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Osteoclast activated FoxP3+ CD8+ T-cells suppress bone resorption in vitro.

Buchwald ZS, Kiesel JR, DiPaolo R, Pagadala MS, Aurora R - PLoS ONE (2012)

Bottom Line: Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation.The suppression did not require direct contact between the Tc(REG) and osteoclasts.Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT

Background: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF)) increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF) produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG)). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption.

Principal findings: To determine the net effect of Tc(REG) on osteoclast activity we used a number of in vitro assays. We found that Tc(REG) can potently and directly suppress bone resorption by osteoclasts. Tc(REG) could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG) suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG) and osteoclasts.

Significance: We have determined that osteoclast-induced Tc(REG) can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology.

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Osteoclast-induced TcREG inhibit osteoclast differentiation, but not survival.A. Osteoclast precursor cells (day 0) were cultured alone, with pre-differentiated TcREG, iTREG, anti-mouse CD3/CD28 re-stimulated iTREG, or with naïve T-cells in the presence of GST-RANKL. After four days, non-adherent cells were removed by aspiration and remaining cells were stained with a fluorescent TRAP substrate ELF-97. Representative images from four experiments are shown in top panel, and quantitated cells counts are shown below. To test if suppression of osteoclastogenesis was mediated solely by IFN-γ, bone marrow cells from IFNγR1−/− mice were used. The results of TRAP staining were confirmed by quantitative real-time PCR using primers for Acp5 (TRAP), Cathepsin K (CTSK) and matrix metalloprotease-9 (MMP-9). β-actin expression was used for normalization. B. Osteoclasts, cultured in the absence or presence of TcREG for five days were counted after TRAP staining with ELF-97. To test for increased apoptosis adherent cells were stained with Annexin V. Each data point is average of three wells per experiment. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 by comparison to untreated (Untx) osteoclast wells.
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pone-0038199-g003: Osteoclast-induced TcREG inhibit osteoclast differentiation, but not survival.A. Osteoclast precursor cells (day 0) were cultured alone, with pre-differentiated TcREG, iTREG, anti-mouse CD3/CD28 re-stimulated iTREG, or with naïve T-cells in the presence of GST-RANKL. After four days, non-adherent cells were removed by aspiration and remaining cells were stained with a fluorescent TRAP substrate ELF-97. Representative images from four experiments are shown in top panel, and quantitated cells counts are shown below. To test if suppression of osteoclastogenesis was mediated solely by IFN-γ, bone marrow cells from IFNγR1−/− mice were used. The results of TRAP staining were confirmed by quantitative real-time PCR using primers for Acp5 (TRAP), Cathepsin K (CTSK) and matrix metalloprotease-9 (MMP-9). β-actin expression was used for normalization. B. Osteoclasts, cultured in the absence or presence of TcREG for five days were counted after TRAP staining with ELF-97. To test for increased apoptosis adherent cells were stained with Annexin V. Each data point is average of three wells per experiment. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 by comparison to untreated (Untx) osteoclast wells.

Mentions: IFN-γ blocks osteoclast differentiation, while RANKL promotes differentiation and survival of the osteoclasts. As the pitting assays described above require 7 to 10 days, one mechanism by which TcREG could mediate a loss of pitting would be to suppress osteoclast differentiation. To test for the effect of TcREG on osteoclastogenesis, we cultured bone marrow cells with M-CSF for three days, and then removed non-adherent cells to enrich for osteoclast precursors; RANKL was then added with TcREG (generated on independent wells with mature osteoclasts). After four days of co-culturing we visualized and enumerated the osteoclasts using the fluorescent substrate ELF-97 for tartrate resistant acid phosphatase (TRAP), a marker of osteoclasts [62]. The results show that no TRAP positive cells were induced by RANKL (top middle panel Fig. 3A) in the presence of TcREG. In contrast, many large TRAP positive osteoclasts were observed in the absence of T-cells (top left), and in the presence of naïve CD8 T-cells (upper right). Sato et al. [8] have previously shown that TGFβ-induced TREG cannot suppress osteoclastogenesis, where as Zaiss et al. [49] obtained the opposite results. Our results (Fig. 3A) show that iTREG cannot suppress osteoclastogenesis unless they are restimulated with anti-CD3 and anti-CD28. To test if IFN-γ is solely responsible for suppression of osteoclast differentiation, we used bone marrow from IFN-γ receptor knockout (IFNγR1−/−) mice. We found that TcREG could efficiently suppress osteoclast differentiation of IFNγR1−/− precursors, indicating that additional cytokines play a role in mediating suppression of osteoclastogenesis. Finally, we confirmed the suppression using quantitative real-time PCR of three osteoclast markers (Fig. 3A bottom right). These marker genes were chosen because they are expressed in mature osteoclasts and absent in T-cells.


