Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

NLRP3 inflammasome-dependent and -independent interleukin-1β release by macrophages exposed to wear and corrosion products from CoCrMo implants

  • Nasteho Abdoulkader,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Mechanical Engineering, Faculty of Engineering, University of Ottawa, Ottawa, Ontario, Canada

    ⨯
  • Jennifer Archibald,

    Roles Data curation, Formal analysis, Investigation, Methodology, Validation

    Affiliation Department of Mechanical Engineering, Faculty of Engineering, University of Ottawa, Ottawa, Ontario, Canada

    ⨯
  • Morgane Lignereux,

    Roles Data curation, Formal analysis, Investigation, Methodology, Validation

    Affiliation Department of Mechanical Engineering, Faculty of Engineering, University of Ottawa, Ottawa, Ontario, Canada

    ⨯
  • Eric A. Lehoux,

    Roles Conceptualization, Formal analysis, Validation, Writing – review & editing

    Affiliation Department of Mechanical Engineering, Faculty of Engineering, University of Ottawa, Ottawa, Ontario, Canada

    ⨯
  • Isabelle Catelas

    Roles Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing

    icatelas@uottawa.ca, icatelas@hotmail.com

    Affiliations Department of Mechanical Engineering, Faculty of Engineering, University of Ottawa, Ottawa, Ontario, Canada, Department of Surgery, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada, Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada

    ⨯

Abstract

Wear particles and metal ions released from cobalt–chromium–molybdenum (CoCrMo) implants can trigger adverse local tissue reactions (ALTR) that can lead to implant failure. Identifying mechanisms involved in ALTR, particularly those underlying the initial inflammatory response elicited by wear particles (Cr2O3 and CoCrMo) and metal ions (Co2+ and Cr3+) is therefore critical. The macrophage pro-inflammatory response to CoCrMo particles, Co2+, and Cr3+ includes interleukin-1β (IL-1β) release, a process putatively linked to the NLPR3 inflammasome. However, the effects of Cr2O3 particles remain largely unknown. The objectives of this study were to determine whether IL-1β release by macrophages exposed to Cr2O3 particles (60 nm), CoCrMo particles (3.4 μm), Co2+, or Cr3+ is dependent on NLRP3 and caspase-1, whether NLRP3-dependent release is mediated by reactive oxygen species (ROS) and/or cathepsin B, and whether caspase-8 is involved when the release is NLRP3 independent. Bone marrow-derived macrophages (BMDM) from wild-type (wt), NLRP3-deficient (Nlrp3-/-), and caspase-1-deficient (Casp1-/-) mice were exposed to the particles or metal ions following priming with lipopolysaccharide. IL-1β release induced by Cr2O3 particles and Cr3+ was shown to be both NLRP3 and caspase-1-dependent. In contrast, IL-1β release induced by CoCrMo particles and Co2+ occurred independently of NLRP3, being caspase-1-independent in response to CoCrMo particles and partially caspase-1-dependent in response to Co2+. Further analysis suggested that NLRP3 inflammasome activation by Cr2O3 particles was cathepsin B dependent and mediated by lysosomal destabilization, whereas activation by Cr3+ was ROS-mediated. NLRP3-independent IL-1β release induced by CoCrMo particles or Co2+ was caspase-8 dependent. Collectively, these findings highlight the diversity and specificity of the mechanisms by which different CoCrMo implant wear particles and metal ions can induce IL-1β release in macrophages. Moreover, they suggest that targeting NLRP3, caspase-1, and/or caspase-8 could help mitigate the IL-1β-mediated component of the inflammatory response triggered by wear particles and metal ions from CoCrMo implants.

Citation: Abdoulkader N, Archibald J, Lignereux M, Lehoux EA, Catelas I (2025) NLRP3 inflammasome-dependent and -independent interleukin-1β release by macrophages exposed to wear and corrosion products from CoCrMo implants. PLoS One 20(11): e0334912. https://doi.org/10.1371/journal.pone.0334912

Editor: David M. Ojcius, University of the Pacific, UNITED STATES OF AMERICA

Received: June 1, 2025; Accepted: October 4, 2025; Published: November 18, 2025

Copyright: © 2025 Abdoulkader et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data included in the manuscript are available from DOI: 10.5683/SP3/IEBNOE.

Funding: This research was supported by funding from the Canadian Institutes of Health Research (CIHR), grant number PJT-169136, to Isabelle Catelas. Nasteho Abdoulkader was a recipient of the Hans K. Uhthoff, MD FRCSC Graduate Fellowship.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cobalt–chromium–molybdenum (CoCrMo) alloys are widely used in medical devices [1], including joint implants. Wear and corrosion of these implants represent a major clinical concern [1], as they have been associated with adverse local tissue reactions (ALTR) [2–4], which can ultimately lead to implant failure [5,6]. ALTR encompass delayed-type IV hypersensitivity-like reactions [7] and pseudotumors [8,9], both frequently accompanied by tissue necrosis [2,10]. Therefore, identifying mechanisms involved in ALTR, particularly those underlying the initial inflammatory response elicited by wear particles and metal ions, may enable the development of non-invasive therapies that improve the lifespan of joint replacements.

Wear of articulating surfaces made of CoCrMo has been reported to primarily generate round to oval-shaped chromium oxide (Cr2O3) nanoparticles (<100 nm) along with a smaller proportion of larger CoCrMo particles [11,12]. In addition to wear, corrosion – particularly at modular interfaces – can lead to the release of metal ions, including Co2+ and Cr3+ [13–15]. Macrophages are considered key mediators of the immune response to implant-derived wear and corrosion products [16,17]. Upon classical activation, they release pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α), which contribute to the induction and maintenance of a pro-inflammatory response. It is well established that macrophage response to metallic wear particles (e.g., CoCrMo and commercially pure [cp] Ti) [18–20] as well as to metal ions (e.g., Co2+, Cr3+, and Ni2+) involve activation of Toll-like receptors (TLRs) [21,22]. More specifically, an upregulation of TLR-1, -4, and -6 in response to CoCrMo particles or a cocktail of metal ions, generated by oxidation of the alloy, was recently reported [23]. In addition, exposure of macrophages to CoCrMo particles, cpTi particles, or metal ions such as Co2+ and Cr3+ has been reported to induce activation of the NLRP3 inflammasome resulting in the release of IL-1β [24–26]. Exposure of macrophages to Cr3+, unlike exposure to Co2+ or Ni2+, has also been shown to induce caspase-1 cleavage in the absence – but not in the presence – of an antioxidant, suggesting that Cr3+ activates the NLRP3 inflammasome through reactive oxygen species (ROS)-dependent mechanisms [27]. However, to the best of our knowledge, the potential involvement of Cr2O3 particles in the activation of the NLRP3 inflammasome and release of IL-1β by macrophages remains unexplored.

The NLRP3 inflammasome consists of a sensor protein (NLR family, pyrin domain-containing 3 [NLRP3]), an adaptor protein (apoptosis-associated speck-like protein containing a caspase recruitment domain [ASC]), and an effector protein (caspase-1). Canonical activation of this tripartite complex requires two distinct signals. The first, known as priming, is initiated when TLRs recognize and bind danger-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide (LPS) – a well-characterized and potent TLR4 agonist derived from Gram-negative bacteria [28]. The second signal, often referred to as a danger signal, is detected by cytosolic NLRP3 and leads to recruitment of the adaptor protein and the zymogen protease, pro-caspase-1 [28,29]. Cellular events capable of serving as this second signal include: ion fluxes (K+ efflux, Ca2+ influx), ROS generation, and lysosomal destabilization [30]. Following dual-signal activation, the NLRP3 inflammasome assembles, inducing auto-catalytic cleavage and activation of caspase-1, which in turn proteolytically processes its substrates, including pro-IL-1β, into their mature biologically active forms [31].

