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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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Effects of BM-MDSCs on arthritis severity and Ag (PG)-specific immune responses in SCID mice with PGIA.(A) Effect of BM-MDSC transfer on arthritis severity. Arthritis was induced in SCID mice via 2 transfers of spleen cells (black arrows) from wild type mice with PGIA as described in the Methods. At the early phase of arthritis, one group of the SCID recipients was co-injected with BM-MDSCs (red arrow). Disease severity scores were monitored until day 34. Arthritis progressed rapidly in the control group (black line), but not in the BM-MDSC-treated group (red line) (*p<0.05, n = 10 mice/group; two-way repeated measures analysis of variance). (B) Joint histopathology of control (left panel) and BM-MDSC-treated (right panel) mice on day 34. The ankle joint of the control mouse demonstrated massive leukocyte infiltration (star) in the joint cavity (JC) and synovial tissue (ST) as well as synovial hyperplasia. The articulating surfaces appeared rough due to cartilage damage. In the ankle joint of the BM-MDSC-treated mouse only mild synovial hyperplasia was seen, suggesting the resolution of initial (previous) inflammation. Representative hematoxylin-eosin-stained tissue sections from both groups are shown. (C) Antigen (PG)-specific T-cell responses of control and BM-MDSC-treated mice. T-cell responses were compared between the two groups on day 34 by measuring spleen cell proliferation in the presence or absence of PG in vitro. Results are expressed as stimulation index (SI), a ratio of [3H]thymidine incorporation by PG-stimulated and non-stimulated cultures. The SI of the BM-MDSC-injected group (red bar) was significantly lower than the SI of the control group (black bar) (*p<0.0001, n = 10 mice/group; Student’s t test). (D) Serum levels of anti-PG antibodies in the control and BM-MDSC-treated groups as determined by ELISA. The levels of IgG1 anti-PG antibodies (top) were significantly lower in the sera of BM-MDSC-injected mice than in control mice (*p<0.01, n = 5 samples/group; Mann-Whitney U test), while the levels of IgG2a anti-PG antibodies (bottom) were similar.
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pone-0111815-g005: Effects of BM-MDSCs on arthritis severity and Ag (PG)-specific immune responses in SCID mice with PGIA.(A) Effect of BM-MDSC transfer on arthritis severity. Arthritis was induced in SCID mice via 2 transfers of spleen cells (black arrows) from wild type mice with PGIA as described in the Methods. At the early phase of arthritis, one group of the SCID recipients was co-injected with BM-MDSCs (red arrow). Disease severity scores were monitored until day 34. Arthritis progressed rapidly in the control group (black line), but not in the BM-MDSC-treated group (red line) (*p<0.05, n = 10 mice/group; two-way repeated measures analysis of variance). (B) Joint histopathology of control (left panel) and BM-MDSC-treated (right panel) mice on day 34. The ankle joint of the control mouse demonstrated massive leukocyte infiltration (star) in the joint cavity (JC) and synovial tissue (ST) as well as synovial hyperplasia. The articulating surfaces appeared rough due to cartilage damage. In the ankle joint of the BM-MDSC-treated mouse only mild synovial hyperplasia was seen, suggesting the resolution of initial (previous) inflammation. Representative hematoxylin-eosin-stained tissue sections from both groups are shown. (C) Antigen (PG)-specific T-cell responses of control and BM-MDSC-treated mice. T-cell responses were compared between the two groups on day 34 by measuring spleen cell proliferation in the presence or absence of PG in vitro. Results are expressed as stimulation index (SI), a ratio of [3H]thymidine incorporation by PG-stimulated and non-stimulated cultures. The SI of the BM-MDSC-injected group (red bar) was significantly lower than the SI of the control group (black bar) (*p<0.0001, n = 10 mice/group; Student’s t test). (D) Serum levels of anti-PG antibodies in the control and BM-MDSC-treated groups as determined by ELISA. The levels of IgG1 anti-PG antibodies (top) were significantly lower in the sera of BM-MDSC-injected mice than in control mice (*p<0.01, n = 5 samples/group; Mann-Whitney U test), while the levels of IgG2a anti-PG antibodies (bottom) were similar.

Mentions: To test whether BM-MDSCs could affect the development of arthritis, an adoptive transfer model of PGIA was employed. On day 0, spleen cells from arthritic wt BALB/c donor mice were injected with Ag (human PG) into SCID recipients. When the clinical signs of arthritis started to develop (15 days after the first injection), the SCID mice were divided into 2 groups with similar mean disease scores, and a second injection was administered. The first (control) group received only arthritic spleen cells and PG, while the second group received the same plus BM-MDSCs. Arthritis severity scores in the control group increased further (Fig. 5A, black line), while, in sharp contrast, the scores of SCID mice transferred with BM-MDSCs remained low until the end (day 34) of the monitoring period (Fig. 5A, red line). Histopathology revealed massive leukocyte infiltration and synovial hyperplasia as well as cartilage erosion in the ankle (tibio-talar) joints of control SCID mice transferred with spleen cells from arthritic donors (Fig. 5B, left panel). In contrast, only mild synovial hyperplasia was observed without evidence of gross inflammation or cartilage damage in the ankle joints of SCID mice co-transferred with spleen cells and BM-MDSCs (Fig. 5B, right panel).


