Unstimulated Dendritic Cells Research Articles

Halophilic Archaea Halorhabdus Rudnickae and Natrinema Salaciae Activate Human Dendritic Cells and Orient T Helper Cell Responses.

Halophilic archaea are procaryotic organisms distinct from bacteria, known to thrive in hypersaline environments, including salt lakes, salterns, brines and salty food. They have also been identified in the human microbiome. The biological significance of halophiles for human health has rarely been examined. The interactions between halophilic archaea and human dendritic cells (DCs) and T cells have not been identified so far. Here, we show for the first time that the halophilic archaea Halorhabdus rudnickae and Natrinema salaciae activate human monocyte-derived DCs, induce DC maturation, cytokine production and autologous T cell activation. In vitro both strains induced DC up-regulation of the cell-surface receptors CD86, CD80 and CD83, and cytokine production, including IL-12p40, IL-10 and TNF-α, but not IL-23 and IL-12p70. Furthermore, autologous CD4+ T cells produced significantly higher amounts of IFN-γ and IL-13, but not IL-17A when co-cultured with halophile-stimulated DCs in comparison to T cells co-cultured with unstimulated DCs. IFN-γ was almost exclusively produced by naïve T cells, while IL-13 was produced by both naïve and memory CD4+ T cells. Our findings thus show that halophilic archaea are recognized by human DCs and are able to induce a balanced cytokine response. The immunomodulatory functions of halophilic archaea and their potential ability to re-establish the immune balance may perhaps participate in the beneficial effects of halotherapies.

Blockade of dendritic cell glutaminolysis induces allergic asthma desensitization via suppression of Tfh13 polarization

Abstract Allergic asthma is a pulmonary disease characterized by airway hyperresponsiveness (AhR) in response to inhaled allergens. AhR is largely mediated by allergen-specific (Ag-specific) IgE induced mast cell (MC) degranulation which drives inflammation of the airways following allergen exposure. Ag-specific IgE production requires T follicular helper 13 cells (Tfh13s) which must first be polarized by dendritic cells (DCs). The mechanisms by which DCs polarize Tfh13s, however, are unclear. We hypothesized that the Tfh13 inducing allergen Alternaria would induce a unique metabolic response compared to non-Tfh13 inducing stimuli. We found, by untargeted metabolomic analysis, that Alternaria induced accumulation of the glutamine-derived TCA cycle metabolite α-ketoglutarate compared to LPS stimulated or unstimulated DCs. Glutamine is converted to α-ketoglutarate by glutaminase (Gls), so we predicted that inhibition of Gls may suppress Tfh13 polarization by DCs. Indeed, we found that treatment with the Gls inhibitor, CB-839, reduced Tfh13 polarization and increased T follicular regulatory cells following Alternaria exposure. This finding was recapitulated in a DC adoptive transfer model, indicating that this mechanism is DC intrinsic. We next tested if CB-839 causes allergen desensitization in a model of allergic asthma. Mice were intranasally sensitized and challenged with Alternaria and intranasally treated with CB-839 or vehicle control prior to challenge. CB-839 treated mice exhibited reduced AhR, Ag-specific IgE, and airway MC degranulation. These data demonstrate that DC glutaminolysis is required for Tfh13 polarization, and that Gls inhibitors may promote allergen desensitization in allergic asthma. This work was funded by VCU's National Center for Advancing Translational Sciences (UL1TR002649) and NIAID (R56AI139658).

Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells

The major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3–7% and ≤ 1%, respectively.

