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Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

Kurkó J, Vida A, Ocskó T, Tryniszewska B, Rauch TA, Glant TT, Szekanecz Z, Mikecz K - PLoS ONE (2014)

Bottom Line: The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide.Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Medicine, Department of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, United States of America; Department of Rheumatology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.

ABSTRACT

Background: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.

Methods: Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.

Results: BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.

Conclusions: Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

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Phenotype and morphology of myeloid-derived suppressor cell (MDSC)-like cells generated in vitro from murine bone marrow (BM) in comparison with synovial fluid (SF) cells.(A) Phenotype of MDSCs arising from growth factor-cytokine treated BM cell cultures as determined by flow cytometry. BM cells were cultured in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each). On day 3, cells were immunostained for CD11b, Ly6C, and Ly6G. Approximately 80% of the cells expressed the common myeloid marker CD11b (gray bar). Gating on CD11b+ cells revealed that the majority of them co-expressed Ly6C (marker of the “monocytic” subset) and Ly6G (marker of the “granulocytic” subset), but cells expressing only one marker were also present (black bars). The results are the means ± SEM of 7 independent BM cultures. (B) In SF, the vast majority of the CD11b+ myeloid population (gray bar) was found to be cells co-expressing Ly6G and Ly6C, and lower proportions of cells expressed Ly6C or Ly6G only (black bars) than in the BM-MDSC cell cultures. The results are the means ± SEM of 7 separate pools of SF cells freshly harvested from arthritic mouse joints. (C) Flow cytometry profile of BM-MDSCs (left panels) is shown as an example of subset identification after gating on CD11b+ cells. Fluorescence image of EGFP+ BM-MDSCs (middle panel) after surface staining with a blue fluorescent antibody to Ly6C and a red fluorescent antibody to Ly6G shows cells expressing one or both markers. BM for culture was obtained from an EGFP-LysM-Tg mouse expressing EGFP (green fluorescence) in myeloid cells. Imaging was performed using two-photon microscopy (TPM). Morphology of BM-MDSCs (right panel) was visualized by Wright-Giemsa staining of a cytospin preparation, which shows both polymorphonuclear granulocyte (neutrophil)-like cells (arrows) and large precursor-like cells (arrowheads). (D) Flow cytometry profile (left) and morphology (right) of SF cells harvested from the arthritic joints of mice with PGIA. While the CD11b+ myeloid population is large in both the BM-MDSC culture and arthritic SF, and is dominated by Ly6C/Ly6G double positive cells in both samples (analyzed simultaneously), BM-MDSCs show greater heterogeneity in morphology than SF cells.
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pone-0111815-g001: Phenotype and morphology of myeloid-derived suppressor cell (MDSC)-like cells generated in vitro from murine bone marrow (BM) in comparison with synovial fluid (SF) cells.(A) Phenotype of MDSCs arising from growth factor-cytokine treated BM cell cultures as determined by flow cytometry. BM cells were cultured in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each). On day 3, cells were immunostained for CD11b, Ly6C, and Ly6G. Approximately 80% of the cells expressed the common myeloid marker CD11b (gray bar). Gating on CD11b+ cells revealed that the majority of them co-expressed Ly6C (marker of the “monocytic” subset) and Ly6G (marker of the “granulocytic” subset), but cells expressing only one marker were also present (black bars). The results are the means ± SEM of 7 independent BM cultures. (B) In SF, the vast majority of the CD11b+ myeloid population (gray bar) was found to be cells co-expressing Ly6G and Ly6C, and lower proportions of cells expressed Ly6C or Ly6G only (black bars) than in the BM-MDSC cell cultures. The results are the means ± SEM of 7 separate pools of SF cells freshly harvested from arthritic mouse joints. (C) Flow cytometry profile of BM-MDSCs (left panels) is shown as an example of subset identification after gating on CD11b+ cells. Fluorescence image of EGFP+ BM-MDSCs (middle panel) after surface staining with a blue fluorescent antibody to Ly6C and a red fluorescent antibody to Ly6G shows cells expressing one or both markers. BM for culture was obtained from an EGFP-LysM-Tg mouse expressing EGFP (green fluorescence) in myeloid cells. Imaging was performed using two-photon microscopy (TPM). Morphology of BM-MDSCs (right panel) was visualized by Wright-Giemsa staining of a cytospin preparation, which shows both polymorphonuclear granulocyte (neutrophil)-like cells (arrows) and large precursor-like cells (arrowheads). (D) Flow cytometry profile (left) and morphology (right) of SF cells harvested from the arthritic joints of mice with PGIA. While the CD11b+ myeloid population is large in both the BM-MDSC culture and arthritic SF, and is dominated by Ly6C/Ly6G double positive cells in both samples (analyzed simultaneously), BM-MDSCs show greater heterogeneity in morphology than SF cells.