The mechanisms by which cytosolic and/or mitochondrial ROS induce assembly of the NLRP3 inflammasome complex remain ill-defined [30]. In contrast, it is well-established that lysosomal destabilization can be triggered by particulates, including silica crystals, aluminum salts, and other agents that compromise lysosomal membrane integrity [32,33]. Membrane destabilization can cause the release of lysosomal cathepsins, including cathepsin B, into the cytosol. This release can be functionally important, as cathepsin B has been shown to interact with NLRP3 at the endoplasmic reticulum and its absence has been shown to inhibit NLRP3 inflammasome complex formation [34]. Interestingly, IL-1β release has also been reported to occur without involvement of the NLRP3 inflammasome. For example, while cathepsins [35] have been shown to promote pro-IL-1β synthesis, granzyme A [36] and caspase-8 [37] have been reported to proteolytically cleave pro-IL-1β into mature biologically active forms.

The mechanisms by which CoCrMo implant wear particles and metal ions induce NLRP3 inflammasome-dependent or -independent release of IL-1β by macrophages remain largely unexplored. Therefore, the objectives of the present study were to determine whether IL-1β release by bone marrow-derived macrophages (BMDM) exposed to Cr2O3 particles (60 nm), CoCrMo particles (3.4 μm), Co2+, or Cr3+ is dependent on NLRP3 and caspase-1, whether NLRP3-dependent release is mediated by ROS and/or cathepsin B, and whether caspase-8 is involved when the release is NLRP3 independent.

Materials and methods

Particles and metal ions

Suspensions of commercially available spherical Cr2O3 nanoparticles (60-nm average diameter, as per the manufacturer; 99% purity; MilliporeSigma, St. Louis, MO, catalog no. 634239) and spherical CoCrMo alloy microparticles (3.4-μm average diameter; d10 = 2.4 μm, d50 = 3.4 μm, d90 = 5.3 μm; composition: Co 59.2%, Cr 30%, Mo 7%, as per the manufacturer; Praxair Surface Technologies, Indianapolis, IN, catalog no. ZCO538-J2010) were prepared as previously described [38]. The mass of particles required to achieve the desired concentrations was calculated based on the density of chromium oxide (5.21 g/cm3) and CoCrMo alloy (4.86 g/cm3) as well as the spherical geometry of the particles. Stock solutions of metal ions were prepared from their corresponding chloride salts, as previously described [27]. Endotoxin levels were assessed using a chromogenic Limulus amebocyte lysate (LAL) assay kit (GenScript, Piscataway, NJ). Briefly, the Cr2O3 and CoCrMo particles (prepared as described above, but in siliconized microcentrifuge tubes; Fisher Scientific, Waltham, MA) were resuspended – at the highest concentrations used in the experiments – in pre-warmed (37°C) endotoxin-free cell culture-grade water (MilliporeSigma) and incubated for 1 h at room temperature with mixing by inversion every 10 min. The suspensions were then centrifuged (21,000 × g for 15 min) and the supernatants were assayed as per the manufacturer’s instructions. Control samples spiked with 0.05 endotoxin unit (EU) of endotoxin, were used to assess and correct for potential interference from the particles. The supernatants, and thus presumably the particle suspensions, contained ≤ 0.01 EU/mL.

Particle-induced cytotoxicity in BMDM was assessed across a range of concentrations using dye-exclusion hemocytometry (S8 Fig). Corresponding data for the metal ions were previously published by our group [27].

Antioxidant and inhibitors

Stock solutions of N-acetyl-L-cysteine (NAC, 180 mM), an antioxidant, were freshly prepared by dissolving NAC (MilliporeSigma) into pre-warmed (37°C) phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Wisent, St-Jean Baptiste, QC). The solutions were sterile-filtered using a syringe filter equipped with a 0.2-μm pore-size cellulose acetate membrane (VWR, Radnor, PA). Stock solutions of CA-074 methyl ester (CA-074Me, 25 mM) were prepared by dissolving CA-074Me (Cayman Chemical, Ann Arbor, MI) into anhydrous dimethyl sulfoxide (DMSO; MilliporeSigma) pre-sparged with argon (Linde Canada, Mississauga, ON). The solutions were sterile-filtered using a syringe filter equipped with a 0.2-μm pore-size nylon membrane (Cytiva, Marlborough, MA). Aliquots, purged with argon, were frozen and stored in airtight polypropylene vials (Simport, Beloeil, QC) at −20°C until use. Finally, stock solutions (5 mM) of Z-Ile-Glu(O-Me)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK), an inhibitor of caspase-8, were prepared by dissolving Z-IETD-FMK (InvivoGen, San Diego, CA) in anhydrous DMSO. The solutions were sterile-filtered using a syringe filter equipped with a 0.2-μm pore-size nylon membrane. Aliquots were frozen and stored at −20°C until use.

Animals

All procedures were approved by the University of Ottawa Animal Care Committee (Protocols ME-2350 and ME-3363). The University of Ottawa Animal Care and Use Program meets the Canadian Council on Animal Care (CCAC) guidelines and is licensed under the Province of Ontario Animals for Research Act. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME), except for the wild-type (wt) mice, used in the experiments reported in Figs 1A, 1C, 1D, 4, and 5, which were purchased from Charles River Laboratories (Montreal, QC). The mice (2–5 per cage) were housed at the Animal Care Facility of the University of Ottawa, a specific-pathogen-free (SPF) facility, in individually ventilated cages (Sealsafe Plus GM500; Techniplast, West Chester, PA) containing 6-mm size corncob bedding (Envigo RMS, Indianapolis, IN), cotton fiber-based nesting material (Ancare, Bellmore, NY), and a shreddable refuge hut (Ketchum, Brockville, ON). They were maintained at 22°C with 40% relative humidity under a 12 h-light:12 h-dark photoperiod with ad libitum access to food (2018 Teklad Global 18% Protein Rodent Diet; Envigo RMS) and water (purified by reverse osmosis and acidified to pH 2.5–3.0 with hydrochloric acid). NLRP3-deficient (Nlrp3-/-), caspase-1-deficient (Casp1-/-), and wt female C57BL/6 mice, 6–17 weeks old (body mass: 20 ± 0.5 g), 4–7 weeks old (body mass 20 ± 0.1 g), and 5–18 weeks old (body mass: 20 ± 1.4 g), respectively, were euthanized between 8h00 and 15h00 by CO2 gas inhalation, followed by cervical dislocation after loss of consciousness – in compliance with CCAC guidelines requiring a secondary physical method to ensure death in small rodents. To minimize distress, euthanasia was performed in the animals’ home cage, using a gradual CO2 fill rate of 20–30% of the chamber volume per min, in accordance with CCAC guidelines and institutional animal care protocols. Euthanized mice were soaked with 70% (v/v) ethanol immediately prior to dissection.

thumbnail
Download:
Fig 1. IL-1β release by BMDM, from wild-type (wt), Nlrp3-/-, and Casp1-/- mice, exposed to Cr2O3 particles or Cr3+.

Bone marrow-derived macrophages (BMDM) were primed with 500 ng/mL of lipopolysaccharide for 3 h, then exposed to either 60-nm spherical Cr2O3 particles (A, C) or Cr3+ (B, D) for 18 h. Interleukin-1β (IL-1β) release was quantified by enzyme-linked immunosorbent assay (ELISA). Two-way analyses of variance (ANOVA) were performed (A, B). Since an interaction (p < 0.001) was detected, Holm-Šídák multiple-comparison post hoc tests were conducted, (A, B). Welch’s t-tests with Benjamini-Hochberg correction were performed (C, D). Data are presented as means ± SEM of 3 (A, B) or 4 (C, D) independent experiments. n.d.: not detected. p/mφ:particles per macrophage.

https://doi.org/10.1371/journal.pone.0334912.g001

thumbnail
Download:
Fig 2. IL-1β release by BMDM exposed to Cr2O3 particles or Cr3+ with or without NAC.

Bone marrow-derived macrophages (BMDM) from wild-type mice were primed with 500 ng/mL of lipopolysaccharide for 3 h, then exposed to 60-nm spherical Cr2O3 particles (A) or Cr3+ (B) for 18 h, in the presence or absence of N-acetyl-L-cysteine (NAC), an antioxidant. Interleukin-1β (IL-1β) release was quantified by enzyme-linked immunosorbent assay (ELISA). Two-way analyses of variance (ANOVA) were performed. Holm-Šídák pairwise post hoc tests were conducted for panel (A). For panel (B), the Holm-Šídák multiple-comparison post hoc test was used following the detection of an interaction (p < 0.001). Data are presented as means ± SEM of 3 independent experiments. p/mφ: particles per macrophage.

https://doi.org/10.1371/journal.pone.0334912.g002

thumbnail
Download:
Fig 3. IL-1β release and galectin-1 puncta formation in BMDM exposed to Cr2O3 particles or Cr3+ with or without CA-074Me.

