Osteoimmunology arose from the recognition that cytokines produced by lymphocytes can affect bone homeostasis. We have previously shown that osteoclasts, cells that resorb bone, act as APCs. Cross-presentation of Ags by osteoclasts leads to expression of CD25 and Foxp3, markers of regulatory T cells in the CD8 T cells. Octeoclast-induced Foxp3+ CD25+ regulatory CD8 T cells (OC-iTcREG) suppress priming of CD4 and CD8 T cells by dendritic cells. OC-iTcREG also limit bone resorption by osteoclasts, forming a negative feedback loop. In this study, we show that OC-iTcREG express concurrently T-bet and Eomesodermin (Eomes) and IFN-γ. Pharmacological inhibition of IκK blocked IFN-γ, T-bet, and Eomes production by TcREG. Furthermore, we show, using chromatin immunoprecipitation, NF-κB enrichment in the T-bet and Eomes promoters. We demonstrate that IFN-γ produced by TcREG is required for suppression of osteoclastogenesis and for degradation of TNFR-associated factor 6 in osteoclast precursors. The latter prevents signaling by receptor activator of NF-κB ligand needed for osteoclastogenesis. Knockout of IFN-γ rendered TcREG inefficient in preventing actin ring formation in osteoclasts, a process required for bone resorption. TcREG generated in vivo using IFN-γ−/− T cells had impaired ability to protect mice from bone resorption and bone loss in response to high-dose receptor activator of NF-κB ligand. The results of this study demonstrate a novel link between NF-κB signaling and induction of IFN-γ in TcREG and establish an important role for IFN-γ in TcREG-mediated protection from bone loss.

Homeostasis describes the mechanisms by which biological systems maintain stability in response to environmental and internal perturbations. Homeostasis in the immune system, needed to minimize damage to healthy tissues, is achieved by a number of mechanisms. Such mechanisms include a balance between effector and regulatory cells. Disruption of this balance leads to autoimmune and inflammatory diseases (1, 2).

Bone homeostasis is maintained by a balance between bone-forming and bone-resorbing activities of osteoblasts and osteoclasts, respectively. This process is tightly regulated by a number of factors, including hormonal changes, mechanical loading, and diet. It has long been noticed that many chronic inflammatory diseases, for example, rheumatoid arthritis, are accompanied by an increase in bone loss (3, 4). Proinflammatory cytokines such as TNF-α and IL-17 have been linked to the promotion of bone catabolism by increasing osteoclastogenesis via upregulation of receptor activator of NF-κB ligand (RANKL) in stromal cells, a cytokine essential for the development and activation of osteoclasts (58). The observation that proinflammatory cytokines produced by activated lymphocytes can perturb bone-remodeling cycle gave rise to the study of osteoimmunology, an emerging field studying the crosstalk between skeletal and immune systems (911).

We have previously identified a novel regulatory mechanism forming a feedback loop between osteoclasts and CD8 T cells (12, 13). Primed by osteoclasts, CD8 T cells differentiate into regulatory T cells expressing CD25 and transcription factor Foxp3 (14), hallmarks of the regulatory cell phenotype (15). Foxp3+ CD25+ regulatory CD8 T cells (TcREG) suppress bone resorption caused by osteoclasts in cell culture (16) and, by administration of high-dose RANKL, in ovariectomized mice, a murine model of postmenopausal-induced osteoporosis (17, 18). T cells produce RANKL upon activation, thus directly inducing osteoclastogenesis and bone loss (19). In contrast, TcREG inhibit bone loss despite the fact that they also express RANKL (14). This discrepancy implies that other functions of TcREG overcome proresorptive effect of their activation, shifting the balance toward bone protection. We, and others, have shown that, similar to classical Foxp3+ CD4 T regulatory cells, regulatory CD8 cells have immunosuppressive activity (2023). Osteoclast-induced TcREG (OC-iTcREG) inhibit proliferation of effector T cells in vitro (14, 17) and reduce the number of effector T cells in the bone marrow of ovariectomized mice (17, 18). Therefore, TcREG directly regulate the levels of osteoclastogenic cytokine-producing cells in the bone marrow, providing a mechanism for TcREG-mediated inhibition of the bone loss (24).

In addition to their immunoregulatory function, TcREG express IFN-γ (14). IFN-γ is usually viewed as a proinflammatory cytokine due to its strong macrophage-activating potential and the ability to drive differentiation of naive CD4 T cells toward a Th1 phenotype (25). However, accumulating evidence now reveals a more complex, bidirectional role for IFN-γ in autoimmune diseases (26). Anti-inflammatory properties of IFN-γ have been demonstrated in inflammatory disorders, such as collagen-induced arthritis (27), experimental autoimmune encephalomyelitis (28), asthma (29), autoimmune myocarditis (30), and others (26). In the context of bone remodeling, IFN-γ was reported to suppress osteoclastogenesis via degradation of receptor activator for NF-κB adapter protein TNFR-associated factor 6 (TRAF6) (31, 32) and induction of apoptosis by Fas–Fas ligand interaction (33). In contrast, IFN-γ stimulates Ag-dependent T cell activation and production of osteoclastogenic factors RANKL and TNF-α (34, 35). It was concluded that in vivo the net effect of these two opposing qualities of IFN-γ is biased toward promoting bone resorption (34).

In this study, we sought to characterize the role of IFN-γ in the context of its production by TcREG. We show that expression of the transcription factors T-bet and Eomesodermin (Eomes) correlates with expression of IFN-γ in CD8 T cells primed by osteoclasts. NF-κB was found to positively regulate expression of T-bet and, to a lesser degree, Eomes in TcREG. We demonstrate that IFN-γ contributes to TcREG-mediated inhibition of osteoclastogenesis through a degradation of TRAF6 in osteoclast precursors and suppression of actin ring formation required for osteoclast-resorbing activity. Upon withdrawal of TcREG, we show that actin ring formation is restored in osteoclasts. Finally, we demonstrate that IFN-γ−/− T cells protect less significantly from RANKL-induced bone loss as compared with wild-type (WT) T cells. Taken together, our results provide further characterization of TcREG population and demonstrate a significant role of IFN-γ in regulation of osteoclastogenesis and osteoclast resorption by these cells.