Mentions: The primary goal of this study was to establish a culture method by which BM cells can be enriched in myeloid cells resembling SF-MDSCs in both their phenotype and function. We chose BM because it is the body’s largest reservoir of myeloid precursors from which large numbers of MDSCs can be generated under appropriate conditions. GM-CSF is essential for the survival and suppressor activity of MDSCs [34], and one study reported successful generation of MDSCs from human blood in 7 days with a combination of GM-CSF and IL-6 [35], factors that are also present in the SF of RA patients [36]. In preliminary experiments, we sought to determine whether BM cells cultured for 3 to 7 days in the presence GM-CSF and IL-6 acquire an SF-MDSC-like phenotype. Although the BM culture became enriched in CD11b+ cells under this condition as determined by flow cytometry, unlike SF-MDSCs, only a small proportion of these myeloid cells expressed Ly6G, a marker of granulocytic MDSCs (data not shown). We added G-CSF as a booster of the granulocytic lineage to the BM culture, which resulted in the rise of cell populations expressing Ly6G alone, or co-expressing Ly6G (at high levels) with low-to-intermediate levels of the monocytic MDSC marker Ly6C (Fig. 1A, and Fig. 1C [left panel]). This overall phenotype was achieved in 3 days of culture in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each); longer culture or higher doses of G-CSF did not result in increases in Ly6G+ or double Ly6C+Ly6G+ cells (data not shown). In comparison with CD11b+ SF cells (Fig. 1B and D), BM-MDSCs contained fewer double Ly6C+Ly6G+ cells and higher proportions of subsets expressing only one of these markers (Fig. 1A and C). However, cells co-expressing Ly6C and Ly6G clearly represented a dominant population in both the BM-MDSC cultures and freshly harvested SF (Fig. 1A–D). Our choice of the combination of growth factors to generate SF-MDSC-like cells from BM was supported by the finding that SF from mice with PGIA contained high amounts of GM-CSF and G-CSF, and detectable amounts of IL-6. In each case, the SF concentrations of these factors exceeded the serum levels (Table S1).


Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

Kurkó J, Vida A, Ocskó T, Tryniszewska B, Rauch TA, Glant TT, Szekanecz Z, Mikecz K - PLoS ONE (2014)

Phenotype and morphology of myeloid-derived suppressor cell (MDSC)-like cells generated in vitro from murine bone marrow (BM) in comparison with synovial fluid (SF) cells.(A) Phenotype of MDSCs arising from growth factor-cytokine treated BM cell cultures as determined by flow cytometry. BM cells were cultured in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each). On day 3, cells were immunostained for CD11b, Ly6C, and Ly6G. Approximately 80% of the cells expressed the common myeloid marker CD11b (gray bar). Gating on CD11b+ cells revealed that the majority of them co-expressed Ly6C (marker of the “monocytic” subset) and Ly6G (marker of the “granulocytic” subset), but cells expressing only one marker were also present (black bars). The results are the means ± SEM of 7 independent BM cultures. (B) In SF, the vast majority of the CD11b+ myeloid population (gray bar) was found to be cells co-expressing Ly6G and Ly6C, and lower proportions of cells expressed Ly6C or Ly6G only (black bars) than in the BM-MDSC cell cultures. The results are the means ± SEM of 7 separate pools of SF cells freshly harvested from arthritic mouse joints. (C) Flow cytometry profile of BM-MDSCs (left panels) is shown as an example of subset identification after gating on CD11b+ cells. Fluorescence image of EGFP+ BM-MDSCs (middle panel) after surface staining with a blue fluorescent antibody to Ly6C and a red fluorescent antibody to Ly6G shows cells expressing one or both markers. BM for culture was obtained from an EGFP-LysM-Tg mouse expressing EGFP (green fluorescence) in myeloid cells. Imaging was performed using two-photon microscopy (TPM). Morphology of BM-MDSCs (right panel) was visualized by Wright-Giemsa staining of a cytospin preparation, which shows both polymorphonuclear granulocyte (neutrophil)-like cells (arrows) and large precursor-like cells (arrowheads). (D) Flow cytometry profile (left) and morphology (right) of SF cells harvested from the arthritic joints of mice with PGIA. While the CD11b+ myeloid population is large in both the BM-MDSC culture and arthritic SF, and is dominated by Ly6C/Ly6G double positive cells in both samples (analyzed simultaneously), BM-MDSCs show greater heterogeneity in morphology than SF cells.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219784&req=5