STING-dependent type-1 interferon restrains schistosome immunopathology via down-regulation of the CD209a lectin receptor

Abstract Infection with the helminth parasite Schistosoma mansoni causes morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens. We now demonstrate that stimulation of dendritic cells (DCs) with schistosome eggs induces robust IFNβ production in a manner dependent on the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon genes (STING) cytosolic DNA sensing pathway, resulting in the suppression of proinflammatory IL-1β and IL-23 production and in Th17 cell activation. Consistent with these results, low-pathology BL/6 mice lacking STING exhibited markedly enhanced hepatic granulomatous inflammation associated with significantly increased Th17 and diminished Th2 cytokine responses. Mechanistically, IFNβ acts by suppressing DC expression and function of CD209a, a C-type lectin receptor associated with severe schistosome immunopathology. Importantly, there was an increased baseline CD209a expression in unstimulated DCs from STING−/− mice, suggesting a role for constitutive IFN signaling. Our findings demonstrate that innate, cGAS/STING-dependent sensing of parasite DNA represents a novel pathway inducing type I IFN production, which protects the host from excessive inflammation and immunopathology in schistosomiasis. This work is supported by NIAID grant R01 AI018919 to MJS.

Effect of Cytomegalovirus Recombinant Phosphoprotein 150 (pp150) on Maturation and Function of Murine Dendritic Cells: an In-Vitro Study.

Tegument protein pp150 of cytomegaloviruses (CMVs) plays a vital role in all stages of viral life cycle, representing the most important tegument protein candidate for HCMV treatment. However, the exact role of pp150 in immune regulation is yet to be elucidated. To examine the effects of pp150 on the maturity and function of murine dendritic cells (DCs). Maturity status (CD40, CD86, and MHC-II expression) and phagocytic capacity of DCs (dextran uptake assay) were characterized. Gene expression profiles of ROR-γ, GATA-3, T-bet, and FOXP-3 as well as the protein expression of INF-γ (Th1), IL-4 (Th2), IL-35 (Treg), IL-17A (Th17), IL-22, TNF-α, IL-6, and IL-2 were evaluated in T cells co-cultured with DCs. A significant increase in CD40, CD86, and CCR7 expression and a reduction in the phagocytosis rate were observed in pp150-stimulated DCs compared with unstimulated DCs. T cells co-cultured with stimulated DCs showed higher expressions of ROR-γ, IL-6, IL-2, IL-17A, IL-22, and TNF-α. Despite improvements in maturity status, pp150-stimulated DCs did not seem to be able to induce Th1 or Th2 immunity. In fact, Th17 and its mediators, IL-17A and IL-22, might be the main inflammatory factors involved in pp150-stimulated DC's mechanism of action. However, it is necessary to conduct further investigations to corroborate these observations.

Las células dendríticas generadas en presencia de vitamina D3 y activadas con lipopolisacáridos incrementan la producción de IL-1β, IL-8 e IL-10 y disminuyen su capacidad de inducir LT CD4+CD25hiFoxp3+

Vitamin D3 (VD3) has been described as a modulator of immune system cells, including dendritic cells (DC). Previous studies have shown its importance in in vitro generation of tolerogenic DC, which have a similar function and phenotype to that of CD141 dermal DCs that produce IL-10 and induce (LTreg) CD4+ T regulator cells. This paper presents a study that compares the phenotype and cytokines produced by DC generated in presence and absence of VD3, which were matured with lipopolysaccharide (LPS), and their ability to induce LTreg from naïve allogeneic CD4+ T cells. In order to compare them, peripheral blood mononuclear cells were isolated to select monocytes CD14+ T cells and differentiate them in vitro from DC in the presence and absence of VD3, and to mature them with LPS. Phenotype and cytokine levels were also analyzed in the culture supernatants. Dendritic cells were then co-cultured with naïve allogeneic CD4+ T cells and the frequencies of LTreg were determined (naïve-activated). The results showed that unstimulated DC generated with VD3 kept the CD14. When activated with LPS, they expressed lower levels of C83, CD83 and CD86; HLA-DR; higher amounts of IL-1β, IL-8, IL-10, and tended to lessen IL-6, IL-12p70 and TGF-β1, compared to DCs not treated with VD3. The frequency of naïve LTreg was similar, although immature DC generated with VD3 tended to induce activated LTregs. Based on these results, it is possible to conclude that DCs generated with VD3 and treated with LPS presented a 'semi-mature' phenotype, and were able to secrete pro-inflammatory and anti-inflammatory cytokines. Besides, they did not increase their capacity to promote the polarization of naïve allogenic CD4+ T cells towards LTregs.

