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NF-κB, p38 MAPK, ERK1/2, mTOR, STAT3 and increased glycolysis regulate stability of paricalcitol/dexamethasone-generated tolerogenic dendritic cells in the inflammatory environment.

Dáňová K, Klapetková A, Kayserová J, Šedivá A, Špíšek R, Jelínková LP - Oncotarget (2015)

Bottom Line: However, the signaling pathways regulating their stable tolerogenic properties are largely unknown.In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-β, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells.In the inflammatory environment, tDCs maintained up-regulated indoleamine 2, 3 dioxygenase but abrogated IκB-α phosphorylation and reduced transcriptional activity of p65/RelA, RelB and c-Rel NF-κB subunits except p50.

View Article: PubMed Central - PubMed

Affiliation: Sotio a.s., Prague, Czech Republic.

ABSTRACT
Tolerogenic dendritic cells (tDCs) may offer an intervention therapy in autoimmune diseases or transplantation. Stable immaturity and tolerogenic function of tDCs after encountering inflammatory environment are prerequisite for positive outcome of immunotherapy. However, the signaling pathways regulating their stable tolerogenic properties are largely unknown. In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-β, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells. In the inflammatory environment, tDCs maintained up-regulated indoleamine 2, 3 dioxygenase but abrogated IκB-α phosphorylation and reduced transcriptional activity of p65/RelA, RelB and c-Rel NF-κB subunits except p50. Mechanistically, p38 MAPK, ERK1/2, mTOR, STAT3 and mTOR-dependent glycolysis regulated expression of ILT-3, PDL-1 and CD86, secretion of IL-10 and T cell stimulatory capacity of tDCs in the inflammatory environment. Stability of tDCs in the inflammatory environment is thus regulated by multiple signaling pathways.

No MeSH data available.


Related in: MedlinePlus

Dex/VitD tDCs maintain reduced T cell stimulatory capacity after restimulationDCs were differentiated in Cell Gro in presence (tDCs) or absence of Dex and VitD2 (cDCs) and activated with MPLA (CG). Then, DCs were washed, recultured in complete RPMI without tolerising factors and treated with cytokine cocktail (CC) described in Figure 1B or LPS (1 μg/ml) or polyI:C (25 μg/ml) or CD40L (1000 ng/ml) or they were left unstimulated (RPMI). After 24 h, tDCs and cDCs were washed and incubated with allogeneic T cells at 1:10 ratio (DCs/T cells). A. Proliferation of T cells was assessed on day 6 by CFSE dilution method. Percentages of proliferating T cells and representative histograms are shown. B. Production of IL-17A in DCs/T cell co-cultures was analyzed by ELISA on day 6. C. Percentages of IFN-γ producing T and D. IL-10 producing T cells was assessed on day 6 or day 9, respectively. Representative dot plots are shown. Data represent mean ± SEM for at least 3 independent experiments and at least 10 donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (paired t-test).
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Figure 2: Dex/VitD tDCs maintain reduced T cell stimulatory capacity after restimulationDCs were differentiated in Cell Gro in presence (tDCs) or absence of Dex and VitD2 (cDCs) and activated with MPLA (CG). Then, DCs were washed, recultured in complete RPMI without tolerising factors and treated with cytokine cocktail (CC) described in Figure 1B or LPS (1 μg/ml) or polyI:C (25 μg/ml) or CD40L (1000 ng/ml) or they were left unstimulated (RPMI). After 24 h, tDCs and cDCs were washed and incubated with allogeneic T cells at 1:10 ratio (DCs/T cells). A. Proliferation of T cells was assessed on day 6 by CFSE dilution method. Percentages of proliferating T cells and representative histograms are shown. B. Production of IL-17A in DCs/T cell co-cultures was analyzed by ELISA on day 6. C. Percentages of IFN-γ producing T and D. IL-10 producing T cells was assessed on day 6 or day 9, respectively. Representative dot plots are shown. Data represent mean ± SEM for at least 3 independent experiments and at least 10 donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (paired t-test).

Mentions: TDCs or cDCs were cultured with allogeneic T cells at a ratio of 1:10. TDCs were weaker inducers of CD4+ as well as CD8+ T cell proliferation, even after the restimulation, irrespective of the maturation agent when compared to cDCs (Figure 2A). In line with this, tDCs induced low IL-17A production by allogeneic T cells even after restimulation in contrast to cDCs that were potent inducers of IL-17A by T cells especially after CC and LPS stimulation (Figure 2B). Moreover, co-incubation of allogeneic T cells with tDCs cultured in Cell Gro skewed the T cell cytokine profile towards reduced IFN-γ and significantly increased IL-10 production by CD4+ as well as CD8+ T cells, in comparison to cDCs (Figure 2C, 2D). In addition, co-incubation of T cells with tDCs restimulated with CC, LPS, polyI:C and CD40L led to marked reduction of CD4+ IFN-γ producing T cells together with stable numbers of CD4+ IL-10 producing T cells. The percentage of CD8+ IFN-γ producing T cells remained stable or slightly decreased after CC and CD40L restimulation of tDCs, while the amount of CD8+ IL-10 producing T cells remained almost the same after restimulation of tDCs with LPS and slightly decreased after restimulation of tDCs with CC, polyI:C and CD40L.


