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 resorption.A. Osteoclasts (day 3) lifted and plated on hydroxyapatite-coated plates. OT-I TcREG or OT-II TGFβ-induced FoxP3+ CD4 T-cells (iTREG) generated in separate dishes were added next day. The co-cultures were re-fed every two days. After seven days, the wells were treated with bleach, photographed, and total pit area was quantified (results in panel B). Representative images from five replicates are shown in panel A. No pitting was observed in the presence of TcREG (top right). Larger pits were observed in the presence of iTREG (bottom left), but TcREG were dominant suppressors (bottom right). B. TcREG suppressed pitting on hydroxyapatite plates without re-stimulation. In contrast, iTREG could only suppress after re-stimulation (see methods for details). IFN-γ producing OT-I T-cells activated by anti-CD3 and anti-CD28 in the presence of IL-2 could partially suppress pitting by osteoclasts. Activated GFP+ CD8 T-cells purified by cell sorting from FoxP3eGFP reporter mice could also suppress osteoclast pitting, while conventional (GFP−) CD8 had no affect on pitting. Statistical significance of area resorbed was assessed by Wilcoxon test: *: P<0.05, **: P<0.01 and ***: P<0.001 relative to osteoclast alone wells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368916&req=5

pone-0038199-g002: TcREG inhibit osteoclast resorption.A. Osteoclasts (day 3) lifted and plated on hydroxyapatite-coated plates. OT-I TcREG or OT-II TGFβ-induced FoxP3+ CD4 T-cells (iTREG) generated in separate dishes were added next day. The co-cultures were re-fed every two days. After seven days, the wells were treated with bleach, photographed, and total pit area was quantified (results in panel B). Representative images from five replicates are shown in panel A. No pitting was observed in the presence of TcREG (top right). Larger pits were observed in the presence of iTREG (bottom left), but TcREG were dominant suppressors (bottom right). B. TcREG suppressed pitting on hydroxyapatite plates without re-stimulation. In contrast, iTREG could only suppress after re-stimulation (see methods for details). IFN-γ producing OT-I T-cells activated by anti-CD3 and anti-CD28 in the presence of IL-2 could partially suppress pitting by osteoclasts. Activated GFP+ CD8 T-cells purified by cell sorting from FoxP3eGFP reporter mice could also suppress osteoclast pitting, while conventional (GFP−) CD8 had no affect on pitting. Statistical significance of area resorbed was assessed by Wilcoxon test: *: P<0.05, **: P<0.01 and ***: P<0.001 relative to osteoclast alone wells.

Mentions: Our results (Fig. 2A, B) show that the in situ produced TcREG suppressed osteoclast resorption activity. We also tested TGFβ-induced OT-II (CD4) TREG (iTREG) in the co-culture assay. Whereas, in the presence of the induced OT-II TREG osteoclast resorption activity modestly increased (1.5-fold in area resorbed), re-stimulation of the TREG with anti-CD3 + anti-CD28 was required to observe suppression. In contrast, the osteoclast-induced TcREG could suppress in the absence of re-stimulation. We also tested if CD8 T-cells when activated by anti-CD3 and anti-CD28 in the presence of IL-2 could suppress resorption by osteoclasts. While CD8 T-cells activated in this manner produce significant levels of IFN-γ, they were only able to partially suppress (∼25% suppression) pitting by osteoclasts. Finally, we tested if invivo generated TcREG could suppress osteoclast activity. TcREG were purified from the bone marrow space by cell sorting to >95% purity, then co-cultured with osteoclasts (at a ratio of 3 osteoclasts to 1 GFP+ T-cell) on hydroxyapatite-coated plates. The CD8+ GFP+ TcREG population was able to efficiently suppress osteoclast resorption activity, whereas the CD8+ GFP− population did not (Fig. 2B).


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 resorption.A. Osteoclasts (day 3) lifted and plated on hydroxyapatite-coated plates. OT-I TcREG or OT-II TGFβ-induced FoxP3+ CD4 T-cells (iTREG) generated in separate dishes were added next day. The co-cultures were re-fed every two days. After seven days, the wells were treated with bleach, photographed, and total pit area was quantified (results in panel B). Representative images from five replicates are shown in panel A. No pitting was observed in the presence of TcREG (top right). Larger pits were observed in the presence of iTREG (bottom left), but TcREG were dominant suppressors (bottom right). B. TcREG suppressed pitting on hydroxyapatite plates without re-stimulation. In contrast, iTREG could only suppress after re-stimulation (see methods for details). IFN-γ producing OT-I T-cells activated by anti-CD3 and anti-CD28 in the presence of IL-2 could partially suppress pitting by osteoclasts. Activated GFP+ CD8 T-cells purified by cell sorting from FoxP3eGFP reporter mice could also suppress osteoclast pitting, while conventional (GFP−) CD8 had no affect on pitting. Statistical significance of area resorbed was assessed by Wilcoxon test: *: P<0.05, **: P<0.01 and ***: P<0.001 relative to osteoclast alone wells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038199-g002: TcREG inhibit osteoclast resorption.A. Osteoclasts (day 3) lifted and plated on hydroxyapatite-coated plates. OT-I TcREG or OT-II TGFβ-induced FoxP3+ CD4 T-cells (iTREG) generated in separate dishes were added next day. The co-cultures were re-fed every two days. After seven days, the wells were treated with bleach, photographed, and total pit area was quantified (results in panel B). Representative images from five replicates are shown in panel A. No pitting was observed in the presence of TcREG (top right). Larger pits were observed in the presence of iTREG (bottom left), but TcREG were dominant suppressors (bottom right). B. TcREG suppressed pitting on hydroxyapatite plates without re-stimulation. In contrast, iTREG could only suppress after re-stimulation (see methods for details). IFN-γ producing OT-I T-cells activated by anti-CD3 and anti-CD28 in the presence of IL-2 could partially suppress pitting by osteoclasts. Activated GFP+ CD8 T-cells purified by cell sorting from FoxP3eGFP reporter mice could also suppress osteoclast pitting, while conventional (GFP−) CD8 had no affect on pitting. Statistical significance of area resorbed was assessed by Wilcoxon test: *: P<0.05, **: P<0.01 and ***: P<0.001 relative to osteoclast alone wells.
Mentions: Our results (Fig. 2A, B) show that the in situ produced TcREG suppressed osteoclast resorption activity. We also tested TGFβ-induced OT-II (CD4) TREG (iTREG) in the co-culture assay. Whereas, in the presence of the induced OT-II TREG osteoclast resorption activity modestly increased (1.5-fold in area resorbed), re-stimulation of the TREG with anti-CD3 + anti-CD28 was required to observe suppression. In contrast, the osteoclast-induced TcREG could suppress in the absence of re-stimulation. We also tested if CD8 T-cells when activated by anti-CD3 and anti-CD28 in the presence of IL-2 could suppress resorption by osteoclasts. While CD8 T-cells activated in this manner produce significant levels of IFN-γ, they were only able to partially suppress (∼25% suppression) pitting by osteoclasts. Finally, we tested if invivo generated TcREG could suppress osteoclast activity. TcREG were purified from the bone marrow space by cell sorting to >95% purity, then co-cultured with osteoclasts (at a ratio of 3 osteoclasts to 1 GFP+ T-cell) on hydroxyapatite-coated plates. The CD8+ GFP+ TcREG population was able to efficiently suppress osteoclast resorption activity, whereas the CD8+ GFP− population did not (Fig. 2B).

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