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The Journal of Immunology, 2008, 181, 5720 -5729
Copyright © 2008 by The American Association of Immunologists, Inc.
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IL-23 Inhibits Osteoclastogenesis Indirectly through Lymphocytes and Is Required for the Maintenance of Bone Mass in Mice1

Julian M. W. Quinn2,*,{dagger}, Natalie A. Sims*,{dagger}, Hasnawati Saleh*,{dagger}, Danijela Mirosa*, Keith Thompson*,{ddagger}, Stelios Bouralexis*, Emma C. Walker*, T. John Martin*,{dagger} and Matthew T. Gillespie*,{dagger}

* St Vincent’s Institute, Fitzroy, Victoria, Australia; {dagger} Department of Medicine, University of Melbourne, St. Vincent’s Hospital, Fitzroy, Victoria, Australia; and {ddagger} Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-23 stimulates the differentiation and function of the Th17 subset of CD4+ T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4+ T lymphocyte-dependent manner. When sufficiently enriched, {gamma}{delta} T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19–/–) had ~30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19–/– mice also had reduced BV/TV. IL-23p19–/– mice had no detectable osteoclast defect in trabecular bone but IL-23p19–/– had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The formation of osteoclasts from myelomonocytic progenitors, a critical point of control of osteolysis, is powerfully influenced by factors released by T cells (1, 2). Osteoclast formation requires two essential factors, namely, M-CSF and receptor activator of NF-{kappa}B ligand (RANKL),3 a TNF-related ligand (3, 4). RANKL also plays important roles in cell-mediated immunity and is essential for lymph node organogenesis (5, 6, 7). Hormonally regulated expression of membrane-bound RANKL by osteoblasts constitutes the major physiological control of osteoclastogenesis (8), but activated T lymphocytes also produce significant amounts of membrane-bound and soluble RANKL (9, 10). Activated T cells can thus stimulate osteoclast formation and osteolysis and contribute to pathological osteolysis in periodontal disease and rheumatoid arthritis (9, 11). Sato et al. (12) found that the major source of T cell-derived RANKL derives from activated Th17 cells, a population recently defined as IL-17-secreting Th cells distinct from Th1 or Th2 subsets and which requires IL-23 for maturation and cytokine secretion. Th17 cells, IL-17, and IL-23 are emerging as critical agents in chronic autoimmune disease models (13, 14), and the common association of chronic inflammation with osteolysis may be explained by activated Th17 cell production of RANKL and the pro-osteolytic cytokine IL-17.

In contrast to activated T cell stimulation of osteolysis, naive or nonactivated T cells mediate inhibitory actions of several cytokines on osteoclasts, including IL-4, IL-18, and IL-12 (15, 16, 17). Some immunosuppressants also cause clinical and experimental bone loss (18). Such observations raise the possibility that under normal physiological conditions T cells mediate significant antiosteolytic effects, but contribute to bone loss under local inflammatory conditions that result in high levels of T cell activation.

IL-23 is a heterodimeric cytokine structurally related to IL-12. IL-12 is a p35/p40 heterodimer which promotes Th1 development and suppresses Th2. IL-12 induces IFN-{gamma} secretion by Th1 and NK cells (19) and synergizes with IL-18 via reciprocal enhancement of specific receptor subunits, which can greatly amplify IL-12 actions (19, 20). IL-23 consists of an IL-12p40 subunit complexed with IL-23 specific p19 subunit (13, 19); receptors for IL-23 and IL-12 are also heterodimers sharing the IL-12Rβ1 subunit and activate many of the same signaling molecules (13, 21). IL-23 strongly stimulates IL-17 production by Th17 cells and stimulates IFN-{gamma} production though less potently than IL-12 (13). IL-23 also induces IL-17 production in {gamma}{delta} T cells (22), a CD4– T cell population with innate immune functions.

The similarities of IL-12 and IL-23 structure and their elicited signaling pathways suggest that IL-23 may also affect osteoclast differentiation. We therefore studied the influence of IL-23 on osteoclast formation and its dependence on T cells. In addition, to determine whether IL-23 influences bone mass and structure, we examined mice lacking the IL-23 p19 subunit. These mice have previously been characterized as resistant to induced autoinflammatory challenge and as lacking Th17 responses. Our results suggest not only that IL-23 does indeed reduce osteoclast formation in the presence of T cells but also that lack of IL-23 leads to significantly lower levels of trabecular (Tb) bone mass in otherwise unchallenged, healthy mice. Although several mechanisms of action of IL-23 could explain this effect, we have identified novel and unusual features of IL-23 null mice that point to a previously unsuspected way in which such a cytokine could affect bone mass and structure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
C57BL/6J mice were obtained from the Animal Resource Centre (Perth, Australia). IL-23p19–/– (C57BL6/J background) mice were provided by Dr N. Ghilardi (Genentech, San Francisco, CA) and have previously been characterized as having an abnormal hypersensitivity response (23, 24, 25). Mice were maintained at St. Vincent’s Hospital Campus (Fitzroy, Australia) and procedures approved by the Animal Ethics Committee at St. Vincent’s Health (Fitzroy, Australia). Littermate controls from heterozygous crosses were used. Cells were cultured in {alpha}-MEM (Life Technologies) with 10% FBS (CSL Biosciences). Recombinant GST-RANKL158–316 (RANKL) was produced using a bacterial construct provided by Dr. F. Patrick Ross (Washington University School of Medicine, St. Louis, MO). Other recombinant proteins were obtained from R&D Systems. 1{alpha},25-dihydroxyvitamin D3 (1,25(OH)2 D3) was obtained from Wako Pure Chemical. Other reagents were analytical grade obtained from Sigma-Aldrich unless noted.