Five- to 10-wk-old C57BL/6 mice from in-house breeding colonies were used for generation of osteoclasts and polyclonal CD8 T cells. OT-I Rag−/− and OT-II Rag−/− mice were purchased from Taconic (model number 4175 and 1896; Germantown, NY). IFN-γ knockout (IFN-γ−/−) mice were from Jackson Laboratories (stock 002287). All animals were maintained in the Department of Comparative Medicine, Saint Louis University School of Medicine, in accordance with institutional and Public Health Service Guidelines. For adoptive transfer experiments, CD8 T cells were isolated from donor mouse bone marrow and spleen (see below) and transferred to a recipient mouse at 1:1 ratio via tail vein in 100 μl PBS.

Osteoclast precursors were isolated, as previously described (18). Briefly, femurs and tibia were harvested after mice were euthanized by CO2 asphyxiation. Each bone was placed in a 0.7-ml microcentrifuge tube that was pierced with a 22 G needle at the bottom. The 0.7-ml tube was placed inside a 1.5-ml microcentrifuge tube and spun for 30 s at 16,000 × g. The bone marrow cells were resuspended and maintained in α-MEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Invitrogen), penicillin-streptomycin-glutamine (Invitrogen), murine rM-CSF (eBioscience, San Diego, CA) at 20 ng/ml, and recombinant murine GST-RANKL (the expression system for GST-RANKL was a gift of S. Teitelbaum, Washington University in St. Louis) at 50 ng/ml. Treatment with Versene (Life Technologies, Carlsbad, CA) for 10 min was used to harvest cells.

Splenocytes were isolated, as previously described (18). Briefly, single-cell suspensions of spleens were prepared in PBS plus 1% FBS and filtered through a 40-μm cell strainer. CD8 T cells were prepared by first enriching for T cells using Pan-T cell beads and then purified by negative selection using appropriate magnetic beads (Miltenyi Biotec, Auburn, CA). To generate TcREG, day 3 osteoclasts were plated at 5 × 105 cells/well in 24-well tissue culture plates (Corning, Corning, NY). Next day, osteoclasts were pulsed with a peptide derived from residues 257–264 (SIINFEKL) of OVA (5 μM; AnaSpec) for 3 h. Freshly isolated, 2.5 × 105 cells/well OT-I splenic CD8 T cells were then added in 2 ml RPMI 1640 supplemented with 10% heat-inactivated FBS, penicillin-streptomycin-glutamine, nonessential amino acids, 10 mM HEPES, and 55 μM 2-ME. M-CSF and RANKL were added at 20 and 100 ng/ml, respectively. For polyclonal CD8 T cells from C57BL/6 mice α-CD3 Ab at 1 μg/ml was used instead of OVA peptide and was added to the T cells before plating them on osteoclasts. In some experiments, γ-secretase inhibitor DAPT and IKK inhibitor IKK-16 (both from Selleckchem) were added to the media at 10 μM concentration.

Whole-cell lysates from TcREG were prepared, according to manufacturer’s instructions (9803; Cell Signaling, Danvers, MA). Proteins were resolved on NuPAGE 4–12% Bis-Tris gel (Life Technologies), transferred on a nitrocellulose membrane, and blocked in TBS containing 5% BSA and 0.1% Tween 20. The membranes were probed overnight with primary Ab against TRAF6 (Millipore) and β-actin (Cell Signaling). Membranes were washed and incubated for 1 h with HRP-labeled secondary Ab (Cell Signaling).

Anti-mouse Abs used for cell staining were from BD Biosciences: CD45-BV711, Foxp3-e450, and CD8-R700; BioLegend: CD3-FITC; and eBiosciences: Tbet-PE, Eomes-PECy7, IFN-γ allophycocyanin, IL-6 FITC, and IL-10 PE. Protein transport inhibitor (1 μg/ml, GolgiStop; BD Biosciences) was added to the medium 3 h prior to collecting the cells for staining to prevent IFN-γ secretion.

For FACS, cells were incubated with anti-mouse FcgRIII/IIR (Fc-block; BD Pharmingen) for 10 min and then stained for 45 min on ice with fluorophore-conjugated Ab. Stained cells were washed, fixed with 1% paraformaldehyde, and analyzed on LSRII (BD Biosciences) instrument. Data were analyzed using FlowJo software (version 8.73; Tree Star, Ashland, OR).

For osteoclast differentiation experiment, control T cells and TcREG made from WT C57BL/6 or IFN-γ−/− mice were cocultured with osteoclast precursors in the presence of M-CSF and RANKL. After 5 d, the T cells were removed and adherent cells were stained with a fluorescent substrate Elf-97 (Invitrogen) for tartrate-resistant acid phosphatase (TRAP) activity in accordance with the manufacturer’s instructions. In some experiments, rIFN-γ (provided by M. Buller, Saint Louis University) was added to the medium at 20 or 100 U/ml at the time of adding T cells.

For actin ring assay, day 3 osteoclasts were plated on bovine bone slices at 3 × 103 cells/well in a 48-well plate. Control naive T cells and TcREG were added at 3 × 103 cells/well on day 5. After 24-h coculture, T cells were removed by aspiration and adherent cells were fixed (4% para-formaldehyde [EMS], 0.2% Triton X-100 [Sigma-Aldrich] in PBS) for 10 min, washed three times with PBS, and then stained with Alexa Fluor 488–conjugated phalloidin (ThermoFisher Scientific) for 15 min. The cells were photographed, and actin ring numbers and sizes were blind scored.

