Interleukin-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signaling

Abstract

Objective: Interleukin-27 (IL-27) has stimulatory and regulatory immune functions and is expressed in rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effects of IL-27 on human osteoclastogenesis, to determine whether IL-27 can stimulate or attenuate the osteoclast-mediated bone resorption that is a hallmark of RA.

Methods: Osteoclasts were generated from blood-derived human CD14+ cells. The effects of IL-27 on osteoclast formation were evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells and measuring the expression of osteoclast-related genes. The induction of nuclear factor of activated T cells c1 (NFATc1) and the activation of signaling pathways downstream of RANK were measured by immunoblotting. The expression of key molecules implicated in osteoclastogenesis (NFATc1, RANK, costimulatory receptors, and immunoreceptor tyrosine-based activation motif-harboring adaptor proteins) was measured by real-time reverse transcription-polymerase chain reaction. Murine osteoclast precursors obtained from mouse bone marrow and synovial fluid macrophages derived from RA patients were also tested for their responsiveness to IL-27.

Results: IL-27 inhibited human osteoclastogenesis, suppressed the induction of NFATc1, down-regulated the expression of RANK and triggering receptor expressed on myeloid cells 2 (TREM-2), and inhibited RANKL-mediated activation of ERK, p38, and NF-kappaB in osteoclast precursors. Synovial fluid macrophages from RA patients were refractory to the effects of IL-27. In contrast to the findings in humans, IL-27 only moderately suppressed murine osteoclastogenesis, and this was likely attributable to low expression of the IL-27 receptor subunit WSX-1 on murine osteoclast precursors.

Conclusion: IL-27 inhibits human osteoclastogenesis by a direct mechanism that suppresses the responses of osteoclast precursors to RANKL. These findings suggest that, in addition to its well-known antiinflammatory effects, IL-27 plays a homeostatic role in restraining bone erosion. This homeostatic function is compromised under conditions of chronic inflammation such as in RA synovitis.

Figure 1. IL-27 inhibits human osteoclastogenesis in a dose- and time-dependent manner

Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) for 48h and RANKL (40ng/ml) was added on Day 3 as described in Materials and Methods. IL-27 (3, 10, 30 and 100ng/ml) was added at the initiation of cultures (Day1) or later (Day 3 or Day 5). (A and B), TRAP positive multinucleated (> 3 nuclei) cells were counted 5 days after RANKL addition and representative data of one out of three independent experiments are shown as mean ±SD from triplicate wells of 96 well plates (* = p>0.05, ** = p<0.05 and *** = p<0.01. p values were calculated by Student's t test). C, Cathepsin K mRNA was measured by using real-time PCR and normalized relative to GAPDH expression. The means ±SD of triplicate determinants in a representative experiment of three independent experiments are shown; small SDs are not readily apparent because of large inductions. D (left panels), freshly isolated human monocytes were cultured as in A and osteoclast function was measured by a resorption pit formation assay. D (right panel), Human CD14⁺ monocytes were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 2days. Cell viability was measured by MTT assay. Representative results of at least three independent experiments are shown.

Figure 2. IL-27 abrogates RANKL-mediated induction of NFATc1

A and B, Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 48h and then were stimulated with RANKL. C and D, Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) for 48h and then were stimulated with RANKL (40ng/ml) in the presence or absence of IL-27 (100ng/ml). NFATc1 protein expression 24, 48 and 72h following RANKL stimulation was measured by immunoblotting (A and C). NFATc1 mRNA was measured using real-time PCR and normalized relative to GAPDH expression (B and D). Representative results of at least three independent experiments are shown.

Figure 3. IL-27 inhibits RANKL-mediated activation of MAPK and NF-κB pathways and induction of c-Jun

Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 48h. Control and IL27-treated cells were stimulated with RANKL (40ng/ml) for 5, 10 and 15 minutes (A and B) or for 0.5, 1 and 3h (C). Immunoblotting was used to measure threonine 202/tyrosine 204 phosphorylation of Erk1/Erk2 and threonine 180/tyrosine 182 phoshorylation of p38 (A), serine 32 phosphorylation of IκBα and total IκBα (B), total TRAF6 (B), and nuclear c-Jun and TBP proteins (C). Representative results of at least five independent experiments are shown. D, RANK mRNA was measured in control and IL-27-treated cells using real-time PCR and normalized relative to GAPDH expression. The means ±SD of triplicate determinants in a representative experiment of three independent experiments are shown; small SDs are not readily apparent because of large inductions.

Figure 4. IL-27 inhibits TREM-2 expression

Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 24 and 48h. TREM-2 mRNA (A), OSCAR mRNA, SIRPβ1 mRNA and ILT7 mRNA (B) and DAP12 and FcRγ mRNA (C) was measured using real-time PCR and normalized relative to GAPDH expression. Representative results of at least four independent experiments are shown.

Figure 5. Synovial fluid macrophages derived from Rheumatoid Arthritis patients are refractory to IL-27

Freshly isolated CD14⁺ cells derived from synovial fluid of five RA patients were stimulated for 15min (A) or 3h (B) with hIFN-γ (100U/ml) or hIL-27 (100ng/ml). A, tyrosine 701 phosphorylation of STAT1 was measured by immunoblotting. Representative results of one out of five independent experiments are shown. B, CXCL10 mRNA expression was measured using real-time PCR and normalized relative to GAPDH expression (p<0.001 by paired Student's t test, n=5).

Figure 6. IL-27 is a moderate inhibitor of in vitro osteoclastogenesis in murine systems

A, Murine monocytes were stimulated for 5, 10 and 20min with mIFN-γ (100U/ml) or mIL-27 (100ng/ml). Immunoblotting was used to measure tyrosine 701 phosphorylation of STAT1 and tyrosine 705 phosphorylation of STAT3. (B-C) Bone marrow-derived osteoclast precursors obtained from C57BL/6J mice were cultured in the presence of MCSF (20ng/ml) and RANKL (80ng/ml) was added as described in Materials and Methods. IL-27 (1, 10 and 100ng/ml) was added 1 day before RANKL (Pretreatment) or simultaneously with RANKL. (B and C), TRAP positive multinucleated (> 3 nuclei) cells were counted 5 days after RANKL addition and representative data of one out of three independent experiments are shown as mean ±SD from triplicate wells of 96 well plates (* = p>0.05 and ** = p<0.05. p values were calculated by Student's t test). D, The expression of WSX-1 m-RNA was measured by PCR in freshly isolated human CD14+ and CD14- cells and in murine BMDM and splenocytes.