Cells and assays

Spleen cells were obtained by spleen disaggregation through a fine wire sieve (16). Lymphocytes were extracted from spleen cells (16) by immunomagnetic Dynabeads (Dynal) coated with Abs to B220 (B cells), Thy1.2 (pan-T cells), L3T4 (CD4+ cells), or Lyt2 (CD8+ cells). {gamma}{delta} T cells were isolated using a Miltenyi Biotec kit according to the manufacturer’s instructions. Bone marrow cells were flushed from long bones with sterile saline (26) and bone marrow macrophages (BMM) prepared as previously described (26). Osteoblasts were prepared from newborn mice calvariae by sequential digestion with 0.1% collagenase/0.2% dispase (Godo Shusei). Osteoclasts were generated in 10-mm diameter culture wells (Greiner Bio-One) seeded with 5 x 105 spleen cells/well or 105 bone marrow or BMM per well and stimulated with RANKL (100 ng/ml) and M-CSF (30 ng/ml) (26); cells were fixed and histochemically stained to identify tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), previously validated as osteoclasts (26). Alternatively, spleen cells or BMM were cocultured with 2 x 104 osteoblasts or Kusa O cells (27) and 10–8 M 1,25(OH)2 D3. To investigate osteoblast mineralization, cells were seeded (5 x 104/well) in 16-mm diameter culture wells (Greiner Bio-One) in MEM/FBS with 50 µg/ml ascorbate, 10 mM β-glycerophosphate, and 10 nM dexamethasone and incubated for 21 days, with medium and mediators changed every 3 days. Cells were ethanol fixed, immersed in 0.5% alizarin red (pH 4.2) for 30 min, rinsed in PBS, and incubated in 10% cetylpyridinium chloride, and eluted alizarin red was measured by spectrophotometer (28).

Flow cytometry determination of {gamma}{delta} T cell numbers

Spleen cells (106) from four mice were incubated in PBS with 2% FBS and anti-mouse FcR Ab (2.4G2 hybridoma supernatant), rinsed, incubated in 1 µg/ml PE-labeled GL3 anti-mouse {gamma}{delta} TCR Ab (BD Bisociences), rinsed, and paraformaldehyde fixed. Forty thousand viable cells per sample (using forward/side scatter gating) were analyzed by single-color flow cytometry (FACSCalibur; BD Biosciences). Negative controls were incubated in anti-FcR Ab alone.

Analysis of mRNA expression

RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was then treated with RNase-free DNase (Roche Diagnostics) for 30 min at 37°C. RNA concentration was determined by spectrophotometer (Nanodrop ND1000). cDNA was synthesized (random hexamers, 10 mM dNTP, 5x First Strand Buffer, 0.1 M DTT, RNaseOUT, and Superscript III RT (200 U/µl)) using 2 µg of starting material under the following conditions: 5 min at 65°C, 5 min at 4°C, 60 min at 50°C, 15 min at 70°C, and 8 min at 4°C (Bio-Rad iCycler). Real-time PCR analysis (Stratagene Mx3000P) of cDNA was performed using Platinum SYBR Green qPCR supermix UDG (Invitrogen) according to the manufacturer’s instructions and the following conditions: 1 cycle for 10 min at 95°C; 40 cycles for 30 s at 95°C, 1 min at 60°C, 30 s at 72°C and 1 cycle for 1 min at 95°C, 30 s at 55°C, 0 s at 95°C normalized to hypoxanthine phosphoribosyltransferase (HPRT). IL-23p19 and IL-23R mRNA were analyzed by semiquantitative RT-PCR as previously described (29). Oligonucleotide primer sequences (Table I) were obtained from Primerbank (30).


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  		Table I. Sequences of oligonucleotide primers used for RT-PCR analysis

 
Peripheral quantitative computer-aided tomography (pQCT) analysis of murine femora

Femoral cortical (Ct) and Tb bone mineral density (BMD), femoral circumference, and femoral Ct thickness were measured by pQCT (Stratec X-CT Research SA+, version 5.5) by methods adapted from Sims et al. (31). Metaphyseal scans of the distal femur were taken at a resolution of 70 µm; Tb and Ct measurements (including circumference) were taken at a distance proximal to the distal growth plate of 5 and 25% of the length of the femur, respectively; Tb BMD was determined as the inner 45% of the total area (peel mode 20).

Bone histomorphometric analysis of murine tibia and femora

Tibiae were collected at 4, 12, or 26 wk of age, fixed in 4% paraformaldehyde in PBS, and embedded in methylmethacrylate (32). Double fluorochrome labeling was performed with calcein injections 10 and 3 days before tissue collection (32). Five-micrometer sections were stained with toluidine blue or stained with xylenol orange for fluorochrome labels according to standard procedures using the Osteomeasure System (OsteoMetrics). Bone and growth plate histomorphometric parameters were measured as previously described (32); safranin O-stained cartilage remnants were measured as described elsewhere (33). Femoral length and width were determined from contact X-rays measured using NIH Image 1.62 (32).