Effect of calcineurin inhibition on phenotypic maturation of dendritic cells in an in-vitro model of invasive aspergillosis in lung transplant recipients

BackgroundInvasive aspergillosis in lung transplant recipients on immunosuppression has high morbidity and mortality. The calcineurin inhibitor FK506 inhibits the calcineurin–NFAT (nucleated factor of activated T cells) axis, which impairs the innate response to fungal infection. Immature dendritic cells (DCs) have a crucial immune signalling role by phagocytosing antigen, and then maturing and stimulating the T cell response. We investigated the effect of FK506 on phenotypic maturation of DCs in response to Aspergillus fumigatus infection. MethodsPeripheral blood mononuclear cells from healthy volunteers were negatively isolated by density gradient centrifugation (Ficoll, GE Healthcare, Amersham, UK) and were differentiated into DCs with molgramostim (GM-CSF) and interleukin 4. Day 5 cells were matured with interferon (IFN) γ. Day 7 cells were treated with FK506 or inoculated with swollen conidia of A fumigatus (multiplicity of infection 1:1). Cells were then stained with PE-bound anti-CD83 (a late maturation marker) and PerCP-Cy5.5-bound anti-CD209 (a DC-specific marker) and analysed by imaging flow cytometry (ImageStream, Amnis, Seattle, USA). Statistical analysis was performed with Graphpad Prism (version 6.0). Findings5000 cells per condition were analysed. The subset of DCs was identified by gating for CD209 positivity. We demonstrated upregulation of CD83 on the surface of DCs with IFNγ stimulation (26 228 mean fluorescence units [SE 1462] vs 12 534 [799], p<0·0001) A fumigatus infection of unstimulated DCs (29 888 [1393] vs 12 534 [799], p<0·0001), and A fumigatus infection of IFNγ-stimulated DCs (36 778 [1356] vs 26 228 [1462], p<0·0001). CD83 mean fluorescence intensity was reduced with FK506 treatment of IFNγ-stimulated DCs (26 228 [1462] vs 20 219 [846], p=0·0004), A fumigatus-infected unstimulated DCs (29 888 [1393] vs 24 289 [1253], p=0·0028), and A fumigatus-infected, IFN γ-stimulated DCs (36 778 [1356] vs 30 159 [1279], p=0·0004), but unchanged for unstimulated, uninfected DCs (12 534 [799] vs 11 942 [762], p=0·5921). InterpretationBoth A fumigatus infection and IFNγ stimulation promote phenotypic maturation of DCs in vitro, and treatment with FK506 inhibits maturation in this context. These findings suggest that FK506 has an inhibitory effect on innate antigen presentation to T cells and that it might impair the adaptive immune response to invasive aspergillosis in lung transplant recipients. FundingMedical Research Council Chain-Florey Clinical Research Fellowship.

Inhibitors of β-catenin affect the immuno-phenotype and functions of dendritic cells in an inhibitor-specific manner

