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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 mediated suppression of T cell proliferation is not mediated by CD11c+ BM-MCs. BM-MCs were magnetically separated with CD11c+ beads. Purity of CD11c+ and CD11c- cells was confirmed and cells analyzed on the BD Accuri C6 Flow cytometer, Purified CD11c+(A) and CD11c-(C) were co-cultured with responder immune cells for 96 hours with media or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture; and the relative percentages of CD11cs+(B) and CD11c-(D) cells expressing maturation markers CD80, CD86 MHCII, PD-L1and PD-L2 were determined. Data are representative of three separate experiments* = p < 0.05.
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Figure 3: RA mediated suppression of T cell proliferation is not mediated by CD11c+ BM-MCs. BM-MCs were magnetically separated with CD11c+ beads. Purity of CD11c+ and CD11c- cells was confirmed and cells analyzed on the BD Accuri C6 Flow cytometer, Purified CD11c+(A) and CD11c-(C) were co-cultured with responder immune cells for 96 hours with media or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture; and the relative percentages of CD11cs+(B) and CD11c-(D) cells expressing maturation markers CD80, CD86 MHCII, PD-L1and PD-L2 were determined. Data are representative of three separate experiments* = p < 0.05.

Mentions: The in vitro differentiation of bone marrow cells with GM-CSF is a commonly used protocol to produce large numbers (>80%) of highly enriched CD11c+ DCs [38,56] that, as a population, are considered immature DCs. However, our data demonstrated that while approximately 80-90% of the cells were CD11c+, the remaining 10-20% were CD11c- but still CD11b+ (Additional file 1: Figure S1A). To determine whether the MCregs induced by RA were DCs, we purified CD11c+ cells from day 7 differentiated cells and cultured them with responder immune cells. Although RA induction of mucosal “DCregs” have been described [9,36,57], we found that RA-treated CD11c+ cells were not the suppressive cell population (Figure 3A). In all experiments, RA-treated CD11c+ cells failed to suppress proliferation and had variable to no effect on proliferation with some experiments actually demonstrating enhanced proliferation (data not shown). Phenotypic evaluation of these CD11c+ cells showed no difference in percentage (Figure 3B) or expression levels of CD80, CD86, MHC class II, PD-L1 and PD-L2 compared to media controls. To determine the source of the suppressive MCregs, we evaluated the CD11c- population and found that the RA CD11c- cells suppressed proliferation of responder cells (Figure 3C). These CD11c- cells had a marked (>30%) increase in the percentage of CD80+, CD86+, MHC class II+ and PD-L1+ cells (with no differences in PD-L2+ cells) (Figure 3D) when differentiated with RA, consistent with an activated regulatory phenotype in these cells described previously [35]. In contrast, levels of CD80, MHC class II and PD-L1 did not change, remaining consistently high (>80%) in RA versus control MCs. (Figure 3B). These data suggest that RA present during GM-CSF differentiation increased an activated regulatory phenotype in the CD11c- (non-DC) populations.


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 mediated suppression of T cell proliferation is not mediated by CD11c+ BM-MCs. BM-MCs were magnetically separated with CD11c+ beads. Purity of CD11c+ and CD11c- cells was confirmed and cells analyzed on the BD Accuri C6 Flow cytometer, Purified CD11c+(A) and CD11c-(C) were co-cultured with responder immune cells for 96 hours with media or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture; and the relative percentages of CD11cs+(B) and CD11c-(D) cells expressing maturation markers CD80, CD86 MHCII, PD-L1and PD-L2 were determined. Data are representative of 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 3: RA mediated suppression of T cell proliferation is not mediated by CD11c+ BM-MCs. BM-MCs were magnetically separated with CD11c+ beads. Purity of CD11c+ and CD11c- cells was confirmed and cells analyzed on the BD Accuri C6 Flow cytometer, Purified CD11c+(A) and CD11c-(C) were co-cultured with responder immune cells for 96 hours with media or anti-CD3 stimulation and then pulsed with H3 thymidine in the final 18 hours of culture; and the relative percentages of CD11cs+(B) and CD11c-(D) cells expressing maturation markers CD80, CD86 MHCII, PD-L1and PD-L2 were determined. Data are representative of three separate experiments* = p < 0.05.
Mentions: The in vitro differentiation of bone marrow cells with GM-CSF is a commonly used protocol to produce large numbers (>80%) of highly enriched CD11c+ DCs [38,56] that, as a population, are considered immature DCs. However, our data demonstrated that while approximately 80-90% of the cells were CD11c+, the remaining 10-20% were CD11c- but still CD11b+ (Additional file 1: Figure S1A). To determine whether the MCregs induced by RA were DCs, we purified CD11c+ cells from day 7 differentiated cells and cultured them with responder immune cells. Although RA induction of mucosal “DCregs” have been described [9,36,57], we found that RA-treated CD11c+ cells were not the suppressive cell population (Figure 3A). In all experiments, RA-treated CD11c+ cells failed to suppress proliferation and had variable to no effect on proliferation with some experiments actually demonstrating enhanced proliferation (data not shown). Phenotypic evaluation of these CD11c+ cells showed no difference in percentage (Figure 3B) or expression levels of CD80, CD86, MHC class II, PD-L1 and PD-L2 compared to media controls. To determine the source of the suppressive MCregs, we evaluated the CD11c- population and found that the RA CD11c- cells suppressed proliferation of responder cells (Figure 3C). These CD11c- cells had a marked (>30%) increase in the percentage of CD80+, CD86+, MHC class II+ and PD-L1+ cells (with no differences in PD-L2+ cells) (Figure 3D) when differentiated with RA, consistent with an activated regulatory phenotype in these cells described previously [35]. In contrast, levels of CD80, MHC class II and PD-L1 did not change, remaining consistently high (>80%) in RA versus control MCs. (Figure 3B). These data suggest that RA present during GM-CSF differentiation increased an activated regulatory phenotype in the CD11c- (non-DC) populations.

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