IL-12p70 Secretion Research Articles

Anin VitroEffect of Coffee on the Antigen-Specific Immune Responses of Naïve Splenocytes

Coffee is a globally consumed beverage with potential health benefits. However, there are few reports about the effects of coffee on immunological functions. We previously reported that in an allergic mouse model, coffee intake prevented allergy development through augmentation of interleukin (IL)-12p40. In order to investigate the anti-allergic activity of coffee, we examined the effect of coffee on antigen (Ag)-specific responses of immune cells in vitro. Coffee treatment suppressed proliferation and IL-2 secretion of mouse splenocytes in the same way as splenocytes from mice administered coffee orally. However, IL-12p40 secretion decreased significantly as a result of in vitro coffee treatment, which was contrary to the results obtained from experiments of mice administered coffee orally. Therefore, modification associated with oral administration might influence the anti-allergic activity of coffee.

Bronchial Epithelial Cell-Derived Prostaglandin E2 Dampens the Reactivity of Dendritic Cells

Airway epithelial cells regulate immune reactivity of local dendritic cells (DCs), thus contributing to microenvironment homeostasis. In this study, we set out to identify factors that mediate this regulatory interaction. We show that tracheal epithelial cells secrete soluble factors that downregulate TNF-α and IL-12p40 secretion by bone marrow-derived DCs but upregulate IL-10 and arginase-1. Size exclusion chromatography identified small secreted molecules having high modulatory activity on DCs. We observed that airway tracheal epithelial cells constitutively release the lipid mediator PGE(2). Blocking the synthesis of PGs within airway epithelial cells relieved DCs from inhibition. Cyclooxygenase-2 was found to be expressed in primary tracheal epithelial cell cultures in vitro and in vivo as shown by microdissection of epithelial cells followed by real-time PCR. Paralleling these findings we observed that DCs treated with an antagonist for E-prostanoid 4 receptor as well as DCs lacking E-prostanoid 4 receptor showed reduced inhibition by airway epithelial cells with respect to secretion of proinflammatory cytokines measured by ELISA. Furthermore, PGE(2) mimicked the effects of epithelial cells on DCs. The results indicate that airway epithelial cell-derived PGE(2) contributes to the modulation of DCs under homeostatic conditions.

Ethanol extract from birch bark (Betula pubescens) suppresses human dendritic cell mediated Th1 responses and directs it towards a Th17 regulatory response in vitro

Extracts and fractions from birch bark have been used to treat various diseases, such as skin disorders and rheumatism, and for analgesic effects. Results from studies in vitro and in vivo have shown that birch bark extracts can have immunoregulatory effects. These effects have mainly been attributed to the various triterpenes found in birch bark. The effects of birch bark from Betula pubescens on immune responses have not been reported. Ethanol extract was prepared from dry birch bark (DBBEE) and five fractions made using various ratios of dichloromethane and methanol (fractions I-V). Human monocyte-derived dendritic cells (DCs) were matured with or without DBBEE or fractions I–V at several concentrations. The effects of the extract and fractions on DC maturation were determined by measuring cytokine secretion by ELISA and expression of surface molecules by flow cytometry. DBBEE and fractions III and IV reduced DC secretion of IL-6, IL-10 and IL-12p40 and expression of CD83, CD86, CCR7 and DC-SIGN compared with control DCs. Proliferation of allogeneic CD4+ T cells co-cultured with DCs matured with fraction IV, as measured by 3H-thymidine incorporation, was similar to proliferation of allogeneic CD4+ T cells co-cultured with control DCs. However, IFN-γ secretion was reduced and IL-10 and IL-17 secretion was increased, a cytokine profile consistent with a Th17 regulatory phenotype. These results indicate that bark from Betula pubescens contains compound(s) that can modulate DCs so that their interaction with T cells leads to an immunoregulatory response.