Statistical analysis

Data are shown as mean ± SE. Cell culture data are pooled from more than three independent experiments. For mRNA expression, triplicate analyses of two independent experiments were performed and statistical significance was determined by Student’s t test. Bone histomorphometric analyses used eight mice per category that were analyzed by one-way ANOVA with Fisher’s post hoc test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-23 inhibits osteoclast formation in the presence of T cells in vitro

When spleen cells were stimulated with RANKL and M-CSF, numerous osteoclasts were formed; this was dose-dependently inhibited by IL-23 (Fig. 1A). Maximal effects were seen at 20 ng/ml, which reduced osteoclast formation by ~80% relative to positive controls; even 50 ng/ml IL-23 did not abolish osteoclast formation (data not shown). In contrast, IL-23 (20 ng/ml) did not affect osteoclast formation in RANKL- and M-CSF-stimulated cultures of bone marrow cells, BMM precursors, or of RAW264.7 cells (Fig. 1A). Similar to previous observations with IL-12 and IL-18 (16, 17), IL-23 inhibited osteoclast formation in spleen cell cultures when present from days 0 to 3, having no effects when added thereafter (Fig. 1B). In all experiments described below, IL-23 was present only during days 0 to 3 of culture. IL-23 did not influence survival of mature osteoclasts (data not shown).


Figure 1
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  		FIGURE 1. IL-23 action on osteoclast formation in vitro. A, IL-23 dose-dependently inhibited osteoclast formation in RANKL/M-CSF-stimulated spleen cell cultures. B, IL-23 had no effect on osteoclast formation in RANKL/M-CSF-stimulated cultures of bone marrow (BM) cells, BMM progenitors, or RAW264.7 cells. C, IL-23 inhibited osteoclast formation in RANKL/M-CSF-stimulated spleen cell cultures when present during days 0–3 of the spleen culture, but had no effect when present during days 3–7. Shown below the figure is the IL-23 dose (in ng/ml) added to cultures. **, p < 0.01 and ***, p < 0.001 relative to positive control ({blacksquare}).

 
To determine whether IL-23 may act via osteoblasts to influence osteoclast formation, we performed cocultures of primary osteoblasts (or the preosteoblastic Kusa O cell line) with bone marrow cells stimulated with 10–8 M 1,25(OH)2 D3 and PGE2; osteoclast formation was not affected by IL-23 treatment (data not shown). Real-time RT-PCR analysis (see Table I for oligonucleotide primer details) of three independent samples of osteoblast and Kusa O cell mRNA was unable to detect expression of IL-23R mRNA (i.e., Ct > 39) although this was easily detected in spleen cell mRNA (Ct = 31.7). This was repeated with a different forward oligonucleotide primer with a similar result. In contrast, the second subunit of the IL-23 receptor, IL-12Rβ1, was detectable (osteoblast Ct = 28.8, Kusa O Ct = 28.2, spleen cell Ct = 21.2).

To determine the mechanism of action of IL-23 inhibition of osteoclast formation in spleen cells, we depleted spleen cell subpopulations. When the nonadherent spleen cell fraction had been removed (leaving predominantly adherent macrophages) or T cells were depleted using Thy1.2-labeled magnetic beads, IL-23 had no effect on osteoclast formation (Fig. 2A). Removal of B cells from the spleen had no effect on IL-23 inhibitory action (data not shown). IL-23 strongly inhibited osteoclast formation in spleen cell cultures in CD8+ depleted cultures, but had no effect in CD4+ depleted cultures (Fig. 2A). This dependency on CD4+ cells was not seen in the action of IL-12 in similar cultures (data not shown), as previously reported (17). To confirm the mediation of IL-23 effects by CD4+ cells, we added T cell subsets into RANKL/M-CSF stimulated cultures of bone marrow cells, a population in which IL-23 did not affect osteoclast formation (Fig. 1A). Consistent with the action of IL-23 through Th17 cells, when either T cells or CD4+ cells (Fig. 2B) were added to cultures of bone marrow cells, IL-23 inhibited osteoclast formation. This was not observed when CD8+ cells were added (Fig. 2B).


Figure 2
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  		FIGURE 2. IL-23 inhibition of osteoclast formation is mediated by CD4+ and {gamma}{delta} T cells. A, IL-23 did not affect osteoclast formation in splenic cell cultures depleted of nonadherent cells, T cells (Thy 1.2+), or CD4+ cell fraction, but did inhibit formation in cultures depleted of CD8+ cells. B, IL-23 inhibited osteoclast activity in bone marrow cultures to which T cells or CD4+ cell fractions were added but not CD8+ cell fractions. C, IL-23 inhibited osteoclast formation in bone marrow cultures to which 104 or more {gamma}{delta} T cells were added. **, p < 0.01 and ***, p < 0.001 relative to positive control ({blacksquare}). Pos co, Positive control.

