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Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells.

VanGundy ZC, Guerau-de-Arellano M, Baker JD, Strange HR, Olivo-Marston S, Muth DC, Papenfuss TL - BMC Immunol. (2014)

Bottom Line: We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells.Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS.These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, College of Veterinary Medicine, 370 Veterinary Medical Academic Building, 1900 Coffey Road, Columbus, OH 43210, USA. papenfuss.1@osu.edu.

ABSTRACT

Background: Myeloid cells (MC) have potent immunoregulatory abilities that can be therapeutically useful to treat inflammatory disease. However, the factors which promote regulatory myeloid cell differentiation remain poorly understood. We have previously shown that estriol (E3) induces mature regulatory dendritic cells in vivo. To determine whether additional steroid hormones could induce mature regulatory myeloid cells, we investigated the effects of retinoic acid (RA) on MCs. Retinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation. We hypothesized that the presence of RA during differentiation would promote the formation of mature regulatory myeloid cells (MCregs).

Methods: To determine RA's ability to induce regulatory myeloid cells, we differentiated bone marrow progenitor cells with granulocytic-macrophage colony-stimulating factor (GM-CSF) under the influence of RA. We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells. Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS.

Conclusion: RA induced mature regulatory myeloid cells that were suppressive and had a CD11b+ CD11c-Ly6C low/intermediate monocyte phenotype. Surprisingly, RA CD11c+ dendritic cells were not suppressive and could contribute to enhanced proliferation. These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

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RA treatment of bone marrow myeloid cells produces a regulatory myeloid cell population. Bone marrow cells were differentiated in the presence of GM-CSF with or without 100 nM of either estriol or retinoic acid over 6–7 days of differentiation to generate MCs, E3 MCregs or RA MCregs. A portion of these cells were also challenged with LPS in the last 24 hours of differentiation. BM-MCs (A) and LPS-stimulated BM-MCs (B) were co-cultured with responder immune cells containing T cell receptor transgenic CD4+ T cells specific for peptide for 96 hours with media, antigen or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture. In MCs, E3 MCregs and RA MCregs, (C) the relative percentage of IL-10+ cells was determined and (D) the ability of these cells (after a 5 day co-culture) to induce FoxP3+ cells from naïve FoxP3-EGFP reporter immune cells was determined by flow cytometry. Data are representative of at least three separate experiments * = p < 0.05.
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Figure 1: RA treatment of bone marrow myeloid cells produces a regulatory myeloid cell population. Bone marrow cells were differentiated in the presence of GM-CSF with or without 100 nM of either estriol or retinoic acid over 6–7 days of differentiation to generate MCs, E3 MCregs or RA MCregs. A portion of these cells were also challenged with LPS in the last 24 hours of differentiation. BM-MCs (A) and LPS-stimulated BM-MCs (B) were co-cultured with responder immune cells containing T cell receptor transgenic CD4+ T cells specific for peptide for 96 hours with media, antigen or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture. In MCs, E3 MCregs and RA MCregs, (C) the relative percentage of IL-10+ cells was determined and (D) the ability of these cells (after a 5 day co-culture) to induce FoxP3+ cells from naïve FoxP3-EGFP reporter immune cells was determined by flow cytometry. Data are representative of at least three separate experiments * = p < 0.05.

Mentions: Given that RA is a regulator of mucosal immunity and influences myelopoiesis, we hypothesized that RA would induce a population of mature MCregs. Day 6–7 BM cells differentiated with GM-CSF in the presence of RA were able to suppress the proliferation of responder immune cells and this suppression was markedly greater than either control or E3 treated cells (Figure 1A). The ability of RA differentiated cells to suppress proliferation was apparent regardless of whether responder immune cells were stimulated with either peptide or anti-CD3. Interestingly, cells treated with E3 suppressed proliferation after stimulation with peptide but not anti-CD3 (Figure 1A). We next determined whether the RA differentiated cells remained regulatory when exposed to the inflammatory stimulus LPS. Figure 1B shows that RA differentiated cells maintained their ability to suppress proliferation even after exposure to LPS challenge and that this was present following stimulation of co-cultures with either peptide or anti-CD3. This effect was entirely lost in E3 treated cells. These results suggest that RA differentiated cells are more potent and stable than E3 differentiated cells and that RA differentiated cells maintain their regulatory ability following exposure to an inflammatory stimulus.


Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells.