Many tumors are characterized by mutation-induced constitutive activation of β-catenin which promotes tumor growth and survival. Consequently, the development of specific β-catenin inhibitors for tumor therapy has come into the focus of drug development. β-Catenin was also shown to contribute to the tolerance-promoting function of unstimulated dendritic cells (DCs). In response to activation, DCs acquire potent T cell stimulatory capacity and induce profound tumor antigen-specific immune responses. Here we asked for effects of pre-clinically established β-catenin inhibitors (CCT-031374, iCRT-5, PNU-75654) on mouse bone marrow-derived (BM)DCs. All three inhibitors moderately increased surface expression of MHCII, CD80, and CD86 on unstimulated DCs, but had no enhancing effect on their capacity to stimulate the proliferation of ovalbumin (OVA) specific CD4+ T cells. CCT-031374 interfered with upregulation of costimulators (CD40, CD86) and cytokines (IL-1β, TNF-α, IL-6, IL-10, IL-12) by LPS-stimulated DCs. Accordingly, this DC population displayed an impaired CD4+ T cell stimulatory activity. iCRT-5 and PNU-75654 had no detrimental effects on the immuno-phenotype of stimulated DCs. Hence, DCs treated with iCRT-5 in the course of stimulation exerted comparably strong T cell proliferation as did control DCs. In contrast, DCs stimulated in the presence of PNU-75654 induced less T cell proliferation than the control population despite enhanced uptake and processing of OVA. Our findings suggest that the differential effects of β-catenin inhibitors on stimulated DCs reflect off target effects. Concerning potential application of β-catenin inhibitors for tumor therapy, iCRT-5 may be most beneficial, since it did not exert detrimental effects on stimulated DCs.

Gene Expression Profiling Implicates Attenuation of NFkB Signalling By Regulatory T Cells in Modulating Dendritic Cell Function

Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a complication of haematopoietic stem cell transplantation and offer an attractive target for therapy. Regulatory T cells (Treg) have a potent immunoregulatory effect on the maturation and the antigen-presenting cell (APC) function of DC and adoptive transfer of Treg is highly efficacious in the induction of tolerance in an experimental model of GvHD and has entered Phase I clinical trials. Several mechanisms of suppression have been proposed, including Treg acting directly on DCs, attenuating their antigen-presenting and co-stimulatory functions by arresting their maturation. However, the molecular basis underpinning these effects in DCs remains ill-defined. We investigated the effect of Treg treatment on DCs by conducting gene expression profiling and confirmed the functional consequences using downstream assays.Immature, mature and Treg-treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC, respectively) and moDC populations were generated using a well-established 6 day culture with GM-CSF and IL-4, followed by 24 hour LPS maturation. Treg were added on day 3 of culture at a 3:1 ratio. All cell populations were harvested on day 7 and sorted via FSC/SSC/CD3neg gating to remove Treg present in the co-culture and control for any changes in gene expression caused by shear stress. Gene expression profiling was carried out using the Illumina HumanHT12 microarray platform. Data was processed using R/Bioconductor workflows and the functional significance of differentially expressed genes was evaluated using Ingenuity Pathway Analysis software.Mat-DC and Treg-DC expression profiles were compared relative to the im-DC for data analysis. Upon LPS treatment, the levels of 1834 unique genes were differentially regulated in mat-DC by at least twofold (862 genes upregulated/972 downregulated) compared to the im-DC counterparts. In the Treg-DC, 1326 unique genes were differentially modulated (633 genes upregulated/693 genes downregulated). Microarray analysis of the CD markers identified a higher expression of the previously identified surface markers CD80, CD83 and CD86 in the mat-DC compared to the Treg-treated counterpart (validated by flow cytometry), confirming the semi-mature phenotype.Novel findings from the dataset include the reduction of the endocytotic-related genes, CD206 and CD209, in the Treg-DCs compared to the im-DC and this reduction manifested functionally in an impaired antigen uptake, as assessed by FITC-Dextran. Additionally, the surface marker, CD38, was downregulated in the Treg-DC compared to the mat-DC, confirmed by flow cytometry. CD38 has been shown to be NFκB-dependent and a marker of maturation in monocyte-derived DCs, further supporting the semi-mature phenotype. Furthermore, CD38 is functionally involved in CD83 expression and IL-12 induction. We assessed IL-12 cytokine secretion by Treg-treated DCs and showed a significantly reduced level of induction compared to mat-DC (p=0.0079).Pathway analysis revealed NFκB-related genes to be downregulated in the Treg-DC compared to the mat-DC. These differentially expressed genes included the TLR-adaptor protein, MYD88, the NFκB subunit, RELB and an inhibitor of NFκB, NFκB1A. This finding, coupled to the importance of NFκB signalling pathway in DC function, prompted us to investigate it at the functional level by measuring levels of phosphorylation of serine 536 of the RelA subunit as a marker of activity in response to LPS stimulation. DC cultured in the absence of Tregs (mat-DC) showed significantly higher levels of Ser536 phosphorylation when compared to those unstimulated cells (im-DC) (p= 0.0018). Concordant with the gene expression data, Treg-treated DCs (Treg-DC), showed a significantly attenuated NFκB activation when compared to their LPS-stimulated DCs counterparts (p = 0.0191), however, signalling was not completely abolished compared to those unstimulated DCs (p= 0.0003).In conclusion, gene expression profiles of Treg-treated DCs are significantly different to their mat-DC and im-DC counterparts. Here, we present the novel finding that Tregs modulate DC function, in part, by attenuation of the NFkB signalling pathway, arresting the DCs at a semi-mature phenotype, as evidenced by expression arrays and functional assays. DisclosuresNo relevant conflicts of interest to declare.