 
A second population of T cells, {gamma}{delta} T cells, has also been reported to respond to IL-23 (22). We found that when isolated splenic {gamma}{delta} T cells were added to bone marrow cells IL-23 significantly inhibited osteoclast formation, but this was only observed when a minimum of 104 {gamma}{delta} T cells/well was added (Fig. 2C). However, osteoclast formation was detected in CD4+ stripped spleen cell populations that contained {gamma}{delta} T cells when treated with IL-23 (Fig. 2A). By FACS analysis, we determined that in our C57BL/6 mouse spleen cell populations the mean proportion of {gamma}{delta} TCR-positive cells was 1.07% (range, 0.90–1.25%) of live cells. Thus, RANKL/M-CSF-stimulated spleen cell cultures described above (which contained 5 x 105 cells in total) contained ~5 x 103 cells, insufficient to mediate IL-23 inhibition.

Mechanism of action of T cell-dependent IL-23 inhibition of osteoclast formation

When spleen cells and bone marrow cells were cultured together but separated by a porous Transwell membrane well, RANKL/M-CSF stimulation caused formation of numerous osteoclasts from the bone marrow cells; this was reduced significantly in the presence of IL-23 (20 ng/ml; Fig. 3A). This suggested that IL-23 elicits the release of a soluble diffusible osteoclast formation inhibitor. IL-23 is known to induce secretion of TNF, IL-17, GM-CSF, and IFN-{gamma} by Th cells (13). TNF and IL-17 are not known to directly inhibit osteoclast formation and in recombinant form (both 10 ng/ml) neither affected osteoclast formation in RANKL/M-CSF-stimulated bone marrow cells (data not shown). IFN-{gamma} action was blocked using an anti-IFN-{gamma}-neutralizing Ab as previously described (17), but no effect on IL-23 inhibition was seen (Fig. 3B). In contrast, a neutralizing Ab to GM-CSF ablated IL-23 action (Fig. 3B). Treatment with both Abs had the same effect as anti-GM-CSF alone (data not shown); as a further control, neither the anti-IFN-{gamma}-neutralizing Ab nor the neutralizing Ab to GM-CSF blocked IL-12 action (Fig. 3B).


Figure 3
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  		FIGURE 3. Mechanism of action of IL-23 inhibition of osteoclast formation. A, Inhibition of osteoclast formation by IL-23 in RANKL/M-CSF-stimulated bone marrow cultures incubated with spleen cells that were separated from the bone marrow cells by a Transwell porous polycarbonate membrane. B, Rescue of IL-23 inhibition (but not IL-12 inhibition) of osteoclast formation in spleen cell cultures by anti-GM-CSF Ab but not anti-IFN-{gamma}. C,. Time course of GM-CSF and IFN-{gamma} mRNA induction by IL-23 (20 ng/ml) and GM-CSF mRNA levels in spleen cells, T cells, and T cell-depleted spleen cells after 24-h IL-23 stimulation. D, Effects of IL-23 on IL-17 mRNA expression levels in spleen cells and T cells at 24 h. E, Effects of 24 h of IL-23 stimulation on GM-CSF mRNA levels in unfractionated spleen cells and spleen-derived T cells; data normalized to respective controls. F, {gamma}{delta} T cell (2 x 104cells/well)-mediated IL-23 inhibition of osteoclast formation in bone marrow cells and was rescued by anti-GM-CSF Ab. **, p < 0.01 and ***, p < 0.001 relative to positive control ({blacksquare}). Co, Control.

 
Since GM-CSF mediated IL-23 action, we investigated GM-CSF mRNA regulation in spleen cells. IL-23 treatment of spleen cells increased GM-CSF mRNA levels ~5-fold relative to control at 24 h by IL-23 (Fig. 3C). A somewhat weaker and transient induction of IFN-{gamma} mRNA levels (<2-fold, peaking at 2 h) was also noted (data not shown). IL-23 treatment enhanced IL-17 mRNA levels very robustly (2500-fold) after 24 h, from a barely detectable level in untreated cells (Fig. 3D). At 24 h, IL-23 induction of GM-CSF mRNA was also observed in magnetic bead-purified T cells (Fig. 3E), with a response magnitude similar to that observed in unfractionated splenic cells.

Because we found that {gamma}{delta} T cells mediated an inhibitory effect of IL-23, we examined the role of GM-CSF in this action. As with CD4+ cell action above, the inhibitory effect mediated by {gamma}{delta} T cell action was ablated by addition of GM-CSF Ab (Fig. 3F).

IL-12 inhibition of osteoclast formation does not depend on IL-23

Although IL-12 powerfully and rapidly stimulated expression of the IL-23p19 subunit (Fig. 4A), IL-12 inhibition of osteoclast formation was not dependent upon the IL-23p19 subunit since IL-12 strongly inhibited osteoclast formation in RANKL-stimulated IL-23p19–/– spleen cells. In addition, IL-12 similarly inhibited osteoclast formation in wild-type (WT) bone marrow cells to which IL-23p19–/– T cells were added (Fig. 4B). Similarly, the inhibitory actions of IL-18 and of IL-18/IL-12 cotreatment were not ablated when IL-23p19–/– T cells were present (data not shown).


