Periprosthetic osteolysis: characterizing the innate immune response to titanium wear-particles

Abstract

Osteolysis of bone following total hip replacement is a major clinical problem. Examination of the areas surrounding failed implants has indicated an increase in the bone-resorption-inducing cytokine, interleukin 1β (IL-1β). NALP3, a NOD-like receptor protein located in the cytosol of macrophages, signals the cleavage of pro-IL-1β into its mature, secreted form, IL-1β. Here we showed that titanium particles stimulate the NALP3 inflammasome. We demonstrated that titanium induces IL-1β secretion from macrophages. This response depended on the expression of components of the NALP3 inflammasome, including NALP3, ASC, and Caspase-1. We also showed that titanium particles trigger the recruitment of neutrophils and that this acute inflammatory response depends on the expression of the IL-1 receptor and IL-1α/β. Moreover, administration of the IL-1 receptor antagonist (IL-1Ra) diminished neutrophil recruitment in response to titanium particles. Together, these results suggest that titanium particle-induced acute inflammation is due to activation of the NALP3 inflammasome, which leads to increased IL-1β secretion and IL-1-associated signaling, including neutrophil recruitment. Efficacy of IL-1Ra treatment introduces the potential for antagonist-based therapies for implant osteolysis.

Figure 1

Neutrophil recruitment following titanium injections in various mouse strains. (a) WT (b) MD2 KO (c) CD14 KO (d) IL-1R KO and (e) IL-1α/β KO. (f) Effect of IL-1Ra treatment on neutrophil recruitment. Graphs show the mean ± s.e.m. from the total number of mice indicated. P-values are shown as *** ≤ 0.0001; ** < 0.01; * < 0.05

Figure 2

IL-1β secretion following titanium stimulation in (a) WT immortalized mouse macrophages and (b) THP-1 human macrophages. Dose-response curves to titanium in (c) WT immortalized mouse macrophages and (d) THP-1 human macrophages. Secreted IL-1β levels are mean ± s.e.m. Significance values are shown relative to Prime levels. P-values are shown as *** ≤ 0.0001

Figure 3

Response to titanium particle uptake is cathepsin B dependent. (a) SEM image of immortalized mouse macrophages incubated with titanium particles for 30 min. (b) Diameter of macrophage-associated particles (n = 82) as determined by confocal microscopy and NIH ImageJ software. (c) IL-1β and (d) IL-6 secretion following titanium stimulation in WT immortalized mouse macrophages treated with the cathepsin-B inhibitor, CA-074-Me. Secreted cytokine levels are mean ± s.e.m. Significance values are shown relative to untreated (0 uM) samples. P-values are shown as *** ≤ 0.0001; ** < 0.01

Figure 4

Titanium-induced IL-1β secretion is inflammasome dependent. (a) IL-1β and (b) IL-6 secretion in WT, NALP3 KO, ASC KO, and CASP KO immortalized mouse macrophages. (c) IL-1β secretion following titanium stimulation in THP-1 human macrophages transfected with siRNA pools specific for NALP3, ASC, or luciferase (NT). Secreted cytokine levels are means ± s.e.m. P-values are shown as *** ≤ 0.0001; ** < 0.01

Figure 5

Titanium triggers inflammasome activation. Titanium particle internalization triggers the release of Cathepsin B from lysosomes, which together activate Nalp3. Activated Nalp3 recruits ASC through PYD domain interactions. This complex triggers cleavage and recruitment of activated Caspase-1 through CARD domain interactions. This inflammasome complex then cleaves pro-IL-1β into its active, secreted form IL-1β, which can trigger downstream IL-1 associated signaling, including neutrophil recruitment, through activation of the IL-1R. This acute inflammatory response can be inhibited with IL-1Ra treatment.