NF-κB, p38 MAPK, ERK1/2, mTOR, STAT3 and increased glycolysis regulate stability of paricalcitol/dexamethasone-generated tolerogenic dendritic cells in the inflammatory environment.

Dáňová K, Klapetková A, Kayserová J, Šedivá A, Špíšek R, Jelínková LP - Oncotarget (2015)

Dex/VitD tDCs maintain reduced T cell stimulatory capacity after restimulationDCs were differentiated in Cell Gro in presence (tDCs) or absence of Dex and VitD2 (cDCs) and activated with MPLA (CG). Then, DCs were washed, recultured in complete RPMI without tolerising factors and treated with cytokine cocktail (CC) described in Figure 1B or LPS (1 μg/ml) or polyI:C (25 μg/ml) or CD40L (1000 ng/ml) or they were left unstimulated (RPMI). After 24 h, tDCs and cDCs were washed and incubated with allogeneic T cells at 1:10 ratio (DCs/T cells). A. Proliferation of T cells was assessed on day 6 by CFSE dilution method. Percentages of proliferating T cells and representative histograms are shown. B. Production of IL-17A in DCs/T cell co-cultures was analyzed by ELISA on day 6. C. Percentages of IFN-γ producing T and D. IL-10 producing T cells was assessed on day 6 or day 9, respectively. Representative dot plots are shown. Data represent mean ± SEM for at least 3 independent experiments and at least 10 donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (paired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4546455&req=5

Figure 2: Dex/VitD tDCs maintain reduced T cell stimulatory capacity after restimulationDCs were differentiated in Cell Gro in presence (tDCs) or absence of Dex and VitD2 (cDCs) and activated with MPLA (CG). Then, DCs were washed, recultured in complete RPMI without tolerising factors and treated with cytokine cocktail (CC) described in Figure 1B or LPS (1 μg/ml) or polyI:C (25 μg/ml) or CD40L (1000 ng/ml) or they were left unstimulated (RPMI). After 24 h, tDCs and cDCs were washed and incubated with allogeneic T cells at 1:10 ratio (DCs/T cells). A. Proliferation of T cells was assessed on day 6 by CFSE dilution method. Percentages of proliferating T cells and representative histograms are shown. B. Production of IL-17A in DCs/T cell co-cultures was analyzed by ELISA on day 6. C. Percentages of IFN-γ producing T and D. IL-10 producing T cells was assessed on day 6 or day 9, respectively. Representative dot plots are shown. Data represent mean ± SEM for at least 3 independent experiments and at least 10 donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (paired t-test).
Mentions: TDCs or cDCs were cultured with allogeneic T cells at a ratio of 1:10. TDCs were weaker inducers of CD4+ as well as CD8+ T cell proliferation, even after the restimulation, irrespective of the maturation agent when compared to cDCs (Figure 2A). In line with this, tDCs induced low IL-17A production by allogeneic T cells even after restimulation in contrast to cDCs that were potent inducers of IL-17A by T cells especially after CC and LPS stimulation (Figure 2B). Moreover, co-incubation of allogeneic T cells with tDCs cultured in Cell Gro skewed the T cell cytokine profile towards reduced IFN-γ and significantly increased IL-10 production by CD4+ as well as CD8+ T cells, in comparison to cDCs (Figure 2C, 2D). In addition, co-incubation of T cells with tDCs restimulated with CC, LPS, polyI:C and CD40L led to marked reduction of CD4+ IFN-γ producing T cells together with stable numbers of CD4+ IL-10 producing T cells. The percentage of CD8+ IFN-γ producing T cells remained stable or slightly decreased after CC and CD40L restimulation of tDCs, while the amount of CD8+ IL-10 producing T cells remained almost the same after restimulation of tDCs with LPS and slightly decreased after restimulation of tDCs with CC, polyI:C and CD40L.

Bottom Line: However, the signaling pathways regulating their stable tolerogenic properties are largely unknown.In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-β, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells.In the inflammatory environment, tDCs maintained up-regulated indoleamine 2, 3 dioxygenase but abrogated IκB-α phosphorylation and reduced transcriptional activity of p65/RelA, RelB and c-Rel NF-κB subunits except p50.

View Article: PubMed Central - PubMed

Affiliation: Sotio a.s., Prague, Czech Republic.

ABSTRACT
Tolerogenic dendritic cells (tDCs) may offer an intervention therapy in autoimmune diseases or transplantation. Stable immaturity and tolerogenic function of tDCs after encountering inflammatory environment are prerequisite for positive outcome of immunotherapy. However, the signaling pathways regulating their stable tolerogenic properties are largely unknown. In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-β, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells. In the inflammatory environment, tDCs maintained up-regulated indoleamine 2, 3 dioxygenase but abrogated IκB-α phosphorylation and reduced transcriptional activity of p65/RelA, RelB and c-Rel NF-κB subunits except p50. Mechanistically, p38 MAPK, ERK1/2, mTOR, STAT3 and mTOR-dependent glycolysis regulated expression of ILT-3, PDL-1 and CD86, secretion of IL-10 and T cell stimulatory capacity of tDCs in the inflammatory environment. Stability of tDCs in the inflammatory environment is thus regulated by multiple signaling pathways.

No MeSH data available.


Related in: MedlinePlus