Figure 4
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  		FIGURE 4. IL-23 interactions with IL-12 and IL-18. A, IL-12 stimulation of IL-23p19 mRNA in spleen cells by semiquantitative RT-PCR. B, IL-12 inhibition of osteoclast formation in RANKL/M-CSF-stimulated cultures of IL-23p19–/– spleen cells and WT bone marrow cells to which IL-23p19–/– T cells were added. **, p < 0.01 relative to positive control ({blacksquare}). C, Dose response of the inhibitory action of IL-18 in the presence of a low concentration (0.01 ng/ml) of IL-23. *, p < 0.05 and **, p < 0.01 relative to untreated control. D, Dose response of the inhibitory action of IL-23 in the presence of a low concentration (0.1 ng/ml) of IL-18. *, p < 0.05 and **, p < 0.01 relative to untreated control. Het, Heterozygote; KO, knockout.

 
Synergy of IL-23 and IL-18 in inhibiting osteoclast differentiation

Previously, we have shown that IL-12 and IL-18 exert synergy in eliciting production of IFN-{gamma} by T cells as well as in their ability to inhibit osteoclast formation (17). The presence of 0.01 ng/ml IL-23 (a concentration at least an order of magnitude below that required to show a measurable effect on osteoclast formation) increased the potency of IL-18 in these assays, shifting the dose-response curve of IL-18 to the left (Fig. 4C). A similar effect on the IL-23 dose-response curve was seen in the presence of 0.1 ng/ml IL-18 (Fig. 4D), a concentration also without effect on osteoclast formation.

Analysis of long bones of WT, IL-23p19+/–, and IL-23p19–/– mice

To determine whether our in vitro findings were indicative of a physiological role of IL-23 in bone, we analyzed the long bones of IL-23p19–/– mice (which lack IL-23 but not IL-12) and compared them with those of heterozygote and WT littermates. By pQCT, low femoral Tb BMD was observed in 12- and 26 (but not 4-)-wk-old male IL-23p19–/– and heterozygote mice (Fig. 5A), pointing to bone defects resulting from lack of IL-23. Female mice have lower Tb BMD than males of the same age; however, IL-23p19–/– females did not have significantly lower Tb BMD than WT female littermates. We then examined tibiae of mice by histomorphometry and, consistent with the pQCT data, found that Tb bone volume (BV/Tb volume (TV)) of male IL-23p19–/– mice was significantly lower than that of WT male mice (Fig. 5, B and C); again, this was seen at 12 and 26 wk of age but not at 4 wk of age (data not shown). Male heterozygotes at 12 and 26 wk age also had lower BV/TV than WT mice. Female IL-23p19–/– mice at 12 wk of age had ~30% lower BV/TV, similar to that in the 12-wk-old males (Fig. 5C). However, low bone mass was not detected in 26-wk-old females (Fig. 5C), probably due to the very low BV/TV of WT female mice rendering a further reduction difficult to observe. Relative to WT controls, Tb thickness was reduced in male IL-23p19–/– mice but was not different in females (Fig. 5D). Tb number was ~30% lower in male and female IL-23p19–/– mice compared with WT mice (Fig. 5E), and a similar difference was also observed in heterozygotes.


Figure 5
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  		FIGURE 5. Structural analysis of bones of 12- and 26-wk-old male and female WT IL-23p19+/– (Het) and IL-23p19–/– (KO) mice. A, pQCT analysis of femoral trabeculae. B, Von Kossa-stained sections of undecalcified tibiae of 12-wk-old WT and IL-23p19–/– male mice, counterstained with eosin. Scale bar, 200 µm. C, Tibial Tb volume (BV/TV). D, Tibial Tb thickness (Tb.Th). E, Tibial Tb number (Tb.N). F, Tibial Tb osteoid surface (OS/BS). G, Tibial osteoblast surface (ObS/BS). H, Periosteal mineral apposition rate in 12-wk-old mice. I, Number of osteoclasts/growth plate perimeter (Noc/GP1Pm). *, p < 0.05 and **, p < 0.01 relative to appropriate WT control ({blacksquare}).

 
Osteoid surface (OS/BS) was not affected in female mice (Fig. 5F) but 26-wk-old male IL-23p19–/– mice had lower osteoid surface than WT mice, a difference not seen in younger mice. Consistent with this, osteoblast surface (ObS/BS) was lower relative to WT mice in the 26-wk-old males (Fig. 5G). No significant differences were observed in osteoclast surface (OcS/BS) or osteoclast numbers (OcN/bone perimeter) (data not shown) in either sex. To assess osteoblast function in these mice, we performed dynamic histomorphometry. In 12-wk-old IL-23p19–/– mice, Tb mineralized surface (MS/BS) and bone formation rate (BFR/BS) were slightly lower in male IL-23p19–/– mice than in WT mice, but the mineral apposition rate was not affected (data not shown); no differences in these parameters were observed in females. Nevertheless, we did observe significantly lower periosteal mineral apposition rates in IL-23p19–/– mice of both sexes (Fig. 5H) and slightly reduced periosteal width in IL-23p19–/– males (data not shown). However, growth plate osteoclast numbers were elevated in females (Fig. 5I).

No differences in any of the above histomorphometric parameters were observed between IL-23p19–/–, heterozygous and WT mice at 4 wk of age (data not shown).

