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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 generated CD11b+ BM-MCs suppress T cell proliferation and express a regulatory phenotype. CD11b+ CD11c- were purified from BM-MCs by sequential purification of CD11b+ cells from the CD11c- fraction and purity determined to be >95%. Cells were routinely stained for cell surface markers and flow cytometry performed using the BD Accuri C6 Flow cytometer. CD11b+ CD11c cells were (A) assessed for expression of maturation markers CD80, CD86, MHCII, PD-L1 and PD-L2 (B) 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, (C) intracellular cytokine staining (IL-17, INF-γ, IL-4) of CD4+ cells performed and (D) co-cultured with activated CTLs and a T lymphoma cell line with cell viability assessed by the MTT assay. Data is representative of three separate experiments. * = p < 0.05 (A-C) Data shown is a representation of 3 experiments (D).
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Figure 4: RA generated CD11b+ BM-MCs suppress T cell proliferation and express a regulatory phenotype. CD11b+ CD11c- were purified from BM-MCs by sequential purification of CD11b+ cells from the CD11c- fraction and purity determined to be >95%. Cells were routinely stained for cell surface markers and flow cytometry performed using the BD Accuri C6 Flow cytometer. CD11b+ CD11c cells were (A) assessed for expression of maturation markers CD80, CD86, MHCII, PD-L1 and PD-L2 (B) 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, (C) intracellular cytokine staining (IL-17, INF-γ, IL-4) of CD4+ cells performed and (D) co-cultured with activated CTLs and a T lymphoma cell line with cell viability assessed by the MTT assay. Data is representative of three separate experiments. * = p < 0.05 (A-C) Data shown is a representation of 3 experiments (D).

Mentions: Both differentiated and precursor populations within the bone marrow are predominantly but not completely CD11b+ (>90%) (Additional file 1: Figure S1A). To definitively isolate the effects of CD11b+ CD11c- cells, we serially purified CD11b+ cells from the CD11c- fraction and evaluated their phenotype and function. As expected, the increases in the percentage of CD80+, CD86+, MHC class II+ and PD-L1+ cells seen in Figure 3D was also seen in the CD11b+ CD11c- serially purified population (Figure 4A). We then went on to evaluate the ability of these cells to influence CD4 and CD8 responses. We found that the CD11b+ CD11c- population was able to suppress the proliferation of responder immune cells (Figure 4B) and could modify the cytokine profile of T cells. The proliferating CD4+ responder immune cells cultured with RA CD11b+ CD11c- cells were also shown to have reduced expression of IL-17 IFN-gamma (Figure 4C) and IL-10 (Additional file 2: Figure S2) but no change in IL-4 production as determined by intracellular cytokine staining (Figure 4C). Intracellular IL-10 and FoxP3+ cells were also increased as expected (Additional file 2: Figure S2A and S2B, respectively). We also evaluated the ability of RA CD11b+ CD11c- cells to influence CD8+ T cell responses. Figure 4D demonstrates reduced cytotoxicity in CD8+ T cells cultured with RA CD11b+ CD11c- cells. Taken together, these results suggest that RA induced an activated regulatory population of CD11b+ CD11c- cells that were able to suppress both CD4+ and CD8+ adaptive immune responses.


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 generated CD11b+ BM-MCs suppress T cell proliferation and express a regulatory phenotype. CD11b+ CD11c- were purified from BM-MCs by sequential purification of CD11b+ cells from the CD11c- fraction and purity determined to be >95%. Cells were routinely stained for cell surface markers and flow cytometry performed using the BD Accuri C6 Flow cytometer. CD11b+ CD11c cells were (A) assessed for expression of maturation markers CD80, CD86, MHCII, PD-L1 and PD-L2 (B) 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, (C) intracellular cytokine staining (IL-17, INF-γ, IL-4) of CD4+ cells performed and (D) co-cultured with activated CTLs and a T lymphoma cell line with cell viability assessed by the MTT assay. Data is representative of three separate experiments. * = p < 0.05 (A-C) Data shown is a representation of 3 experiments (D).
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Figure 4: RA generated CD11b+ BM-MCs suppress T cell proliferation and express a regulatory phenotype. CD11b+ CD11c- were purified from BM-MCs by sequential purification of CD11b+ cells from the CD11c- fraction and purity determined to be >95%. Cells were routinely stained for cell surface markers and flow cytometry performed using the BD Accuri C6 Flow cytometer. CD11b+ CD11c cells were (A) assessed for expression of maturation markers CD80, CD86, MHCII, PD-L1 and PD-L2 (B) 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, (C) intracellular cytokine staining (IL-17, INF-γ, IL-4) of CD4+ cells performed and (D) co-cultured with activated CTLs and a T lymphoma cell line with cell viability assessed by the MTT assay. Data is representative of three separate experiments. * = p < 0.05 (A-C) Data shown is a representation of 3 experiments (D).
Mentions: Both differentiated and precursor populations within the bone marrow are predominantly but not completely CD11b+ (>90%) (Additional file 1: Figure S1A). To definitively isolate the effects of CD11b+ CD11c- cells, we serially purified CD11b+ cells from the CD11c- fraction and evaluated their phenotype and function. As expected, the increases in the percentage of CD80+, CD86+, MHC class II+ and PD-L1+ cells seen in Figure 3D was also seen in the CD11b+ CD11c- serially purified population (Figure 4A). We then went on to evaluate the ability of these cells to influence CD4 and CD8 responses. We found that the CD11b+ CD11c- population was able to suppress the proliferation of responder immune cells (Figure 4B) and could modify the cytokine profile of T cells. The proliferating CD4+ responder immune cells cultured with RA CD11b+ CD11c- cells were also shown to have reduced expression of IL-17 IFN-gamma (Figure 4C) and IL-10 (Additional file 2: Figure S2) but no change in IL-4 production as determined by intracellular cytokine staining (Figure 4C). Intracellular IL-10 and FoxP3+ cells were also increased as expected (Additional file 2: Figure S2A and S2B, respectively). We also evaluated the ability of RA CD11b+ CD11c- cells to influence CD8+ T cell responses. Figure 4D demonstrates reduced cytotoxicity in CD8+ T cells cultured with RA CD11b+ CD11c- cells. Taken together, these results suggest that RA induced an activated regulatory population of CD11b+ CD11c- cells that were able to suppress both CD4+ and CD8+ adaptive immune responses.

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