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. 2018 Nov 13;476(12):2442–2453. doi: 10.1097/CORR.0000000000000548

Wear Particle-induced Priming of the NLRP3 Inflammasome Depends on Adherent Pathogen-associated Molecular Patterns and Their Cognate Toll-like Receptors: An In Vitro Study

Givenchy W Manzano 1,2,3, Brian P Fort 1,2,3, George R Dubyak 1,2,3, Edward M Greenfield 1,2,3,✉
PMCID: PMC6259896  PMID: 30427314

Abstract

Background

Orthopaedic wear particles activate the NLRP3 inflammasome to produce active interleukin 1β (IL1β). However, the NLRP3 inflammasome must be primed before it can be activated, and it is unknown whether wear particles induce priming. Toll-like receptors (TLRs) are thought to mediate particle bioactivity. It remains controversial whether pathogen-associated molecular patterns (PAMPs) and/or alarmins are responsible for TLR activation by wear particles.

Questions/purposes

(1) Does priming of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? (2) Does priming of the NLRP3 inflammasome by wear particles depend on TLRs and TIRAP/Mal? (3) Does priming of the NLRP3 inflammasome by wear particles depend on cognate TLRs? (4) Does activation of the NLRP3 inflammasome by wear particles depend on adherent PAMPs?

Methods

Immortalized murine macrophages were stimulated by as-received titanium particles with adherent bacterial debris, endotoxin-free titanium particles, or titanium particles with adherent ultrapure lipopolysaccharide. To study priming, NLRP3 and IL1β mRNA and IL1β protein levels were assessed in wild-type, TLR4-/-, TLR2-/-, and TIRAP/Mal-/- macrophages. To study activation, IL1β protein secretion was assessed in wild-type macrophages preprimed with ultrapure lipopolysaccharide.

Results

Compared with titanium particles with adherent bacterial debris, endotoxin-free titanium particles induced 86% less NLRP3 mRNA (0.05 ± 0.03 versus 0.35 ± 0.01 NLRP3/GAPDH, p < 0.001) and 91% less IL1β mRNA (0.02 ± 0.01 versus 0.22 ± 0.03 IL1β/GAPDH, p < 0.001). ProIL1β protein level was robustly increased in wild-type macrophages stimulated by particles with adherent PAMPs but was not detectably produced in macrophages stimulated by endotoxin-free particles. Adherence of ultrapure lipopolysaccharide to endotoxin-free particles reconstituted stimulation of NLRP3 and IL1β mRNA. Particles with adherent bacterial debris induced 79% less NLRP3 mRNA (0.09 ± 0.004 versus 0.43 ± 0.13 NLRP3/GAPDH, p < 0.001) and 40% less IL1β mRNA (0.09 ± 0.04 versus 0.15 ± 0.03 IL1β/GAPDH, p = 0.005) in TLR4-/- macrophages than in wild-type. Similarly, those particles induced 49% less NLRP3 mRNA (0.22 ± 0.10 versus 0.43 ± 0.13 NLRP3/GAPDH, p = 0.004) and 47% less IL1β mRNA (0.08 ± 0.02 versus 0.15 ± 0.03 IL1β/GAPDH, p = 0.012) in TIRAP/Mal-/- macrophages than in wild-type. Particles with adherent ultrapure lipopolysaccharide induced 96% less NLRP3 mRNA (0.012 ± 0.001 versus 0.27 ± 0.05 NLRP3/GAPDH, p = 0.003) and 91% less IL1β mRNA (0.03 ± 0.01 versus 0.34 ± 0.07 IL1β/GAPDH, p < 0.001) expression in TLR4-/- macrophages than in wild-type. In contrast, those particles did not induce less NLRP3 and IL1β mRNA in TLR2-/- macrophages. IL1β protein secretion was equivalently induced by particles with adherent bacterial debris or by endotoxin-free particles in a time-dependent manner in wild-type macrophages. For example, particles with adherent bacterial debris induced 99% ± 2% of maximal IL1β secretion after 12 hours, whereas endotoxin-free particles induced 92% ± 11% (p > 0.5).

Conclusions

This cell culture study showed that adherent PAMPs are required for priming of the NLRP3 inflammasome by wear particles and this process is dependent on their cognate TLRs and TIRAP/Mal. In contrast, activation of the NLRP3 inflammasome by titanium particles is not dependent on adherent PAMPs. Animal and implant retrieval studies are needed to determine whether wear particles have similar effects on the NLRP3 inflammasome in vivo.

Clinical Relevance

Our findings, together with recent findings that aseptic loosening associates with polymorphisms in the TIRAP/Mal locus, support that adherent PAMPs may contribute to aseptic loosening in patients undergoing arthroplasty.