Bone marrow-derived macrophages (BMDM) from wild-type mice were primed with 500 ng/mL of lipopolysaccharide for 3 h, then exposed to 60-nm spherical Cr2O3 particles or Cr3+ for 18 h, in the presence or absence of CA-074 methyl ester (CA-074Me), a cathepsin B inhibitor. Interleukin-1β (IL-1β) release (A, B) was quantified by enzyme-linked immunosorbent assay (ELISA), and galectin-1 puncta formation (C) was visualized using fluorescently labeled anti-lysosome-associated membrane protein 2 (LAMP-2, red) and anti-galectin-1 (green) antibodies. Nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue). L-leucyl-L-leucine methyl ester (LL-OMe) was used as a positive control for galectin-1 puncta formation. Note that image brightness and contrast were digitally optimized for visual clarity. Therefore, signal intensity is not directly comparable between images. Arrows point to galectin-1 puncta. Two-way analyses of variance (ANOVA) were performed. Holm-Šídák pairwise post hoc tests were conducted for panel (A). For panel (B), the Holm-Šídák multiple-comparison post hoc test was used following the detection of an interaction (p < 0.001). Data are presented as means ± SEM of 3 independent experiments. p/mφ: particles per macrophage. Scale bar: 10 μm.

https://doi.org/10.1371/journal.pone.0334912.g003

Bone marrow-derived macrophages

Bone marrow cells were harvested from tibiae and femora isolated from euthanized mice and prepared as previously described [27], except that Dulbecco’s modified Eagle medium (DMEM)-based complete growth medium (CGM) was used. The CGM consisted of DMEM (Wisent) supplemented with 8% (v/v) heat-inactivated fetal bovine serum (FBS; < 0.15 EU/mL endotoxin; NorthBio, Toronto, ON) and 100 U/mL each of penicillin and streptomycin (Cytiva). Cell counts were performed using dye-exclusion hemocytometry with 0.04% (w/v, final) trypan blue (MilliporeSigma) and an Improved Neubauer hemocytometer (Hausser Scientific, Horsham, PA). Cells were then seeded at ca. 230,000 cells/cm2 in polystyrene Petri dishes (Greiner Bio-One, Monroe, NC) coated with recombinant macrophage colony stimulating factor (M-CSF, 0.85 ug/cm2; R&D systems, Minneapolis, MN), as described by Sadh et al. [39]. The seeded cells were incubated for 6 days in CGM supplemented with β-mercaptoethanol (55 μM final; ThermoFisher Scientific, Rockford, IL), under cell culture conditions (37°C, humidified atmosphere of 95% air and 5% CO2). At the end of the incubation, non-adherent cells were removed by rinsing with CGM, and BMDM were detached either by pipetting using a 10-mL Class A volumetric glass pipette (Sibata Scientific Technology, Soka, Japan) or using a 2-cm-blade polyethylene cell lifter (Fisher Scientific). BMDM were collected by centrifugation (300 × g for 10 min) and resuspended at 1.0 × 106 cells/mL in pre-warmed (37°C) CGM supplemented (Figs 2, 3, 6, S2 Fig, S3 Fig, and S6 Fig) or not (Figs 1, 4, 5, and S8 Fig) with M-CSF (5 ng/mL final). Supplementation was implemented following the observation that it enhanced cell viability during the recovery period. Flow cytometric immunophenotyping (S1 Materials and methods) indicated that ca. 95% of the cells in the BMDM preparations were macrophages (S2 Fig).

Interleukin-1β and tumor necrosis factor-α release

BMDM were seeded at 1.6 × 105 cells/cm2 in 24-well tissue culture-treated polystyrene plates (Greiner Bio-One) and incubated overnight in CGM, supplemented or not with M-CSF (5 ng/mL final), to allow recovery from harvesting. The following day, BMDM were primed in CGM freshly supplemented with LPS from E. coli O55:B5 (500 ng/mL; MilliporeSigma) for 3 h under cell culture conditions, as previously described [27]. The effects of priming with different LPS concentrations on IL-1β release are shown in S3 Fig. Primed BMDM from wt mice were pre-incubated for 1 h under cell culture conditions in fresh CGM, unsupplemented (negative control), or supplemented with 2 mM NAC, 20 μM CA-074Me, or 10 μM Z-IETD-FMK. The cells were then exposed to Cr2O3 particles (0–2.4 × 106 particles/macrophage [p/mφ]), CoCrMo particles (0–70 p/mφ), Cr3+ (300 ppm), Co2+ (18 ppm), or CGM (vehicle) for 18 h. Where appropriate (Figs 1, 4, and 5), nigericin (15 μM; Cayman Chemical) – a potassium ionophore and a potent NLRP3 inflammasome inducer – was used as a positive control. The incubation period (18 h) with the particles, metal ions, or nigericin was selected as a compromise that allows sufficient time for stimulation while minimizing cell death. At the end of the incubation, culture supernatants were collected, centrifuged (21,000 × g for 10 min), flash-frozen in liquid nitrogen, and stored at −80°C for subsequent cytokine analysis. IL-1β and TNF-α were analyzed using enzyme-linked immunosorbent assays (ELISA; ThermoFisher Scientific, catalog nos. 88-7013A-77 and 88-7324-88; nominal detection limit: 8 pg/mL and 15.6 pg/mL, respectively), as per the manufacturer’s instructions. Interference from CGM, Co2+, or Cr3+ with IL-1β quantification by ELISA was ca. 6% (negative interference), 5% (positive interference), and 14% (negative interference), respectively, as shown in S4 Fig. No correction was applied to the data since the magnitude of the interference was relatively small, and the primary aim of the present study was to identify mechanisms underlying IL-1β release rather than to precisely quantify its absolute levels, which can vary depending on experimental conditions.

Galectin puncta formation

Lysosomal membrane permeabilization was assessed using the galectin puncta formation assay [40]. Briefly, BMDM from wt mice were seeded, as described above, in 18-well chamber slides (no. 1.5 tissue-culture treated proprietary polymer coverslip; Ibidi, Gräfelfing, Germany), and incubated overnight with 5 ng/mL M-CSF. The cells were then primed as described above and exposed to Cr2O3 particles (2.5 × 106 p/mφ), Cr3+ (300 ppm), or CGM (vehicle) for 6 h, or to L-leucyl-L-leucine methyl ester (LL-OMe; 2 mM; Cayman Chemical) for 2 h as a positive control for lysosomal membrane permeabilization, all under cell culture conditions. At the end of the exposure, attached cells were fixed in buffered paraformaldehyde solution (4% w/v in PBS; ThermoFisher Scientific) for 10 min, then washed with PBS without Ca2+ and Mg2+ (Wisent). The fixed cells were incubated for 20 min in blocking buffer (1% w/v bovine serum albumin [MilliporeSigma], 0.3% v/v Triton X-100 [MilliporeSigma], and 5% v/v FBS, in PBS without Ca2+ and Mg2+), followed by another wash with PBS without Ca2+ and Mg2+. They were then co-immunostained by overnight incubation at 4°C with recombinant rabbit anti-galectin-1 monoclonal antibody [EPR3205] (1:1000 dilution in blocking buffer; Abcam, Cambridge, UK; catalog no. ab108389) and rat anti-lysosome-associated-membrane protein 2 (LAMP-2) monoclonal antibody [GL2A7] (1:1000 dilution in blocking buffer; Abcam, catalog no. ab13524). The cells were then washed three times with staining buffer (0.25% w/v bovine serum albumin [BSA; Millipore Sigma] and 0.1% v/v Triton X-100 in PBS without Ca2+ and Mg2+) and co-incubated for 1 h with polyclonal secondary antibodies: donkey anti-rabbit IgG (H + L) conjugated to Alexa Fluor 488 (1:1000 dilution in staining buffer; ThermoFisher Scientific, RRID: AB 2535792) and donkey anti-rat IgG (H + L) conjugated to Alexa Fluor 594 (1:1000 dilution in staining buffer; ThermoFisher Scientific, RRID: AB 2535795). Nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 0.1% w/v in PBS without Ca2+ and Mg2+; MilliporeSigma) for 10 min. Galectin puncta formation was visualized using an inverted epifluorescence widefield microscope (Axio Observer Z1; Zeiss, Oberkochen, Germany). The stand was equipped with a light-emitting diode (LED) light source (X-Cite Xylis; Excelitas Technologies; Pittsburgh, PA) and a monochrome charge-coupled device (CCD) camera (Axiocam MRm, Zeiss). Image acquisition parameters were configured using ZEN (Blue Edition) v2.3 software (Zeiss), as detailed below. Images were acquired using a Plan-Apochromat 63 × oil-immersion differential interference contrast objective with a 1.4 numerical aperture (Zeiss). Fluorescence imaging was performed using the following filter cubes: blue (G 365 nm LED peak excitation, farb teiler [FT] 395 nm dichromatic beamsplitter, long-pass [LP] 420 nm emission) for DAPI, green (band-pass [BP] 470/40 nm excitation, FT 495 nm, BP 525/50 nm emission) for Alexa Fluor 488, and orange (BP 546/12 nm [high efficiency; HE] excitation, FT 560 nm [HE], BP 607/80 nm [HE] emission) for Alexa Fluor 594. The light source power output and the exposure time were 8% and 100 ms for DAPI, 20% and 250 ms for Alexa Fluor 488, and 20% and 300 ms, for Alexa Fluor 594, respectively. z-stacking was performed with a step size of 120 nm.

