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Toll-like receptor 4 engagement drives differentiation of human and murine dendritic cells from a pro- into an anti-inflammatory mode.

Luger R, Valookaran S, Knapp N, Vizzardelli C, Dohnal AM, Felzmann T - PLoS ONE (2013)

Bottom Line: T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels.When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity.We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response.

View Article: PubMed Central - PubMed

Affiliation: St. Anna Children's Cancer Research Institute, Laboratory of Tumor Immunology, Department of Pediatrics, Medical University Vienna, Austria.

ABSTRACT
The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs' initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

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Production of IL-12 and IL-10 on a single-cell level in DCs.(A) Real-Time PCR was used to assess IL-12p35, IL-12p40 and IL-10 mRNA on single cell levels before and after 6 or 12 hours exposure to LPS. All b2m positive sorted single cells were further analyzed for IL-12p35, IL12-p40 and IL-10. Every single cell is depicted (sample ID) for signal (black), no signal (white) and mRNA signal of all three targets (arrow). (B) The summary of 3 independent experiments is depicted as median±SEM. Bars represent the percentage of IL-10 (grey bars), IL-12 (white bars, p35/p40 double positive signals) or IL-10/IL-12 double producing cells (black bars). (C) The concentration of freshly secreted IL-12 (circles), IL-10 (squares), and soluble IL-2RA (triangles) from LPS/IFN-γ activated human DCs is shown in 6 hours intervals after LPS/IFN-γ maturation. At each time point the entire medium was exchanged in order to assess only freshly produced cytokines during the respective time intervals. One representative out of 3 experiments is shown. (D) Intracellular staining of IL-10 and IL-12 protein before and after 6, 24, and 48 hours of exposure to LPS or LPS/IFN-γ. Quadrants are adjusted to the fluorescence of antibody-labeled immature DCs. One representative experiment of 2 is shown. DCs from Donor A and F were used in experiment (A) to (D).
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pone-0054879-g005: Production of IL-12 and IL-10 on a single-cell level in DCs.(A) Real-Time PCR was used to assess IL-12p35, IL-12p40 and IL-10 mRNA on single cell levels before and after 6 or 12 hours exposure to LPS. All b2m positive sorted single cells were further analyzed for IL-12p35, IL12-p40 and IL-10. Every single cell is depicted (sample ID) for signal (black), no signal (white) and mRNA signal of all three targets (arrow). (B) The summary of 3 independent experiments is depicted as median±SEM. Bars represent the percentage of IL-10 (grey bars), IL-12 (white bars, p35/p40 double positive signals) or IL-10/IL-12 double producing cells (black bars). (C) The concentration of freshly secreted IL-12 (circles), IL-10 (squares), and soluble IL-2RA (triangles) from LPS/IFN-γ activated human DCs is shown in 6 hours intervals after LPS/IFN-γ maturation. At each time point the entire medium was exchanged in order to assess only freshly produced cytokines during the respective time intervals. One representative out of 3 experiments is shown. (D) Intracellular staining of IL-10 and IL-12 protein before and after 6, 24, and 48 hours of exposure to LPS or LPS/IFN-γ. Quadrants are adjusted to the fluorescence of antibody-labeled immature DCs. One representative experiment of 2 is shown. DCs from Donor A and F were used in experiment (A) to (D).

Mentions: The sequential secretion of cytokines raised the question of whether they were secreted from the same DC undergoing continuous differentiation; or from distinct DC subpopulations, each of which is characterized by the secretion of different cytokines. In order to address this question we investigated the sequential expression of IL-12 and IL-10 within the same cell using quantitative RT-PCR of flow-sorted single DCs after LPS stimulation. Only 5% of immature DCs exhibited signs of simultaneous IL-12p35, IL-12p40, and IL-10 mRNA expression (figure 5A & B). Six hours after LPS exposure, induction of both IL-12p35 and IL-12p40 were detected in 22% of all single DCs, whereas 48% of all single DCs expressed IL-10 mRNA; 19% of all cells did not express any cytokine-related mRNA. However, 11% of these cells produced both IL-12 and IL-10 mRNA at this time point. After 12 hours of LPS activation, 58% of all single DCs expressed IL-12p35 and IL-12p40, and 72% expressed IL-10 mRNA; 47% of single DCs expressed IL-12 as well as IL-10 mRNA 12 hours after activation. The IL-2 receptor α-chain (CD25) may not only be detected as a DC membrane molecule [26], but was also found in the culture supernatant as sIL-2RA. It became detectable 18 hours after the exposure of DCs to LPS/IFN-γ and reached its plateau expression at 48 hours (figure 5C).