Serum levels of C-terminal cross-linking telopeptide of type I collagen (CTX) were measured as a marker of bone resorption (36) by ELISA, according to the manufacturer’s instructions (Immunodiagnostic Systems). Food was withdrawn for 6–10 h prior to collecting blood via submandibular vein. After 1-h incubation at room temperature, clotted blood was spun to obtain serum.

To measure bone volume density over total volume (BV/TV), the bones were scanned in μCT 40 (Scanco Medical) at 55 kVp, 145 μA, and a resolution of 16 μm. Gauss σ of 1.2, Gauss support of 2, lower threshold of 237, and upper threshold of 1000 were used for all the analyses. Regions of interest were selected 50 slices below the growth plate of proximal tibia to evaluate the trabecular compartment. BV/TV was obtained by quantitative μCT using Scanco Phantoms for calibration (37).

Chromatin preparation and immunoprecipitation were done, as previously described (38). Briefly, TcREG collected at 24 h or freshly isolated naive CD8 T cells were fixed for 10 min at room temperature with 1% formaldehyde at 2 × 106 cells/ml in α-MEM. Fixation was stopped by addition of glycine to 0.125 M. Fixed cells were centrifuged at 1000 × g for 5 min at 4°C, washed twice with cold PBS, frozen on dry ice, and stored at −80°C. For chromatin isolation, 5 × 106 fixed cells were resuspended in 300 μl sonication buffer (10 mM Tris HCl [pH 7.6], 0.4% NaDodSO4 [SDS]), containing freshly added protease inhibitors (Pierce; EDTA free), and incubated on ice for 20 min. The cell suspension was sonicated (Branson 250 Sonifier, tip 102) for 14–16 cycles (30 s on, 30 s off) at 4°C to shear the chromatin to an average DNA length of ∼400 bp as determined by gel electrophoresis after reversing the cross-links. A total of 900 μl adjustment buffer (10 mM Tris HCl [pH 7.6], 1.3 mM EDTA, 0.133% sodium deoxycholate, 1.33% Triton X-100) was added to cell lysates, followed by incubation on ice for 20 min, and then clarified by centrifugation at 16,000 × g for 10 min at 4°C. Glycerol was added to supernatant to 5% and snap frozen and stored at −80°C.

To perform chromatin immunoprecipitation (ChIP), 900 μl chromatin was precleared by incubating with 40 μl protein G dynabeads (Life Technologies; prewashed with PBS/0.5% BSA) for 1 h at 4°C. Precleared chromatin was incubated with anti–NF-κB p65 (Millipore), anti–T-bet (Santa Cruz), or anti-Eomes (Abcam) Ab overnight with gentle rotation. Prewashed protein G dynabeads (40 μl) were added to the chromatin–Ab mixture and rotated for 1.5 h at 4°C. The beads were washed twice at 4°C in the following: radioimmunoprecipitation assay buffer (10 mM Tris HCl [pH 7.6], 1 mM EDTA [pH 8.0], 0.1% sodium deoxycholate, 1% Triton X-100) and radioimmunoprecipitation assay buffer plus 0.3 M NaCl, LiCl buffer (0.25 M LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate). The beads were further washed in 10 mM Tris HCl (pH 7.6), 1 mM EDTA (pH 8.0), and 0.2% Triton X-100 and finally resuspended in Tris-EDTA (TE) buffer (10 mM Tris HCl [pH 7.6], 1 mM EDTA [pH 8.0]). To reverse the cross-links, the beads (150 μl in TE buffer) or input chromatin were incubated at 65°C overnight after addition of 5 μl 10% SDS and 7 μl proteinase K (20 mg/ml). The supernatant containing eluted DNA was collected, and the beads were washed once with 150 μl TE buffer containing 0.5 M NaCl and pooled with the first supernatant. The DNA was extracted with phenol:chloroform, precipitated with 20 μg glycogen, 0.5 vol 7.5 M ammonium acetate, and 2.5 vol ethanol. Precipitated DNA was dissolved in 30 μl 3 mM Tris HCl (pH 8.0) and stored at −20°C until further analysis. Enrichment of NF-κB p65, T-bet, and Eomes binding sites containing DNA sequences in the eluted DNA was determined by quantitative real-time PCR using Power SYBR Green PCR Master Mix (4367659; Applied Biosystems, Foster City, CA). Sequences of oligonucleotides and annealing temperatures used for quantitative PCR (qPCR) are shown in Table I. The average raw cycle threshold (CT) values are provided in Supplemental Table I, and the relative fold enrichment was calculated as follows: 2^(CT InputTcREG − CT ChIPTcREG)/2^(CT InputNaive − CT ChIPNaive). The ΔΔCT was further normalized to ADP-ribosylation factor 2 (Arf2) enrichment levels (oligonucleotides for Arf2 promoter qPCR: forward, 5′-AGGCTTATGTACCTCCCCGT-3′; reverse, 5′-CTGACTGACTGCAGCTCCAA-3′).