Maturation of monocyte derived dendritic cells with OK432 boosts IL-12p70 secretion and conveys strong T-cell responses

BackgroundDesign of tumour specific immunotherapies using the patients' own dendritic cells (DC) is a fast advancing scientific field. The functional qualities of the DC generated in vitro are critical, and today's gold standard for maturation is a cytokine cocktail consisting of IL-1β, IL-6, TNF-α and PGE2 generating cells lacking IL-12p70 production. OK432 is an immunotherapeutic agent derived from killed Streptococcus pyogenes that has been used clinically to treat malignant and benign neoplasms for decades.MethodsIn this study, we analysed the effects of OK432 on DC maturation, DC migration, cytokine and chemokine secretion as well as T-cell stimulatory capacity, and compared it to the cytokine cocktail alone and combinations of OK432 with the cytokine cocktail.ResultsOK432 induced a marked up-regulation of CD40 on the cell surface as well as a strong inflammatory response from the DC with significantly more secretion of 19 different cytokines and chemokines compared to the cytokine cocktail. Interestingly, secretion of IL-15 and IL-12p70 was detected at high concentrations after maturation of DC with OK432. However, the OK432 treated DC did not migrate as well as DC treated with cytokine cocktail in a transwell migration assay. During allogeneic T-cell stimulation OK432 treated DC induced proliferation of over 50 percent of CD4 and 30 percent of CD8 T-cells for more than two cell divisions, whereas cytokine cocktail treated DC induced proliferation of 12 and 11 percent of CD4 and CD8 T-cells, respectively.ConclusionsThe clinically approved compound OK432 has interesting properties that warrants its use in DC immunotherapy and should be considered as a potential immunomodulating agent in cancer immunotherapy.

Lys-des[Arg9]–Bradykinin Alters Migration and Production of Interleukin-12 in Monocyte-Derived Dendritic Cells

This study tested the hypothesis that proinflammatory kinin peptides are involved in modulating human dendritic cell (DC) function. Inflammation is accompanied by an increased maturation of DCs and the generation of kinins, particularly Lys-des[Arg(9)]-bradykinin (Lda-BK). We assessed the role of Lda-BK in the activation and migration of human monocyte-derived DCs (hMo-DCs) matured through the use of LPS, TNF-α + IL-1β, or CD40 ligand. Kinin B(1) and B(2) receptor mRNA and protein expression were assessed by confocal microscopy, flow cytometry, and RT-PCR. The effects of Lda-BK on the migration of mature hMo-DCs were assessed in Transwell chambers, whereas the expression of costimulatory molecules and the secretion of IL-12 were assessed by flow cytometry and ELISA, respectively. The expression of the kinin B(1) receptor (B(1)R) was down-regulated during the maturation of hMo-DCs, whereas the expression of B(2)R was unchanged. The B(1)R agonist Lda-BK was not chemotactic for hMo-DCs matured using LPS, TNF-α + IL-1β, or CD40 ligand, but Lda-BK enhanced the secretion of IL-12p70 and inhibited the secretion of IL-12p40 by mature hMo-DCs. However, the exposure of hMo-DCs matured with TNF-α + IL-1β to Lda-BK for 6 hours decreased subsequent migration in response to Lda-BK, the chemokine CCL19, or Lda-BK combined with CCL19. The expression of B(1)R was increased in hMo-DCs from subjects with asthma compared with subjects without asthma, in keeping with a tendency toward increased in vitro migration of asthmatic hMo-DCs in response to Lda-BK. The increased formation of Lda-BK and the enhanced expression of B(1)R as a consequence of inflammation may alter the migration of mature, antigen-laden DCs to regional lymph nodes in response to CCL19, may modulate the secretion of cytokines by these DCs, and may contribute to the accumulation of mature DCs in the lungs of patients with asthma.

Bifidobacterium animalis AHC7 protects against pathogen-induced NF-κB activation in vivo