MiR-155 Upregulation in Dendritic Cells Is Sufficient To Break Tolerance In Vivo by Negatively Regulating SHIP1.

TLR-induced maturation of dendritic cells (DCs) leads to the production of proinflammatory cytokines as well as the upregulation of various molecules involved in T cell activation. These are believed to be the critical events that account for the induction of the adaptive immune response. In this study, we have examined the role of miR-155 in DC function and the induction of immunity. Using a model in which the transfer of self-Ag-pulsed, TLR-matured DCs can induce a functional CD8 T cell response and autoimmunity, we find that DCs lacking miR-155 have an impaired ability to break immune tolerance. Importantly, transfer of self- Ag-pulsed DCs overexpressing miR-155 was sufficient to break tolerance in the absence of TLR stimuli. Although these unstimulated DCs induced T cell function in vivo, there was no evidence for the upregulation of costimulatory ligands or cytokine secretion. Further analysis showed that miR-155 influenced the level of the phosphatase SHIP1 in DCs and that the lack of SHIP1 in DCs was sufficient to break T cell tolerance in vivo, again in the absence of TLR-induced DC maturation. Our study demonstrates that the overexpression of miR-155 in DCs is a critical event that is alone sufficient to break self-tolerance and promote a CD8-mediated autoimmune response in vivo. This process is independent of the induction of conventional DC maturation markers, indicating that miR-155 regulation of SHIP represents a unique axis that regulates DC function in vivo.

Correction: Corrigendum: Dendritic cells from aged subjects contribute to chronic airway inflammation by activating bronchial epithelial cells under steady state

Correction to: Mucosal Immunology advance online publication 23 April 2014; doi:10.1038/mi.2014.28 In this article, published on 23 April 2014, the fifth author's name was misspelled as ‘BenMohamad’. The correct spelling is ‘BenMohamed’. The authors regret the error. Correction to: Mucosal Immunology advance online publication 23 April 2014; doi:10.1038/mi.2014.28 In this article, published on 23 April 2014, the fifth author's name was misspelled as ‘BenMohamad’. The correct spelling is ‘BenMohamed’. The authors regret the error. Correction to:Mucosal Immunology advance online publication 23 April 2014; doi:10.1038/mi.2014.28 In this article, published on 23 April 2014, the fifth author's name was misspelled as ‘BenMohamad’. The correct spelling is ‘BenMohamed’. The authors regret the error. Dendritic cells from aged subjects contribute to chronic airway inflammation by activating bronchial epithelial cells under steady stateMucosal ImmunologyVol. 7Issue 6PreviewThe mechanisms underlying the increased susceptibility of the elderly to respiratory infections are not well understood. The crosstalk between the dendritic cells (DCs) and epithelial cells is essential in maintaining tolerance as well as in generating immunity in the respiratory mucosa. DCs from aged subjects display an enhanced basal level of activation, which can affect the function of epithelial cells. Our results suggest that this is indeed the scenario as exposure of primary bronchial epithelial cells (PBECs) to supernatants from unstimulated DCs of aged subjects resulted in activation of PBECs. Full-Text PDF Open Archive

