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CCL3 and MMP-9 Are Induced by TL1A During Death Receptor 3 (TNFRSF25)-dependent Osteoclast Function and Systemic Bone Loss

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CCL3 and MMP-9 Are Induced by TL1A During Death Receptor 3 (TNFRSF25)-dependent Osteoclast Function and Systemic Bone Loss

Fraser L Collins et al. Bone.

Abstract

Reduced bone density and secondary osteoporosis, resulting in increased risk of fracture, is a significant complicating factor in the inflammatory arthritides. While the exact etiology of systemic bone loss is not fully elucidated, recent insights into the tumor necrosis factor super family (TNFSF) revealed a potential role for death receptor 3 (DR3/TNFRSF25) and one of its ligands, TNF-like protein 1A (TL1A/TNFSF15). The mechanisms by which DR3/TL1A signalling modulates bone loss are unclear. We investigated the effect of DR3/TL1A signalling upon osteoclast-dependent chemokine and MMP production to unravel novel mechanisms whereby this pathway regulates OC formation and OC-dependent bone resorption. Collagen induced arthritis (CIA) was established in DR3wt and DR3ko mice, joints were sectioned and analysed histologically for bone damage while systemic trabecular bone loss distal to the affected joints was compared by micro-CT. Ablation of DR3 protected DBA/1 mice against the development and progression of CIA. In DR3ko, joints of the ankle and mid-foot were almost free of bone erosions and long bones of mice with CIA were protected against systemic trabecular bone loss. In vitro, expression of DR3 was confirmed on primary human CD14+ osteoclast precursors by flow cytometry. These cells were treated with TL1A in osteoclast differentiation medium and TRAP+ osteoclasts, bone resorption, levels of osteoclast-associated chemokines (CCL3, CCL2 and CXCL8) and MMP-9 measured. TL1A intensified human osteoclast differentiation and bone resorption and increased osteoclast-associated production of CCL3 and MMP-9. Our data reveals the DR3 pathway as an attractive therapeutic target to combat adverse bone pathology associated with inflammatory arthritis. We demonstrate that DR3 is critical in the pathogenesis of murine CIA and associated secondary osteoporosis. Furthermore, we identify a novel mechanism by which the DR3/TL1A pathway directly enhances human OC formation and resorptive activity, controlling expression and activation of CCL3 and MMP-9.

Keywords: CCL3; Collagen-induced arthritis; MMP-9; Osteoclast; Osteoporosis; TNFRSF25; TNFSF15.

Figures

Fig. 1
Fig. 1
Absence of DR3 protects against collagen-induced arthritis. CIA was induced in male DR3wt and DR3ko mice bred onto a DBA/1 background. a) Arthritis severity was determined by level of paw swelling. DR3wt CIA mice had significantly worse arthritis severity (p < 0.05) compared to the DR3wt baseline control. No significant difference was observed between the DR3ko CIA and DR3ko baseline controls. b) Summary data of pathology by an arthritis index determined by scoring for erosion, cellular infiltrates, exudate and hyperplasia. Arthritis index was significantly lower in DR3ko CIA compared to DR3wt CIA mice (p < 0.01); n = 5–15 mice per group. c) Representative micrographs of joints from DR3wt and DR3ko mice following CIA induction: e = erosion; p = pannus formation; sh = synovial hyperplasia; si = synovial infiltration; l = loss of joint space. Scale bar = 0.1 mm. Statistical analysis performed with unpaired students t-test.
Fig. 2
Fig. 2
Absence of DR3 protects against systemic bone loss associated with collagen-induced arthritis. CIA was induced in male DR3wt and DR3ko mice. Representative images of a. i) control and ii) severe arthritis ankle joints showing erosions. Arrows indicate areas of erosion. b) Representative image of distal femoral trabecular region analysed by μCT. c) Distal femoral trabecular BV/TV was determined by μCT. Both DR3wt (p < 0.0001) and DR3ko (p < 0.01) CIA mice significantly lost trabecular bone compared to relevant baseline control. DR3wt CIA mice lost significantly more trabecular bone than DR3ko counterparts (p < 0.05). d) Representative μCT isosurface images. Scale bar = 1 mm. n = 5–14 mice per group. Statistical analysis performed with 1-Way ANOVA and Holm-Šídák post-test.
Fig. 3
Fig. 3
Expression of DR3 on human CD14+ monocyte osteoclast precursors. CD14+ monocytes were isolated from pre-menopausal females (n = 7) and cultured on ivory discs for 7 days in media + MCSF. DR3 expression was determined by flow cytometry. a) DR3 was not detected on freshly isolated CD14+ monocytes but confirmed after 7 days culture in MCSF (light grey line = isotype, shaded peak = DR3). b) DR3 expression was maintained in cultures following addition of RANKL (black line = isotype, grey line = DR3).
Fig. 4
Fig. 4
TL1A concentration-dependently increases osteoclast formation and osteoclast resorption. CD14+ monocytes were isolated from pre-menopausal females (n = 7) and cultured on ivory discs for 7 days in media + MCSF. Cells were differentiated for 7, 10 or 14 days in the presence of MCSF (M) and RANKL (R) ± TL1A (T) at 10 ng/ml or 100 ng/ml. At experiment end-point cells were stained for TRAP. a) TL1A had no effect on total cell number. b) A significant increase in OC numbers was observed in the 10 ng/ml (p < 0.01) and the 100 ng/ml (p < 0.0001) TL1A cultures across the time-course. c) Representative images of TRAP stained i) control and ii) 100 ng/ml TL1A cultures at day 14. Scale bar = 250 μm. Arrows indicate multinucleated OC. d) Discs were stained with toluidine blue and % area resorbed calculated. Significantly increased resorption was observed in the 10 ng/ml (p < 0.0001) and 100 ng/ml (p < 0.01) TL1A cultures across the time-course compared to control. e) Representative day 14 confocal images of i) control, ii) 10 ng/ml and iii) 100 ng/ml TL1A culture resorption pits. Statistical analysis performed with 2-Way ANOVA and Bonferroni post-test.
Fig. 5
Fig. 5
TL1A concentration-dependently increases expression of the osteoclastogenic chemokine CCL3 and gelatinase MMP-9. CD14+ monocytes were isolated from pre-menopausal females (n = 7). Cells were cultured on ivory discs for 7 days in media + MCSF and further differentiated for 7, 10 or 14 days in the presence of MCSF (M) and RANKL (R) ± TL1A (T) at 10 ng/ml or 100 ng/ml. Culture supernatants were collected at indicated time-points and tested for a) i) CCL2, ii) CXCL8 and iii) CCL3. Significantly increased CCL3 levels were detected across the time-course in 100 ng/ml TL1A cultures compared to control (p < 0.01). No differences were observed in CCL2 and CXCL8 levels. b) CCL3 levels significantly correlated with OC numbers at (i) day 7, (ii) day 10 and (ii) day 14. c) i) Significantly increased total MMP-9 expression was observed in the 100 ng/ml TL1A cultures across the time-course compared to control (p < 0.0001) and 10 ng/ml TL1A cultures (p < 0.05). ii) Levels of total MMP-9 significantly correlated with % area resorbed at day 14. d) Levels of pro- and active-MMP-9 in cultures were determined by gelatine zymography (n = 4). Representative zymograms for control (MR) and 100 ng/ml TL1A (MRT) cultures. Pro-MMP-9 was detected in both cultures across the time-course. Active MMP-9 was first detected at day 3 in the 100 ng/ml TL1A OC cultures and day 7 in control cultures. Samples run in duplicate. Statistical analysis performed with 2-Way ANOVA and Spearman correlation.

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