BackgroundBifidobacteria and lactobacilli are among the early and important colonizers of the gastrointestinal tract and are generally considered to be part of a normal, healthy microbiota. It is believed that specific strains within the microbiota can influence host immune-reactivity and may play a role in protection from infection and aberrant inflammatory activity. One such strain, Bifidobacterium animalis AHC7, has been previously shown to protect against Salmonella typhimurium infection in mice and helps resolve acute idiopathic diarrhea in dogs. The aim of this study was to investigate the potential molecular and cellular mechanisms underpinning the Bifidobacterium animalis AHC7 protective effect.ResultsFollowing 4 hours of infection with Salmonella typhimurium, NF-κB activation was significantly elevated in vivo in placebo and Enterococcus faecium-fed animals while Bifidobacterium animalis AHC7 consumption significantly attenuated the NF-κB response. In vitro anti-CD3/CD28 stimulated Peyer's patch cells secreted significantly less TNF-α and IFN-γ following Bifidobacterium animalis AHC7 consumption. Stimulated cells released more IL-12p70 but this difference did not reach statistical significance. No alteration in mucosal IL-6, IL-10 or MCP-1 levels were observed. No statistically significant change in the cytokine profile of mesenteric lymph node cells was noted. In vitro, Bifidobacterium animalis AHC7 was bound by dendritic cells and induced secretion of both IL-10 and IL-12p70. In addition, co-culture of CD4+ T cells with Bifidobacterium animalis AHC7-stimulated dendritic cells resulted in a significant increase in CD25+Foxp3+ T cell numbers.ConclusionBifidobacterium animalis AHC7 exerts an anti-inflammatory effect via the attenuation of pro-inflammatory transcription factor activation in response to an infectious insult associated with modulation of pro-inflammatory cytokine production within the mucosa. The cellular mechanism underpinning Bifidobacterium animalis AHC7 mediated attenuation of NF-κB activation may include recognition of the bacterium by dendritic cells and induction of CD25+Foxp3+ T cells.

Perifosine Affects Phenotype and Function of Human Myeloid Dendritic Cells

AbstractAbstract 3911Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase III clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. Dendritic cells (DCs) play a crucial role in immune system via mediating antigen-specific immune responses as well as regulating the innate and adaptive immunity through secreted cytokines. In this study, effect of perifosine on phenotype, antigen uptake, processing and presentation, and cytokine production on human monocyte-derived dendritic cells (DCs) were evaluated at clinically relevant concentrations (2.5μM to 10μM). The effect of perifosine on survival of DCs was tested by annexin V and PI staining. We observed that up to 48 hours of perifosine treatment had no effect on viability of both immature DCs and DCs during maturation by LPS (100ng/ml) or poly(IC) (25μg/ml) (>90%). Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40), antigen presentation (HLA-ABC) and maturation (CD83) on both immature and mature DCs at 24 and 48 hours. We next evaluated whether perifosine affected the antigen uptake capacity by immature DCs, using various sizes and forms of antigens. Compared to control, 24 hours-perifosine (10μM) treatment significantly reduced uptake capacity of both protein antigens (Alexa Fluor 488-conjugated 45-KDa protein A and 20-KDa protein G) and polysaccharide antigen (Alexa Fluor 488-conjugated 40-KD dextran) by immature DCs. Importantly, we systematically investigated the impact of perifosine on DCs involved in both classic (MHC-mediated) and non-classic (CD1d-mediated) antigen specific immune responses and report a significant impairment upon perifosine treatment. Perifosine inhibited DC-mediated T cell activation. Following 24 hours treatment, control or perifosine (10μM)-treated immature DCs were pulsed with tetanus toxoid (0.5μg/ml) overnight in the presence of LPS, and then used to stimulate autologous T cells. T cell response was inhibited as evidenced by significantly reduced IFN-gamma production detected by ELISA (control vs treatment = 15106 pg/ml vs 8332 pg/ml). We also confirmed that perifosine pretreatment of freshly isolated blood myeloid DCs led to significant inhibition of allogenic T cell immune responses (control vs treatment = 982 pg/ml vs 605 pg/ml). In addtion to presenting peptide antigens, DCs also present glycolipids to activate invariant NKT (iNKT) cells. Our data demonstrated that perifosine impairs DCs-mediated iNKT cell activation. The production of both Th1-type cytokines and Th2-type cytokines by iNKT cells were significantly repressed upon perifosine (10μM)-treated alpha-GalCer-pulsing DCs, compared to control (IFN-gamma production (control vs treatment= 11631 pg/ml vs 6768 pg/ml) and IL-4 production (control vs treatment= 1285 pg/ml vs 783 pg/ml). Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (control vs treatment = 192 pg/ml vs 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM)), as well as inhibited IL-10 production (control vs treatment = 472 pg/ml vs 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM)). Mechanism study revealed that RelB, a component of NF-kappaB signaling pathway, was downregulated in immature DCs by 18 hours of perifosine treatment (10μM). We have also evaluated the effect of perifosine on the expression of IDO, a repressor of DCs activation. However, we did not observe the significant alteration of perifosine treatment compared to the control. In summary, our preclinical data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses, indicating a need to monitor immune functions in patients under the Akt inhibitor treatment. Disclosures:No relevant conflicts of interest to declare.

