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

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.

Figure 1. 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 FoxP3^eGFP reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of Tc_REG (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 Tc_REG 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.

Figure 2. TcREG inhibit osteoclast resorption.

A. Osteoclasts (day 3) lifted and plated on hydroxyapatite-coated plates. OT-I Tc_REG or OT-II TGFβ-induced FoxP3⁺ CD4 T-cells (iT_REG) 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 Tc_REG (top right). Larger pits were observed in the presence of iT_REG (bottom left), but Tc_REG were dominant suppressors (bottom right). B. Tc_REG suppressed pitting on hydroxyapatite plates without re-stimulation. In contrast, iT_REG 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 FoxP3^eGFP 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.

Figure 3. Osteoclast-induced Tc_REG inhibit osteoclast differentiation, but not survival.

A. Osteoclast precursor cells (day 0) were cultured alone, with pre-differentiated Tc_REG, iT_REG, anti-mouse CD3/CD28 re-stimulated iT_REG, 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 Tc_REG 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.

Figure 4. Tc_REG suppress mature osteoclasts by effecting the cytoskeletal reorganization:

Osteoclasts were cultured either alone or with Tc_REG 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.

Figure 5. Tc_REG 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 Tc_REG 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. Tc_REG 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 Tc_REG 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.