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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 mature myeloid cells. 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. Cells were routinely stained for flow cytometry using antibodies against CD11c-, CD11b+, CD80, CD86, MHC class II, PD-L1 and PD-L2, run of a BD Accuri C6 flow cytometer and phenotypic profiles evaluated using the Cflow plus software program. Cells were evaluated for the presence of maturation (activation) markers (A) CD80, CD86 MHCII, and the inhibitory co-stimulatory markers (B) PD-L1and PD-L2. (C) Mean fluorescence intensity (MFI) was determined and relative expression shown by overlays of cell treatment groups and MFI values (D). Data are representative of at least three separate experiments.
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Figure 2: RA treatment of bone marrow myeloid cells produces mature myeloid cells. 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. Cells were routinely stained for flow cytometry using antibodies against CD11c-, CD11b+, CD80, CD86, MHC class II, PD-L1 and PD-L2, run of a BD Accuri C6 flow cytometer and phenotypic profiles evaluated using the Cflow plus software program. Cells were evaluated for the presence of maturation (activation) markers (A) CD80, CD86 MHCII, and the inhibitory co-stimulatory markers (B) PD-L1and PD-L2. (C) Mean fluorescence intensity (MFI) was determined and relative expression shown by overlays of cell treatment groups and MFI values (D). Data are representative of at least three separate experiments.

Mentions: To determine whether these RA differentiated cells were mature, we evaluated the cell surface expression of maturation markers CD80, CD86 and MHC class II and inhibitory markers PD-L1 and PD-L2. RA differentiated cells demonstrated an increased percentage of CD80+, CD86+ and MHC class II+ (Figure 2A), indicating that an increased proportion of the cells were mature and/or activated in comparison to E3 or control cells. Additionally, there were increases in the mean fluorescence intensity (MFI) of CD80, CD86 and MHC class II in RA differentiated cells as depicted in Figures 2C and D, indicating that the relative expression levels on a per cell basis were increased in RA differentiated cells. Although E3 differentiated cells had mildly increased expression levels of CD80, CD86 and MHC class II, RA differentiated cells had consistently higher levels than either E3 differentiated or control cells. To confirm that RA differentiated cells demonstrated an “activated regulatory” phenotype as previously described for E3, we evaluated the expression of inhibitory co-stimulatory molecules PD-L1 and PD-L2 [35]. RA increased the percentage of PD-L1+ cells (but not PD-L2+) (Figure 2B) and the MFI of both PD-L1 and PD-L2 (Figure 2C, D) compared to E3 or media controls. These results demonstrate that during differentiation RA induces a population of mature activated MCregs that suppress the proliferation of responder immune cells even in the face of inflammatory challenge. Additionally, our data shows that although both RA and E3 may induce MCregs which suppress proliferation (Figure 1A) RA MCregs appear to have superior regulatory abilities compared to E3 MCregs.


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 mature myeloid cells. 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. Cells were routinely stained for flow cytometry using antibodies against CD11c-, CD11b+, CD80, CD86, MHC class II, PD-L1 and PD-L2, run of a BD Accuri C6 flow cytometer and phenotypic profiles evaluated using the Cflow plus software program. Cells were evaluated for the presence of maturation (activation) markers (A) CD80, CD86 MHCII, and the inhibitory co-stimulatory markers (B) PD-L1and PD-L2. (C) Mean fluorescence intensity (MFI) was determined and relative expression shown by overlays of cell treatment groups and MFI values (D). Data are representative of at least three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: RA treatment of bone marrow myeloid cells produces mature myeloid cells. 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. Cells were routinely stained for flow cytometry using antibodies against CD11c-, CD11b+, CD80, CD86, MHC class II, PD-L1 and PD-L2, run of a BD Accuri C6 flow cytometer and phenotypic profiles evaluated using the Cflow plus software program. Cells were evaluated for the presence of maturation (activation) markers (A) CD80, CD86 MHCII, and the inhibitory co-stimulatory markers (B) PD-L1and PD-L2. (C) Mean fluorescence intensity (MFI) was determined and relative expression shown by overlays of cell treatment groups and MFI values (D). Data are representative of at least three separate experiments.
Mentions: To determine whether these RA differentiated cells were mature, we evaluated the cell surface expression of maturation markers CD80, CD86 and MHC class II and inhibitory markers PD-L1 and PD-L2. RA differentiated cells demonstrated an increased percentage of CD80+, CD86+ and MHC class II+ (Figure 2A), indicating that an increased proportion of the cells were mature and/or activated in comparison to E3 or control cells. Additionally, there were increases in the mean fluorescence intensity (MFI) of CD80, CD86 and MHC class II in RA differentiated cells as depicted in Figures 2C and D, indicating that the relative expression levels on a per cell basis were increased in RA differentiated cells. Although E3 differentiated cells had mildly increased expression levels of CD80, CD86 and MHC class II, RA differentiated cells had consistently higher levels than either E3 differentiated or control cells. To confirm that RA differentiated cells demonstrated an “activated regulatory” phenotype as previously described for E3, we evaluated the expression of inhibitory co-stimulatory molecules PD-L1 and PD-L2 [35]. RA increased the percentage of PD-L1+ cells (but not PD-L2+) (Figure 2B) and the MFI of both PD-L1 and PD-L2 (Figure 2C, D) compared to E3 or media controls. These results demonstrate that during differentiation RA induces a population of mature activated MCregs that suppress the proliferation of responder immune cells even in the face of inflammatory challenge. Additionally, our data shows that although both RA and E3 may induce MCregs which suppress proliferation (Figure 1A) RA MCregs appear to have superior regulatory abilities compared to E3 MCregs.

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