Analysis of bone length, growth plate, and cartilage remnants

Slightly shorter (Fig. 6A) and narrower (data not shown) femora were noted in 26-wk-old IL-23p19–/– male mice compared with WT, but this difference was not detected earlier. The difference in length was small (8.6%) but significant and suggested that IL-23p19–/– mice could have an abnormality in bone growth at or after 12 wk of age. Indeed, we found that at 12 wk of age (but not 4 wk), the IL-23p19–/– and heterozygote tibial growth plate was significantly narrower in both sexes than in WT controls (Fig. 6, B and C), with a greater extent of growth plate closure (data not shown). The narrower growth plates were characterized by a smaller hypertrophic zone but not proliferating zone (Fig. 6C), suggesting that chondrocyte proliferation was not affected by IL-23 deletion. The smaller hypertrophic zone may be explained by increased resorption of the hypertrophic cartilage as it mineralizes. Consistent with this, the length of the primary spongiosa was strikingly truncated in IL-23p19–/– males (Fig. 6D) suggesting that this layer of primitive, woven bone is removed more rapidly than in WT. This also suggests a local increase in bone resorption. Osteoclasts are abundant in this region in both WT and IL-23p19–/– mice, but differences in osteoclast numbers proved impossible to interpret due to grossly different surface areas of primary spongiosa and growth plate between the two genotypes. Female IL-23p19–/– and heterozygote mice also had truncated growth plate hypertrophic zones, but did not display significantly shorter primary spongiosa. Nevertheless, female IL-23p19–/– and heterozygotes had reduced cartilage remnants within TB bone (Fig. 6E), consistent with increased bone resorption; this was not observed in males, suggesting that their elevated bone resorption was confined to the growth plate region.


Figure 6
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  		FIGURE 6. IL-23p19–/– male mice develop shorter limb bones than WT: analysis of femoral length, growth plate, primary spongiosa, and cartilage remnants. A, Femoral length and width. *, p < 0.05 relative to WT control. B, Growth plate region of tibia showing reduced hypertrophic zones in IL-23p19–/– mice at 12 wk. Prol, Chondrocyte proliferation zone; Hyp, hypotrophic zone; Prim, primary spongiosa; b, mature Tb bone; m, bone marrow. C, Relative widths of proliferating zone ({blacksquare}) and hypertrophic zone ({square}) of growth plates of 12-wk-old mice. *, p < 0.05 and **, p < 0.01 relative to hypertrophic zone of WT control. D, Length of primary spongiosa. E, Cartilage remnants in Tb bone (proportion of area). F, Ex vivo cultures of hemopoietic cells from WT and IL-23p19–/– mice: osteoclast formation in RANKL/M-CSF- stimulated cultures of spleen and bone marrow cells. *, p < 0.05 and **, p < 0.01 relative to appropriate WT control. Het, Heterozygote IL-23p19+/–; KO, knockout IL-23p19–/–.

 
Ex vivo cultures of cells from WT and IL-23p19–/– mice

Both bone marrow cells and spleen cells from 10-wk-old male IL-23p19–/– mice formed a greater number of osteoclasts when stimulated with RANKL and M-CSF in vitro compared with age-matched male WT mice (Fig. 6F). We found no difference between calvarial osteoblasts derived from WT and IL-23p19–/– mice in their ability to mineralize in vitro (data not shown).

IL-23 treatment does not affect osteoblasts directly or indirectly via lymphocytes

Consistent with the lack of IL-23R mRNA expression (see above), IL-23 did not affect the ability of WT or IL-23p19–/– osteoblasts or Kusa O cells to mineralize matrix in vitro or induce osteocalcin expression in ascorbate-treated cultures (data not shown). IL-23 also had no effect on osteocalcin mRNA expression in ascorbate-treated Kusa O osteoblastic cells grown in the presence or absence of T cells (or unfractionated spleen cells) in direct coculture or separated by Transwell membrane (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IL-23 is emerging as a cytokine of critical importance in chronic inflammation. Elevated incidence of ankylosing spondylitis, inflammatory bowel disease, psoriasis, and myocardial infarction also associate with variants of the gene encoding IL-23R (34, 35, 36, 37). Bone destruction notably occurs at or near sites affected by chronic inflammation, and IL-23 may influence this by participating in processes that maintain inflammation. However, examining bone loss secondary to inflammation does not shed light on the role of IL-23 in normal bone homeostasis. To clarify this, we investigated actions of rIL-23 on osteoclasts in vitro and the bone phenotype of IL-23p19–/– mice which lacks functional IL-23.

IL-23p19–/– mice displayed a progressively developing osteopenia, evident at 12 wk of age. The progressive nature of this osteopenia may explain a report of normal bone mass in 10-wk-old IL-23p19 null mice (12). Tb BMD and bone volume were reduced in male IL-23p19–/– mice, the latter due mainly to low Tb number, although Tb thickness was reduced in older males. Surprisingly, bone mass was also reduced in heterozygotes, suggesting that low IL-23 levels (assuming it occurs in heterozygotes) significantly affect bone, and is consistent with data on IL-23R variants. In contrast to males, female IL-23p19–/– mice had low Tb bone mass at 12 wk but had otherwise a less pronounced bone phenotype. As commonly observed, female WT mice had less bone mass than males, which possibly makes further reduced bone mass harder to observe. Indeed, this is likely in 26-wk-old females with their extremely low bone mass; Tb BMD was also normal in female IL-23p19–/– mice. As well as lower bone mass, female mice have a higher bone remodeling rate than males (and consequently a shorter primary spongiosa) as well as different patterns of bone apposition and longitudinal bone growth. Such differences between the sexes may result in differing patterns of response to cytokine ablation that influence bone turnover rate, even where sex hormone responsiveness is not targeted.

