image_7_Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.PDF

<p>Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-β) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-β present in semen, suggesting that PGE2 overrides the influence exerted by TGF-β. No previous studies have analyzed the joint actions induced by PGE2 and TGF-β on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-β and PGE2. DC differentiation guided by TGF-β alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-β and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-β signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-β during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-β and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.</p

image_2_Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.PDF

<p>Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-β) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-β present in semen, suggesting that PGE2 overrides the influence exerted by TGF-β. No previous studies have analyzed the joint actions induced by PGE2 and TGF-β on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-β and PGE2. DC differentiation guided by TGF-β alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-β and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-β signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-β during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-β and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.</p

image_3_Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.PDF

<p>Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-β) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-β present in semen, suggesting that PGE2 overrides the influence exerted by TGF-β. No previous studies have analyzed the joint actions induced by PGE2 and TGF-β on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-β and PGE2. DC differentiation guided by TGF-β alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-β and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-β signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-β during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-β and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.</p

image_1_Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.PDF

<p>Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-β) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-β present in semen, suggesting that PGE2 overrides the influence exerted by TGF-β. No previous studies have analyzed the joint actions induced by PGE2 and TGF-β on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-β and PGE2. DC differentiation guided by TGF-β alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-β and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-β signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-β during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-β and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.</p

image_5_Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.PDF

<p>Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-β) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-β present in semen, suggesting that PGE2 overrides the influence exerted by TGF-β. No previous studies have analyzed the joint actions induced by PGE2 and TGF-β on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-β and PGE2. DC differentiation guided by TGF-β alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-β and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-β signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-β during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-β and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.</p

image_4_Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.PDF

<p>Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-β) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-β present in semen, suggesting that PGE2 overrides the influence exerted by TGF-β. No previous studies have analyzed the joint actions induced by PGE2 and TGF-β on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-β and PGE2. DC differentiation guided by TGF-β alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-β and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-β signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-β during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-β and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.</p

Epithelial cells induce the phenotypic maturation of pDCs in a cell contact-dependent manner.

<p>Experiments were performed using 24-transwell chambers with a polycarbonate filter (0.2 µm pore size). HT-29 cells were grown to confluence on the filter. pDCs (3×10⁵) were cultured alone in the lower chamber or in contact with the monolayer of epithelial cells, in the upper chamber. Control cells were cultured in the upper chamber without epithelial cells. After 12 h of culture, pDCs were harvested and the expression of HLA-DR, CD83, CD86, and MHC class I was analyzed in the gate of CD123⁺ cells by flow cytometry. The relative mean fluorescence intensity (MFI) of isotype controls were in all cases lower than 5 (not shown). The MFI for control cells (for all the markers analyzed) was assigned to the value of 100, and the MFI for pDCs cultured alone in the lower chamber (X) or those cultured in contact with the monolayer of epithelial cells in the upper chamber (Y) was calculated using the equation: X or Y×100/MFI of control pDCs. Histograms show a representative experiment (n = 4–8). Graph bars show the MFI of HLA-DR, CD83, CD86, and MHC class I in the gate of CD123⁺ cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028709#s2" target="_blank">Results</a> are the mean ± SEM of 6–7 experiments. (* p<0.05 vs control).</p

Primary human renal tubular epithelial cells (HRTEC) induce the activation of pDCs.

<p>Primary human renal proximal tubular cells, obtained as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028709#s4" target="_blank">Materials and Methods</a>, were grown to confluence in 96 well, flat bottom plates. pDCs (1×10⁵/well) were cultured for 12 h alone (controls), with confluent HRTEC, or with supernatants collected from confluent HRTEC incubated alone for 12 h. Then, the phenotype of pDCs in the gate of CD123+ cells was analyzed by flow (A) Histograms of representative experiments (n = 6) are shown. The MFI of isotype controls were in all cases lower than 5 (not shown). Graph bars show the relative mean fluorescence intensity (MFI) of HLA-DR, CD83, and CD80 for pDCs cultured alone or in the presence of epithelial cells or epithelial cell supernatants. The MFI of pDCs cultured alone is assigned the value of 100. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028709#s2" target="_blank">Results</a> are the mean ± SEM of 7–8 experiments performed in duplicate. (* p<0.05 vs pDCs). (B) The production of TNF-α, IL-6, and IL-1β were assessed in cell supernatants by ELISA. *p<0.05 vs pDCs.</p

Epithelial cells induce neither the phenotypic maturation nor the stimulation of the production of inflammatory cytokines by cDCs.