Introduction

Aseptic loosening caused by polymeric and metallic wear particles [7] is the most common long-term reason for revision surgery after total joint arthroplasty despite the advent of crosslinked polyethylene [5]. Particles stimulate macrophages to release proinflammatory cytokines, which induce osteoclast differentiation and osteolysis [30, 33, 40, 57]. Interleukin 1β (IL1β; Table 1), along with IL1α, IL6, and TNFα, mediates particle-induced osteoclast differentiation in vitro [71]. IL1Ra, which antagonizes both IL1α and IL1β, reduces particle-induced osteolysis in mice [75] and a IL1Ra polymorphism is associated with aseptic loosening in patients [29], suggesting that IL1β plays a major role in aseptic loosening.

Table 1.

Abbreviations

graphic file with name abjs-476-2442-g001.jpg

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The Nod-like-receptor-protein-3 (NLRP3, also known as NALP3) inflammasome processes inactive proIL1β to active IL1β [26]. The inflammasome consists of a sensor protein (NLRP3), an adaptor (apoptosis-associated-speck-like-protein [ASC]), and an effector protease (caspase-1). The NLRP3 inflammasome is regulated by a two-checkpoint system known as priming and activation (Fig. 1). Priming involves transcription and translation of NLRP3 and proIL1β. Priming can be induced by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide and lipoteichoic acid and other inflammatory mediators that also stimulate the NF-κB signaling pathway [8, 12, 26, 44, 52, 58]. Activation of the NLRP3 inflammasome involves assembly of the complex and processing of proIL1β to IL1β. Activation can be induced by ATP, nonorthopaedic particles, and numerous other stimuli [8, 12, 26, 39, 44, 52, 67]. Caspase-1, the effector protease of all known inflammasomes, contributes to particle-induced osteolysis [13]. The NLRP3 inflammasome processes IL1β in response to orthopaedic particles [3, 13-15, 42, 49, 50, 63, 68]. However, those studies focused on activation of the NLRP3 inflammasome in preprimed macrophages and did not examine whether the particles can prime the NLRP3 inflammasome, which is important because priming is a prerequisite for activation [8, 12, 26, 44, 52, 58].

Fig. 1.

Fig. 1

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This diagram illustrates the pathways responsible for priming (blue) and activation (green) of the NLRP3 inflammasome. See text for details.

Priming the NLRP3 inflammasome can be induced by stimulation of toll-like receptors (TLRs) [8, 12, 26, 44, 52, 58], which are also associated with aseptic loosening [30, 45, 70]. Polymorphisms in the TLR pathway associate with aseptic loosening [48] and TLR-positive macrophages are increased in periprosthetic tissue of patients with aseptic loosening [45]. TLRs can be activated by exogenous PAMPs [1] or endogenous danger signals known as alarmins or danger-associated molecular patterns [11, 51]. For example, particle-induced osteolysis in mice is dependent on adherent PAMPs, their cognate TLRs (TLR2 and TLR4), and Toll-interleukin 1 receptor domain-containing adapter protein/Myd88 adapter-like protein (TIRAP/Mal), a member of the MyD88 family of adaptor proteins that is uniquely specific to TLR2 and TLR4 [9, 10, 32]. The requirement for a PAMP-TLR cognate pair supports the conclusion that alarmins are not sufficient and therefore adherent PAMPs are needed to induce TLR activation in cell culture and mouse models of particle-induced osteolysis [9, 32]. Consistent with that conclusion, the idea that bacterial PAMPs contribute to aseptic loosening has received considerable interest [30, 31, 38, 47, 56, 69, 74] but remains controversial [30, 45, 61, 69, 70, 74]. It is unknown whether adherent PAMPs are needed for orthopaedic particles to prime and/or activate the NLRP3 inflammasome, because the previous studies on wear particles and the NLRP3 inflammasome did not compare the effects of particles with and without adherent PAMPs [3, 13, 14, 42, 49, 50, 61, 63, 68].

This study was therefore designed to determine whether orthopaedic particles can prime the NLRP3 inflammasome and whether priming and/or activation of the NLRP3 inflammasome by orthopaedic particles depend on adherent PAMPs, their cognate TLRs, and TIRAP/Mal. Specifically we asked: (1) Does priming of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? (2) Does priming of the NLRP3 inflammasome by wear particles depend on TLRs and TIRAP/Mal? (3) Does priming of the NLRP3 inflammasome by wear particles depend on cognate TLRs? (4) Does activation of the NLRP3 inflammasome by wear particles depend on adherent PAMPs?

