Introduction:. Wear particles cause aseptic loosening by stimulating macrophages to produce inflammatory cytokines. Recent studies indicate that Toll-like receptor 2 (TLR2) and TLR4 mediate macrophage responses to the wear particles [1–3]. TLR2 and TLR4 uniquely activate MyD88-dependent signaling via an additional adapter protein known as TIRAP/Mal [4]. Del Vescovo et al reported that three single nucleotide polymorphisms (SNPs) within the TIRAP/Mal gene associate with aseptic loosening in THA patients [5]. The goal of the current study was therefore to determine whether TIRAP/Mal mediates responses to orthopaedic wear particles. Methods:. Immortalized wild type (WT) and TIRAP/Mal knockout (KO) murine macrophages (Mfs) were incubated in the presence or absence of titanium (Ti) particles (1 × 10. 8. particles/cm. 2. [2]. Three types of particles were used as described previously [1,2]: Ti particles with adherent bacterial debris (38.3 Endotoxin Units/10. 9. particles), endotoxin-free Ti particles (<0.1 EU/10. 9. particles), and Ti particles with adherent lipopolysacharide (LPS, 32.8 EU/10. 9. particles). TNFa, IL-1b, and IL-6 mRNAs were measured by real-time PCR and the secreted cytokines were measured by ELISA. Particle-induced osteolysis in calvaria of TIRAP/Mal KO and WT mice was measured 7 days after particle implantation [1,2]. In vitro results are presented as mean ± SEM of 3–4 replicate experiments analyzed by two-way ANOVA with Bonferroni post-hoc corrections. In vivo results are presented as means of individual parietal bones ± SEM (n = 22) and analyzed by one-way ANOVA on ranks with Student Neuman-Keuls post-hoc corrections. * denotes p < 0.5, ** denotes p < 0.01, *** denotes p < 0. Results:. Ti particles with adherent bacterial debris induced substantial osteolysis and expression of TNFa, IL-1b, and IL-6 at both the mRNA and protein levels and all of those responses were significantly inhibited by TIRAP/Mal KO (Fig 1 & Fig 2). Endotoxin-free Ti particles had a small effect on osteolysis and cytokine mRNA expression that was not dependent on TIRAP/Mal (Fig 1 & data not shown). Adherence of highly purified LPS to the endotoxin-free particles reconstituted the stimulation of osteolysis and cytokine expression as well as the dependence on TIRAP/MAL (Fig 1 & data not shown). Specificity of the effects of TIRAP/Mal KO was demonstrated since responses induced by recombinant murine IL-1b were unaffected (data not shown). Discussion:. Our results are the first demonstration that TIRAP/Mal mediates effects of orthopaedic wear particles. TIRAP/Mal KO inhibited expression of TNFa by ∼50% and almost completely inhibited particle-induced osteolysis, as well as expression of IL-1b and IL-6. Our results, coupled with the genetic association of SNPs in human TIRAP/Mal with aseptic loosening [5], lead to two conclusions. First, activation of TIRAP/Mal likely contributes to aseptic loosening in patients. Second, pathogen-associated molecular patterns (PAMPs) also likely contribute to aseptic loosening since the results with endotoxin-free Ti particles demonstrate that adherent PAMPs are required for activation of TIRAP/Mal