Table I.
Primers used for ChIP–qPCR
ChIP AbPromoterSiteForward PrimerReverse PrimerTa (°C)a
NF-κB p65 IFN-γ 5′-AGTGTGTGTCTAGTGCTGGA-3′ 5′-GTCAACGTGCCCAGAAAGAA-3′ 58 
IL-10 5′-ATTATGACCTGGGAGTGCGT-3′ 5′-TGTGGCTTTGGTAGTGCAAG-3′ 56 
T-bet 5′-GAGTTTCAGGTGGCAGGTTG-3′ 5′-TCCTGGGCTTTCTCTGCAG-3′ 58 
Eomes 5′-AGTCTCCCTCCTCTTCATACCT-3′ 5′-AGGCCATGAATTTGAGAGGGG-3′ 56 
5′-TAAGCCACGGAAAACCAGGC-3′ 5′-GGTGTCTCAGGCACACTTTAAAAT-3′ 56 
5′-GTCAGCCCGAGTTCTCTGAG-3′ 5′-AAGCTTTCCAACCTGGGTCC-3′ 56 
T-bet/Eomes IFN-γ CNS -22 5′-CCAGGACAGAGGTGTTAAGCCA-3′ 5′-GCAACTTCTTTCTTCTCAGGGTG-3′ 58 
CNS -34 5′-GGTATGCATCATCCCGGG-3′ 5′-TGGCCTGTCTTCAGAAGTTTGC-3′ 58 
IL-10 5′-GTTACACGTCTCCAAGGCTG-3′ 5′-GCAGTTGGTCAGAGGAGAGT-3′ 58 
ChIP AbPromoterSiteForward PrimerReverse PrimerTa (°C)a
NF-κB p65 IFN-γ 5′-AGTGTGTGTCTAGTGCTGGA-3′ 5′-GTCAACGTGCCCAGAAAGAA-3′ 58 
IL-10 5′-ATTATGACCTGGGAGTGCGT-3′ 5′-TGTGGCTTTGGTAGTGCAAG-3′ 56 
T-bet 5′-GAGTTTCAGGTGGCAGGTTG-3′ 5′-TCCTGGGCTTTCTCTGCAG-3′ 58 
Eomes 5′-AGTCTCCCTCCTCTTCATACCT-3′ 5′-AGGCCATGAATTTGAGAGGGG-3′ 56 
5′-TAAGCCACGGAAAACCAGGC-3′ 5′-GGTGTCTCAGGCACACTTTAAAAT-3′ 56 
5′-GTCAGCCCGAGTTCTCTGAG-3′ 5′-AAGCTTTCCAACCTGGGTCC-3′ 56 
T-bet/Eomes IFN-γ CNS -22 5′-CCAGGACAGAGGTGTTAAGCCA-3′ 5′-GCAACTTCTTTCTTCTCAGGGTG-3′ 58 
CNS -34 5′-GGTATGCATCATCCCGGG-3′ 5′-TGGCCTGTCTTCAGAAGTTTGC-3′ 58 
IL-10 5′-GTTACACGTCTCCAAGGCTG-3′ 5′-GCAGTTGGTCAGAGGAGAGT-3′ 58 
a

Ta is the annealing temperature used for PCR.

CNS, conserved nucleotide sequence (across multiple species) in the IFN-γ enhancer.

Statistical significance was assessed by paired two-tailed Mann-Whitney U test in GraphPad Prism 5. The p values <0.05 were considered significant.

IFN-γ was reported to negatively regulate osteoclastogenesis when directly added to osteoclast precursor culture (34) or when secreted by activated γδ T cells (39). CD8 T cells primed by osteoclasts express Foxp3 and IFN-γ as measured by intracellular fluorescent staining of TcREG (Fig. 1A). Additionally, we have previously demonstrated the presence of IFN-γ in the medium conditioned by TcREG (14, 16). To test whether TcREG-mediated inhibition of osteoclast differentiation is dependent on IFN-γ, we generated TcREG from WT or IFN-γ−/− mice and cocultured them with osteoclast precursors for 5 d in the presence of M-CSF and RANKL. After removal of nonadherent T cells, the adherent cells were stained for osteoclast-specific TRAP activity (Fig. 1B). WT TcREG significantly reduced osteoclast formation, whereas lack of IFN-γ partially rescued osteoclastogenesis, significantly increasing TRAP-specific fluorescence. IFN-γ produced by T cells was shown to mediate inhibition of osteoclastogenesis by causing degradation of receptor activator for NF-κB adapter protein TRAF6 (32). We found that TRAF6 was reduced in bone marrow macrophages 24 h after coculture with WT TcREG (Fig. 1C). Treatment with 20 or 100 U/ml rIFN-γ resulted in similar levels of TRAF6 inhibition. In contrast, IFN-γ−/− TcREG did not affect TRAF6. Together, these results demonstrate that one of the mechanisms used by TcREG to inhibit osteoclastogenesis is IFN-γ–mediated suppression of TRAF6 in osteoclast precursors.

FIGURE 1.

TcREG inhibit osteoclastogenesis via IFN-γ. (A) Representative flow cytometry plots showing that OT-I CD8 T cells primed by osteoclasts pulsed with SIINFEKL express Foxp3 and IFN-γ. T cells were collected for staining after 48-h coculture with osteoclasts. Protein transport inhibitor GolgiStop was added to the cultures during last 3 h of incubation. Cells were gated on CD45+CD3+CD8+ population to determine Foxp3 and IFN-γ induction. (B) IFN-γ−/− TcREG have reduced antiosteoclastogenic activity. Osteoclast-specific TRAP mean fluorescence intensity values are shown for osteoclast precursors after coculturing them for 5 d with TcREG generated from WT or IFN-γ−/− splenic CD8 T cells (n = 5–8 wells/group). (C) TcREG induced TRAF6 degradation in macrophages via IFN-γ. Bone marrow macrophages were cultured in the presence of WT or IFN-γ−/− TcREG, or rIFN-γ (20 or 100 U/ml) for 24 h. Adherent cells were removed, and whole-cell lysates of the macrophages were subjected to Western blotting for TRAF6 and β-actin. Band intensities were quantitated to show normalized TRAF6 levels. Data are representative of three independent experiments. MØ, macrophage; N, naive CD8 T cells. Statistical significance was assessed by nonparametric paired t test: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

TcREG inhibit osteoclastogenesis via IFN-γ. (A) Representative flow cytometry plots showing that OT-I CD8 T cells primed by osteoclasts pulsed with SIINFEKL express Foxp3 and IFN-γ. T cells were collected for staining after 48-h coculture with osteoclasts. Protein transport inhibitor GolgiStop was added to the cultures during last 3 h of incubation. Cells were gated on CD45+CD3+CD8+ population to determine Foxp3 and IFN-γ induction. (B) IFN-γ−/− TcREG have reduced antiosteoclastogenic activity. Osteoclast-specific TRAP mean fluorescence intensity values are shown for osteoclast precursors after coculturing them for 5 d with TcREG generated from WT or IFN-γ−/− splenic CD8 T cells (n = 5–8 wells/group). (C) TcREG induced TRAF6 degradation in macrophages via IFN-γ. Bone marrow macrophages were cultured in the presence of WT or IFN-γ−/− TcREG, or rIFN-γ (20 or 100 U/ml) for 24 h. Adherent cells were removed, and whole-cell lysates of the macrophages were subjected to Western blotting for TRAF6 and β-actin. Band intensities were quantitated to show normalized TRAF6 levels. Data are representative of three independent experiments. MØ, macrophage; N, naive CD8 T cells. Statistical significance was assessed by nonparametric paired t test: *p < 0.05, **p < 0.01, ***p < 0.001.