Human mesenchymal stem cells and renal tubular epithelial cells differentially influence monocyte-derived dendritic cell differentiation and maturation

Mesenchymal stem cells (MSCs) possess immunosuppressive properties. But also fully differentiated human renal tubular epithelial cells (RTECs) are able to modulate T-cell proliferation in vitro. In this study we compared two MSC populations, human adipose derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs), and RTECs regarding their potential to inhibit monocyte-derived dendritic cell (DC) differentiation and maturation in indirect co-culture. In the presence of hAMSCs and RTECs, monocytes stimulated to undergo DC differentiation were inhibited to acquire surface phenotype of immature and mature DCs. In contrast, ASCs showed only limited suppressive capacity. Secretion of IL-12p70 was suppressed in hAMSC co-cultures and high IL-10 levels were detected in all co-cultures. Prostaglandin E 2 was found in ASC and hAMSC co-cultures, whereas soluble human leukocyte antigen-G was highly elevated only in RTEC co-cultures. Thus, inhibition of DC generation by MSCs and RTECs might be mediated by different soluble factors.

Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies

BackgroundPegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.MethodsTwelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. In vitro-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.ResultsPegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated in vitro after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.ConclusionsPegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.

Surface modification of poly(D,L‐lactic‐co‐glycolic acid) nanoparticles with protamine enhanced cross‐presentation of encapsulated ovalbumin by bone marrow‐derived dendritic cells

Cross-presentation is the key process in stimulation of cytotoxic T lymphocyte (CTL) immune response in eliminating many infectious diseases and tumors. Previous studies have shown that surface modification of poly(D,L-lactic-co-glycolic acid) (PLGA) particles with polycations enhanced their adjuvant ability resulting in a strong antibody response to the encapsulated antigen. However, the in vitro cross-presentation by protamine-coated PLGA nanoparticles (NPs) has not been addressed yet. In this study, a model antigen ovalbumin (OVA) was encapsulated into PLGA nanoparticles, with (OVA-NPs/protamine) or without protamine coating (OVA-NPs). These nanoparticles were then used to stimulate murine bone marrow-derived dendritic cells (BMDCs). Flow cytometry analysis revealed an increase in endocytosis of protamine-coated PLGA nanoparticles by BMDCs at 37°C. Compared with OVA-NPs-treated BMDCs, stimulation with OVA-NPs/protamine led to significantly upregulation of CD80, CD86, and CD83, increased secretion of IL-12p70, and decreased production of IL-4 by BMDCs. Furthermore, OVA-NPs/protamine-treated BMDCs also showed an enhanced cross-presentation to B3Z T cell hybridoma in vitro. Transmission electron microscopy (TEM) study showed that protamine-coated PLGA nanoparticles escaped from lysosomes through the interaction with lysosomal membrane. These results demonstrated that protamine-coated PLGA nanoparticles could enhance the cross-presentation of encapsulated exogenous antigen by facilitating antigen uptake and lysosomal escape, suggesting the feasibility to be a potent adjuvant for cellular vaccines.

Vitamin C-treated murine bone marrow-derived dendritic cells preferentially drive naïve T cells into Th1 cells by increased IL-12 secretions

Vitamin C has been reported to shift immune responses toward Th1. In this study, we evaluated whether this effect was by way of dendritic cells. Murine dendritic cells (DCs) were prepared from bone marrow precursors. DCs treated with vitamin C secreted an increased amount of IL-12p70 after activation with LPS. These cells rendered naïve T cells to secrete more Th1 cytokine, IFN-γ, and less Th2-cytokine, IL-5 in the culture supernatants. Vitamin C-treatment also increased phosphorylation of p38 and ERK1/2 in DCs. p38 inhibitor in culture media suppressed the effect of vitamin C to elevate IL-12p70 secretion. In contrast, ERK inhibitor elevated IL-12p70 secretion. In summary, vitamin C taken up into DCs increased IL-12p70 secretion of these cells by modulating the activation of signal molecules, and thus shifted immune responses toward Th1. These data provide us a new insight on the role of vitamin C in modulating immune responses.