VanGundy ZC, Guerau-de-Arellano M, Baker JD, Strange HR, Olivo-Marston S, Muth DC, Papenfuss TL - BMC Immunol. (2014)

RA treatment of bone marrow myeloid cells produces a regulatory myeloid cell population. Bone marrow cells were differentiated in the presence of GM-CSF with or without 100 nM of either estriol or retinoic acid over 6–7 days of differentiation to generate MCs, E3 MCregs or RA MCregs. A portion of these cells were also challenged with LPS in the last 24 hours of differentiation. BM-MCs (A) and LPS-stimulated BM-MCs (B) were co-cultured with responder immune cells containing T cell receptor transgenic CD4+ T cells specific for peptide for 96 hours with media, antigen or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture. In MCs, E3 MCregs and RA MCregs, (C) the relative percentage of IL-10+ cells was determined and (D) the ability of these cells (after a 5 day co-culture) to induce FoxP3+ cells from naïve FoxP3-EGFP reporter immune cells was determined by flow cytometry. Data are representative of at least three separate experiments * = p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016499&req=5

Figure 1: RA treatment of bone marrow myeloid cells produces a regulatory myeloid cell population. Bone marrow cells were differentiated in the presence of GM-CSF with or without 100 nM of either estriol or retinoic acid over 6–7 days of differentiation to generate MCs, E3 MCregs or RA MCregs. A portion of these cells were also challenged with LPS in the last 24 hours of differentiation. BM-MCs (A) and LPS-stimulated BM-MCs (B) were co-cultured with responder immune cells containing T cell receptor transgenic CD4+ T cells specific for peptide for 96 hours with media, antigen or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture. In MCs, E3 MCregs and RA MCregs, (C) the relative percentage of IL-10+ cells was determined and (D) the ability of these cells (after a 5 day co-culture) to induce FoxP3+ cells from naïve FoxP3-EGFP reporter immune cells was determined by flow cytometry. Data are representative of at least three separate experiments * = p < 0.05.
Mentions: Given that RA is a regulator of mucosal immunity and influences myelopoiesis, we hypothesized that RA would induce a population of mature MCregs. Day 6–7 BM cells differentiated with GM-CSF in the presence of RA were able to suppress the proliferation of responder immune cells and this suppression was markedly greater than either control or E3 treated cells (Figure 1A). The ability of RA differentiated cells to suppress proliferation was apparent regardless of whether responder immune cells were stimulated with either peptide or anti-CD3. Interestingly, cells treated with E3 suppressed proliferation after stimulation with peptide but not anti-CD3 (Figure 1A). We next determined whether the RA differentiated cells remained regulatory when exposed to the inflammatory stimulus LPS. Figure 1B shows that RA differentiated cells maintained their ability to suppress proliferation even after exposure to LPS challenge and that this was present following stimulation of co-cultures with either peptide or anti-CD3. This effect was entirely lost in E3 treated cells. These results suggest that RA differentiated cells are more potent and stable than E3 differentiated cells and that RA differentiated cells maintain their regulatory ability following exposure to an inflammatory stimulus.

Bottom Line: We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells.Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS.These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, College of Veterinary Medicine, 370 Veterinary Medical Academic Building, 1900 Coffey Road, Columbus, OH 43210, USA. papenfuss.1@osu.edu.

ABSTRACT

Background: Myeloid cells (MC) have potent immunoregulatory abilities that can be therapeutically useful to treat inflammatory disease. However, the factors which promote regulatory myeloid cell differentiation remain poorly understood. We have previously shown that estriol (E3) induces mature regulatory dendritic cells in vivo. To determine whether additional steroid hormones could induce mature regulatory myeloid cells, we investigated the effects of retinoic acid (RA) on MCs. Retinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation. We hypothesized that the presence of RA during differentiation would promote the formation of mature regulatory myeloid cells (MCregs).

Methods: To determine RA's ability to induce regulatory myeloid cells, we differentiated bone marrow progenitor cells with granulocytic-macrophage colony-stimulating factor (GM-CSF) under the influence of RA. We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells. Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS.

Conclusion: RA induced mature regulatory myeloid cells that were suppressive and had a CD11b+ CD11c-Ly6C low/intermediate monocyte phenotype. Surprisingly, RA CD11c+ dendritic cells were not suppressive and could contribute to enhanced proliferation. These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

Show MeSH
Related in: MedlinePlus