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In order to resorb, osteoclasts adhere to the bone surface and form sealing zones composed of multiple actin ring structures (4042). We tested whether IFN-γ is required for TcREG to inhibit actin ring formation in mature (day 5) osteoclasts. Coculturing day 5 osteoclasts (grown on bovine bone discs with WT TcREG) dramatically reduced number and size of actin rings in the osteoclasts, whereas IFN-γ−/− TcREG did not affect actin ring formation (Fig. 2A, 2B). These data show that IFN-γ is a crucial mediator of antiresorbing function of TcREG.

FIGURE 2.

TcREG suppress actin ring formation via IFN-γ reversibly. (A) IFN-γ is required for TcREG to suppress actin ring formation. Osteoclasts grown on bovine bone slices were cultured for 24 h with WT or IFN-γ−/− TcREG, followed by removal of T cells and staining of osteoclasts with fluorophore-conjugated phalloidin to assay for actin ring formation. n = 8 wells/group. (B) Representative images of the actin rings from (A) are shown. Original magnification ×200. (C) Representative images showing actin ring structures in osteoclasts after coculture with naive T cells or TcREG for 24 h (upper panel) or 48 h after removing T cells (bottom panel). (D) Quantitative results from the experiment described in (C) were obtained by blind scoring of actin ring formation. Scoring in (B) and (C) is on a scale from 0 to 5, based on staining intensity, the number, and thickness of rings per osteoclast considering all osteoclasts observed in a field (n = 7–9 wells/group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

TcREG suppress actin ring formation via IFN-γ reversibly. (A) IFN-γ is required for TcREG to suppress actin ring formation. Osteoclasts grown on bovine bone slices were cultured for 24 h with WT or IFN-γ−/− TcREG, followed by removal of T cells and staining of osteoclasts with fluorophore-conjugated phalloidin to assay for actin ring formation. n = 8 wells/group. (B) Representative images of the actin rings from (A) are shown. Original magnification ×200. (C) Representative images showing actin ring structures in osteoclasts after coculture with naive T cells or TcREG for 24 h (upper panel) or 48 h after removing T cells (bottom panel). (D) Quantitative results from the experiment described in (C) were obtained by blind scoring of actin ring formation. Scoring in (B) and (C) is on a scale from 0 to 5, based on staining intensity, the number, and thickness of rings per osteoclast considering all osteoclasts observed in a field (n = 7–9 wells/group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We have previously reported that TcREG do not inhibit survival of osteoclasts (16). To investigate whether suppressive effect of TcREG could be reversed, we cocultured osteoclasts for 24 h with TcREG, removed them (by washing), and then continued culturing the osteoclasts for additional 48 h before staining with phalloidin to visualize actin rings. In contrast to naive T cells, TcREG significantly suppressed actin ring formation (Fig. 2C, 2D). Interestingly, when osteoclasts were allowed to grow for 48 h after removal of TcREG, the actin ring formation was partially restored (Fig. 2C, 2D). These data suggest that suppression of actin ring formation in osteoclasts (and consequently their resorbing activity) requires sustained signaling by IFN-γ to degrade TRAF6.

T-box transcription factor T-bet was originally described as a critical factor for Th1 polarization during viral infections and expression of IFN-γ in CD4, but not in CD8 T cells (43). It was postulated that T-bet paralogue Eomes controls CD8 T cell effector functions, including IFN-γ production (44). Therefore, we measured T-bet and Eomes levels in OC-iTcREG. In the presence of Ag (SIINFEKL), OT-1 CD8 T cells stained double positive for T-bet and Eomes (Fig. 3A, left). In contrast, naive T cells did not express substantial levels of either of the factors. IFN-γ was expressed in T-bet–positive cells with the highest levels in T-bet+ Eomes+ double-positive CD8 T cells (Fig. 3A, right). These results suggest that T-bet and Eomes regulate IFN-γ production in osteoclast-primed CD8 T cells.

FIGURE 3.

Osteoclasts induce the transcription factors T-bet and Eomes in CD8 T cells needed for IFN-γ production by TcREG. (A) Flow cytometry plots showing stimulation of T-bet and Eomes expression in OT-I CD8 T cells cultured under TcREG-inducing conditions (cocultured with osteoclasts for 48 h in the presence of the OVA-derived peptide Ag SIINFEKL). Cells were gated on CD45+CD3+CD8+ populations prior to determining T-bet and Eomes expression. Data are representative of five independent experiments. (B) Left panel, Inhibition of IKK, by IKK-16, but not Notch signaling, by DAPT, blocks IFN-γ production by TcREG. OT-I CD8 T cells were primed by osteoclasts for 48 h in the presence of 10 μM γ-secretase inhibitor DAPT or 10 μM IKK inhibitor IKK-16. GolgiStop was added to the cultures during last 3 h of incubation. T cells were collected and stained for flow cytometry analysis. Right panel, Effect of the inhibitors on Foxp3 expression in CD8 T cells. For both panels, cells were gated on CD45+CD3+CD8+ population to determine IFN-γ or Foxp3 expression. (C) Reduction in IFN-γ production in TcREG by IKK-16 coincides with decrease in T-bet and Eomes expression levels. Flow cytometry plots showing cells from the experiment described in (B) and analyzed for T-bet and Eomes in CD45+CD3+CD8+ population. (D) Effect of IKK-16 on expression of IL-6 and IL-10 by TcREG. Flow cytometry plots showing that IKK inhibitor does not prevent induction of IL-6, whereas expression of IL-10 is blocked by IKK-16. Cells were gated on CD45+CD3+CD8+ population to determine expression of the cytokines.