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)

Effects of BM-MDSCs on arthritis severity and Ag (PG)-specific immune responses in SCID mice with PGIA.(A) Effect of BM-MDSC transfer on arthritis severity. Arthritis was induced in SCID mice via 2 transfers of spleen cells (black arrows) from wild type mice with PGIA as described in the Methods. At the early phase of arthritis, one group of the SCID recipients was co-injected with BM-MDSCs (red arrow). Disease severity scores were monitored until day 34. Arthritis progressed rapidly in the control group (black line), but not in the BM-MDSC-treated group (red line) (*p<0.05, n = 10 mice/group; two-way repeated measures analysis of variance). (B) Joint histopathology of control (left panel) and BM-MDSC-treated (right panel) mice on day 34. The ankle joint of the control mouse demonstrated massive leukocyte infiltration (star) in the joint cavity (JC) and synovial tissue (ST) as well as synovial hyperplasia. The articulating surfaces appeared rough due to cartilage damage. In the ankle joint of the BM-MDSC-treated mouse only mild synovial hyperplasia was seen, suggesting the resolution of initial (previous) inflammation. Representative hematoxylin-eosin-stained tissue sections from both groups are shown. (C) Antigen (PG)-specific T-cell responses of control and BM-MDSC-treated mice. T-cell responses were compared between the two groups on day 34 by measuring spleen cell proliferation in the presence or absence of PG in vitro. Results are expressed as stimulation index (SI), a ratio of [3H]thymidine incorporation by PG-stimulated and non-stimulated cultures. The SI of the BM-MDSC-injected group (red bar) was significantly lower than the SI of the control group (black bar) (*p<0.0001, n = 10 mice/group; Student’s t test). (D) Serum levels of anti-PG antibodies in the control and BM-MDSC-treated groups as determined by ELISA. The levels of IgG1 anti-PG antibodies (top) were significantly lower in the sera of BM-MDSC-injected mice than in control mice (*p<0.01, n = 5 samples/group; Mann-Whitney U test), while the levels of IgG2a anti-PG antibodies (bottom) were similar.
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pone-0111815-g005: Effects of BM-MDSCs on arthritis severity and Ag (PG)-specific immune responses in SCID mice with PGIA.(A) Effect of BM-MDSC transfer on arthritis severity. Arthritis was induced in SCID mice via 2 transfers of spleen cells (black arrows) from wild type mice with PGIA as described in the Methods. At the early phase of arthritis, one group of the SCID recipients was co-injected with BM-MDSCs (red arrow). Disease severity scores were monitored until day 34. Arthritis progressed rapidly in the control group (black line), but not in the BM-MDSC-treated group (red line) (*p<0.05, n = 10 mice/group; two-way repeated measures analysis of variance). (B) Joint histopathology of control (left panel) and BM-MDSC-treated (right panel) mice on day 34. The ankle joint of the control mouse demonstrated massive leukocyte infiltration (star) in the joint cavity (JC) and synovial tissue (ST) as well as synovial hyperplasia. The articulating surfaces appeared rough due to cartilage damage. In the ankle joint of the BM-MDSC-treated mouse only mild synovial hyperplasia was seen, suggesting the resolution of initial (previous) inflammation. Representative hematoxylin-eosin-stained tissue sections from both groups are shown. (C) Antigen (PG)-specific T-cell responses of control and BM-MDSC-treated mice. T-cell responses were compared between the two groups on day 34 by measuring spleen cell proliferation in the presence or absence of PG in vitro. Results are expressed as stimulation index (SI), a ratio of [3H]thymidine incorporation by PG-stimulated and non-stimulated cultures. The SI of the BM-MDSC-injected group (red bar) was significantly lower than the SI of the control group (black bar) (*p<0.0001, n = 10 mice/group; Student’s t test). (D) Serum levels of anti-PG antibodies in the control and BM-MDSC-treated groups as determined by ELISA. The levels of IgG1 anti-PG antibodies (top) were significantly lower in the sera of BM-MDSC-injected mice than in control mice (*p<0.01, n = 5 samples/group; Mann-Whitney U test), while the levels of IgG2a anti-PG antibodies (bottom) were similar.
Mentions: To test whether BM-MDSCs could affect the development of arthritis, an adoptive transfer model of PGIA was employed. On day 0, spleen cells from arthritic wt BALB/c donor mice were injected with Ag (human PG) into SCID recipients. When the clinical signs of arthritis started to develop (15 days after the first injection), the SCID mice were divided into 2 groups with similar mean disease scores, and a second injection was administered. The first (control) group received only arthritic spleen cells and PG, while the second group received the same plus BM-MDSCs. Arthritis severity scores in the control group increased further (Fig. 5A, black line), while, in sharp contrast, the scores of SCID mice transferred with BM-MDSCs remained low until the end (day 34) of the monitoring period (Fig. 5A, red line). Histopathology revealed massive leukocyte infiltration and synovial hyperplasia as well as cartilage erosion in the ankle (tibio-talar) joints of control SCID mice transferred with spleen cells from arthritic donors (Fig. 5B, left panel). In contrast, only mild synovial hyperplasia was observed without evidence of gross inflammation or cartilage damage in the ankle joints of SCID mice co-transferred with spleen cells and BM-MDSCs (Fig. 5B, right panel).

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