Materials and Methods

Study Design

We first validated the current cell culture model system by determining whether it reproduces previous studies showing that stimulation of IL1β secretion by wear particles depends on the NLRP3 inflammasome and on adherent PAMPs. To address the first study question, we compared priming in response to titanium particles with adherent bacterial debris and endotoxin-free titanium particles (Fig. 2). To address the second question, we compared priming by wild-type, TLR2-/-, TLR4-/-, and TIRAP/Mal-/- macrophages in response to titanium particles with adherent bacterial debris (Fig. 2). To address the third question, we compared priming in response to titanium particles with adherent ultrapure lipopolysaccharide by wild-type, TLR2-/-, and TLR4-/- macrophages (Fig. 2). To address the final question, we compared activation by preprimed cells in response to titanium particles with adherent bacterial debris and endotoxin-free titanium particles (Fig. 2).

Fig. 2.

Fig. 2

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This diagram illustrates the overall study design. See text for details.

Titanium Particles

As-received titanium particles (catalog number 00681, lot F06Q16; Johnson Matthey, Royston, UK) have substantial adherent bacterial debris (34 EU/109 particles) [32, 59]. Endotoxin-free titanium particles (< 0.3 EU/109 particles) were prepared by removing > 99% of the endotoxin from the as-received particles as we previously described [59]. Titanium particles with adherent lipopolysaccharide (33 EU/109 particles) were prepared by incubating endotoxin-free titanium particles with 50 μg/mL ultrapure lipopolysaccharide (tlrl-eblps; InvivoGen, San Diego, CA, USA) for 4 days in phosphate-buffered saline (PBS) containing 1.1 mM calcium chloride [4, 32]. Those particles were then washed 10 times in PBS with calcium chloride to remove any unbound, soluble lipopolysaccharide. The final wash was confirmed to be endotoxin-free, documenting removal of unbound lipopolysaccharide. Endotoxin in the particle suspensions was measured using a chromogenic Limulus amebocyte lysate assay (50-647U; Lonza, Basel, Switzerland) as we previously described [55]. Thus, false-negatives resulting from assay inhibition were eliminated by assaying particle suspensions spiked with known amounts of endotoxin [55]. β-glucan blocker (Lonza) was used to eliminate false-positives resulting from β-glucan [55]. As-received particles, endotoxin-free particles, and particles with adherent ultrapure lipopolysaccharide were used for all experiments.

Cell Culture

Immortalized wild-type, NLRP3-/-, and TIRAP/Mal-/- macrophages were a gift from Dr Katherine Fitzgerald (University of Massachusetts Medical School). Immortalized TLR2-/- and TLR4-/- macrophages were obtained from BEI Resources (Manassas, VA, USA). The macrophage lines were immortalized as previously described and accurately reflect the phenotype of freshly isolated macrophages [9, 35, 54]. Cells were maintained in Minimal Essential Medium (Hyclone, South Logan, UT, USA) with 10% heat-inactivated fetal bovine serum (Hyclone), nonessential amino acids (Mediatech, Manassas, VA, USA), L-glutamine (Mediatech), streptomycin (Mediatech), and penicillin (Mediatech). Macrophages were plated at 2.5 x 105 cells/cm2 in 24-well culture plates (Falcon, Tewksbury, MA, USA) for mRNA and enzyme-linked immunosorbent assay (ELISA) experiments or in six-well plates (Falcon) for Western blot experiments.

To study priming of the NLRP3 inflammasome, cells were stimulated with 1 x 108 particles/cm2 of titanium particles with adherent bacterial debris, titanium particles with adherent ultrapure lipopolysaccharide, endotoxin-free titanium particles, 4 μg/mL ultrapure lipopolysaccharide as a positive control, or a negative control (culture medium supplemented as previously described). Priming the NLRP3 inflammasome was determined by measuring NLRP3 and IL1β mRNA by real-time polymerase chain reaction and proIL1β protein by Western blot (described subsequently).

To study NLRP3 inflammasome activation, cells were preprimed with 4 μg/mL ultrapure lipopolysaccharide for 4 hours. Cells were washed three times with PBS and subsequently stimulated with titanium particles with adherent bacterial debris, endotoxin-free titanium particles, 5 mM ATP as a positive control, or a negative control (culture medium supplemented as described previously). Activation of the NLRP3 inflammasome was determined by measuring secreted IL1β protein by ELISA.