Caspase-8 processing

Caspase-8 processing was analyzed by immunoblotting. BMDM from wt mice were seeded, as above, in 6-well tissue culture-treated polystyrene plates (Greiner Bio-One), incubated overnight in CGM supplemented with 5 ng/mL M-CSF, primed as described above, then incubated for 18 h with either CoCrMo particles (100 p/mφ), Co2+ (18 ppm), or CGM (vehicle). At the end of the incubation, the plates were placed on ice and the cells washed twice with ice-cold PBS without Ca2+ and Mg2+. The cells were then detached in ice-cold PBS without Ca2+ and Mg2+, supplemented with an ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (cOmplete; MilliporeSigma), as per the manufacturer’s instructions, using a cell lifter. Cells were collected by centrifugation (10,000 × g for 5 min at 4°C), and resuspended in 50 µL of ice-cold lysis buffer composed of 125 mM Tris-HCl (pH 6.8; Fisher Scientific), 2% (w/v) sodium dodecyl sulfate (SDS; Fisher Scientific), 10% (v/v) glycerol (MilliporeSigma), and 100 mM DL-1,4-dithiothreitol (VWR). Cells lysis was performed by sonication on ice for 15 s using an ultrasonic processor (Model 120 Sonic Dismembrator; Fisher Scientific) set to 20% amplitude, fitted with a 1/8-inch probe microtip (Model CL-18; Fisher Scientific). The lysates were centrifuged (20,000 × g for 15 min at 4°C) and the supernatants were collected and stored at −20°C until use. Aliquots containing 40 μg of total protein, as determined using a colorimetric assay with BSA as the protein standard (Pierce 660 nm Protein Assay with Ionic Detergent Compatibility Reagent; ThermoFisher Scientific), were mixed with 4 × protein loading buffer (LI-COR, Lincoln, NE), as per the manufacturer’s instructions. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using precast mini-format Tris-glycine gradient gels (8–16%; Bio-Rad; Hercules, CA). A prestained protein ladder (visible and near-infrared [NIR]; LI-COR) served as molecular weight marker. Following electrophoresis, proteins were electrotransferred onto a 0.45-μm pore size fluorescent-grade polyvinylidene fluoride membrane (Immobilon-FL; MilliporeSigma). The membrane was then reversibly stained for total protein with acid blue (REVERT Total Protein Stain; LI-COR), as per the manufacturer’s instructions, and imaged at 700 nm using a NIR fluorescence imaging system (Odyssey Fc; LI-COR). The stained membrane was incubated in commercial blocking buffer (Intercept [PBS] Blocking Buffer; LI-COR) for 1 h at room temperature. Immunodetection was performed using a rabbit anti-mouse cleaved caspase-8 antibody (Cell Signaling Technology, catalog no. 9429) diluted 1:1000 in commercial antibody diluent (Intercept T20 [PBS] Antibody Diluent) supplemented with an additional 0.1% (v/v) polysorbate 20 (MilliporeSigma), with overnight incubation at 4°C. The secondary antibody used was a goat anti-rabbit IgG (H + L) fluorescently labeled with 800CW (LI-COR, catalog no. 926–32211), diluted 1:10,000 in commercial antibody diluent (Intercept T20 [PBS] Antibody Diluent) supplemented with 0.01% (w/v) SDS and polysorbate 20 at a final concentration of 0.2% (v/v), and incubated 1 h with the membrane at room temperature. The membrane was washed in PBS without Ca2+ and Mg2+, air-dried in the dark, and then imaged at 800 nm using the above LI-COR NIR fluorescence imaging system.

Statistical analysis

Unless otherwise specified, statistical analysis was performed using GraphPad Prism v9.4.1 for macOS. Data were considered to meet the assumptions of normality, and homoscedasticity was assessed using Levene’s test, implemented in R v2025.1.513 [41]. When the assumption of homogeneity was violated, the Welch t-test was performed, and the resulting p values were adjusted using the Benjamini-Hochberg procedure to control the false discovery rate (FDR) at 0.05. Otherwise, two-way analyses of variance (ANOVA) were performed, followed by the Holm-Šídák test: pairwise tests to analyze main effects, or multiple comparison tests to assess simple main effects when an interaction was found. p < 0.05 was considered significant. Significant differences are reported exclusively for the following three types of comparisons: 1) Particle/ion exposure – BMDM (from either wt or knockout mice) exposed to a specific particle or metal ion concentration were compared to the negative control (cells unexposed to exogenous particles or metal ions), in the absence of an inhibitor or antioxidant; 2) Genetic manipulation – BMDM from wt mice were compared to those from knockout mice, both exposed to the same particle or metal ion concentration; and 3) Inhibitor/antioxidant treatment – BMDM exposed to a specific particle or metal ion concentration were compared in the presence or absence of an inhibitor or antioxidant. Effect sizes, computed manually in Microsoft Excel, are presented in S5 Table as Cohen’s d with 95% confidence intervals (CI). Unless otherwise specified, data are presented as mean ± SEM of at least 3 independent experiments, each performed with 3 replicate samples per condition.

Results

NLRP3 and caspase-1 dependence of Cr2O3 particle- and Cr3+-induced IL-1β release

To determine the NLRP3 and/or caspase-1 dependence of IL-1β release by macrophages exposed to Cr2O3 nanoparticles or Cr3+, we analyzed IL-1β release by BMDM, from wt, Nlrp3-/-, or Casp1-/- mice, exposed to 60-nm spherical Cr2O3 particles or Cr3+ (Fig 1). Exposure of BMDM from wt mice to the particles induced a particle concentration-dependent increase in IL-1β release, reaching ca. 400% with 2.4 × 106 p/mφ (p < 0.001), relative to the negative control (BMDM unexposed to Cr2O3 particles; Fig 1A). Similarly, exposure to Cr3+ (300 ppm) induced an increase in IL-1β release of ca. 870% (p < 0.001), relative to the negative control (BMDM unexposed to Cr3+; Fig 1B). In contrast, IL-1β release by BMDM from Nlrp3-/- mice was less than 7% of that observed by BMDM from wt mice when the cells were exposed to Cr2O3 particles (e.g., 3 ± 1% with 2.4 × 106 p/mφ; p < 0.001) or Cr3+ (300 ppm; 6 ± 2%; p < 0.001). Similarly, IL-1β release by BMDM from Casp1-/- mice was ca. 17% (p = 0.005) of that by BMDM from wt mice when the cells were exposed to 1.7 × 106 Cr2O3 p/mφ (Fig 1C) and ca. 13% (p < 0.001) of that by BMDM from wt mice when exposed to Cr3+ (300 ppm; Fig 1D). Finally, exposure of BMDM from wt mice to the positive control, nigericin (15 μM), induced a large increase in IL-1β release (p < 0.001), but had no statistically significant effect on BMDM from Nlrp3-/- mice (S6 Fig).

