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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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Reversal of the suppressive effect of BM-MDSCs on T-cell proliferation by inhibitors of inducible nitric oxide synthase (iNOS).Various inhibitors of MDSC effector molecules, including the arginase 1 inhibitor nor-NOHA, iNOS inhibitors L-NMMA and 1400W, the reactive oxygen species (ROS) scavenger catalase, and the caspase/apoptosis inhibitor Z-VAD-FMK, were used to inhibit the BM-MDSC-mediated suppression of (A) Ag (rhG1)-induced/DC-dependent and (B) anti-CD3/CD28-induced proliferation of PG-TCR-Tg T cells. The results (compiled from 2 independent series of experiments, each with 2 co-cultures) are expressed as percent suppression of T-cell proliferation in the presence (black bars) or absence (red bar) of inhibitors. While suppression of T-cell proliferation in both induction systems was significantly reversed by the iNOS inhibitors L-NMMA and 1400W (*p<0.0001 in all cases; Kruskal-Wallis test followed by Dunn’s multiple comparisons test), none of the other inhibitors had a significant effect on BM-MDSC-mediated suppression of T cells.
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pone-0111815-g003: Reversal of the suppressive effect of BM-MDSCs on T-cell proliferation by inhibitors of inducible nitric oxide synthase (iNOS).Various inhibitors of MDSC effector molecules, including the arginase 1 inhibitor nor-NOHA, iNOS inhibitors L-NMMA and 1400W, the reactive oxygen species (ROS) scavenger catalase, and the caspase/apoptosis inhibitor Z-VAD-FMK, were used to inhibit the BM-MDSC-mediated suppression of (A) Ag (rhG1)-induced/DC-dependent and (B) anti-CD3/CD28-induced proliferation of PG-TCR-Tg T cells. The results (compiled from 2 independent series of experiments, each with 2 co-cultures) are expressed as percent suppression of T-cell proliferation in the presence (black bars) or absence (red bar) of inhibitors. While suppression of T-cell proliferation in both induction systems was significantly reversed by the iNOS inhibitors L-NMMA and 1400W (*p<0.0001 in all cases; Kruskal-Wallis test followed by Dunn’s multiple comparisons test), none of the other inhibitors had a significant effect on BM-MDSC-mediated suppression of T cells.

Mentions: To reveal the possible mechanism of the suppressive activity of the BM-MDSCs, we repeated the Ag-dependent and Ag-independent T-cell proliferation assays with and without various inhibitors of MDSC-related effector molecules such as arginase 1 (nor-NOHA), iNOS (L-NMMA and the more selective 1400W), and ROS (catalase). A caspase (apoptosis) inhibitor (Z-VAD-FMK) was used as a MDSC-unrelated control. Both Ag (rhG1)- and anti-CD3/CD28-induced T-cell proliferation remained suppressed in the presence of the arginase 1 inhibitor, the ROS scavenger, or the caspase inhibitor (Fig. 3A and B). However, BM-MDSCs lost much of their ability to suppress T-cell proliferation in both induction systems in the presence of the iNOS inhibitors (Fig. 3), suggesting that the main MDSC product mediating T-cell suppression was NO.


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)

Reversal of the suppressive effect of BM-MDSCs on T-cell proliferation by inhibitors of inducible nitric oxide synthase (iNOS).Various inhibitors of MDSC effector molecules, including the arginase 1 inhibitor nor-NOHA, iNOS inhibitors L-NMMA and 1400W, the reactive oxygen species (ROS) scavenger catalase, and the caspase/apoptosis inhibitor Z-VAD-FMK, were used to inhibit the BM-MDSC-mediated suppression of (A) Ag (rhG1)-induced/DC-dependent and (B) anti-CD3/CD28-induced proliferation of PG-TCR-Tg T cells. The results (compiled from 2 independent series of experiments, each with 2 co-cultures) are expressed as percent suppression of T-cell proliferation in the presence (black bars) or absence (red bar) of inhibitors. While suppression of T-cell proliferation in both induction systems was significantly reversed by the iNOS inhibitors L-NMMA and 1400W (*p<0.0001 in all cases; Kruskal-Wallis test followed by Dunn’s multiple comparisons test), none of the other inhibitors had a significant effect on BM-MDSC-mediated suppression of T cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111815-g003: Reversal of the suppressive effect of BM-MDSCs on T-cell proliferation by inhibitors of inducible nitric oxide synthase (iNOS).Various inhibitors of MDSC effector molecules, including the arginase 1 inhibitor nor-NOHA, iNOS inhibitors L-NMMA and 1400W, the reactive oxygen species (ROS) scavenger catalase, and the caspase/apoptosis inhibitor Z-VAD-FMK, were used to inhibit the BM-MDSC-mediated suppression of (A) Ag (rhG1)-induced/DC-dependent and (B) anti-CD3/CD28-induced proliferation of PG-TCR-Tg T cells. The results (compiled from 2 independent series of experiments, each with 2 co-cultures) are expressed as percent suppression of T-cell proliferation in the presence (black bars) or absence (red bar) of inhibitors. While suppression of T-cell proliferation in both induction systems was significantly reversed by the iNOS inhibitors L-NMMA and 1400W (*p<0.0001 in all cases; Kruskal-Wallis test followed by Dunn’s multiple comparisons test), none of the other inhibitors had a significant effect on BM-MDSC-mediated suppression of T cells.
Mentions: To reveal the possible mechanism of the suppressive activity of the BM-MDSCs, we repeated the Ag-dependent and Ag-independent T-cell proliferation assays with and without various inhibitors of MDSC-related effector molecules such as arginase 1 (nor-NOHA), iNOS (L-NMMA and the more selective 1400W), and ROS (catalase). A caspase (apoptosis) inhibitor (Z-VAD-FMK) was used as a MDSC-unrelated control. Both Ag (rhG1)- and anti-CD3/CD28-induced T-cell proliferation remained suppressed in the presence of the arginase 1 inhibitor, the ROS scavenger, or the caspase inhibitor (Fig. 3A and B). However, BM-MDSCs lost much of their ability to suppress T-cell proliferation in both induction systems in the presence of the iNOS inhibitors (Fig. 3), suggesting that the main MDSC product mediating T-cell suppression was NO.

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