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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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Related in: MedlinePlus

TcREG suppress mature osteoclasts by effecting the cytoskeletal reorganization:Osteoclasts were cultured either alone or with TcREG on bovine bone chips for 24 hrs. T-cells were then removed, and osteoclasts were stained with phalloidin-Texas Red to visualize actin rings. Representative images are shown in panel A. Quantitation of three independent experiments is shown in panel B. Panel C is quantitation of phalloidin staining of osteoclasts plated on tissue culture treated dishes. Statistical significance of actin ring area was assessed by non-parametric paired T test: **: P<0.01 in comparison to osteoclast alone.
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pone-0038199-g004: TcREG suppress mature osteoclasts by effecting the cytoskeletal reorganization:Osteoclasts were cultured either alone or with TcREG on bovine bone chips for 24 hrs. T-cells were then removed, and osteoclasts were stained with phalloidin-Texas Red to visualize actin rings. Representative images are shown in panel A. Quantitation of three independent experiments is shown in panel B. Panel C is quantitation of phalloidin staining of osteoclasts plated on tissue culture treated dishes. Statistical significance of actin ring area was assessed by non-parametric paired T test: **: P<0.01 in comparison to osteoclast alone.

Mentions: Osteoclasts must adhere to the bone surface and migrate along it to resorb [66]; cytoskeletal reorganization is critical for this process [67], [68]. Therefore, we stained for actin rings in osteoclasts in the presence and absence of TcREG. TcREG were cultured with mature osteoclasts seeded on bone, or on plastic for 24 h. T-cells were then removed and the actin rings were visualized using flour-conjugated phalloidin. In the presence of TcREG, the actin ring was either eliminated on bone (Fig. 4A and B), or (the actin belt) was reduced in size on plastic (Fig. 4C).


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

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

TcREG suppress mature osteoclasts by effecting the cytoskeletal reorganization:Osteoclasts were cultured either alone or with TcREG on bovine bone chips for 24 hrs. T-cells were then removed, and osteoclasts were stained with phalloidin-Texas Red to visualize actin rings. Representative images are shown in panel A. Quantitation of three independent experiments is shown in panel B. Panel C is quantitation of phalloidin staining of osteoclasts plated on tissue culture treated dishes. Statistical significance of actin ring area was assessed by non-parametric paired T test: **: P<0.01 in comparison to osteoclast alone.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038199-g004: TcREG suppress mature osteoclasts by effecting the cytoskeletal reorganization:Osteoclasts were cultured either alone or with TcREG on bovine bone chips for 24 hrs. T-cells were then removed, and osteoclasts were stained with phalloidin-Texas Red to visualize actin rings. Representative images are shown in panel A. Quantitation of three independent experiments is shown in panel B. Panel C is quantitation of phalloidin staining of osteoclasts plated on tissue culture treated dishes. Statistical significance of actin ring area was assessed by non-parametric paired T test: **: P<0.01 in comparison to osteoclast alone.
Mentions: Osteoclasts must adhere to the bone surface and migrate along it to resorb [66]; cytoskeletal reorganization is critical for this process [67], [68]. Therefore, we stained for actin rings in osteoclasts in the presence and absence of TcREG. TcREG were cultured with mature osteoclasts seeded on bone, or on plastic for 24 h. T-cells were then removed and the actin rings were visualized using flour-conjugated phalloidin. In the presence of TcREG, the actin ring was either eliminated on bone (Fig. 4A and B), or (the actin belt) was reduced in size on plastic (Fig. 4C).

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