FIGURE 3.

Osteoclasts induce the transcription factors T-bet and Eomes in CD8 T cells needed for IFN-γ production by TcREG. (A) Flow cytometry plots showing stimulation of T-bet and Eomes expression in OT-I CD8 T cells cultured under TcREG-inducing conditions (cocultured with osteoclasts for 48 h in the presence of the OVA-derived peptide Ag SIINFEKL). Cells were gated on CD45+CD3+CD8+ populations prior to determining T-bet and Eomes expression. Data are representative of five independent experiments. (B) Left panel, Inhibition of IKK, by IKK-16, but not Notch signaling, by DAPT, blocks IFN-γ production by TcREG. OT-I CD8 T cells were primed by osteoclasts for 48 h in the presence of 10 μM γ-secretase inhibitor DAPT or 10 μM IKK inhibitor IKK-16. GolgiStop was added to the cultures during last 3 h of incubation. T cells were collected and stained for flow cytometry analysis. Right panel, Effect of the inhibitors on Foxp3 expression in CD8 T cells. For both panels, cells were gated on CD45+CD3+CD8+ population to determine IFN-γ or Foxp3 expression. (C) Reduction in IFN-γ production in TcREG by IKK-16 coincides with decrease in T-bet and Eomes expression levels. Flow cytometry plots showing cells from the experiment described in (B) and analyzed for T-bet and Eomes in CD45+CD3+CD8+ population. (D) Effect of IKK-16 on expression of IL-6 and IL-10 by TcREG. Flow cytometry plots showing that IKK inhibitor does not prevent induction of IL-6, whereas expression of IL-10 is blocked by IKK-16. Cells were gated on CD45+CD3+CD8+ population to determine expression of the cytokines.

Close modal

Notch signaling was implicated in the regulation of IFN-γ expression in γδ T cells (45), CD8 T cells stimulated with anti-CD3 and anti-CD28 (46), and peripheral T cells (47, 48). Multiple cross-interactions have been described for Notch and NF-κB pathways in various experimental models (49). Therefore, we used the γ-secretase inhibitor DAPT and IKK inhibitor IKK-16 to measure the effect of Notch and NF-κB signaling, respectively, on IFN-γ expression. Treatment with DAPT had little to no effect on expression of IFN-γ, whereas IKK-16 completely blocked its production (Fig. 3B, left). Additionally, we found that IKK-16 significantly reduced FoxP3 expression in CD8 T cells (Fig. 3B, right). Although T-bet and Eomes levels were not affected by DAPT, they were drastically reduced in the presence of IKK-16 (Fig. 3C). Expression of T-bet was found to be more sensitive to NF-κB inhibition (93.8% versus 36.2% reduction in the levels of T-bet and Eomes, respectively, as compared with TcREG not treated with IKK-16). These results indicate that NF-κB positively regulates expression of T-bet and Eomes.

In addition to IFN-γ, TcREG also secrete IL-6 and IL-10 (14, 16). To test whether NF-κB signaling plays a role in induction of these cytokines, we examined the expression of IL-6 and IL-10 in TcREG in the presence of IKK-16. IL-6 expression was not significantly affected by IKK-16, whereas IL-10 expression was blocked by the inhibitor (Fig. 3D). Together, these data indicate that NF-κB signaling regulates expression of T-bet, Eomes, IFN-γ, and IL-10.

To determine whether NF-κB directly regulates expression of T-bet, Eomes, IFN-γ, and IL-10, we performed ChIP using anti-p65 Ab, followed by qPCR using OC-iTcREG and naive CD8 T cells. We found enrichment of p65 on NF-κB binding sites in TcREG relative to naive T cells within T-bet, Eomes, and IL-10 promoters, but not in IFN-γ promoter (Fig. 4A, Table I, Supplemental Table I).

FIGURE 4.

Gene regulatory network. (A) Measurements of occupancy of the p65 subunit of NF-κB on T-bet, Eomes, and IL-10 promoters in TcREG. Left panel, A diagram showing location of primers used for qPCR to detect enrichment of p65-binding sites. Right panel, Enrichment of p65-binding DNA sequences in the promoters of indicated genes of TcREG relative to naive CD8 T cells. TcREG chromatin for use in ChIP was prepared after 24 h of incubation with osteoclasts. (B) T-bet and Eomes bind to distal conserved sequences CNS-22 and CNS-34 of IFN-γ gene in TcREG. Chromatin from naive CD8 T cells (negative control) and from 24-h TcREG was immunoprecipitated with anti–T-bet or anti-Eomes Ab. Enrichment of T-bet and Eomes binding sites at −22K and −34K of IFN-γ gene was assessed by qPCR. (C) T-bet and Eomes do not bind IL-10 promoter. Chromatin prepared and immunoprecipitated, as described in (B), was subjected to qPCR with primers, which flank putative T-bet/Eomes binding sites in IL-10 promoter. These results show no significant enrichment for these sites in TcREG relative to naive CD8 T cells.

FIGURE 4.