ROS dependence of Cr2O3 particle- and Cr3+-induced NLRP3 inflammasome activation

ROS are established mediators of NLRP3 inflammasome activation. To assess whether IL-1β release by macrophages exposed to Cr2O3 nanoparticles or Cr3+ is ROS dependent, we analyzed the effect of NAC, an antioxidant, on this release. IL-1β release by BMDM, from wt mice, exposed to 60-nm Cr2O3 particles (up to 2.4 × 106 p/mφ) was not statistically significantly affected by NAC (Fig 2A). In sharp contrast, IL-1β release by BMDM, from wt mice, exposed to Cr3+ (300 ppm) was ca. 90% lower (p < 0.001; Fig 2B) in the presence of NAC, i.e., similar to that of the negative control.

Cathepsin B dependence of Cr2O3 particle- and Cr3+-induced IL-1β release

Cathepsin B is a known mediator of NLRP3 inflammasome activation. To determine whether IL-1β release by macrophages exposed to Cr2O3 nanoparticles or Cr3+ is dependent on cathepsin B activity, we analyzed the effect of CA-074Me, a cathepsin B inhibitor, on this release. In the presence of CA-074Me, IL-1β release by BMDM, from wt mice, exposed to 60-nm Cr2O3 particles (2.5 × 106 p/mφ) or Cr3+ (300 ppm) was inhibited by ca. 70% (p = 0.01) and 84% (p < 0.001), respectively – resulting in levels similar to that of the negative control (Fig 3A and B, respectively). To assess whether the cathepsin B dependence of IL-1β release by macrophages exposed to 60-nm Cr2O3 particles or Cr3+ results from lysosomal leakage of the enzyme, we examined galectin-1 puncta formation – an indicator of lysosomal destabilization – in BMDM from wt mice. Upon lysosomal permeabilization, galectin-1, a small cytosolic carbohydrate-binding protein, translocates to the inner surface of the lysosomal membrane. This translocation can be visualized by immunofluorescence as a shift from diffuse to punctate staining [40]. As shown in Fig 3C, galectin-1 puncta formation was observed in BMDM exposed to Cr2O3 particles (2.5 × 106 p/mφ), but not in those exposed to Cr3+ (300 ppm).

NLRP3 independence of CoCrMo particle- and Co2+-induced IL-1β release

To analyze the NLRP3 dependence of IL-1β release by macrophages exposed to CoCrMo microparticles or Co2+, we compared the release of IL-1β by BMDM from wt and Nlrp3-/- mice. Exposure of the BMDM from wt mice to 3.4-μm CoCrMo particles induced a particle concentration-dependent increase in IL-1β release reaching ca. 525% with 70 p/mφ (p < 0.001), relative to the negative control (BMDM unexposed to CoCrMo particles; Fig 4A). Similarly, exposure of BMDM, from wt mice, to Co2+ (18 ppm) induced an increase in IL-1β release of ca. 175% (p = 0.02), relative to the negative control (BMDM unexposed to Co2+; Fig 4B). Knocking out Nlrp3 did not significantly affect IL-1β release by BMDM exposed to CoCrMo particles, and IL-1β release by BMDM from both wt and Nlrp3-/- mice peaked with 35–70 p/mφ (Fig 4A). Similarly, IL-1β release by BMDM exposed to Co2+ was not significantly affected by the absence of NLRP3. Interestingly, knocking out Nlrp3 increased IL-1β release induced by CoCrMo particles and Co2+ by up to 60% with 70 p/mφ (p < 0.001; Fig 4A) and 116% with 18 ppm (p = 0.001; Fig 4B), respectively.

Caspase-1 dependence of CoCrMo particle- and Co2+-induced IL-1β release

To analyze the caspase-1 dependence of IL-1β release by macrophages exposed to CoCrMo microparticles or Co2+, we compared the release of IL-1β by BMDM from wt and Casp1-/- mice. Knocking out Casp1 did not significantly affect IL-1β release by BMDM exposed to 3.4-μm CoCrMo particles (Fig 5A). In contrast, the absence of caspase-1 resulted in ca. 43% lower IL-1β release by BMDM exposed to Co2+ (18 ppm; p < 0.001; Fig 5B).

Caspase-8 dependence of CoCrMo- and Co2+-induced IL-1β release

Caspase-8 has been shown to cleave pro-IL-1β into its biologically active form and be able to do so independently of the NLRP3 inflammasome [37]. To assess whether IL-1β release by macrophages exposed to CoCrMo microparticles or Co2+ depends on caspase-8, we analyzed the effects of Z-IETD-FMK, a caspase-8 inhibitor, on this release. In the presence of Z-IETD-FMK, IL-1β release by BMDM, from wt mice, exposed to 3.4-μm CoCrMo particles (100 p/mφ) or Co2+ (18 ppm) was inhibited by ca. 70% (p = 0.006 and p = 0.004, respectively), resulting in levels similar to that of the negative control (Fig 6A and B, respectively). To confirm that CoCrMo particles and Co2+ induced caspase-8 activation, we analyzed caspase-8 cleavage in BMDM from wt mice. Immunoblotting revealed that exposure to CoCrMo particles (100 p/mφ) or Co2+ (18 ppm) resulted in caspase-8 processing, as evidenced by the detection of the 43 kDa cleaved caspase-8 fragment (Fig 6C).

Discussion

The present study aimed to elucidate molecular mechanisms by which wear particles (Cr2O3 and CoCrMo) and metal ions (Co2+ and Cr3+) from CoCrMo implants lead to IL-1β release by murine BMDM. Distinct activation pathways were observed depending on the stimulus. The release of IL-1β induced by spherical Cr2O3 nanoparticles or Cr3+ was dependent on both NLRP3 and caspase-1, whereas that induced by spherical CoCrMo microparticles or Co2+ was NLRP3 independent and either caspase-1 independent (in the case of CoCrMo particles) or partially caspase-1 dependent (for Co2+). Further analysis suggested that IL-1β release in response to the Cr2O3 particles was cathepsin B dependent, implicating cathepsin B-mediated NLRP3 inflammasome activation. In contrast, Cr3+-induced IL-1β release was ROS dependent, as shown by antioxidant treatment, suggesting ROS-mediated NLRP3 inflammasome activation. The NLRP3-independent IL-1β release induced by the CoCrMo particles was caspase-8 dependent, based on inhibition and processing evidence, but caspase-1 independent. In contrast, Co2+-induced IL-1β release was caspase-8 dependent and partially caspase-1 dependent.

Macrophages have been shown to accumulate in periprosthetic tissues of failed hip implants [17] and play a central role in the immune response to implant wear and corrosion products [42]. Their activation in response to these products leads to the release of pro-inflammatory cytokines, including IL-1β [24,25,27,43]. Murine BMDM, the most common macrophage model in immunological studies [44], were used in the present study for reasons detailed elsewhere [44,45], including their easy availability from genetically modified mouse strains.

The size, shape, concentration, and composition of wear particles are known to influence macrophage response in vitro and tissue reactions in vivo [12,24,46,47]. In the present study, commercially available spherical Cr2O3 nanoparticles (60 nm; 0–3.5 × 106 p/mφ) and CoCrMo microparticles (3.4 μm; 0–70 p/mφ) were used. The size and shape of Cr2O3 particles are consistent with those isolated from periprosthetic tissues of patients with a metal-on-metal hip implant [11,48,49], as well as those generated by hip simulators [11,50]. While the CoCrMo particles are exclusively spherical and larger than those typically found in vivo, their size remains within the endocytosable range [51], and their composition is physiologically relevant [11,48,49]. A range of particle concentrations was selected to assess dose-dependent responses, based on the conceptual premise that macrophages in vitro require higher concentrations of stimulating agents than they do in vivo, where they are exposed to multiple cues. The concentrations of Co2+ (18 ppm) and Cr3+ (300 ppm) were selected to induce peak IL-1β release, as determined by previous work from our group [27].

