Limits...
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.

Show MeSH

Related in: MedlinePlus

TcREG inhibit osteoclast activity in an antigen- and contact-independent manner by secreted cytokines.A. Osteoclasts were seeded on hydroxyapatite-coated plates and allowed to adhere overnight. The osteoclasts were pulsed with SIINFEKL peptide (+Antigen) or a control FLAG peptide (-Antigen). OT-I TcREG or naïve CD8 T-cells were added and co-cultured for 7 days. Cells were re-fed with medium containing M-CSF and RANKL every 2 to 3 days. The plates were then treated with bleach solution, washed, dried and photographed. Representative photomicrographs are shown on the left. Quantitation from four experiments of three wells each is shown on the left. B. TcREG were added to top-insert of transwell (0.45-µ membrane) separated from the osteoclast plated on 24-well Corning Osteo-Assay plate. Osteoclast activity was determined by quantifying total pit area resorbed following 10 days of co-incubation. C. In a culture of both osteoclast and TcREG on Corning Osteo-Assay plates, neutralizing antibodies against IL-10 (25 µg/ml), IFN-γ (50 µg/ml), IL-6 (20 µg/ml) or CTLA-4 (10 µg/ml) were added to determine their impact on osteoclast activity. The cells were co-cultured for 10 additional days, and re-fed with media containing M-CSF, RANKL every three days. Re-feed media with antibodies were added on days 3 and 6. D. Pitting assay as described in Panel C were conducted in parallel using osteoclasts from either wild type (WT) or IFNγR1−/− mice. Statistical significance of area resorbed was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 in comparison to osteoclast alone wells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368916&req=5

pone-0038199-g005: TcREG inhibit osteoclast activity in an antigen- and contact-independent manner by secreted cytokines.A. Osteoclasts were seeded on hydroxyapatite-coated plates and allowed to adhere overnight. The osteoclasts were pulsed with SIINFEKL peptide (+Antigen) or a control FLAG peptide (-Antigen). OT-I TcREG or naïve CD8 T-cells were added and co-cultured for 7 days. Cells were re-fed with medium containing M-CSF and RANKL every 2 to 3 days. The plates were then treated with bleach solution, washed, dried and photographed. Representative photomicrographs are shown on the left. Quantitation from four experiments of three wells each is shown on the left. B. TcREG were added to top-insert of transwell (0.45-µ membrane) separated from the osteoclast plated on 24-well Corning Osteo-Assay plate. Osteoclast activity was determined by quantifying total pit area resorbed following 10 days of co-incubation. C. In a culture of both osteoclast and TcREG on Corning Osteo-Assay plates, neutralizing antibodies against IL-10 (25 µg/ml), IFN-γ (50 µg/ml), IL-6 (20 µg/ml) or CTLA-4 (10 µg/ml) were added to determine their impact on osteoclast activity. The cells were co-cultured for 10 additional days, and re-fed with media containing M-CSF, RANKL every three days. Re-feed media with antibodies were added on days 3 and 6. D. Pitting assay as described in Panel C were conducted in parallel using osteoclasts from either wild type (WT) or IFNγR1−/− mice. Statistical significance of area resorbed was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 in comparison to osteoclast alone wells.

Mentions: To test if the regulation of osteoclast activity was antigen-dependent, OT-I FoxP3+ T-cells were cultured with osteoclasts in the presence or absence of OVA peptide. As shown in Fig. 5A, TcREG could suppress osteoclasts to similar levels in the presence or absence of antigen presentation. Therefore, we next tested if direct contact between osteoclasts and TcREG was required to mediate suppression. TcREG were able to suppress osteoclast activity to a significant level when separated by a membrane with a 0.45 μ pore (Fig. 5B). These results indicated that the secreted cytokines were, to a large extent, responsible for the suppressive activity.


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 inhibit osteoclast activity in an antigen- and contact-independent manner by secreted cytokines.A. Osteoclasts were seeded on hydroxyapatite-coated plates and allowed to adhere overnight. The osteoclasts were pulsed with SIINFEKL peptide (+Antigen) or a control FLAG peptide (-Antigen). OT-I TcREG or naïve CD8 T-cells were added and co-cultured for 7 days. Cells were re-fed with medium containing M-CSF and RANKL every 2 to 3 days. The plates were then treated with bleach solution, washed, dried and photographed. Representative photomicrographs are shown on the left. Quantitation from four experiments of three wells each is shown on the left. B. TcREG were added to top-insert of transwell (0.45-µ membrane) separated from the osteoclast plated on 24-well Corning Osteo-Assay plate. Osteoclast activity was determined by quantifying total pit area resorbed following 10 days of co-incubation. C. In a culture of both osteoclast and TcREG on Corning Osteo-Assay plates, neutralizing antibodies against IL-10 (25 µg/ml), IFN-γ (50 µg/ml), IL-6 (20 µg/ml) or CTLA-4 (10 µg/ml) were added to determine their impact on osteoclast activity. The cells were co-cultured for 10 additional days, and re-fed with media containing M-CSF, RANKL every three days. Re-feed media with antibodies were added on days 3 and 6. D. Pitting assay as described in Panel C were conducted in parallel using osteoclasts from either wild type (WT) or IFNγR1−/− mice. Statistical significance of area resorbed was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 in comparison to osteoclast alone wells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038199-g005: TcREG inhibit osteoclast activity in an antigen- and contact-independent manner by secreted cytokines.A. Osteoclasts were seeded on hydroxyapatite-coated plates and allowed to adhere overnight. The osteoclasts were pulsed with SIINFEKL peptide (+Antigen) or a control FLAG peptide (-Antigen). OT-I TcREG or naïve CD8 T-cells were added and co-cultured for 7 days. Cells were re-fed with medium containing M-CSF and RANKL every 2 to 3 days. The plates were then treated with bleach solution, washed, dried and photographed. Representative photomicrographs are shown on the left. Quantitation from four experiments of three wells each is shown on the left. B. TcREG were added to top-insert of transwell (0.45-µ membrane) separated from the osteoclast plated on 24-well Corning Osteo-Assay plate. Osteoclast activity was determined by quantifying total pit area resorbed following 10 days of co-incubation. C. In a culture of both osteoclast and TcREG on Corning Osteo-Assay plates, neutralizing antibodies against IL-10 (25 µg/ml), IFN-γ (50 µg/ml), IL-6 (20 µg/ml) or CTLA-4 (10 µg/ml) were added to determine their impact on osteoclast activity. The cells were co-cultured for 10 additional days, and re-fed with media containing M-CSF, RANKL every three days. Re-feed media with antibodies were added on days 3 and 6. D. Pitting assay as described in Panel C were conducted in parallel using osteoclasts from either wild type (WT) or IFNγR1−/− mice. Statistical significance of area resorbed was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 in comparison to osteoclast alone wells.
Mentions: To test if the regulation of osteoclast activity was antigen-dependent, OT-I FoxP3+ T-cells were cultured with osteoclasts in the presence or absence of OVA peptide. As shown in Fig. 5A, TcREG could suppress osteoclasts to similar levels in the presence or absence of antigen presentation. Therefore, we next tested if direct contact between osteoclasts and TcREG was required to mediate suppression. TcREG were able to suppress osteoclast activity to a significant level when separated by a membrane with a 0.45 μ pore (Fig. 5B). These results indicated that the secreted cytokines were, to a large extent, responsible for the suppressive activity.

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