Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles

Abstract

The inflammatory response to prosthetic implant-derived wear particles is the primary cause of bone loss and aseptic loosening of implants, but the mechanisms by which macrophages recognize and respond to particles remain unknown. Studies of innate immunity demonstrate that Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPS). All TLRs signal through myeloid differentiation factor 88 (MyD88), except TLR3 which signals through TIR domain containing adapter inducing interferon-beta (TRIF), and TLR4 which signals through both MyD88 and TRIF. We hypothesized that wear-debris particles may act as PAMPs/DAMPs and activate macrophages via TLRs. To test this hypothesis, we first demonstrated that inhibition of MyD88 decreases polymethylmethacrylate (PMMA) particle-induced production of TNF-α in RAW 264.7 macrophages. Next we compared particle-induced production of TNF-α among MyD88 knockout (MyD88(-/-)), TRIF knockout (TRIF(-/-)), and wild type (WT) murine macrophages. Relative to WT, disruption of MyD88 signaling diminished, and disruption of TRIF amplified the particle-induced production of TNF-α. Gene expression data indicated that this latter increase in TNF-α was due to a compensatory increase in expression of MyD88 associated components of the TLR pathway. Finally, using an in vivo model, MyD88(-/-) mice developed less particle-induced osteolysis than WT mice. These results indicate that the response to PMMA particles is partly dependent on MyD88, presumably as part of TLR signaling; MyD88 may represent a therapeutic target for prevention of wear debris-induced periprosthetic osteolysis.

Fig. 1

Macrophage recognition of PMMA particles involves MyD88-dependent TLRs and is independent of adherent endotoxin TNF-α production by RAW 264.7 murine macrophages cultured with (a) PMMA particles at a concentration of 0.03, 0.075, 0.15 and 0.30% v/v (n = 3); (b) PMMA particles, PMMA particles and Polymyxin B, LPS, LPS and Polymyxin B (4 independent trials, n = 3–6 per trial); (c) PMMA particles, PMMA particles plus a MyD88 inhibitory peptide, PMMA particles plus a control peptide. (2 independent trials, n = 3–6 per trial). % v/v = volume of particle (mL) per 100 mL of solution. * = P < 0.05, ** = P < 0.01.

Fig. 2

Inflammatory response to PMMA particles is mediated by MyD88-dependent and TRIF-independent TLRs The inflammatory response toward PMMA particles is diminished in MyD88 knockout (MyD88^−/−) relative to wild type (WT) bone marrow derived macrophages at the both the level of TNF-α (a) protein production (4 independent trials, n = 3–4 per trial); and (b) mRNA expression (2 independent trials, n = 3 per trial). Fold change is relative to the mRNA expression level of each cell type prior to PMMA particle exposure. (c) TRIF knockout (TRIF^−/−) macrophages demonstrate enhanced TNF-α production relative to WT bone marrow derived macrophages in response to PMMA particles (2 independent trials, n = 3–4 per trial). (d) RT-PCR comparing the expression of numerous genes involved in the TLR signaling pathway by MyD88^−/− and (e) TRIF^−/− BMDMs prior to and following 12 h of PMMA particle exposure. Fold change is expressed relative to WT cells. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Fig. 2

Inflammatory response to PMMA particles is mediated by MyD88-dependent and TRIF-independent TLRs The inflammatory response toward PMMA particles is diminished in MyD88 knockout (MyD88^−/−) relative to wild type (WT) bone marrow derived macrophages at the both the level of TNF-α (a) protein production (4 independent trials, n = 3–4 per trial); and (b) mRNA expression (2 independent trials, n = 3 per trial). Fold change is relative to the mRNA expression level of each cell type prior to PMMA particle exposure. (c) TRIF knockout (TRIF^−/−) macrophages demonstrate enhanced TNF-α production relative to WT bone marrow derived macrophages in response to PMMA particles (2 independent trials, n = 3–4 per trial). (d) RT-PCR comparing the expression of numerous genes involved in the TLR signaling pathway by MyD88^−/− and (e) TRIF^−/− BMDMs prior to and following 12 h of PMMA particle exposure. Fold change is expressed relative to WT cells. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Fig. 3

Disruption of MyD88-dependent signaling diminishes PMMA particle-induced osteolysis (a) PMMA particle-induced osteolysis in WT and MyD88^−/− mice as assessed within the volume of interest by longitudinal 3D micro-computed tomography (μCT). The volume of interest is indicated by the yellow shaded region (top left panel), with the 2-dimensional borders indicated in the coronal plane (top right panel), axial plane (bottom left panel) and sagittal plane (bottom right panel). (b) Graphical representation of micro-computed tomography quantifying the percent change in bone volume (BV) induced by injecting PMMA particles onto the calvarium of WT (n = 7) and MyD88^−/− mice (n = 5). * = P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)