Toll-like receptor 4 engagement drives differentiation of human and murine dendritic cells from a pro- into an anti-inflammatory mode.

Luger R, Valookaran S, Knapp N, Vizzardelli C, Dohnal AM, Felzmann T - PLoS ONE (2013)

Production of IL-12 and IL-10 on a single-cell level in DCs.(A) Real-Time PCR was used to assess IL-12p35, IL-12p40 and IL-10 mRNA on single cell levels before and after 6 or 12 hours exposure to LPS. All b2m positive sorted single cells were further analyzed for IL-12p35, IL12-p40 and IL-10. Every single cell is depicted (sample ID) for signal (black), no signal (white) and mRNA signal of all three targets (arrow). (B) The summary of 3 independent experiments is depicted as median±SEM. Bars represent the percentage of IL-10 (grey bars), IL-12 (white bars, p35/p40 double positive signals) or IL-10/IL-12 double producing cells (black bars). (C) The concentration of freshly secreted IL-12 (circles), IL-10 (squares), and soluble IL-2RA (triangles) from LPS/IFN-γ activated human DCs is shown in 6 hours intervals after LPS/IFN-γ maturation. At each time point the entire medium was exchanged in order to assess only freshly produced cytokines during the respective time intervals. One representative out of 3 experiments is shown. (D) Intracellular staining of IL-10 and IL-12 protein before and after 6, 24, and 48 hours of exposure to LPS or LPS/IFN-γ. Quadrants are adjusted to the fluorescence of antibody-labeled immature DCs. One representative experiment of 2 is shown. DCs from Donor A and F were used in experiment (A) to (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569454&req=5

pone-0054879-g005: Production of IL-12 and IL-10 on a single-cell level in DCs.(A) Real-Time PCR was used to assess IL-12p35, IL-12p40 and IL-10 mRNA on single cell levels before and after 6 or 12 hours exposure to LPS. All b2m positive sorted single cells were further analyzed for IL-12p35, IL12-p40 and IL-10. Every single cell is depicted (sample ID) for signal (black), no signal (white) and mRNA signal of all three targets (arrow). (B) The summary of 3 independent experiments is depicted as median±SEM. Bars represent the percentage of IL-10 (grey bars), IL-12 (white bars, p35/p40 double positive signals) or IL-10/IL-12 double producing cells (black bars). (C) The concentration of freshly secreted IL-12 (circles), IL-10 (squares), and soluble IL-2RA (triangles) from LPS/IFN-γ activated human DCs is shown in 6 hours intervals after LPS/IFN-γ maturation. At each time point the entire medium was exchanged in order to assess only freshly produced cytokines during the respective time intervals. One representative out of 3 experiments is shown. (D) Intracellular staining of IL-10 and IL-12 protein before and after 6, 24, and 48 hours of exposure to LPS or LPS/IFN-γ. Quadrants are adjusted to the fluorescence of antibody-labeled immature DCs. One representative experiment of 2 is shown. DCs from Donor A and F were used in experiment (A) to (D).
Mentions: The sequential secretion of cytokines raised the question of whether they were secreted from the same DC undergoing continuous differentiation; or from distinct DC subpopulations, each of which is characterized by the secretion of different cytokines. In order to address this question we investigated the sequential expression of IL-12 and IL-10 within the same cell using quantitative RT-PCR of flow-sorted single DCs after LPS stimulation. Only 5% of immature DCs exhibited signs of simultaneous IL-12p35, IL-12p40, and IL-10 mRNA expression (figure 5A & B). Six hours after LPS exposure, induction of both IL-12p35 and IL-12p40 were detected in 22% of all single DCs, whereas 48% of all single DCs expressed IL-10 mRNA; 19% of all cells did not express any cytokine-related mRNA. However, 11% of these cells produced both IL-12 and IL-10 mRNA at this time point. After 12 hours of LPS activation, 58% of all single DCs expressed IL-12p35 and IL-12p40, and 72% expressed IL-10 mRNA; 47% of single DCs expressed IL-12 as well as IL-10 mRNA 12 hours after activation. The IL-2 receptor α-chain (CD25) may not only be detected as a DC membrane molecule [26], but was also found in the culture supernatant as sIL-2RA. It became detectable 18 hours after the exposure of DCs to LPS/IFN-γ and reached its plateau expression at 48 hours (figure 5C).

Bottom Line: T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels.When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity.We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response.

View Article: PubMed Central - PubMed

Affiliation: St. Anna Children's Cancer Research Institute, Laboratory of Tumor Immunology, Department of Pediatrics, Medical University Vienna, Austria.

ABSTRACT
The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs' initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

Show MeSH
Related in: MedlinePlus