The bone abnormalities in IL-23p19–/– mice could not be explained by high osteoclast or decreased osteoblast numbers, although osteoblast numbers and osteoid were mildly reduced in 26-wk-old males. We were alerted to another explanation by finding that IL-23p19–/– femora are slightly shorter than those of WT, suggesting bone growth defects. Indeed, IL-23p19–/– mice of both sexes had abnormally narrow growth plates, but this was restricted to the hypertrophic zone, indicating that the shortened bone length was not a result of impaired chondrocyte proliferation. Bone growth occurs due to growth plate expansion; as well as expansion due to chondrocyte proliferation, mineralized cartilage at the hypertrophic zone is resorbed by osteoclasts and replaced by low-density primary spongiosa (Fig. 7A). This woven bone is also resorbed, forming a template for denser (lamellar) Tb bone formation (secondary spongiosa) that we routinely examine by pQCT and histomorphometry. Unresorbed cartilage remnants are detectable in Tb bone and excessive persistence of such remnants occurs when bone resorption is impaired (33).


Figure 7
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  		FIGURE 7. A, Tb formation by bone modeling and remodeling below the growth plate, left to right. In the hypertrophic zone (Hyp) of the growth plate, calcified cartilage (light gray) is resorbed by osteoclasts (OC). Resulting small processes form scaffolds for woven bone formation (primary spongiosa, darker gray) by osteoblasts (OB). This is later remodeled by osteoclasts and osteoblasts to form mature Tb (darkest gray). Remnants of the original cartilage are left embedded in trabeculae. In IL-23p19–/– mice, the width of the calcified cartilage layer (both sexes) and primary spongiosa (in males) is reduced. This suggests greater osteoclast activity in these regions resulting in formation of smaller Tb structures. B, Model of IL-23 indirect actions on bone cells. IL-23 has actions upon T cells, resulting in expansion of Th17 subsets. Upon Ag presentation, Th17 cells express RANKL as well as IL-17, which elicits osteoblast-derived RANKL. Both sources of RANKL would stimulate osteoclast differentiation. In contrast, IL-23 acts upon Th17 cells (and {gamma}{delta} T cells), which results in increased GM-CSF production, reducing osteoclast differentiation.

 
The thin growth plate hypertrophic zones in IL-23p19–/– tibia could be due to abnormal chondrocyte differentiation or excessive osteoclast action. The latter was strongly suggested by the truncated primary spongiosa in male IL-23p19–/– mice. This feature is rarely reported but is apparent in 13-wk-old male osteoprotegerin null (OPG–/–) and OPG+/– mice (38). The primary spongiosa region is abundant in osteoclasts, but due to its complex morphology and gross differences in bone surfaces (BS) (on which osteoclasts form) between genotypes, it was impossible to determine whether osteoclast numbers were elevated in IL-23p19–/– mice. Nevertheless, since Tb bone is built on primary spongiosa which in turn is built on calcified cartilage, it is likely that a reduction in primary spongiosa results in less Tb bone (summarized in Fig. 7A) and/or reduced initiation of new trabeculae. In females, the primary spongiosa was not shorter but cartilage remnants were reduced, which also points to elevated osteolysis and may also suggest that increased resorption is less localized than in males. It should also be noted that the primary spongiosa is shorter in WT females than in matched males due to higher bone remodeling rates in females and the elevation in osteolysis due to IL-23 ablation may not have as pronounced effects on female primary spongiosa width. Collectively, these observations indicate that IL-23 particularly influences resorption near the growth plate and that in other models displaying osteopenia (particularly involving lymphocyte-mediated cytokines) defects at this location should be examined.

In spleen cell cultures, we characterized antiosteoclastogenic and potentially antiosteolytic actions of IL-23 that resemble those of IL-12 and IL-18 (16, 17). A number of cell types respond to IL-23, including subpopulations of macrophages, CD4+ and CD8+ T cells (39), and {gamma}{delta} T cells. BMM cultures (devoid of T cells) and T cell depletion and addition experiments demonstrated that CD4+ T cells mediated these inhibitory actions of IL-23. This suggests that the mediating cells are Th17 cells, which are characterized by IL-23-elicited secretion of IL-17 and IL-6 (13). However, CD4– {gamma}{delta} T cells also secrete IL-17 in response to IL-23 (22). {gamma}{delta} T cells are a poorly understood T cell subpopulation participating in innate immune functions and compose only a small fraction of T cells in spleen (22). Addition of 104 or more {gamma}{delta} T cells to bone marrow cell cultures resulted in sensitivity to IL-23 inhibition. {gamma}{delta} T cells were present in our splenic cell cultures, including CD4+ cell-depleted splenic cultures; these were insensitive to IL-23 due to insufficient {gamma}{delta} T cells (5 x 103 per culture) to mediate significant inhibition. {gamma}{delta} T cells can accumulate in pathological conditions and exert both positive and negative effects on joint inflammation progression. Our data point to an unsuspected influence of these cells on bone.