Viral Inhibitory Peptide of TLR4, a Peptide Derived from Vaccinia Protein A46, Specifically Inhibits TLR4 by Directly Targeting MyD88 Adaptor-Like and TRIF-Related Adaptor Molecule

TLRs are critical pattern recognition receptors that recognize bacterial and viral pathogen-associated molecular patterns leading to innate and adaptive immune responses. TLRs signal via homotypic interactions between their cytoplasmic Toll/IL-1R (TIR) domains and TIR domain-containing adaptor proteins. Over the course of evolution, viruses have developed various immune evasion strategies, one of which involves inhibiting TLR signaling pathways to avoid immune detection. Thus, vaccinia virus encodes the A46 protein, which binds to multiple TIR-domain containing proteins, ultimately preventing TLRs from signaling. We have identified an 11-aa-long peptide from A46 (termed viral inhibitor peptide of TLR4, or VIPER), which, when fused to a cell-penetrating delivery sequence, potently inhibits TLR4-mediated responses. VIPER was TLR4 specific, being inert toward other TLR pathways, and was active in murine and human cells and in vivo, where it inhibited LPS-induced IL-12p40 secretion. VIPER also prevented TLR4-mediated MAPK and transcription factor activation, suggesting it acted close to the TLR4 complex. Indeed, VIPER directly interacted with the TLR4 adaptor proteins MyD88 adaptor-like (Mal) and TRIF-related adaptor molecule (TRAM). Viral proteins target host proteins using evolutionary optimized binding surfaces. Thus, VIPER possibly represents a surface domain of A46 that specifically inhibits TLR4 by masking critical binding sites on Mal and TRAM. Apart from its potential therapeutic and experimental use in suppressing TLR4 function, identification of VIPER's specific binding sites on TRAM and Mal may reveal novel therapeutic target sites. Overall, we demonstrate for the first time disruption of a specific TLR signaling pathway by a short virally derived peptide.

TLR1/2, TLR7, and TLR9 Signals Directly Activate Human Peripheral Blood Naive and Memory B Cell Subsets to Produce Cytokines, Chemokines, and Hematopoietic Growth Factors

Recently, it has been reported that using multiple signals, murine and human B cells secrete several cytokines with pro-inflammatory and immunoregulatory properties. We present the first comprehensive analysis of 24 cytokines, chemokines, and hematopoietic growth factors production by purified human peripheral blood B cells (CD19+), and naive (CD19+CD27-) and memory (CD19+CD27+) B cells in response to direct and exclusive signaling provided by toll-like receptor (TLR) ligands Pam3CSK (TLR1/TLR2), Imiquimod (TLR7), and GpG-ODN2006 (TLR9). All three TLR ligands stimulated B cells (CD19+) to produce cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-13, and IL-10, and chemokines MIP-1α, MIP-1β, MCP-1, IP-10, and IL-8. However, GM-CSF and G-CSF production was predominantly induced by TLR2 agonist. Most cytokines/chemokines/hematopoietic growth factors were predominantly or exclusively produced by memory B cells, and in general, TLR2 signal was more powerful than signal provided viaTLR7 and TLR9. No significant secretion of eotaxin, IFN-α, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-7, IL-15, IL-17, IL-12p40, IL-12p70, and TNF-β (lymphotoxin) was observed. These data demonstrate that human B cells can be directly activated viaTLR1/TLR2, TLR7, and TLR9 to induce secretion of cytokines, chemokines, and hematopoietic growth factors and suggest a role of B cells in immune response against microbial pathogenesis and immune homeostasis.

Immunomodulating effects of extracts from Icelandic marine invertebrates

Iceland has a unique position in the North Atlantic, with marine biodiversity largely unexplored with respect to chemical constituents. In particular, the confluence of cold-water currents from Atlantic gyres and warm-water geothermal activity accounts for a unique marine biogeography that has never been evaluated for the potential of marine natural product diversity. The aim of this project is to investigate the immunomodulating effects of extracts and fractions obtained from marine invertebrates collected around Iceland. As dendritic cells play an important role as a bridge between the innate and adaptive immune response the effects of extracts from marine organisms were tested by analysing the cytokine secretion and expression of surface molecules on human monocyte-derived dendritic cells. Sixty extracts were prepared by solvent extraction [dichloromethane/methanol (1:1)] and screened for immunomodulating effects. Four extracts at the concentration 100µg/mL were shown to be cytotoxic and seven reduced both IL-12p40 and IL-10 secretion and CD86 expression. An active extract of the sponge, Isodictya palmata, was further fractionized using modified Kupchan partition and the chloroform and hexane fractions were shown to be active in the dendritic cell model. Further fractionation and characterization is in progress.