IL-1β release has been classically linked with activation of inflammasome complexes, particularly the NLRP3 inflammasome. Sterile inflammation inducers have also frequently been associated with NLRP3 inflammasome activation and subsequent IL-1β release [52]. In the present study, we investigated the dependence of IL-1β release on NLRP3-inflammasome signaling in macrophages exposed to spherical Cr2O3 (60 nm) or CoCrMo (3.4 μm) particles, or Co2+ or Cr3+. Exposure of the primed BMDM to CoCrMo particles, Co2+, or Cr3+ induced IL-1β release, in agreement with previous studies [27,43]. Notably, Cr2O3 particles, despite their relatively inert ceramic composition, also induced a concentration-dependent IL-1β release. This finding is consistent with earlier reports showing that exposure of THP-1 human monocytes to nearly spherical Cr2O3 particles (60-nm) [53] or spherical CrOOH particles (15-nm) [54] increased IL-1β gene expression and IL-1β release, respectively. The release of IL-1β induced by Cr2O3 particles and Cr3+ was both NLRP3- and caspase-1-dependent, suggesting NLRP3 inflammasome dependence.

Given the established role of ROS and lysosomal destabilization in NLRP3 inflammasome activation, we investigated whether Cr2O3 particles and Cr3+ can trigger these upstream events. Specifically, the involvement of ROS in Cr2O3 particle- and Cr3+-induced NLRP3-dependent IL-1β release was assessed by measuring the effects of the antioxidant NAC on IL-1β release. Results showed that IL-1β release induced by Cr3+, but not by the Cr2O3 particles, was marginal in the presence of NAC, thereby suggesting a ROS-dependent response. This finding is consistent with a previous study from our group [27], which demonstrated that Cr3+-induced IL-1β release by BMDM was marginal in the presence of ascorbic acid, another antioxidant, and that processed caspase-1 (p20 subunit) was detectable only in its absence. Moreover, the marginal IL-1β release induced by Cr3+ in the presence of either NAC or ascorbic acid suggests that these effects were primarily due to their antioxidant properties rather than their ability to chelate metal ions [55,56] – as the concentrations were insufficient to extensively chelate 300 ppm of Cr3+ – or the role of ascorbic acid as an enzymatic cofactor. Although the mechanisms by which Cr3+ induce ROS production remain unclear, proteomics data have shown that Cr3+ can directly bind to cellular redox enzymes [57]. Cr3+ have also been shown to significantly impair the catalytic activity of catalase, reducing their ability to decompose hydrogen peroxide (H₂O₂) in human erythrocytes [57]. Additionally, Cr3+ have been reported to lower glutathione levels in the liver and kidney of goldfish [58]. Together, these findings suggest that Cr3+ may increase ROS levels by disrupting key elements of the cellular antioxidant defense system. Interestingly, Salloum et al. [59] reported that Cr3+ exposure did not significantly increase ROS levels or oxidative stress in RAW 264.7 murine macrophages. This apparent inconsistency with the present findings may be attributed to ROS compartmentalization, the hyperoxic conditions of standard cell culture [60], or methodological distinctions such as the use of primary non-neoplastic cells vs. neoplastic cells.

To investigate the potential role of cathepsin B in NLRP3-dependent IL-1β release induced by the Cr2O3 particles and Cr3+, we assessed the effects of cathepsin B inhibitor CA-074Me on IL-1β release. Results showed that CA-074Me prevented IL-1β release induced by the Cr2O3 particles as well as by Cr3+, suggesting dependence on cathepsin B for both stimuli. We further assessed the effects of Cr2O3 particles and Cr3+ on galectin puncta formation – an indicator of lysosomal destabilization that can lead to cytosolic release of cathepsin B. The particles, but not the ions, induced galectin puncta formation. Together, these results suggest that 60-nm spherical Cr2O3 particles can induce lysosomal destabilization, leading to cathepsin B leakage and subsequent NLRP3 inflammasome activation. In contrast, cathepsin B-dependent IL-1β release induced by Cr3+ occurred without detectable lysosomal destabilization, possibly due to spatially and temporally restricted lysosomal release of limited quantities of cathepsin B into the cytosol [34,61]. Interestingly, Cr3+ elicited a stronger cathepsin B-dependent IL-1β release than the Cr2O3 particles, despite the absence of overt lysosomal destabilization, suggesting the existence of complementary mechanisms. The ROS-dependence of Cr3+ -induced IL-1β release further suggests that ROS production and other synergistic pathways may potentiate cathepsin B-mediated inflammasome activation. By contrast, lysosomal rupture may lack these synergistic signals or could even activate negative regulatory pathways that limit IL-1β release despite the release of cathepsin B [62]. A controlled release of cathepsin B could occur in proximity to inflammasome assembly sites, such as mitochondria-associated endoplasmic reticulum (ER) membranes, thereby enhancing the efficiency of inflammasome activation [34]. In contrast, lysosomal rupture might result in the diffuse or mislocalized release of cathepsins, reducing their effective concentration at critical signalling hubs.

Exposure of primary human monocytes and THP-1 (human) macrophages to spherical or irregularly-shaped 6–7-μm CoCrMo particles [24,25], as well as to Co2+, has been reported to activate the NLRP3 inflammasome and trigger the subsequent release of IL-1β [25]. More specifically, Caicedo et al. [24] reported that increasing size (up to 6 μm diameter) and shape irregularity of CoCrMo particles resulted in greater lysosomal damage and enhanced NLRP3 inflammasome activation. However, the present study found that both 3.4-μm spherical CoCrMo particles and Co2+ induced IL-1β release independently of the NLRP3 inflammasome. As a result, the potential of CoCrMo particles or Co2+ to cause lysosomal damage was not investigated. The apparent inconsistency between studies may be attributed to differences in cell type (human peripheral blood monocytes vs. murine BMDM), particle characteristics (for CoCrMo particles only, including, size and shape), and/or experimental design – for example, the use of small interfering RNA (siRNA) for Nlrp3 knockdown compared to genetic knockout in the present study. Moreover, exposing NLRP3-deficient BMDM to CoCrMo particles increased IL-1β release, suggesting the activation of compensatory pathways in the absence of NLRP3.

Experiments using BMDM from Casp1-/- mice showed that IL-1β release induced by the 3.4-μm CoCrMo particles was caspase-1 independent, whereas that induced by Co2+ was partially caspase-1 dependent. Interestingly, NLRP3-independent but caspase-1-dependent IL-1β release has also been reported in 3T3-L1 adipocytes exposed to TNF-α [63], as well as in RAW 264.7 macrophages stimulated with monosodium urate crystals [64]. Since exposure to Co2+ induced TNF-α release by BMDM (S3 Fig), the NLRP3-independent but partially caspase-1-dependent IL-1β release observed in response to Co2+ could involve TNF-α signaling. Although the mechanisms by which Co2+ induce the cleavage of inactive pro-caspase-1 into its mature form remain largely unknown, potential NLRP3-independent mechanisms have been identified. For example, in RAW 264.7 macrophages exposed to monosodium urate crystal, epidermal growth factor receptor pathway substrate 8 (Eps8) has been reported to form a complex with pro-caspase-1 and be required for its activation – suggesting that the Eps8-pro-caspase-1 complex facilitates cleavage [64].