pone-0111815-g001: Phenotype and morphology of myeloid-derived suppressor cell (MDSC)-like cells generated in vitro from murine bone marrow (BM) in comparison with synovial fluid (SF) cells.(A) Phenotype of MDSCs arising from growth factor-cytokine treated BM cell cultures as determined by flow cytometry. BM cells were cultured in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each). On day 3, cells were immunostained for CD11b, Ly6C, and Ly6G. Approximately 80% of the cells expressed the common myeloid marker CD11b (gray bar). Gating on CD11b+ cells revealed that the majority of them co-expressed Ly6C (marker of the “monocytic” subset) and Ly6G (marker of the “granulocytic” subset), but cells expressing only one marker were also present (black bars). The results are the means ± SEM of 7 independent BM cultures. (B) In SF, the vast majority of the CD11b+ myeloid population (gray bar) was found to be cells co-expressing Ly6G and Ly6C, and lower proportions of cells expressed Ly6C or Ly6G only (black bars) than in the BM-MDSC cell cultures. The results are the means ± SEM of 7 separate pools of SF cells freshly harvested from arthritic mouse joints. (C) Flow cytometry profile of BM-MDSCs (left panels) is shown as an example of subset identification after gating on CD11b+ cells. Fluorescence image of EGFP+ BM-MDSCs (middle panel) after surface staining with a blue fluorescent antibody to Ly6C and a red fluorescent antibody to Ly6G shows cells expressing one or both markers. BM for culture was obtained from an EGFP-LysM-Tg mouse expressing EGFP (green fluorescence) in myeloid cells. Imaging was performed using two-photon microscopy (TPM). Morphology of BM-MDSCs (right panel) was visualized by Wright-Giemsa staining of a cytospin preparation, which shows both polymorphonuclear granulocyte (neutrophil)-like cells (arrows) and large precursor-like cells (arrowheads). (D) Flow cytometry profile (left) and morphology (right) of SF cells harvested from the arthritic joints of mice with PGIA. While the CD11b+ myeloid population is large in both the BM-MDSC culture and arthritic SF, and is dominated by Ly6C/Ly6G double positive cells in both samples (analyzed simultaneously), BM-MDSCs show greater heterogeneity in morphology than SF cells.
Mentions: The primary goal of this study was to establish a culture method by which BM cells can be enriched in myeloid cells resembling SF-MDSCs in both their phenotype and function. We chose BM because it is the body’s largest reservoir of myeloid precursors from which large numbers of MDSCs can be generated under appropriate conditions. GM-CSF is essential for the survival and suppressor activity of MDSCs [34], and one study reported successful generation of MDSCs from human blood in 7 days with a combination of GM-CSF and IL-6 [35], factors that are also present in the SF of RA patients [36]. In preliminary experiments, we sought to determine whether BM cells cultured for 3 to 7 days in the presence GM-CSF and IL-6 acquire an SF-MDSC-like phenotype. Although the BM culture became enriched in CD11b+ cells under this condition as determined by flow cytometry, unlike SF-MDSCs, only a small proportion of these myeloid cells expressed Ly6G, a marker of granulocytic MDSCs (data not shown). We added G-CSF as a booster of the granulocytic lineage to the BM culture, which resulted in the rise of cell populations expressing Ly6G alone, or co-expressing Ly6G (at high levels) with low-to-intermediate levels of the monocytic MDSC marker Ly6C (Fig. 1A, and Fig. 1C [left panel]). This overall phenotype was achieved in 3 days of culture in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each); longer culture or higher doses of G-CSF did not result in increases in Ly6G+ or double Ly6C+Ly6G+ cells (data not shown). In comparison with CD11b+ SF cells (Fig. 1B and D), BM-MDSCs contained fewer double Ly6C+Ly6G+ cells and higher proportions of subsets expressing only one of these markers (Fig. 1A and C). However, cells co-expressing Ly6C and Ly6G clearly represented a dominant population in both the BM-MDSC cultures and freshly harvested SF (Fig. 1A–D). Our choice of the combination of growth factors to generate SF-MDSC-like cells from BM was supported by the finding that SF from mice with PGIA contained high amounts of GM-CSF and G-CSF, and detectable amounts of IL-6. In each case, the SF concentrations of these factors exceeded the serum levels (Table S1).

Bottom Line: The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide.Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Medicine, Department of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, United States of America; Department of Rheumatology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.

ABSTRACT

Background: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.

Methods: Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.

Results: BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.

Conclusions: Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

Show MeSH
Related in: MedlinePlus