Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction

Abstract

In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-gamma remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23-IL-17 axis, rather than the IL-12-IFN-gamma axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.

Figure 1.

Effects of Th1, Th2, and T reg cells on in vitro osteoclastogenesis. (A) Schematics of two culture systems for osteoclast differentiation and Th cell addition. In the RANKL–M-CSF system, mouse nonadherent BMCs were stimulated with M-CSF for 2 d and adherent cells were used as BMMs. After BMMs were stimulated with recombinant RANKL and M-CSF for 3 d, the formation of TRAP⁺ MNCs was analyzed. In the co-culture system, BMCs were co-cultured with osteoblasts stimulated with VitD₃ and PGE₂, and the formation of TRAP⁺ MNCs was observed 7 d after the addition of BMCs. (B) Inhibitory effects of Th1 and Th2 cells on TRAP⁺ MNC formation in the RANKL–M-CSF system. Th cells (4,000 or 20,000 cells/ml) were added at the same time as RANKL (day 0) with (black bars) or without (white bars) anti-CD3 mAb. n.d., not detected. (C) Inhibitory effects of Th1 and Th2 cells on TRAP⁺ MNC formation in the co-culture system. The same number of T cells as in B was added 2 d after BMC addition (day 2). (D) Microphotographs of the in vitro osteoclast formation systems in the presence of Th1 or Th2 cells (20,000 cells/ml) with anti-CD3 mAb (TRAP staining). (E) Cytokine profile of culture supernatants in the presence of Th cells and 1 μg/ml of soluble anti-CD3 mAb (the RANKL–M-CSF system on day 2). Without restimulation by anti-CD3 mAb, cytokine production was much less than this result and was difficult to detect after 2-d culture with osteoclast precursor cells (not depicted). (F) Effects of Th1 and Th2 cells (20,000 cells/ml plus anti-CD3 mAb) on WT or IFN-γ receptor–deficient (Ifngr1 ^−/−) osteoclast precursor cells. (G) Effects of isolated CD4⁺CD25⁺ T reg cells (4,000 or 20,000 cells/ml plus anti-CD3 mAb) on osteoclastogenesis in vitro. n.s., not significantly different. The survival of a considerable number of T reg cells after 3 d was confirmed by CFSE staining (not depicted).

Figure 2.

Formation of multinuclear cells with no bone-resorbing activity induced by Th2 cells and IL-4. (A) Inhibitory effects of Th1 and Th2 cells on osteoclastogenesis are reduced when T cells are added 1 d later. Th cells (20,000 cells/ml plus anti-CD3 mAb) were added on days 0 (at the same time as RANKL, gray bars) or 1 (black bars) to the RANKL–M-CSF system and on days 2 (2 d after BMC addition, gray bars) or 3 (black bars) to the co-culture system. (B) Microphotographs and (C) quantification of in vitro osteoclast formation (left, TRAP staining) and resorption pit formation (right). Th1 and Th2 cells (20,000 cells/ml plus anti-CD3 mAb) were added to WT or Stat6 ^−/− osteoclast precursor cells on day 1. (D) Effect of IL-4 on mRNA expression of osteoclast-related genes in osteoclast precursor cells (GeneChip analysis). Osteoclast precursor cells were stimulated by 10 ng/ml IL-4 from day 1 in the RANKL–M-CSF system and harvested on day 3. Fold mRNA difference was calculated by dividing the average difference of the IL-4–treated sample by that of the control sample. The expressions of most of the osteoclast-specific genes are down-regulated. (E) Reduced expression of NFATc1 protein in the cells treated with IL-4. Osteoclast precursor cells were stimulated by 10 ng/ml IL-4 from day 1 in the RANKL–M-CSF system, fixed on day 3, and stained with anti-NFATc1 antibody followed by Alexa Fluoro 488–labeled secondary antibody.

Figure 3.

Enhanced osteoclastogenesis by Th17 cells in the co-culture system but not in the RANKL–M-CSF system. (A) Effects of Th1 and Th17 cells on the osteoclast differentiation systems. T cells (4,000 or 20,000 cells/ml plus anti-CD3 mAb) were added on day 1 to the RANKL–M-CSF system and on day 3 to the co-culture system. When the Th17 cells were added 1 d earlier, or in the absence of soluble anti-CD3 mAb, enhancement of osteoclastogenesis was not observed even in the co-culture system (not depicted). (B) Cytokine profile of the culture supernatants obtained on day 3 from the RANKL–M-CSF system in the presence of Th1 and Th17 cells derived from either WT or Il17 ^−/− mice under the conditions described in A. (C) Effects of Th1 and Th17 cells derived from either WT or Il17 ^−/− mice on the formation of TRAP⁺ MNCs or TRAP⁺ cells in the co-culture system in the absence of VitD₃ and PGE₂. T cells (20,000 cells/ml plus anti-CD3 mAb) were added on day 3. (D) Effects of recombinant IL-17 and IL-23 (2 or 10 ng/ml) on osteoclastogenesis in vitro. (E) Expression of RANKL on Th subsets. CD4⁺ T cells cultured in each of the Th conditions for 3 d were restimulated with 1 μg/ml of plate-bound anti-CD3 mAb for 4 h and subjected to flow cytometric analysis using anti-RANKL mAb. Without the restimulation by anti-CD3 mAb, RANKL expression was barely detectable (not depicted).

Figure 4.

Contribution of IL-17 and IL-23 to the physiological and pathological bone resorption. (A) Bone mineral densities (measured in 20 longitudinal divisions of the femurs), (B) micro-computed tomography (at 10% length above the distal epiphyseal plate), and (C) bone morphometic analyses of WT, Il17 ^−/−, and Il23a ^−/− mice at the age of 12 wk. (D) Histological examination of calvarial bones of WT, Il17 ^−/−, and Il23a ^−/− mice treated with LPS (hematoxylin and TRAP staining). The degree of bone destruction was analyzed by the number of osteoclasts and the area of the eroded surface (%).

Figure 5.

Regulation of RANKL-mediated osteoclastogenesis by the IL-23–IL-17 axis in the RA synovial tissue. (A) Correlation of the mRNA expression level of RANKL with that of IL23A (p19), IL12A (p35), or IL12B (p40) in the synovium of RA patients. The relative expressions of RANKL, IL23A, IL12A, and IL12B were all standardized using that of GAPDH. (B) Model of Th17-mediated bone destruction in autoimmune arthritis. Th17 cells function as an osteoclastogenic Th cell subset by stimulating local inflammation, inducing RANKL on osteoclastogenesis-supporting cells, and expressing RANKL on themselves and stimulating local inflammation, all of which contribute to an acceleration of osteoclastogenesis. It is notable that RANKL on Th17 cells alone is not sufficient for the induction of osteoclast differentiation (a dotted line). See Discussion for the details. Op, osteoclast precursor cell.