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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 produce IL-10, IL-6, and IFN-γ.Mature osteoclasts (day 4) or osteoclast precursors were cultured with no Ag or OVA protein and then used to prime CD8 T-cells from an OT-I mouse. A. T-cells were collected at 48 h following initiation of co-culture. T-cells were stained for CD44, CD25 and FoxP3 and then analyzed by flow cytometry. T-cells co-cultured with osteoclasts in the absence of antigen do not express FoxP3+ or CD25 (top); FoxP3 and CD25 were induced in the presence of antigen as shown in the representative flow plot. The expression of FoxP3 was confirmed by reverse-transcription of RNA isolated from the co-culture and subsequent PCR of cDNA. GFP sorted cells from FoxP3eGFP reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of TcREG (right panel). B. Anti-mouse TGFβ was added to co-cultures at the dose indicated (left). Addition of recombinant murine TGFβ1 to co-cultures of CD8 T-cells and osteoclasts at concentration indicated (right). The percent of input T-cells converted to FoxP3+ are plotted in both panels. No statistically significant effect was observed on TcREG induction with either the addition of neutralizing antibody or recombinant TGFβ. C. Media was collected and cytokine quantitated by multiplexed ELISA. After 48 h of co-culture, cells were treated with Golgi stop and PMA plus ionomycin for 6 h. The cells were permeabilized, stained and evaluated for cytokine production by flow cytometry. While the CD11b+ osteoclasts were negative for all cytokines, the CD8+ T-cells stained triple positive for IL-10, IL-6, and IFN-γ. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01, ***: P<0.001 and ****: P<0.0001.
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pone-0038199-g001: Osteoclast-induced TcREG produce IL-10, IL-6, and IFN-γ.Mature osteoclasts (day 4) or osteoclast precursors were cultured with no Ag or OVA protein and then used to prime CD8 T-cells from an OT-I mouse. A. T-cells were collected at 48 h following initiation of co-culture. T-cells were stained for CD44, CD25 and FoxP3 and then analyzed by flow cytometry. T-cells co-cultured with osteoclasts in the absence of antigen do not express FoxP3+ or CD25 (top); FoxP3 and CD25 were induced in the presence of antigen as shown in the representative flow plot. The expression of FoxP3 was confirmed by reverse-transcription of RNA isolated from the co-culture and subsequent PCR of cDNA. GFP sorted cells from FoxP3eGFP reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of TcREG (right panel). B. Anti-mouse TGFβ was added to co-cultures at the dose indicated (left). Addition of recombinant murine TGFβ1 to co-cultures of CD8 T-cells and osteoclasts at concentration indicated (right). The percent of input T-cells converted to FoxP3+ are plotted in both panels. No statistically significant effect was observed on TcREG induction with either the addition of neutralizing antibody or recombinant TGFβ. C. Media was collected and cytokine quantitated by multiplexed ELISA. After 48 h of co-culture, cells were treated with Golgi stop and PMA plus ionomycin for 6 h. The cells were permeabilized, stained and evaluated for cytokine production by flow cytometry. While the CD11b+ osteoclasts were negative for all cytokines, the CD8+ T-cells stained triple positive for IL-10, IL-6, and IFN-γ. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01, ***: P<0.001 and ****: P<0.0001.