Osteoclast activated FoxP3+ CD8+ T-cells suppress bone resorption in vitro.

Buchwald ZS, Kiesel JR, DiPaolo R, Pagadala MS, Aurora R - PLoS ONE (2012)

Osteoclast-induced TcREG inhibit osteoclast differentiation, but not survival.A. Osteoclast precursor cells (day 0) were cultured alone, with pre-differentiated TcREG, iTREG, anti-mouse CD3/CD28 re-stimulated iTREG, or with naïve T-cells in the presence of GST-RANKL. After four days, non-adherent cells were removed by aspiration and remaining cells were stained with a fluorescent TRAP substrate ELF-97. Representative images from four experiments are shown in top panel, and quantitated cells counts are shown below. To test if suppression of osteoclastogenesis was mediated solely by IFN-γ, bone marrow cells from IFNγR1−/− mice were used. The results of TRAP staining were confirmed by quantitative real-time PCR using primers for Acp5 (TRAP), Cathepsin K (CTSK) and matrix metalloprotease-9 (MMP-9). β-actin expression was used for normalization. B. Osteoclasts, cultured in the absence or presence of TcREG for five days were counted after TRAP staining with ELF-97. To test for increased apoptosis adherent cells were stained with Annexin V. Each data point is average of three wells per experiment. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 by comparison to untreated (Untx) osteoclast wells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368916&req=5

pone-0038199-g003: Osteoclast-induced TcREG inhibit osteoclast differentiation, but not survival.A. Osteoclast precursor cells (day 0) were cultured alone, with pre-differentiated TcREG, iTREG, anti-mouse CD3/CD28 re-stimulated iTREG, or with naïve T-cells in the presence of GST-RANKL. After four days, non-adherent cells were removed by aspiration and remaining cells were stained with a fluorescent TRAP substrate ELF-97. Representative images from four experiments are shown in top panel, and quantitated cells counts are shown below. To test if suppression of osteoclastogenesis was mediated solely by IFN-γ, bone marrow cells from IFNγR1−/− mice were used. The results of TRAP staining were confirmed by quantitative real-time PCR using primers for Acp5 (TRAP), Cathepsin K (CTSK) and matrix metalloprotease-9 (MMP-9). β-actin expression was used for normalization. B. Osteoclasts, cultured in the absence or presence of TcREG for five days were counted after TRAP staining with ELF-97. To test for increased apoptosis adherent cells were stained with Annexin V. Each data point is average of three wells per experiment. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 by comparison to untreated (Untx) osteoclast wells.
Mentions: IFN-γ blocks osteoclast differentiation, while RANKL promotes differentiation and survival of the osteoclasts. As the pitting assays described above require 7 to 10 days, one mechanism by which TcREG could mediate a loss of pitting would be to suppress osteoclast differentiation. To test for the effect of TcREG on osteoclastogenesis, we cultured bone marrow cells with M-CSF for three days, and then removed non-adherent cells to enrich for osteoclast precursors; RANKL was then added with TcREG (generated on independent wells with mature osteoclasts). After four days of co-culturing we visualized and enumerated the osteoclasts using the fluorescent substrate ELF-97 for tartrate resistant acid phosphatase (TRAP), a marker of osteoclasts [62]. The results show that no TRAP positive cells were induced by RANKL (top middle panel Fig. 3A) in the presence of TcREG. In contrast, many large TRAP positive osteoclasts were observed in the absence of T-cells (top left), and in the presence of naïve CD8 T-cells (upper right). Sato et al. [8] have previously shown that TGFβ-induced TREG cannot suppress osteoclastogenesis, where as Zaiss et al. [49] obtained the opposite results. Our results (Fig. 3A) show that iTREG cannot suppress osteoclastogenesis unless they are restimulated with anti-CD3 and anti-CD28. To test if IFN-γ is solely responsible for suppression of osteoclast differentiation, we used bone marrow from IFN-γ receptor knockout (IFNγR1−/−) mice. We found that TcREG could efficiently suppress osteoclast differentiation of IFNγR1−/− precursors, indicating that additional cytokines play a role in mediating suppression of osteoclastogenesis. Finally, we confirmed the suppression using quantitative real-time PCR of three osteoclast markers (Fig. 3A bottom right). These marker genes were chosen because they are expressed in mature osteoclasts and absent in T-cells.

Bottom Line: Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation.The suppression did not require direct contact between the Tc(REG) and osteoclasts.Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT

Background: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF)) increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF) produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG)). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption.

Principal findings: To determine the net effect of Tc(REG) on osteoclast activity we used a number of in vitro assays. We found that Tc(REG) can potently and directly suppress bone resorption by osteoclasts. Tc(REG) could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG) suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG) and osteoclasts.

Significance: We have determined that osteoclast-induced Tc(REG) can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology.

Show MeSH
Related in: MedlinePlus