Geldanamycin-mediated inhibition of heat shock protein 90 partially activates dendritic cells, but interferes with their full maturation, accompanied by impaired upregulation of RelB

BackgroundThe chaperon heat shock protein 90 (HSP90) constitutes an important target for anti-tumor therapy due to its essential role in the stabilization of oncogenes. However, HSP90 is ubiquitously active to orchestrate protein turnover, chemotherapeutics that target HSP90 may affect immune cells as a significant side effect. Therefore, we asked for potential effects of pharmacological HSP90 inhibition at a therapeutically relevant concentration on human dendritic cells (DCs) as main inducers of both cellular and humoral immune responses, and on human CD4+ T cells as directly activated by DCs and essential to confer B cell help.MethodsUnstimulated human monocyte-derived DCs (MO-DCs) were treated with the prototypical HSP90 inhibitor geldanamycin (GA). Based on dose titration studies performed to assess cytotoxic effects, GA was applied at a rather low concentration, comparable to serum levels of clinically used HSP90 inhibitors. The immuno-phenotype (surface markers, cytokines), migratory capacity, allo T cell stimulatory and polarizing properties (proliferation, cytokine pattern) of GA-treated MO-DCs were assessed. Moreover, effects of GA on resting and differentially stimulated CD4+ T cells in terms of cytotoxicity and proliferation were analysed.ResultsGA induced partial activation of unstimulated MO-DCs. In contrast, when coapplied in the course of MO-DC stimulation, GA prevented the acquisition of a fully mature DC phenotype. Consequently, this MO-DC population exerted lower allo CD4+ T cell stimulation and cytokine production. Furthermore, GA exerted no cytotoxic effect on resting T cells, but abrogated proliferation of T cells stimulated by MO-DCs at either state of activation or by stimulatory antibodies.ConclusionHSP90 inhibitors at clinically relevant concentrations may modulate adaptive immune responses both on the level of DC activation and T cell proliferation. Surprisingly, unstimulated DCs may be partially activated by that agent. However, due to the potent detrimental effects of HSP90 inhibitors on stimulated CD4+ T cells, as an outcome a patients T cell responses might be impaired. Therefore, HSP90 inhibitors most probably are not suitable for treatment in combination with immunotherapeutic approaches aimed to induce DC/T cell activation.

Activation of NOD2 signalling pathway stimulates the function of human dendritic cells loaded with leukemia cell lysates

The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide (MDP) on the immunomodulation effect of human monocyte-derived dendritic cells (DC) loaded with leukemia cell lysates. Peripheral blood mononuclear cells (PBMNC) were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 (p40) . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC+MDP group, which was significantly higher than that in the DC+MDP group and sensitized DC without MDP stimulation, the difference was statistically significant (P < 0.05). Besides, the expression of surface molecules (HLA-DR, CD80, CD83, CD86, CD40) in the group of DC loaded with leukemia cell lysate and stimulated by MDP (sensitized DC+MDP) reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest (P < 0.05). Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level (573.86 ± 32.09 pg/ml) in DC+MDP group was higher than that in group of sensitized DC (365.03 ± 28.86 pg/ml) (P < 0.05), and it in sensitized DC+MDP group reached the highest (898.30 ± 61.08) pg/ml, compared to other groups (P < 0.05). It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC, at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.