Measurement of Specific mRNAs

Total RNA was isolated using the Promega SV Total RNA Isolation System and measured spectrophotometrically (Nanodrop™; Thermo Fisher Scientific, Waltham, MA, USA). Two hundred nanograms of total RNA were converted to cDNA using the Superscript First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Real-time polymerase chain reaction (PCR) was performed using SYBR™ Green (BioRad, Hercules, CA, USA) with the following primers: NLRP3 forward 5′-gcaacctccagaaactgtggt-3′, reverse 5′-tgggtccttcatcttttcaca-3′; IL-1β forward 5′-gaccccaaaagatgaaggg-3′, reverse 5′- aggtgctcatgtcctcatcc-3′; GAPDH forward 5′-atgggaagctggtcatcaac-3′, reverse 5′-gtggttcacacccatcacaa-3′. To exclude false-positives from genomic DNA, 3′ termini of the PCR primers overlap exon-exon junctions [16], except for the GAPDH amplicon, which is encoded within a single exon. All primers were validated by sequencing of PCR amplicons. Gene expression was determined from a standard curve [18] and normalized to GAPDH levels. Specificity of each reaction was verified by melt curve analysis and agarose gel electrophoresis.

Measurement of IL-1β Proteins

Culture supernatants were harvested, and cells were lysed with RIPA buffer (Thermo Fisher, Waltham, MA, USA) and both were stored at -80° C. IL1β ELISAs were performed on supernatants (Biolegend, San Diego, CA, USA). For Western blots, proteins of cell lysates were separated on 15% polyacrylamide gels (Lonza) and transferred onto polyvinylidene fluoride membranes (BioRad) at 24 V for 1 hour at 4° C. The membranes were then blocked with 5% nonfat milk in Tris-buffered-saline for 1 hour at room temperature and incubated with primary antibody overnight at 4° C followed by a secondary antibody for 1 hour at room temperature. Thereafter, membranes were incubated with chemoluminescent solution (Prime-ECL™; GE Healthcare, Chicago, IL, USA). Membranes were washed with Tris-buffered saline with 0.1% Tween (Sigma, St Louis, MO, USA) before administration of a secondary antibody and chemoluminescent solution. Membranes were imaged digitally (Kodak Image Station In-Vivo FX, Rochester, NY, USA). All Western blots are representative images of three independent experiments.

Primary antibodies were murine antihuman IL1β (3ZD, dilution-1:40,000; National Institutes of Health, Bethesda, MD, USA) and goat antimurine actin (Santa Cruz Biotechnology, Dallas, TX, USA; sc-1615, dilution-1:5000). Secondary antibodies conjugated to horseradish peroxidase were goat antimurine IgG and donkey antigoat IgG (Santa Cruz Biotechnology; sc-2005 and sc-2020, dilution-1:5000).

Statistics

All quantitative data represent means ± SD from n = 3 to 5 independent experiments. Each experiment included triplicate cell culture wells per group, each assayed in triplicate. Because data passed normality as assessed by the Shapiro-Wilk test and equal variance as assessed by the Brown Forsythe test, analysis was by two-way analysis of variance with one-sided post hoc Bonferroni corrections (Prism; GraphPad Software, La Jolla, CA, USA). Significance is denoted by *p < 0.05, **p < 0.01, *** p < 0.001. Error bars represent SD.

Results

Validation of Cell Culture Model System for Investigation of Effects of Wear Particles on the NLRP3 Inflammasome

Compared with the vehicle control, particles with adherent bacterial debris stimulated robust secretion of IL1β by wild-type macrophages (Fig. 3; mean difference 97.6% of maximal secretion [95% confidence interval {CI}, 96.6-98.6], p < 0.001). In contrast, NLRP3-/- macrophages secreted 93% less IL1β in response to particles with adherent bacterial debris (Fig. 3 [95% CI, 85-100], 7.4% ± 4.6% versus 100% of maximal secretion, p < 0.001). Moreover, endotoxin-free particles induced 94% less IL1β secretion by wild-type macrophages than particles with adherent bacterial debris (Fig. 3 [95% CI, 91-96], 6.3% ± 1.7% versus 100% of maximal secretion, p < 0.001). These results are consistent with previous studies showing that secretion of mature IL1β in response to wear particles depends on the NLRP3 inflammasome [3, 13-15, 42, 49, 50, 63, 68] and on adherent PAMPs [2, 9, 10, 17, 19] and therefore validated our cell culture model system for investigation of priming and activation of the NLRP3 inflammasome by orthopaedic wear particles.

Fig. 3.