Gene regulatory network. (A) Measurements of occupancy of the p65 subunit of NF-κB on T-bet, Eomes, and IL-10 promoters in TcREG. Left panel, A diagram showing location of primers used for qPCR to detect enrichment of p65-binding sites. Right panel, Enrichment of p65-binding DNA sequences in the promoters of indicated genes of TcREG relative to naive CD8 T cells. TcREG chromatin for use in ChIP was prepared after 24 h of incubation with osteoclasts. (B) T-bet and Eomes bind to distal conserved sequences CNS-22 and CNS-34 of IFN-γ gene in TcREG. Chromatin from naive CD8 T cells (negative control) and from 24-h TcREG was immunoprecipitated with anti–T-bet or anti-Eomes Ab. Enrichment of T-bet and Eomes binding sites at −22K and −34K of IFN-γ gene was assessed by qPCR. (C) T-bet and Eomes do not bind IL-10 promoter. Chromatin prepared and immunoprecipitated, as described in (B), was subjected to qPCR with primers, which flank putative T-bet/Eomes binding sites in IL-10 promoter. These results show no significant enrichment for these sites in TcREG relative to naive CD8 T cells.

Close modal

It has been reported previously that T-bet and Eomes bind to distal conserved noncoding sequences to regulate histone modification and expression of IFN-γ gene (5052). Consistent with these results, we found that in TcREG T-bet and Eomes bind to conserved noncoding sequences located at −22K and −34K from IFN-γ gene transcription start (Fig. 4B). In contrast, no enrichment by ChIP-qPCR was found for putative T-bet and Eomes binding site in IL-10 promoter (Fig. 4C). Our data demonstrate that in TcREG IFN-γ production is regulated by T-bet and Eomes; expression of these transcription factors is p65 dependent. In contrast, IL-10 is regulated by p65 and not by T-bet or Eomes.

We have previously demonstrated that TcREG limit bone loss induced in mice by administration of high dose (1 mg/kg) of RANKL (17). As IFN-γ has pleiotropic effects in the context of other cell types, we tested the role of IFN-γ in TcREG-mediated reduction of bone loss in vivo. To this end, we adoptively transferred bone marrow and splenic CD8 T cells from WT and IFN-γ−/− mice to OT-II Rag−/− mice, followed by two daily doses of 1 mg/kg RANKL or buffer. OT-II Rag−/− mice were used as recipients because these mice lack endogenous TcREG, because they are not lymphopenic, and because we could track the donor CD8 cells. Bone resorption levels were assessed by serum CTX levels 50 h after first injection of RANKL. Administration of WT CD8 T cells significantly reduced high-dose RANKL-induced bone resorption (Fig. 5A). This is consistent with our previous results demonstrating induction of TcREG in the bone marrow in response to RANKL administration (18). Interestingly, reconstitution of OT-II mice with IFN-γ−/− CD8 T cells also decreased bone resorption as compared with control mice (which received only high-dose RANKL), but significantly less efficient relative to WT T cells (Fig. 5A). Similarly, mice injected with WT T cells had increased bone volume as compared with control RANKL-treated animals (Fig. 5B). Adoptive transfer of IFN-γ−/− T cells increased BV/TV, but to lower extent, relative to WT T cell treatment (Fig. 5B, 5C). Together, these results indicate that IFN-γ produced by TcREG in vivo is partially responsible for the bone-protective effect of TcREG, and indicate that additional factors also contribute.

FIGURE 5.

IFN-γ is important for the bone-protective effect of TcREG in vivo. (A) WT or IFN-γ−/− CD8 T cells were adoptively transferred into OT-II mice (n = 4–8 mice/group), followed 24 h later by administration of 1 mg/kg RANKL daily for 2 d. Serum CTX levels at 50 h after first RANKL injections are shown to assess relative bone resorption levels. IFN-γ−/− CD8 T cells have reduced ability to suppress RANKL-induced bone resorption as compared with WT CD8 T cells. (B) μCT of proximal tibia of mice from the experiment described in (A). Reconstitution of OT-II mice with IFN-γ−/− CD8 T cells partially protected mice, relative to WT T-cells, from losing bone volume in response to 1 mg/kg RANKL, resulting in intermediate levels of BV/TV. Consistent with these results, measurement of bone mineral density gave corresponding results. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative μCT images from the experiment described in (B). (D) A model summarizing the feedback loop between osteoclasts and CD8 T cells. Left panel, Osteoclast are APCs that provide TCR activation, costimulation, and differentiation (Δ-like ligand 4 [DLL4]) signals to naive CD8 T cells. This leads to induction of Foxp3 and IFN-γ in the CD8 T cell. IFN-γ in turn causes degradation of TRAF6 and suppression of actin ring formation in osteoclasts inhibiting bone resorption. Right panel, A proposed model of events triggered in CD8 T cells. Upon interaction with osteoclasts, NF-κB signaling is activated in the T cells, leading to expression of transcription factors T-bet and Eomes, which then activate transcription of IFN-γ.

FIGURE 5.

IFN-γ is important for the bone-protective effect of TcREG in vivo. (A) WT or IFN-γ−/− CD8 T cells were adoptively transferred into OT-II mice (n = 4–8 mice/group), followed 24 h later by administration of 1 mg/kg RANKL daily for 2 d. Serum CTX levels at 50 h after first RANKL injections are shown to assess relative bone resorption levels. IFN-γ−/− CD8 T cells have reduced ability to suppress RANKL-induced bone resorption as compared with WT CD8 T cells. (B) μCT of proximal tibia of mice from the experiment described in (A). Reconstitution of OT-II mice with IFN-γ−/− CD8 T cells partially protected mice, relative to WT T-cells, from losing bone volume in response to 1 mg/kg RANKL, resulting in intermediate levels of BV/TV. Consistent with these results, measurement of bone mineral density gave corresponding results. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative μCT images from the experiment described in (B). (D) A model summarizing the feedback loop between osteoclasts and CD8 T cells. Left panel, Osteoclast are APCs that provide TCR activation, costimulation, and differentiation (Δ-like ligand 4 [DLL4]) signals to naive CD8 T cells. This leads to induction of Foxp3 and IFN-γ in the CD8 T cell. IFN-γ in turn causes degradation of TRAF6 and suppression of actin ring formation in osteoclasts inhibiting bone resorption. Right panel, A proposed model of events triggered in CD8 T cells. Upon interaction with osteoclasts, NF-κB signaling is activated in the T cells, leading to expression of transcription factors T-bet and Eomes, which then activate transcription of IFN-γ.