Longitudinal expression of Toll-like receptors on dendritic cells in uncomplicated pregnancy and postpartum

Toll-like receptors (TLRs) are integral parts of the innate immune system and have been implicated in complications of pregnancy. The longitudinal expression of TLRs on dendritic cells in the maternal circulation during uncomplicated pregnancies is unknown. The objective of this study was to prospectively evaluate TLRs 1-9 as expressed on dendritic cells in the maternal circulation at defined intervals throughout pregnancy and postpartum. This was a prospective cohort of 30 pregnant women with uncomplicated pregnancies and 30 nonpregnant controls. TLRs and cytokine expression was measured in unstimulated dendritic cells at 4 defined intervals during pregnancy and postpartum. Basal expression of TLRs and cytokines was measured by multicolor flow cytometry. The percent-positive dendritic cells for each TLRs were compared with both nonpregnant and postpartum levels with multivariate linear regression. TLRs 1, 7, and 9 were elevated compared with nonpregnant controls with persistent elevation of TLR 1 and interleukin-12 (IL-12) into the postpartum period. Concordantly, levels of IL-6, IL-12, interferon alpha, and tumor necrosis factor alpha increased during pregnancy and returned to levels similar to nonpregnant controls during the postpartum period. The elevated levels of TLR 1 and IL-12 were persistent postpartum, challenging notions that immunologic changes during pregnancy resolve after the prototypical postpartum period. Normal pregnancy is associated with time-dependent changes in TLR expression compared with nonpregnant controls; these findings may help elucidate immunologic dysfunction in complicated pregnancies.

Application of dendritic cells stimulated with Trichinella spiralis excretory–secretory antigens alleviates experimental autoimmune encephalomyelitis

The parasitic nematode, Trichinella spiralis (T. spiralis), exerts an immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from encysted muscle larvae. Our model of combined T. spiralis infection and experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats demonstrated a significant reduction in EAE severity in infected animals. Recently, we have created an immune status characteristic for the live infection by in vivo application of dendritic cells (DCs) stimulated with ES L1 products of T. spiralis muscle larvae. Moreover, these cells were able to ameliorate EAE when applied 7days before EAE induction. ES L1-stimulated DCs increased production of IL-4, IL-10 and TGF-β, and decreased production of IFN-γ and IL-17, both at the systemic level and in target organs. A significant increase in the proportion of CD4+CD25+Foxp3+ T cells was found among spleen cells, and CNS infiltrates from DA rats treated with ES L1-stimulated DCs before EAE induction, compared to controls injected with unstimulated DCs. Regulatory T cells, together with elevated levels of IL-10 and TGF-β, are most likely involved in restraining the production of Th1 and Th17 cytokines responsible for autoimmunity and thus are responsible for the beneficial effect of ES L1-educated DCs on the course of EAE. Our results show that ES L1 antigen-stimulated DCs are able not only to provoke, but also to sustain anti-inflammatory and regulatory responses regardless of EAE induction, with subsequent amelioration of EAE, or even protection from the disease.

The effects of anabolic steroids on dendritic cells

Event Abstract Back to Event The effects of anabolic steroids on dendritic cells Liam M. Cosgrave1, Ekua W. Brenu1, Teilah K. Huth1*, Bon Gray2 and Sonya M. Marshall-Gradisnik1 1 Griffith University, Griffith Health Institute, Australia 2 Bond University, Australia To date there is limited information on the effects of Anabolic Androgenic Steroids (AAS) on antigen presentation function of dendritic cells (DC). Co-stimulatory molecules CD80 and CD86 are important during antigen presentation as they associate with CD28 on T cells which is important for T cell survival. Various factors including AAS may alter these processes, particularly at high levels. The objective of this study was to investigate the effects of a commonly used AAS, testosterone cypionate (TC), on DC antigen presentation over time in vitro. Ten healthy male university students (mean age = 22.9 ± 0.7SD) were recruited for this study. Blood samples from the participants were incubated with or without 3µM of TC and PMA for 4, 12 and 24 hours. Following incubation, the samples were fluorescently labelled with CD80, CD86, CD3, CD14, CD19, CD20, CD56 and CD11c to facilitate preferential identification of DCs from other cells. Flow cytometry analysis determined the expression of CD80/CD86 surface markers on stimulated and unstimulated DC. A repeated measures ANOVA identified significance where p<0.05.TC treated samples demonstrated a significant increase in CD80 expression over time for both stimulated and unstimulated DCs. CD86 expression in unstimulated samples was significantly decreased. However, TC treated samples had significantly higher expression of CD86 compared to controls at 12 and 24 hours post incubation. These studies have highlighted the effects of testosterone on DCs which may have important implications in individuals who administer different types of AAS for recreational purposes. Keywords: Dendritic Cells, Testosterone, Steroids, Antigen Presentation, co-stimulatory molecules Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Cosgrave LM, Brenu EW, Huth TK, Gray B and Marshall-Gradisnik SM (2013). The effects of anabolic steroids on dendritic cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00926 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Miss. Teilah K Huth, Griffith University, Griffith Health Institute, Gold Coast, Queensland, 4222, Australia, t.huth@griffith.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Liam M Cosgrave Ekua W Brenu Teilah K Huth Bon Gray Sonya M Marshall-Gradisnik Google Liam M Cosgrave Ekua W Brenu Teilah K Huth Bon Gray Sonya M Marshall-Gradisnik Google Scholar Liam M Cosgrave Ekua W Brenu Teilah K Huth Bon Gray Sonya M Marshall-Gradisnik PubMed Liam M Cosgrave Ekua W Brenu Teilah K Huth Bon Gray Sonya M Marshall-Gradisnik Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