Our observation that IL-1β release induced by 3.4-μm spherical CoCrMo particles and Co2+ occurred independently of NLRP3 and was either fully (CoCrMo particles) or partially (Co2+) caspase-1 independent, led us to investigate the potential involvement of caspase-8 in this release. Caspase-8 non-apoptotic functions have been shown to include the cleavage of IL-1β into its mature active form, either directly [37,65] or via activation of the NLRP3 inflammasome [65,66]. Furthermore, elevated levels of caspase-8 have been found in the interface membranes of aseptically loosened total hip implants, but not in control tissues (e.g., from hip dysplasia or mechanical loosening) [67–69]. In the present study, we showed that a caspase-8 inhibitor (Z-IETD-FMK) prevented IL-1β release induced by CoCrMo particles and Co2+, suggesting that these processes are fully caspase-8-dependent. This is particularly interesting because Co2+-induced IL-1β release is also partially dependent on caspase-1, even though it is independent of the NLRP3 inflammasome. We propose that Co2+ may be inducing IL-1β release by causing transient caspase-1 dimerization, a mechanism described by Boucher et al. [70]. Additionally, the enhanced release of IL-1β observed in NLPR3-deficient BMDM exposed to the CoCrMo particles or Co2+ may be attributed to increased caspase-8 synthesis or activity when NLRP3 is absent. The observed dependence on caspase-8 might be explained by the potential of Co2+ to induce the proapoptotic Bax/Bak proteins [67], which can lead to caspase-8 activation [71]. It should be noted that our findings contrast with those of Petit et al. [67], who observed that Cr3+, but not Co2+, increased caspase-8 expression and activity in U937 human macrophages. However, an important caveat to their study is that, unlike in non-neoplastic macrophages such as BMDM, mature caspase-8 was constitutively expressed in the U937 cells used [67]. Finally, it should be noted that caspase-8, which is capable of activating the NLRP3 inflammasome [66,72–74], may also have contributed to the NLRP3-dependent IL-1β release induced by the Cr2O3 particles or Cr3+.

Limitations of the study

BMDM used in the present study were generated exclusively from female mice due to housing considerations. Although sex-based differences are not expected to alter the core immune mechanisms under investigation, they may influence the magnitude of the response. Murine BMDM are a widely used model for studying inflammatory mechanisms in vitro; however, species-specific differences between murine and human macrophages should be considered when extrapolating these findings to humans. Most notably, unlike in murine monocytes, human monocytes do not always require a two-step process for NLRP3 inflammasome activation. TLR4 stimulation alone has been shown to be sufficient to induce the release of IL-1β [75,76]. In the present in vitro study, BMDM were primed with LPS, a commonly used priming agent in NLRP3 inflammasome studies. In vivo, however, priming may be mediated by a variety of endogenous factors (such as DAMPs or alarmins) [19] or exogenous PAMPs (like LPS) that originate from a subclinical bacterial biofilm on the implant material surface [77,78].

The commercial availability of CoCrMo nanoparticles is limited by challenges in consistent generation, instability due to spontaneous oxidation, and the occupational hazards presented by their inherent biological toxicity. As a result, the CoCrMo particles used in the present study were larger than those typically isolated from periprosthetic tissues [11]. It should also be noted that, although both spherical and elongated CoCrMo particles are found in periprosthetic tissues, spherical particles are generally less abundant [11]. Although particle size and shape can be important factors influencing IL-1β release and lysosomal destabilization [24], particle composition is also a critical determinant. For example, our results demonstrate that Cr2O3 particles and Cr3+ activated similar inflammatory mechanisms, as did CoCrMo particles and Co2+. Given that CoCrMo alloys can release Co2+ through degradation, the similarity in responses induced by CoCrMo particles and Co2+ may reflect ion release as a contributing factor. In contrast, Cr2O3 is considered relatively chemically inert and is therefore unlikely to generate Cr3+ under physiological conditions. The similarity of the mechanisms activated by Cr2O3 particles and Cr3+ may thus be attributed to their shared elemental composition (Cr) and size congruity. Our results also demonstrated that IL-1β release by BMDM, from Casp1-/- mice, exposed to CoCrMo particles was caspase-1 independent, whereas IL-1β release in response to Co2+ was partially dependent on caspase-1. This divergence suggests that the effects observed with CoCrMo particles were, at least in part, attributable to the particles per se rather than to potential release of Co2+.

Finally, it should be noted that although the inhibitors used in the present study are generally considered specific for their intended targets, the possibility of off-target effects cannot be fully excluded.

Conclusion

This study provides novel insight into the inflammatory response of macrophages triggered by CoCrMo implant wear and corrosion products. It highlights the specificity and diversity of the underlying mechanisms, revealing distinct pathways through which CoCrMo and Cr2O3 particles, as well as Co2+ and Cr3+, can induce IL-1β release in macrophages (Fig. 7). Among these pathways, NLRP3, caspase-1, and caspase-8 emerge as key mediators, positioning them as potential molecular targets for future therapeutic strategies. The activation of stimulus-specific signaling cascades underscores the complexities of implant-related inflammation while also presenting opportunities for more precise tailored therapeutic approaches. Although IL-1β represents only one component of a much broader multifaceted response, it plays a central role in periprosthetic inflammation. By elucidating the mechanisms through which wear particles and metal ions promote IL-1β-mediated inflammatory response in macrophages, this research provides a critical foundation for the development of targeted strategies to reduce inflammation and ultimately extend the functional lifespan of CoCrMo-based implants.

thumbnail
Download:
Fig 4. IL-1β release by BMDM, from wild-type (wt) and Nlrp3-/- mice, exposed to CoCrMo particles or Co2+.

Bone marrow-derived macrophages (BMDM) were primed with 500 ng/mL of lipopolysaccharide for 3 h, then exposed to 3.4-μm spherical CoCrMo particles (A) or Co2+ (B) for 18 h. Interleukin-1β (IL-1β) release was quantified by enzyme-linked immunosorbent assay (ELISA). Two-way analyses of variance (ANOVA) were performed. Since an interaction (p = 0.003 [A] and p = 0.001 [B]) was detected, Holm-Šídák multiple-comparison post hoc tests were conducted (A, B). Data are presented as means ± SEM of 3 independent experiments. p/mφ: particles per macrophage.

https://doi.org/10.1371/journal.pone.0334912.g004

thumbnail
Download:
Fig 5. IL-1β release by BMDM, from wild-type (wt) and Casp1-/- mice, exposed to CoCrMo particles or Co2+.

Bone marrow-derived macrophages (BMDM) were primed with 500 ng/mL of lipopolysaccharide for 3 h, then exposed to 3.4-μm spherical CoCrMo particles (A) or Co2+ (B) for 18 h. Interleukin-1β (IL-1β) release was quantified by enzyme-linked immunosorbent assay (ELISA). Two-way analyses of variance (ANOVA) were performed. Holm-Šídák pairwise post hoc tests were conducted for panel (A). For panel (B), the Holm-Šídák multiple-comparison post hoc test was used following the detection of an interaction (p = 0.003). Data are presented as means ± SEM of 3 independent experiments. p/mφ: particles per macrophage.

https://doi.org/10.1371/journal.pone.0334912.g005

thumbnail
Download:
Fig 6. IL-1β release, with and without a caspase 8 inhibitor, and caspase-8 cleavage in BMDM exposed to CoCrMo particles or Co2+.

Bone marrow-derived macrophages (BMDM) from wild-type mice were primed with 500 ng/mL of lipopolysaccharide for 3 h, then exposed to 3.4-μm spherical CoCrMo particles or Co2+ for 18 h in the presence or absence of a caspase-8 inhibitor (Z-IETD-FMK). Interleukin-1β (IL-1β) release (A, B) was quantified by enzyme-linked immunosorbent assay (ELISA). Cell lysates were analyzed for caspase-8 processing (C) by immunoblotting. A representative immunoblot is shown. All (full-size) immunoblot replicates (including total protein staining) are shown in S7 Fig. Two-way analyses of variance (ANOVA) were performed. Since an interaction (p = 0.05 [A] and p = 0.04 [B]) was detected, Holm-Šídák multiple-comparison post hoc tests were conducted (A, B). Data are presented as means ± SEM of 3 independent experiments. neg. ctrl: negative control (vehicle). p/mφ: particles per macrophage.

https://doi.org/10.1371/journal.pone.0334912.g006

thumbnail
Download:
Fig 7. Schematic summary of the mechanisms involved in the release of IL-1β by BMDM characterized in the present study.