Close modal

An increasingly complex interaction between the skeletal and immune systems has been observed in multiple studies in recent years. This led to the development of a new interdisciplinary field of research termed osteoimmunology. The immune system plays an important role in regulating bone metabolism with various bone-affecting diseases spawning from immunologic imbalance. In contrast, the immune system also protects bone and maintains homeostasis, indicating that regulatory feedback loops must have evolved between the skeletal and immune compartments of the vertebrates. In this study, we extend our previous study of a negative feedback loop between osteoclasts and CD8 T lymphocytes, which not only limits bone resorption (Fig. 5D), but also limits activation of the immune system (13). In the current study, we focused on the mechanism by which osteoclasts induce IFN-γ and the role of IFN-γ produced by TcREG in the antiresorptive activity of these cells.

We demonstrate that TcREG generated from IFN-γ−/− mice have reduced ability to suppress osteoclastogenesis and are unable to cause degradation of TRAF6 in osteoclast precursors relative to their WT counterparts. Furthermore, we show that IFN-γ−/− TcREG fail to suppress the formation of actin rings in osteoclasts, which is in contrast to the effect produced by WT TcREG (Fig. 5D). These results suggest that TcREG regulate actin reorganization in osteoclasts via IFN-γ. Withdrawal of WT TcREG from osteoclast culture for 48 h led to a significant restoration of actin ring formation. Actin ring structures reflect the establishment of sealing zones between osteoclasts and bone that are necessary for resorption (53). Signals provided by αvβ3 integrins are required for inducing actin reorganization and attachment of osteoclasts to the bone matrix (54, 55). Mice lacking the β3 integrin have dysfunctional osteoclasts and show protection from bone loss induced by ovariectomy (56, 57). Interestingly, IFN-γ has been implicated in a suppression of αvβ3 expression and adhesion properties of various cell types (5861). It is likely, therefore, that TcREG directly inhibit osteoclast resorbing through a blockade of αvβ3 signaling by IFN-γ.

We show that expression of transcription factors T-bet and Eomes accompanies production of IFN-γ by TcREG. Inhibition of Notch signaling by DAPT did not affect either IFN-γ or T-bet and Eomes expression. In contrast, we found that inhibition of IKK completely blocked IFN-γ production that was associated with 93.8 and 36.2% reduction in the levels of T-bet and Eomes, respectively. The IKK kinase complex is the core element of the NF-κB signaling cascade, which regulates the expression of genes critical for immune development and immune responses, cell survival, and proliferation (62). Our data indicate that IFN-γ production by TcREG is controlled by NF-κB signaling. Based on our data and published studies (52, 63, 64), we interpret these results to indicate that NF-κB contributes to (or regulates) the expression of T-bet and Eomes in TcREG, which then leads to IFN-γ production (Fig. 5D).

Several studies have reported opposing effects of rIFN-γ on bone development and homeostasis (34, 65, 66). IFN-γ directly targets osteoclast precursors, thereby suppressing osteoclast differentiation, but indirectly promotes bone resorption by stimulating T cell activation and secretion of pro-osteoclastogenic cytokines RANKL and TNF-α (34). The net effect of IFN-γ treatment on the bone therefore appears to be highly dependent on the context and dose. We show in this work that adoptive transfer of WT CD8 T cells into OT-II mice (to generate TcREG in vivo) inhibits RANKL-induced bone loss, whereas lack of IFN-γ renders TcREG less protective. However, the absence of IFN-γ did not completely block the antiresorptive effects of the treatment. These results indicate that IFN-γ is not the sole mediator of the bone-protecting activity of TcREG, but nevertheless a contributing factor. We have previously shown that, in addition to IFN-γ, TcREG secrete IL-10 and IL-6 (14). IL-10 is a potent anti-inflammatory cytokine that inhibits production of cytokines by Th1 cells and is involved in regulation of bone metabolism (67). IL-6 is a pleiotropic cytokine that has both pro- and anti-inflammatory activities depending on a particular environment. Several studies have demonstrated IL-6–mediated inhibition of osteoclastogenesis through a number of mechanisms, including suppression of RANKL signaling pathway and downregulation of IL-10 expression (68, 69). Together, our results indicate that TcREG limit bone resorption via multiple cytokines.

In summary, we have characterized the induction of IFN-γ expression by OC-iTcREG and demonstrated its important role in bone protection. The results of this study add to our understanding of how TcREG function to prevent excessive bone catabolism and maintain a healthy homeostasis. By exhibiting two separate functions that include direct inhibition of osteoclastogenesis via IFN-γ and regulation of the immune system, TcREG are able to shift the balance toward bone anabolic environment.

We thank Dr. Ryan Teague for helpful discussions and Dr. R. Mark Buller for providing rIFN-γ. We also thank Sheri Koehm and Joy Eslick in the flow cytometry core (Saint Louis University School of Medicine, St. Louis, MO).

This work was supported in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health Awards RO1AR064821 and RO1AR068438. The Washington University Musculoskeletal Research Core (National Instititutes of Health Grant P30 AR057235) also partially supported this study.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BV/TV

bone volume density over total volume

ChIP

chromatin immunoprecipitation

CT

cycle threshold

CTX

C-terminal cross-linking telopeptide of type I collagen

Eomes

Eomesodermin

OC-iTcREG

octeoclast-induced TcREG

qPCR

quantitative PCR

RANKL

receptor activator of NF-κB ligand

TcREG

Foxp3+ CD25+ regulatory CD8 T cell

TE

Tris-EDTA

TRAF6

TNFR-associated factor 6

TRAP

tartrate-resistant acid phosphatase

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data