Netrin-1 is highly expressed and required in inflammatory infiltrates in wear particle-induced osteolysis

Abstract

Objective: Netrin-1 is a chemorepulsant and matrix protein expressed during and required for osteoclast differentiation, which also plays a role in inflammation by preventing macrophage egress. Because wear particle-induced osteolysis requires osteoclast-mediated destruction of bone, we hypothesised that blockade of Netrin-1 or Unc5b, a receptor for Netrin-1, may diminish this pathological condition.

Methods: C57BL/6 mice, 6-8 weeks old, had 3 mg of ultrahigh-molecular-weight polyethylene particles implanted over the calvaria and then received 10 µg of monoclonal antibodies for Netrin-1 or its receptors, Unc5b and deleted in colon cancer (DCC), injected intraperitoneally on a weekly basis. After 2 weeks, micro-computed tomography and histology analysis were performed. Netrin-1 expression was analysed in human tissue obtained following primary prosthesis implantation or after prosthesis revision for peri-implant osteolysis and aseptic implant loosening.

Results: Weekly injection of anti-Netrin-1 or anti-Unc5b-antibodies significantly reduced particle-induced bone pitting in calvaria exposed to wear particles (46±4% and 49±3% of control bone pitting, respectively, p<0.001) but anti-DCC antibody did not affect inflammatory osteolysis (80±7% of control bone pitting, p=ns). Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibody treatment markedly reduced the inflammatory infiltrate and the number of tartrate resistance acid phosphatase (TRAP)-positive osteoclasts (7±1, 4±1 and 14±1 cells/high power field (hpf), respectively, vs 12±1 cells/hpf for control, p<0.001), with no significant changes in alkaline phosphatase-positive osteoblasts on bone-forming surfaces in any antibody-treated group. Netrin-1 immunostaining colocalised with CD68 staining for macrophages. The peri-implant tissues of patients undergoing prosthesis revision surgery showed an increase in Netrin-1 expression, whereas there was little Netrin-1 expression in soft tissues removed at the time of primary joint replacement.

Conclusions: These results demonstrate a unique role for Netrin-1 in osteoclast biology and inflammation and may be a novel target for prevention/treatment of inflammatory osteolysis.

Keywords: Bone Mineral Density; Inflammation; Treatment.

Figure 1. Netrin-1 is highly expressed in inflammatory infiltrates in wear particle-induced osteolysis

A) The figures show representative images for H&E, Netrin-1 immunostaining and TRAP staining in human tissue biopsies from implant revision and primary implants (n=5 each). B) The figures show representative images for Netrin-1, Unc5b and DCC immunostaining in a murine UHMWPE particle-induced osteolysis model (n=5 mice per group). Images were taken at 100X and 400X magnification. Scale bar indicates 50 and 100 μm. ***p<0.001, compared to untreated (Sham) (ANOVA).

Figure 2. Blocking Netrin-1 using monoclonal antibody injections decreases bone pitting, inflammation and osteoclasts in mouse calvariae after exposure to wear particles

A) The figures show representative microCT images of calvaria of mice treated with UHMWPE wear particles combined with anti-IgG (Control), anti-Netrin-1, anti-Unc5b or anti-DCC blocking antibodies at 10μg (n=5 mice per group). B) Calvariae were stained with hematoxylin & eosin to determine the presence of inflammation on the outer bone surface. The area of inflammatory infiltrate was quantified and expressed as a percentage of the area of the control particle-exposed mice (n = 5 mice per group). Shown are representative images for TRAP staining for osteoclasts in mice calvariae and the mean (±SEM, n=5 mice per group) number of osteoclasts/hpf. Images were taken at 400X magnification. Scale bar indicates 50 μm. Data were expressed as mean±SEM (n=5 per group). * p<0.05, **p<0.001, ***p<0.001 compared to control (ANOVA).

Figure 3. Immunohistochemistry for markers of osteoclasts

Calvaria were processed and immunohistologic staining carried out. Shown are representative H&E sections of calvariae (from n=5 mice per group) stained for Cathepsin K (green). Nuclei are shown in blue (Dapi). Quantification of the number of positive cellst/hpf was done. Data are means±SEM (n=5 mice per group). All images were taken at the same magnification 200X and 400X. Scale bar indicates 50 μm. ***p<0.001 (ANOVA).

Figure 4. Immunohistochemistry for markers of osteoblasts

Calvariae were processed and immunohistologic staining carried out. Shown are representative representative H&E sections of calvaria (from n=5 mice per group) stained for Alkaline Phosphatase (green). Nuclei are shown in blue (Dapi). Quantification of the number of positive cellst/hpf was done. Data are means±SEM (n=5 mice per group). All images were taken at the same magnification 200X and 400X. Scale bar indicates 50 μm. ***p<0.001 (ANOVA).