Type‐1 polarized dendritic cells loaded with apoptotic prostate cancer cells are potent inducers of CD8+ T cells against prostate cancer cells and defined prostate cancer‐specific epitopes

In order to develop improved vaccines for patients with recurrent prostate cancer (PCa), we tested the feasibility of using type-1 polarized dendritic cells (αDC1s) to cross-present antigens from allogeneic PCa cells and to induce functional CD8(+) T cell responses against PCa cells and against defined MHC class I-restricted PCa-relevant epitopes. Monocyte-derived DCs from PCa patients were matured using the "standard" cytokine cocktail (IL-1β/TNFα/IL-6/PGE₂) or using the αDC1-polarizing cocktail (IL-1β/TNFα/IFNα/IFNγ/poly-I:C), loaded with UV-irradiated LNCaP cells, and used to sensitize autologous CD8(+) T cells. αDC1s from PCa patients secreted 10-30 times higher levels of IL-12p70 than sDCs. Importantly this elevated capacity for IL-12p70 secretion was not inhibited by loading with apoptotic tumor cells. Compared to standard DCs, αDC1s induced higher numbers of CD8(+) T cells capable of recognizing both the original PCa cells as well as another PCa cell line, DU145, in MHC class I-restricted fashion. Furthermore, αDC1s induced higher numbers of CD8(+) T cells recognizing defined PCa-specific class I-restricted peptide epitopes of prostate-specific antigen and prostatic acid phosphatase: PAP(135-143) (average 49-fold higher), PAP(112-120) (average 24-fold), PSA(141-150) (average 5.5-fold), and PSA(146-154) (average 11-fold). Type-1 polarization of GM-CSF/IL-4-generated DCs enhances their ability to present allogeneic tumor cells and to induce CD8(+) T cells recognizing different PCa cells and multiple defined PCa-specific epitopes. These observations help to develop improved immunotherapies of PCa for patients with different HLA types and lacking autologous tumor material.

In vitro assessment of immunomodulating activity of the two Lactobacillus strains isolated from traditional fermented milk

The objective of this study was to evaluate the in vitro immunomodulating capacity of Lactobacilluscoryniformis subsp torquens T3L (L.coryniformis T3L) isolated from traditional fermented yak’s milk in Tibet, China, and Lactobacillus paracasei supsp. paracasei M5L (L. paracasei M5L)isolated from kumiss in Sinkiang, China was used as control. The effects of live bacteria, cell wall and genomic DNA of the two Lactobacillus strains on human peripheral blood mononuclear cells (PBMCs) proliferation, production of interleukin-12 (IL-12 p70), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and natural killer (NK) cell activity were assessed. The live bacteria, cell wall and genomic DNA of the two lactobacilli exerted proliferative effects on PBMCs. Live bacteria at 1 × 106 c.f.u. ml−1, cell wall at 20 μg protein ml−1 and DNA at 50 μg DNA ml−1 of the strainS induced the secretion of IL-12 (p70), IFN-γ and TNF-α by PBMCs. NK cell activities increased after cultivation of PBMCs with live bacteria, cell wall and DNA of the strains. Overall, these results demonstrate that the live bacteria, cell wall and genomic DNA of the two Lactobacillus strains exhibit immunomodulating activity.

Iloprost has potent anti‐inflammatory properties on human monocyte‐derived dendritic cells

The stable prostaglandin I2 analogue (iloprost) iloprost has been shown to inhibit allergic airway inflammation in mice by modulating the function of myeloid dendritic cells (DCs). The aim of the current study was to investigate the biological activity of iloprost on human monocyte-derived DCs. I prostanoid (IP) receptor expression was analysed by RT-PCR. Cytokine secretion by DCs and CD4+ T cells was measured by ELISA. The expression of the transcription factor FoxP3 after co-culture of DCs with CD4+ CD45RA+ T cells was analysed by flow cytometry. Human monocyte-derived DCs were found to express mRNA specific for the PGI2 receptor IP, and stimulation with iloprost resulted in increased cyclic AMP levels in both immature DCs (iDCs) and mature DCs (mDCs). Moreover, iloprost dose dependently inhibited the secretion of TNF-alpha, IL-6, IL-8 and IL-12p70 in mDCs, while it enhanced IL-10 production. Changes in cytokine secretion were paralleled by an altered T-cell priming capacity of DCs: in co-culture experiments of iloprost-treated mDC and naïve CD45RA+ T cells, an induction of regulatory T cells could be observed, as demonstrated by increased intracellular FoxP3 expression and IL-10 production. Additionally, iloprost inhibited the MIP-3beta-induced migration of mDCs. In summary, our results provide evidence that iloprost profoundly affects the function of human myeloid DCs. Therefore, iloprost might also be a new therapeutical option for the treatment of asthma in humans.