Mentions: We first show (Fig. 1A) that mature osteoclasts can induce FoxP3 and CD25 in ovalbumin (OVA)-specific OT-I CD8 T-cells only in the presence of antigen. The induction of FoxP3 in the presence of antigen was assayed by both multicolor flow cytometry (top panels) and by reverse transcription (RT) of RNA isolated from the co-culture, followed by PCR (middle panel). As controls, we isolated RNA from purified GFP positive and negative CD4 and CD8 T-cells from the transgenic FoxP3eGFP reporter mouse [52], [53]. These reporter mice co-express the enhanced green fluorescent protein (eGFP) and transcription factor FoxP3 under control of the endogenous promoter. We show that only mature osteoclasts (incubated in the presence of RANKL for 4 days) have the ability to induce FoxP3 as the osteoclast precursors (bone marrow derived monocytes) could not induce the regulatory phenotype in CD8 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 produce IL-10, IL-6, and IFN-γ.Mature osteoclasts (day 4) or osteoclast precursors were cultured with no Ag or OVA protein and then used to prime CD8 T-cells from an OT-I mouse. A. T-cells were collected at 48 h following initiation of co-culture. T-cells were stained for CD44, CD25 and FoxP3 and then analyzed by flow cytometry. T-cells co-cultured with osteoclasts in the absence of antigen do not express FoxP3+ or CD25 (top); FoxP3 and CD25 were induced in the presence of antigen as shown in the representative flow plot. The expression of FoxP3 was confirmed by reverse-transcription of RNA isolated from the co-culture and subsequent PCR of cDNA. GFP sorted cells from FoxP3eGFP reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of TcREG (right panel). B. Anti-mouse TGFβ was added to co-cultures at the dose indicated (left). Addition of recombinant murine TGFβ1 to co-cultures of CD8 T-cells and osteoclasts at concentration indicated (right). The percent of input T-cells converted to FoxP3+ are plotted in both panels. No statistically significant effect was observed on TcREG induction with either the addition of neutralizing antibody or recombinant TGFβ. C. Media was collected and cytokine quantitated by multiplexed ELISA. After 48 h of co-culture, cells were treated with Golgi stop and PMA plus ionomycin for 6 h. The cells were permeabilized, stained and evaluated for cytokine production by flow cytometry. While the CD11b+ osteoclasts were negative for all cytokines, the CD8+ T-cells stained triple positive for IL-10, IL-6, and IFN-γ. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01, ***: P<0.001 and ****: P<0.0001.
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Related In: Results  -  Collection

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pone-0038199-g001: Osteoclast-induced TcREG produce IL-10, IL-6, and IFN-γ.Mature osteoclasts (day 4) or osteoclast precursors were cultured with no Ag or OVA protein and then used to prime CD8 T-cells from an OT-I mouse. A. T-cells were collected at 48 h following initiation of co-culture. T-cells were stained for CD44, CD25 and FoxP3 and then analyzed by flow cytometry. T-cells co-cultured with osteoclasts in the absence of antigen do not express FoxP3+ or CD25 (top); FoxP3 and CD25 were induced in the presence of antigen as shown in the representative flow plot. The expression of FoxP3 was confirmed by reverse-transcription of RNA isolated from the co-culture and subsequent PCR of cDNA. GFP sorted cells from FoxP3eGFP reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of TcREG (right panel). B. Anti-mouse TGFβ was added to co-cultures at the dose indicated (left). Addition of recombinant murine TGFβ1 to co-cultures of CD8 T-cells and osteoclasts at concentration indicated (right). The percent of input T-cells converted to FoxP3+ are plotted in both panels. No statistically significant effect was observed on TcREG induction with either the addition of neutralizing antibody or recombinant TGFβ. C. Media was collected and cytokine quantitated by multiplexed ELISA. After 48 h of co-culture, cells were treated with Golgi stop and PMA plus ionomycin for 6 h. The cells were permeabilized, stained and evaluated for cytokine production by flow cytometry. While the CD11b+ osteoclasts were negative for all cytokines, the CD8+ T-cells stained triple positive for IL-10, IL-6, and IFN-γ. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01, ***: P<0.001 and ****: P<0.0001.
Mentions: We first show (Fig. 1A) that mature osteoclasts can induce FoxP3 and CD25 in ovalbumin (OVA)-specific OT-I CD8 T-cells only in the presence of antigen. The induction of FoxP3 in the presence of antigen was assayed by both multicolor flow cytometry (top panels) and by reverse transcription (RT) of RNA isolated from the co-culture, followed by PCR (middle panel). As controls, we isolated RNA from purified GFP positive and negative CD4 and CD8 T-cells from the transgenic FoxP3eGFP reporter mouse [52], [53]. These reporter mice co-express the enhanced green fluorescent protein (eGFP) and transcription factor FoxP3 under control of the endogenous promoter. We show that only mature osteoclasts (incubated in the presence of RANKL for 4 days) have the ability to induce FoxP3 as the osteoclast precursors (bone marrow derived monocytes) could not induce the regulatory phenotype in CD8 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