Fig. 3

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IL1β protein secretion depends on the NLRP3 inflammasome and adherent PAMPs. Wild-type and NLRP3-/- macrophages were treated with a vehicle control (blue triangles), endotoxin-free titanium particles (green squares), or titanium particles with adherent bacterial debris (red circles) for 8 hours. IL1β was measured in supernatant using ELISA. All values are shown as percent of maximal IL1β secretion in that experiment, which are the wild-type macrophages stimulated by titanium particles with adherent bacterial debris. N = 3 independent experiments were performed. Each experiment included triplicate cell culture wells per group, each assayed in triplicate. Error bars represent SD. Statistical significance was determined by one-sided analysis of variance. ***p < 0.001. Ti = titanium; NLRP3 = Nod-like-receptor-protein-3.

Priming of the NLRP3 Inflammasome by Wear Particles Depends on Adherent PAMPs

NLRP3 and IL1β mRNAs were robustly expressed in macrophages stimulated by particles with adherent bacterial debris, but not in macrophages stimulated by endotoxin-free titanium particles (Fig. 4A-B). For example, compared with titanium particles with adherent bacterial debris at the 2-hour time point, endotoxin-free titanium particles induced 86% less NLRP3 mRNA (Fig. 4A; 0.05 ± 0.03 versus 0.35 ± 0.01 NLRP3/GAPDH, mean difference 0.30 [95% CI, 0.21-0.40], p < 0.001) and 91% less IL1β mRNA (Fig. 4B; 0.02 ± 0.01 versus 0.22 ± 0.03 IL1β/GAPDH, mean difference 0.20 [95% CI, 0.11-0.30], p < 0.001). ProIL1β protein level was robustly increased in wild-type macrophages stimulated by particles with adherent PAMPs but was not detectably produced in macrophages stimulated by endotoxin-free particles (Fig. 4C). Adherence of ultrapure lipopolysaccharide to endotoxin-free particles reconstituted stimulation of NLRP3 mRNA (Fig. 5A; 0.27 ± 0.05 versus 0.02 ± 0.001 NLRP3/GAPDH, mean difference 0.25 [95% CI, 0.21-0.30], p < 0.001) and IL1β mRNA (Fig. 5B; 0.34 ± 0.07 versus 0.05 ± 0.004 IL1β/GAPDH, mean difference 0.29 [95% CI, 0.22-0.36], p < 0.001). Together, these results show that priming of the NLRP3 inflammasome by wear particles depends on adherent PAMPs.

Fig. 4 A-C.

Fig. 4 A-C

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Priming of the NLRP3 inflammasome depends on adherent PAMPs at the mRNA (A-B) and protein (C) levels. Wild-type macrophages were treated with either endotoxin-free titanium particles (A-B, green squares) or titanium particles with adherent bacterial debris (A-B, red circles). Markers of priming (NLRP3 and IL1β mRNA and pro-IL1β protein) were measured in cell lysates at indicated time points and the mRNA values were normalized to GAPDH mRNA. N = 3 independent experiments were performed (A-B). Each experiment included triplicate cell culture wells per group, each assayed in triplicate. Error bars represent SD. Statistical significance was determined by one-sided analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001 with comparisons made between titanium particles with adherent bacterial debris and endotoxin-free titanium particles at each indicated time point. Pro-IL1β protein was detected by Western blot (C). The pictured gel is representative of three independent experiments. Ti = titanium; NLRP3 = Nod-like-receptor-protein-3; mRNA = messenger RNA; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Fig. 5 A-B.

Fig. 5 A-B

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Priming of the NLRP3 inflammasome depends on adherent PAMPs, their cognate TLRs, and TIRAP/Mal. Wild-type (red circle), TLR4-/- (green square), TLR2-/- (purple triangle), and TIRAP/Mal-/- (brown diamond) macrophages were treated with vehicle control, endotoxin-free titanium particles, titanium particles with adherent bacterial debris, or titanium particles with adherent uLPS for 2 hours. NLRP3 and IL1β mRNA were measured in cell lysates and the mRNA values were normalized to GAPDH mRNA. N = 5 independent experiments were performed. Each experiment included triplicate cell culture wells per group, each assayed in triplicate. Error bars represent SD. Statistical significance was determined by one-sided analysis of variance. *p < 0.05, ** p < 0.01, ***p < 0.001.