We determined that IL-23 effects on osteoclast formation via CD4+/{gamma}{delta} T cells were dependent on GM-CSF production; IL-23 induction of GM-CSF protein in IL-2-treated CD4+ cells has been noted by Aggarwal et al. (25). No mediation by IFN-{gamma} Abs was noted, although IFN-{gamma} is better characterized as a target of IL-23 action (13). The effect of IL-23 resembles that of IL-18 (16), which also exerts actions on osteoclasts via T cell-derived GM-CSF; consistent with this, we found synergy between IL-23 and IL-18. Thus, IL-23 effects may be strongly influenced by other local cytokines. In addition, production of IL-23 by dendritic cells (13) (which form in the presence of GM-CSF) and GM-CSF-stimulated macrophages (40) suggest that IL-23 and GM-CSF may cause local positive feedback enhancement of each other’s production.

The T cell populations we used were not activated and in the absence of IL-23 did not affect osteoclasts. However, we previously found that T cell activation results in RANKL production and osteoclast formation (9), and in an important extension to those observations Sato et al. (12) showed anti-CD3/anti-CD28-activated Th17 cells are the RANKL-producing T cell subset. This implies that IL-23 treatment of activated T cells has a net osteolytic effect. GM-CSF powerfully inhibits osteoclast formation, but only in progenitors exposed to both GM-CSF with RANKL (16). When GM-CSF treatment is withdrawn before RANKL exposure, no inhibition occurs (41); indeed, osteoclast progenitors expand (42). IL-23 elicits T cell IL-17 and IL-6 production, both stimulators of osteoblast RANKL expression (43). IL-23 stimulus thus results in exposure to both RANKL and GM-CSF, the latter potentially blocking action of the former (Fig. 7B). In contrast, in inflammatory lesions with high levels of T cell activation, IL-23 expansion of Th17 cells may result in levels of T cell-derived RANKL high enough to overcome GM-CSF effects.

We found no evidence that IL-23 influenced osteoblasts in vitro and could not detect their expression of IL-23R mRNA. IL-23 may indirectly affect osteoblasts, which is suggested by low periosteal mineral apposition in IL-23p19–/– mice; notably, these cells are of a different origin than the osteoblasts on Tb bone. It is unclear how this effect on periosteal osteoblasts may occur, although IL-23 acts on cell types that may influence stromal cells (21, 24, 44).

In contrast, IL-23-elicited GM-CSF powerfully affects the osteoclast lineage; however, as no GM-CSF null bone phenotype has been reported and GM-CSF-transgenic mice have a complex inflammatory syndrome (45) (rendering its influence on bone hard to interpret), the contribution of GM-CSF to the IL-23p19–/– phenotype mice is difficult to assess. Indeed, although our in vitro observations suggest that IL-23-elicited GM-CSF is a plausible mechanism to explain the lower bone mass in IL-23p19–/– mice, IL-23 ablation may also result in a lack of Th17 cells (and perhaps other subpopulations yet to be defined) that may reduce bone mass due to lack of some other osteoclast inhibitory influence. IL-23 may also elicit other T cell-derived osteoclast inhibitors in vivo that it does not under our in vitro culture conditions.

Thus, with these considerations in mind and since the localization of IL-23 responsive or IL-23-dependent T cells in long bones are unknown, we cannot fully explain the localization of the IL-23p19–/– bone defect. However, the abundance of osteoclasts at the growth plate may render this site particularly sensitive to inhibitors such as GM-CSF. Alternatively, differences in osteoclast progenitors found at different sites or in proximity of Th17 cells may account for our observations, but further work is needed to clarify this.

In summary, we have identified that lack of IL-23 has significant negative consequences for bone mass and found powerful T cell-mediated effects of IL-23 on osteoclast formation. This provides new evidence regarding lymphocyte influence on physiological bone homeostasis and suggests this influence differs from that exerted when activated lymphocytes are present under inflammatory circumstances.


   Acknowledgments
 
We thank Nico Ghilardi of Genentech Inc. (South San Francisco, CA) for supplying us with IL-23p19–/– mice. We thank Dr. David Izon (St. Vincent’s Institute, Fitzroy, Australia) for advice with flow cytometry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Program Grant 345401 from the National Health and Medical Research Council of Australia. Back

2 Address correspondence and reprint requests to Dr. Julian Quinn, St. Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065, Australia. E-mail address: jquinn{at}svi.edu.au Back

3 Abbreviations used in this paper: RANKL, receptor activator of NF-{kappa}B ligand; 1,25(OH)2 D3, 1{alpha},25-dihydroxyvitamin D3; BMM, bone marrow macrophage; pQCT, peripheral quantitative computer-aided tomography; Ct, cortical; Tb, trabecular; WT, wild type; BMD, bone mineral density; TRAP, tartrate-resistant acid phosphatase; MNC, multinucleated cell; BV, bone volume; TV, Tb volume. Back

Received for publication April 1, 2008. Accepted for publication August 17, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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