Mechanisms of IL-12 Synthesis by Human Dendritic Cells Treated with the Chemical Sensitizer NiSO4

Allergic contact dermatitis, caused by metallic ions, is a T cell-mediated inflammatory skin disease. IL-12 is a 70-kDa heterodimeric protein composed of IL-12p40 and IL-12p35, playing a major role in the generation of allergen-specific T cell responses. Dendritic cells (DCs) are APCs involved in the induction of primary immune responses, as they possess the ability to stimulate naive T cells. In this study, we address the question whether the sensitizer nickel sulfate (NiSO(4)) itself or in synergy with other signals can induce the secretion of IL-12p70 in human monocyte-derived DCs (Mo-DCs). We found that IL-12p40 was produced by Mo-DC in response to NiSO(4) stimulation. Addition of IFN-gamma concomitantly to NiSO(4) leads to IL-12p70 synthesis. NiSO(4) treatment leads to the activation of MAPK, NF-kappaB pathways, and IFN regulatory factor 1 (IRF-1). We investigated the role of these signaling pathways in IL-12 production using known pharmacological inhibitors of MAPK and NF-kappaB pathways and RNA interference-mediated silencing of IRF-1. Our results showed that p38 MAPK, NF-kappaB, and IRF-1 were involved in IL-12p40 production induced by NiSO(4). Moreover, IRF-1 silencing nearly totally abrogated IL-12p40 and IL-12p70 production provoked by NiSO(4) and IFN-gamma. In response to NiSO(4), we observed that STAT-1 was phosphorylated on both serine and tyrosine residues and participated to NiSO(4)-induced IRF-1 activation. N-acetylcysteine abolished STAT-1 phosphorylation, suggesting that STAT-1 activation may be dependent on NiSO(4)-induced alteration of the redox status of the cell. These results indicate that p38 MAPK, NF-kappaB, and IRF-1 are activated by NiSO(4) in Mo-DC and cooperate for IL-12 production.

MTOR and GSK-3 shape the CD4+ T-cell stimulatory and differentiation capacity of myeloid DCs after exposure to LPS

AbstractProlonged inhibition of the kinase, mammalian target of rapamycin (mTOR), during myeloid dendritic cell (DC) generation confers resistance to maturation. Recently, however, mTOR inhibition immediately before Toll-like receptor ligation has been found to exert proinflammatory effects on myeloid cells, notably enhanced IL-12p40/p70 production. We show, for the first time, that mouse or human DCs generated under mTOR inhibition exhibit markedly enhanced IL-12p70 production after lipopolysaccharide (LPS) stimulation, despite impaired costimulatory molecule expression and poor T-cell stimulatory ability. Consistent with this finding, we reveal that increased IL-12p40 production occurs predominantly in CD86lo immature DCs. High IL-12p40/p70 production by CD86lo DC resulted from failed down-regulation of glycogen synthase kinase-3 (GSK-3) activity and could not be ascribed to enhanced Akt function. Despite high IL-12p70 secretion, rapamycin-conditioned, LPS-stimulated DCs remained poor T-cell stimulators, failing to enhance allogeneic Th1 cell responses. We also report that inhibition of GSK-3 impedes the ability of LPS-stimulated DCs to induce forkhead box p3 in CD4+CD25− T cells, as does the absence of IL-12p40/p70. Thus, GSK-3 activity in DC is regulated via signaling linked to mTOR and modulates their capacity both to produce IL-12p40/p70 and induce forkhead box p3 in CD4+ T cells under inflammatory conditions.

NALP3 is not necessary for early protection against experimental tuberculosis

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1β and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1α, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.