Priming of the NLRP3 Inflammasome by Wear Particles Depends on TLR4 and TIRAP/Mal

Particles with adherent bacterial debris induced 79% less NLRP3 mRNA (Fig. 5A; 0.09 ± 0.004 versus 0.43 ± 0.13 NLRP3/GAPDH, mean difference 0.34 [95% CI, 0.17-0.51], p < 0.001) and 40% less IL1β mRNA (Fig. 5B; 0.09 ± 0.04 versus 0.15 ± 0.03 IL1β/GAPDH, mean difference 0.06 [95% CI, 0.02-0.10], p = 0.005) in TLR4-/- macrophages than in wild-type macrophages. Similarly, those particles induced 49% less NLRP3 mRNA (Fig. 5A; 0.22 ± 0.10 versus 0.43 ± 0.13 NLRP3/GAPDH, mean difference 0.21 [95% CI, 0.05-0.38], p = 0.004) and 47% less IL1β mRNA (Fig. 5B; 0.08 ± 0.02 versus 0.15 ± 0.03 IL1β/GAPDH, mean difference 0.07 [95% CI, 0.03-0.11], p = 0.012) in TIRAP/Mal-/- macrophages than in wild-type macrophages. In contrast, deletion of TLR2 did not detectably reduce NLRP3 expression (Fig. 5A; 0.30 ± 0.06 versus 0.43 ± 0.13 NLRP3/GAPDH, mean difference 0.13 [95% CI, -0.06 to 0.33], p = 0.13) or IL1β expression (Fig. 5B; 0.25 ± 0.12 versus 0.15 ± 0.03 IL1β/GAPDH, mean difference 0.10 [95% CI, -0.04 to 0.24], p = 0.13) in response to particles with adherent bacterial debris. Together, these results show that priming of the NLRP3 inflammasome by wear particles depends on TLR4 and TIRAP/Mal.

Priming of the NLRP3 Inflammasome by Wear Particles Depends on Cognate TLRs

Particles with adherent ultrapure liposaccharide induced 96% less NLRP3 mRNA (Fig. 5A; 0.012 ± 0.001 versus 0.27 ± 0.05 NLRP3/GAPDH, mean difference 0.26 [95% CI, 0.06-0.46], p = 0.003) and 91% less IL1β mRNA (Fig. 5B; 0.03 ± 0.01 versus 0.34 ± 0.07 IL1β/GAPDH, mean difference 0.31 [95% CI, 0.16-0.44], p < 0.001) expression in TLR4-/- macrophages than in wild-type macrophages. In contrast, those particles did not induce less NLRP3 mRNA (Fig. 5A; 0.31 ± 0.14 versus 0.27 ± 0.05 NLRP3/GAPDH, nonsignificant [NS]) or IL1β mRNA (Fig. 5B; 0.55 ± 0.15 versus 0.34 ± 0.07 IL1β/GAPDH, NS) in TLR2-/- macrophages when compared with wild-type macrophages. Together, these results show that priming of the NLRP3 inflammasome by particles with adherent uLPS depends on the cognate TLR4, but not on the noncognate TLR2.

Activation of the NLRP3 Inflammasome by Wear Particles Does Not Depend on Adherent PAMPs

IL1β protein secretion was equivalently induced by particles with adherent bacterial debris or by endotoxin-free particles in a time-dependent manner in wild-type macrophages that had been preprimed with lipopolysaccharide (Fig. 6). For example, particles with adherent bacterial debris induced 99% ± 2% of maximal IL1β secretion after 12 hours, whereas endotoxin-free particles induced 92% ± 11% (mean difference 7 [95% CI, -36 to 23], p > 0.5 at all time points). Activation induced by the endotoxin-free particles is unlikely the result of residual lipopolysaccharide from the prepriming step because the vehicle control group did not induce detectable activation (Fig. 6; 0.83 ± 0.03 versus 99% ± 2% of maximal IL1β secretion after 12 hours, mean difference 98 [95% CI, 95-101], p < 0.001). Together, these results show that activation of the NLRP3 inflammasome by wear particles does not depend on adherent PAMPs.

Fig. 6.

Fig. 6

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Activation of the NLRP3 inflammasome does not depend on adherent PAMPs. Wild-type macrophages were primed with uLPS for 4 hours and subsequently treated with a vehicle control (blue triangle), endotoxin-free titanium particles (green squares), or titanium particles with adherent bacterial debris (red circles). IL1β protein was measured in culture supernatants at the indicated time points using ELISA. All values are shown as percent of maximal IL1β secretion in that experiment. N = 3 independent experiments were performed. Each experiment included triplicate cell culture wells per group, each assayed in triplicate. Error bars represent SD. Statistical significance was determined by one-sided analysis of variance. p > 0.5 at all time points when comparing titanium particle with adherent bacterial debris and endotoxin-free titanium particles at each indicated time point. Ti = titanium.

