were derived from TTP−/− and WT bone marrow progenitor cells (BM-DCs). Unstimulated BM-DCs of TTP−/− mice expressed lower levels of mRNAs encoding the costimulatory molecules CD40 and CD86 and surprisingly also the canonical TTP targets TNF-α and IL-10 as compared with WT DCs. On the protein level, both DC populations expressed comparable amounts of CD80 and CD86 and of either cytokine, but TTP−/− DCs expressed less MHCII than WT DCs. On the other hand, TTP−/− DCs displayed elevated expression of other TTP target mRNAs like IL-1β, c-fos and Mkp-1. Stimulation of BM-DCs of either genotype with lipopolysaccharide resulted in a rapid upregulation to a comparable extent of all molecules monitored so far, except for c-fos mRNA. Subsequent mRNA decay analysis revealed gene-specific differences in mRNA stability, which was influenced by the presence of TTP and the activation state of the DCs. Unstimulated TTP−/− DCs exerted a markedly lower allogeneic T cell stimulatory potential than WT DCs. Moreover, TTP−/− DCs induced an altered cytokine pattern in cocultures of DCs and T cells. However, allogeneic T cells primed by unstimulated DCs of either genotype were equally refractory to restimulation and suppressed the proliferation of naive T cells to the same extent. Thus, the findings of this study lend support to the interpretation that without external stimulation antigen presenting activity in DCs in the presence of TTP is more pronounced than in its absence and that posttranscriptional regulation contributes to the control of gene expression in DCs.

Aging‐dependent upregulation of IL‐23p19 gene expression in dendritic cells is associated with differential transcription factor binding and histone modifications

Age-associated changes in immune response increase the risk of infection and promote inflammation and autoimmunity in older adults. The newly discovered cytokine IL-23 contributes to the maintenance and expansion of Th-17 cells, which promote proinflammatory responses. Our preliminary findings suggested that Th-17 responses are increased in aged mice. IL-23 consists of p40 and p19 subunits. Expression of the p19 subunit is regulated at the transcriptional level by NF-kappaB p65 and c-Rel transcription factors. Using bone-marrow-derived dendritic cells (DCs) from C57BL/6 mice, we show that IL-23 protein production and p19 subunit mRNA levels are significantly increased in DCs from aged mice after activation with TLR ligands (LPS + R848) when compared with DCs of young adult mice. We found that the increase in p19 expression in aged cells is associated with chromatin remodeling characterized by di- and tri-methylation of histone H3K4 and binding of mainly c-Rel at the p19 promoter. In young DCs, the promoter is tri-methylated only at H3K4 and bound by both p65 and c-Rel. C-Rel knockdown restores p65 binding in aged cells but does not activate p19 expression, suggesting that c-Rel is critical for p19 expression. In addition, p65 knockdown significantly increases c-Rel binding and p19 expression in young DCs to levels close to those detected in old cells. Furthermore, the decrease in p65 binding at the p19 promoter in old DCs was specific to the p19 gene since p65 binding to the IL-12p40 promoter was not significantly different between old and young DCs. Our results demonstrate that selective changes in H3K4 methylation, and c-Rel and p65 binding at the p19 promoter occur in DCs and contribute to the upregulation of the p19 subunit expression and IL-23 protein production observed in aged mice. This suggests epigenetic and transcriptional mechanisms contribute to dysregulated inflammatory and autoimmune responses associated with aging.

Immunosuppressive effects of an ethyl acetate extract from Urtica dentata Hand on skin allograft rejection

Aim of the study To investigate the immunosuppressive effects of HPLC qualitied ethyl acetate extract (EAE) from Urtica dentate Hand on skin allograft rejection in a murine model. Materials and methods Allo-skin transplantation model was established by placing skin allograft of C57BL/6 mice in the wound bed which was on the back of Balb/c mice. We used FACS to study the effects of EAE on dendritic cells (DCs) maturation and CD4 +CD25 +T regulatory cells (Tregs) differentiation. We also studied spleen lymphocyte proliferation and T-bet gene expression in DCs. Concentration of Th1/Th2 cytokines was monitored as markers of Th1/Th2 responses by ELISA. Results A significant prolongation of skin allografts survival was observed as a dose-dependent manner in the animals treated with EAE. By FACS, we found that treatment with EAE (200 mg kg −1) resulted in an immature statement of DCs and stimulated the differentiation of CD4 +CD25 +Tregs. Additionally, the expression of T-bet gene and the proliferation of spleen lymphocytes were efficiently abated in EAE treated mice. Comparing to the model control, EAE-treated recipients showed a significant down-regulation ( P < 0.01) of Th1 cytokines (IL-2, IFN-γ) and an obviously increase ( P < 0.01) of Th2 cytokine (IL-10) in the serum, which presented in a dose-related way. Conclusions The anti-allograft rejection effect of EAE by enhancing CD4 +CD25 +Tregs differentiation and sustaining DCs immaturation makes EAE to be a possible choice for treating autoimmune diseases in a way of inducing a stable immunological tolerance state.

Immune modulation through RNA interference-mediated silencing of CD40 in dendritic cells

RNA interference (RNAi) is an exciting mechanism for knocking down any target gene in transcriptional level. It is now clear that small interfering RNA (siRNA), a 19–21 nt long dsRNA, can trigger a degradation process (RNAi) that specifically silences the expression of a cognate mRNA. Our findings in this study showed that down regulation of CD40 gene expression in dendritic cells (DCs) by RNAi culminated to immune modulation. Effective delivery of siRNA into DCs would be a reasonable method for the blocking of CD40 gene expression at the cell surface without any effect on other genes and cell cytotoxicity. The effects of siRNA against CD40 mRNA on the function and phenotype of DCs were investigated. The DCs were separated from the mice spleen and then cultured in vitro. By the means of Lipofectamine2000, siRNA was delivered to the cells and the efficacy of transfection was estimated by flow cytometry. By Annexine V and Propidium Iodide staining, we could evaluate the transfected cells viability. Also, the mRNA expression and protein synthesis were assessed by real-time PCR and flow cytometry, respectively. Knocking down the CD40 gene in the DCs caused an increase in IL-4 production, decrease in IL-12 production and allostimulation activity. All together, these effects would stimulate Th2 cytokines production from allogenic T-cells in vitro.