<p>Conventional DCs were obtained from human monocytes (>85% purity) cultured for 5 days with GM-CSF plus IL-4. The epithelial cell lines HT-29 and Caco-2 were grown to confluence in 96 well, flat bottom plates. DCs (1×10⁵/200 µl) were cultured alone for 12 h, in the absence or presence of 100 ng/ml of LPS or in the presence of confluent monolayers of HT-29 or Caco-2 cells, in 96 well flat bottom plates. (<b>A</b>) Dot-plots illustrating the purity of cDCs and the expression of CD1a and HLA-DR. (<b>B</b>) The expression of CD83, MHC class I, and CD86 was analyzed by flow cytometry in the gate of CD1a+ cells and a representative experiment (n = 5–7) is shown. Graph bars show the MFI of CD83, MHC class I, and CD86. The MFI of cDCs cultured alone is assigned the value of 100. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028709#s2" target="_blank">Results</a> are the mean ± SEM of 7–10 experiments. (* p<0.05 vs cDCs). (<b>C</b>) The production of TNF-α, IL-12p70, and IL-10 was evaluated in cell supernatants by ELISA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028709#s2" target="_blank">Results</a> are the mean ± SEM of 7–10 experiments performed in duplicate. (* p<0.05 vs controls).</p

<i>Candida albicans</i> abrogates the production of virus particles by HIV-1-challenged macrophages.

<p>(<b>A</b>) Macrophages (5x10⁵/0.5 ml) were cultured in 24 flat-bottom plats with the R5 tropic virus HIV-1_BaL (50 ng p24/ml) for 2 h. Then, cells were washed and the production of HIV-1 was evaluated at days 0, 3, 5 and 7 post-infection by measuring the amount of p24 antigen in cell supernatants (n=4). (<b>B</b>) Macrophages were cultured with HIV-1_BaL (50 ng p24/ml) for 2 h in the absence or presence of CA (macrophage: CA ratio of 1:10) or zymosan (100 µg/0.5 ml). Then, cells were washed and the production of HIV-1 was evaluated after 7 days (n=9). (<b>C</b>) Macrophages were cultured for 7 days in the absence or presence of HIV-1_BaL (50 ng p24/ml), CA (macrophage: CA ratio of 1:10) or zymosan (100 µg/0.5 ml). Then, the viability of macrophages was analyzed by flow cytometry using Annexin V and propidium iodide. Positive control of necrosis represents macrophages cultured for 3 days in protein-free medium (n=3). (<b>D</b>) Histograms illustrate the phenotype of DCs used in our experiments (n=5). (<b>E</b>) DCs (5x10⁵/0.5 ml) were cultured with HIV-1_BaL (50 ng p24/ml) for 2 h. Then, cells were washed and the production of HIV-1 was evaluated at days 0, 3, 5 and 7 post-infection (n=4). (<b>F</b>) DCs were cultured with HIV-1_BaL (50 ng p24/ml) for 2 h in the absence or presence of CA (macrophage: CA ratio of 1:10) or zymosan (100 µg/0.5 ml). Then, cells were washed and the production of HIV-1 was evaluated after 7 days of culture (n=5). (<b>G</b>) The T cell line Jurkat (5x10⁵/0.5 ml) was incubated with the X4 tropic virus HIV-1_IIIB (50 ng p24/ml) and CA (Jurkat: CA ratios of 1:10, 1:1, and 1:0.1) for 2 h. Cells were then washed and HIV infection was revealed after 7 days (n=3). In all cases, results are expressed as the arithmetic means ± SEM of n experiments or are illustrated as representative dot-plot or histograms. *p<0.05 vs HIV.</p