Discussion

Previous studies showed that orthopaedic wear particles can activate the NLRP3 inflammasome to process proIL1β to bioactive IL1β but did not examine whether the particles can prime the NLRP3 inflammasome, which is a prerequisite for activation [8, 12, 26, 44, 52, 58]. Moreover, it was unknown whether adherent PAMPs are needed for orthopaedic particles to prime and/or activate the NLRP3 inflammasome, because the previous studies did not compare the effects of particles with and without adherent PAMPs [3, 13-15, 42, 49, 50, 63, 68]. Our aim was therefore to evaluate whether orthopaedic particles prime the NLRP3 inflammasome and whether priming and/or activation are dependent on adherent PAMPs. Our results document that adherent PAMPs are required for particles to prime the NLRP3 inflammasome and that this process is dependent on their cognate TLRs and TIRAP/Mal (Fig. 7). In contrast, our results document that activation of the NLRP3 inflammasome by particles is not dependent on adherent PAMPs (Fig. 7).

Fig. 7.

Fig. 7

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This diagram illustrates the current working model. Priming of the NLRP3 inflammasome by wear particles depends on adherent PAMPs and their cognate TLRs (blue). Activation of the NLRP3 inflammasome by wear particles does not depend on adherent PAMPs (green). See text for details.

One limitation is that any cell culture study may not reflect the in vivo situation. For example, the NLRP3 inflammasome might be primed in vivo in a subset of macrophages by alarmins released either constitutively or in response to particle-induced tissue damage [51]. Wear particles would then be expected to activate the NLRP3 inflammasome in those macrophages even in the absence of PAMPs. Such a mechanism may account for our previous results that endotoxin-free titanium particles induce approximately 50% as much osteolysis in murine calvaria as is induced by titanium particles with adherent bacterial debris [32]. However, osteolysis induced by endotoxin-free particles is not affected by deletion of TLR2 or TLR4, either alone or together [32]. Thus, if alarmins contribute to particle-induced osteolysis in murine calvaria, they do so independently of TLR2 and TLR4. A second limitation is that the PAMP removal process [59] may alter particle surface chemistry and thereby impair cellular responses [70]. However, no differences are detected in shape, size, or surface chemical composition [22, 59]. Moreover, changes in chemical properties (hydrophobicity, oxidation, ionization, charge, etc) would likely be reversible during the extensive PBS washes that follow PAMP removal [59]. In addition, other methods of PAMP removal that would be expected to have different effects on surface chemistry also substantially reduce particle bioactivity [17, 22, 64, 65, 73]. Finally, results that adherence of lipopolysaccharide [28, 32, 37, 64] or lipoteichoic acid [32, 53] reconstitutes the bioactivity and TLR dependence of particles strongly argues against an impaired response resulting from surface chemistry. Some authors have claimed that the particles with adherent lipopolysaccharide expose macrophages to levels of lipopolysaccharide found during sepsis [61, 62]. However, those authors did not note that most of the lipopolysaccharide is removed from the particle suspensions during the extensive PBS washes [59]. A third limitation is the sole use of titanium particles, because polyethylene is the predominant wear debris in most patients with aseptic loosening [7, 60]. Investigation of polyethylene particles in cell culture is not feasible because they float in culture media, preventing interactions with the macrophages. However, adherent PAMPs increase the bioactivity of all types of orthopaedic particles [30, 31, 37, 53, 61, 62], including polyethylene [30, 32, 76]. Moreover, titanium and polyethylene particles produce indistinguishable responses in the mouse calvarial model [43, 46, 72] and in patients with aseptic loosening [34].

The initial experiments in this study showed that IL1β secretion in response to titanium particles depends on the NLRP3 inflammasome and adherent PAMPs. Those results are consistent with previous studies that documented dependence of IL1β secretion in response to wear particles on the NLRP3 inflammasome [3, 13-15, 42, 49, 50, 63, 68] and on adherent PAMPs [2, 9, 10, 17, 19] and therefore validated our cell culture model system for investigation of priming and activation of the NLRP3 inflammasome by orthopaedic wear particles.