PMO-231 Ileal and colonic mucosal dendritic cell cytokine profiles differ at rest and after in vitro bacteria and pro-biotic challenge in postoperative Crohn's disease patients

IntroductionPostoperative Crohn's disease (CD) recurrence predominantly affects the ileal mucosa at the ileo-colonic anastomosis with the colonic side often spared. Altered immune responses to bacterial flora are thought to be...

Nuclear factor-κB1 controls the functional maturation of dendritic cells and prevents the activation of autoreactive T cells

Mature dendritic cells (DCs) are crucial for the induction of adaptive immune responses and perturbed DC homeostasis can result in autoimmune disease. Either uncontrolled expansion or enhanced survival of DCs can result in a variety of autoimmune diseases in mouse models. In addition, increased maturation signals, through overexpression of surface Toll-like receptors (TLRs) or stimulation by type I interferon (IFN), has been associated with systemic autoimmunity. Whereas recent studies have focused on identifying factors required for initiating the maturation process, the possibility that resting DCs also express molecules that 'hold' them in an immature state has generally not been considered. Here we show that nuclear factor-κB1 (NF-κB1) is crucial for maintaining the resting state of DCs. Self-antigen-pulsed unstimulated DCs that do not express NF-κB1 were able to activate CD8(+) T lymphocytes and induce autoimmunity. We further show that NF-κB1 negatively regulates the spontaneous production of tumor necrosis factor-α (TNF-α), which is associated with increased granzyme B expression in cytotoxic T lymphocytes (CTLs). These findings provide a new perspective on functional DC maturation and a potential mechanism that may account for pathologic T cell activation.

The gene expression profile of unstimulated dendritic cells can be used as a predictor of function

Dendritic cells (DCs) represent a subset of professional antigen presenting cell (APC) whose role is to elicit immune responses against harmful antigens. They have been used in DC vaccines to stimulate the immune system to kill cancer cells. However, successes in clinical trials have been limited, which may be attributed to a lack of appreciation of the quality of DCs used. In the present study, whole human genome microarrays were used to examine alterations in gene expression of monocyte-derived DCs after stimulation with supernatants derived from tumours. Our primary aim was to investigate the possibility of a gene signature for DCs that could be used to forecast responsiveness to tumour stimuli. Results showed that DCs are divided into two groups based on their ability to increase costimulatory markers and to trigger T-cell responses. The gene profiles of the immature DCs from these two groups were distinct, with particular divergence in genes from the interleukin (IL) 8 and thrombospondin-1 hubs. A subpanel of genes was identified, whose signature of expression was capable of predicting DC-stimulatory capacity. Overall, these studies have highlighted a gene-based screen that predicts DC function, which could be used to guide DC-vaccine trials.

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.