This study found that inflammasome priming by orthopaedic particles depends on adherent PAMPs, their cognate TLRs, and TIRAP/Mal. The following evidence documents that alarmins are insufficient to activate TLRs and prime the NLRP3 inflammasome and that this process is dependent on adherent PAMPs. First, we found that endotoxin-free particles, in contrast to particles with adherent bacterial debris, are unable to prime the NLRP3 inflammasome. Those results are consistent with previous findings that adherence of lipopolysaccharide [9, 28, 32, 37] or lipoteichoic acid [32, 53] substantially increases stimulation by particles of mRNAs encoding IL1β or NLRP3. Second, noncognate TLRs do not contribute to priming by particles with adherent PAMPs as would be expected if alarmins were sufficient for TLR-dependent priming [30]. For example, we found that priming by particles with adherent ultrapure lipopolysaccharide is not affected by deletion of the noncognate TLR2 but is potently inhibited by deletion of either the cognate TLR4 or TIRAP/Mal. Thus, if alarmins are released in response to the particles, they are not sufficient and adherent PAMPs are needed to activate TLRs during priming of the NLRP3 inflammasome. Similarly, alarmins are not sufficient to activate TLRs during particle-induced cytokine production or particle-induced osteolysis [9, 32]. Alarmins may nonetheless contribute to aseptic loosening either by acting together with PAMPs to activate TLRs or by mechanisms independent of TLRs [25, 30]. Our results may appear to conflict with reports that orthopaedic particles that lack adherent PAMPs can activate the NLRP3 inflammasome [3, 13-15, 42, 49, 50, 63, 68]. However, priming with soluble PAMPs was needed in those studies to allow activation by the particles. One study reported that cobalt-chrome particles activate the NLRP3 inflammasome in the absence of priming [61]. However, that study relied on macrophages pretreated with 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA) or thioglycolate, either of which potently prime the NLRP3 inflammasome [6, 23, 41].

The findings described in the previous paragraph that inflammasome priming by orthopaedic particles depends on adherent PAMPs, their cognate TLRs, and TIRAP/Mal further supports the controversial hypothesis that bacterial PAMPs contribute to aseptic loosening. Multiple other lines of evidence also support that hypothesis [30, 31, 38, 47, 56, 69, 74]. Clinically, antibiotics can reduce aseptic loosening [24, 30], and PAMPs are found in periprosthetic tissue from patients with aseptic loosening [36, 55]. Possible PAMP sources include bacterial flora in the gastrointestinal tract and oral cavity from which bacteria and PAMPs episodically translocate to the systemic circulation, potentially reaching the implant [30]. Subclinical, low-grade bacterial biofilms on implant surfaces are another possible source of PAMPs [20, 21, 30, 38, 47, 56, 66, 74], and the presence of biofilms strongly correlates with increased osteolysis [66]. Additionally, TLR pathway polymorphisms associated with aseptic loosening [48] and increased TLR-positive macrophages were reported in periprosthetic tissue of patients with aseptic loosening [45]. In cell culture and the murine calvarial model of particle-induced osteolysis, titanium particle-induced inflammation and osteolysis are partially dependent on TLR2 and TLR4 but only if their cognate PAMPS are adherent to the particles [32]. Our current results extend those previous findings to inflammasome processing of proIL1β and thereby further support the hypothesis that bacterial PAMPs contribute to aseptic loosening [30, 31, 38, 47, 56, 69, 74].

In contrast to priming, activation of the NLRP3 inflammasome does not depend on adherent PAMPs, as demonstrated by equivalent secretion of IL1β protein by preprimed macrophages subsequently stimulated by either endotoxin-free titanium particles or titanium particles with adherent bacterial debris. Thus, it is possible that particles with and without adherent PAMPs can work together by sequentially priming (PAMP-dependent) and then activating (PAMP-independent) the NLRP3 inflammasome. Of all the macrophage responses induced by wear particles that have been studied [9, 10, 22, 28, 30-32, 37, 38, 47, 53, 56, 69, 72, 74, 76], cell death [22, 64] and activation of the NLRP3 inflammasome are the only ones that are not increased by adherent PAMPs. Intriguingly, cell death can also activate the NLRP3 inflammasome [27]. Future studies should therefore determine whether cell death contributes to activation of the NLRP3 inflammasome by orthopaedic wear particles.

This cell culture study showed that orthopaedic wear particles with adherent PAMPs can prime the NLRP3 inflammasome and that the particles can then activate the NLRP3 inflammasome independently of adherent PAMPs. Future animal and implant retrieval studies are needed to determine whether wear particles have similar effects on the NLRP3 inflammasome in vivo. Nonetheless, the current results add further support to the controversial concept that bacterial PAMPs may contribute to aseptic loosening.

Acknowledgments

We thank Dr Katherine Fitzgerald for the wild-type, NLRP3-/-, and TIRAP/Mal-/- immortalized macrophages and Bryan Hausman for assisting with experiments.

Footnotes

One or more of the authors received funding from the Dudley P. Allen Fellowship (GWM); the National Institutes of Health TL1 TR000441 (BPF), T32 AR007505 (BPF), and R21 AR069785 (EMG); and the Harry E. Figgie III MD Professorship (EMG).

Clinical Orthopaedics and Related Research® neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDA approval status, of any drug or device before clinical use.

Each author certifies that his institution waived approval for the reporting of this investigation and that all investigations were conducted in conformity with ethical principles of research.

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