IL-1β: interleukin-1β; BMDM: bone marrow-derived macrophages. Created in BioRender. Abdoulkader, N. (2025) https://BioRender.com/s84o209.

https://doi.org/10.1371/journal.pone.0334912.g007

Supporting information

S1 Materials and methods. Purity of bone marrow-derived macrophage preparations.

https://doi.org/10.1371/journal.pone.0334912.s001

(PDF)

S2 Fig. Characterization of BMDM cell preparation purity.

https://doi.org/10.1371/journal.pone.0334912.s002

(PDF)

S3 Fig. IL-1β (A) and TNF-α (B) release by BMDM primed with various concentrations of LPS, then exposed to Co2+.

https://doi.org/10.1371/journal.pone.0334912.s003

(PDF)

S4 Fig. Effects of Co2+ and Cr3+ on the quantification of IL-1β by ELISA.

https://doi.org/10.1371/journal.pone.0334912.s004

(PDF)

S5 Table. Cohen’s d values and confidence intervals (CI).

https://doi.org/10.1371/journal.pone.0334912.s005

(PDF)

S6 Fig. IL-1β release by BMDM, from wild-type (wt) and Nlrp3-/- mice, exposed to nigericin.

https://doi.org/10.1371/journal.pone.0334912.s006

(PDF)

S7 Fig. Raw immunoblot images.

https://doi.org/10.1371/journal.pone.0334912.s007

(PDF)

S8 Fig. Trypan blue staining of BMDM detached after exposure to CoCrMo particles (A) or Cr2O3 particles (B).

https://doi.org/10.1371/journal.pone.0334912.s008

(PDF)

Acknowledgments

The authors thank Dr. Liyuan Wang from the Cell Biology and Image Acquisition (CBIA) Core Facility at the University of Ottawa.

References

  1. 1. Knecht C, Polakof L, Behrens J, Goodman SB. Wear debris in metal-on-metal bearings and modular junctions : What have we learned from the last decades? Orthopadie (Heidelb). 2023;52(3):206–13. pmid:36820850
  2. 2. Esposito CI, Wright TM, Goodman SB, Berry DJ, Clinical, Biological and Bioengineering Study Groups from Carl T. Brighton Workshop. What is the trouble with trunnions? Clin Orthop Relat Res. 2014;472(12):3652–8. pmid:24980639
  3. 3. Kolatat K, Perino G, Wilner G, Kaplowitz E, Ricciardi BF, Boettner F, et al. Adverse local tissue reaction (ALTR) associated with corrosion products in metal-on-metal and dual modular neck total hip replacements is associated with upregulation of interferon gamma-mediated chemokine signaling. J Orthop Res. 2015;33(10):1487–97. pmid:25940887
  4. 4. Laaksonen I, Galea VP, Donahue GS, Matuszak SJ, Muratoglu O, Malchau H. The cobalt/chromium ratio provides similar diagnostic value to a low cobalt threshold in predicting adverse local tissue reactions in patients with metal-on-metal hip arthroplasty. J Arthroplasty. 2018;33(9):3020–4. pmid:29716769
  5. 5. Grammatopoulos G, Pandit H, Kamali A, Maggiani F, Glyn-Jones S, Gill HS, et al. The correlation of wear with histological features after failed hip resurfacing arthroplasty. J Bone Joint Surg Am. 2013;95(12):e81. pmid:23783212
  6. 6. Grammatopoulos G, Munemoto M, Pollalis A, Athanasou NA. Correlation of serum metal ion levels with pathological changes of ARMD in failed metal-on-metal-hip-resurfacing arthroplasties. Arch Orthop Trauma Surg. 2017;137(8):1129–37. pmid:28660477
  7. 7. Willert H-G, Buchhorn GH, Fayyazi A, Flury R, Windler M, Köster G, et al. Metal-on-metal bearings and hypersensitivity in patients with artificial hip joints. A clinical and histomorphological study. J Bone Joint Surg Am. 2005;87(1):28–36. pmid:15637030
  8. 8. Campbell P, Ebramzadeh E, Nelson S, Takamura K, De Smet K, Amstutz HC. Histological features of pseudotumor-like tissues from metal-on-metal hips. Clin Orthop Relat Res. 2010;468(9):2321–7. pmid:20458645
  9. 9. Pandit H, Glyn-Jones S, McLardy-Smith P, Gundle R, Whitwell D, Gibbons CLM, et al. Pseudotumours associated with metal-on-metal hip resurfacings. J Bone Joint Surg Br. 2008;90(7):847–51. pmid:18591590
  10. 10. Mahendra G, Pandit H, Kliskey K, Murray D, Gill HS, Athanasou N. Necrotic and inflammatory changes in metal-on-metal resurfacing hip arthroplasties. Acta Orthop. 2009;80(6):653–9. pmid:19995315
  11. 11. Catelas I, Medley JB, Campbell PA, Huk OL, Bobyn JD. Comparison of in vitro with in vivo characteristics of wear particles from metal-metal hip implants. J Biomed Mater Res B Appl Biomater. 2004;70(2):167–78. pmid:15264297
  12. 12. Madl AK, Liong M, Kovochich M, Finley BL, Paustenbach DJ, Oberdörster G. Toxicology of wear particles of cobalt-chromium alloy metal-on-metal hip implants Part I: physicochemical properties in patient and simulator studies. Nanomedicine. 2015;11(5):1201–15. pmid:25744761
  13. 13. Maezawa K, Nozawa M, Hirose T, Matsuda K, Yasuma M, Shitoto K, et al. Cobalt and chromium concentrations in patients with metal-on-metal and other cementless total hip arthroplasty. Arch Orthop Trauma Surg. 2002;122(5):283–7. pmid:12070648
  14. 14. Harding I, Bonomo A, Crawford R, Psychoyios V, Delves T, Murray D, et al. Serum levels of cobalt and chromium in a complex modular total hip arthroplasty system. J Arthroplasty. 2002;17(7):893–5. pmid:12375249
  15. 15. Lhotka C, Szekeres T, Steffan I, Zhuber K, Zweymüller K. Four-year study of cobalt and chromium blood levels in patients managed with two different metal-on-metal total hip replacements. J Orthop Res. 2003;21(2):189–95. pmid:12568948
  16. 16. Nich C, Takakubo Y, Pajarinen J, Ainola M, Salem A, Sillat T, et al. Macrophages–Key cells in the response to wear debris from joint replacements. J Biomed Mater Res A. 2013;101(10):3033–45. pmid:23568608
  17. 17. Campbell P, Park S-H, Ebramzadeh E. Semi-quantitative histology confirms that the macrophage is the predominant cell type in metal-on-metal hip tissues. J Orthop Res. 2022;40(2):387–95. pmid:33749015
  18. 18. Samelko L, Landgraeber S, McAllister K, Jacobs J, Hallab NJ. TLR4 (not TLR2) dominate cognate TLR activity associated with CoCrMo implant particles. J Orthop Res. 2017;35(5):1007–17. pmid:27416075
  19. 19. Takagi M, Takakubo Y, Pajarinen J, Naganuma Y, Oki H, Maruyama M, et al. Danger of frustrated sensors: Role of Toll-like receptors and NOD-like receptors in aseptic and septic inflammations around total hip replacements. J Orthop Translat. 2017;10:68–85. pmid:29130033
  20. 20. Naganuma Y, Takakubo Y, Hirayama T, Tamaki Y, Pajarinen J, Sasaki K, et al. Lipoteichoic acid modulates inflammatory response in macrophages after phagocytosis of titanium particles through Toll-like receptor 2 cascade and inflammasomes. J Biomed Mater Res A. 2016;104(2):435–44. pmid:26440284
  21. 21. Schmidt M, Raghavan B, Müller V, Vogl T, Fejer G, Tchaptchet S, et al. Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel. Nat Immunol. 2010;11(9):814–9. pmid:20711192
  22. 22. Rachmawati D, Bontkes HJ, Verstege MI, Muris J, von Blomberg BME, Scheper RJ, et al. Transition metal sensing by Toll-like receptor-4: next to nickel, cobalt and palladium are potent human dendritic cell stimulators. Contact Dermatitis. 2013;68(6):331–8. pmid:23692033
  23. 23. Cheng X, Jansson V, Kretzer JP, Bader R, Utzschneider S, Paulus AC. The expression levels of Toll-like receptors after metallic particle and ion exposition in the synovium of a murine model. J Clin Med. 2021;10(16):3489. pmid:34441785
  24. 24. Caicedo MS, Samelko L, McAllister K, Jacobs JJ, Hallab NJ. Increasing both CoCrMo-alloy particle size and surface irregularity induces increased macrophage inflammasome activation in vitro potentially through lysosomal destabilization mechanisms. J Orthop Res. 2013;31(10):1633–42. pmid:23794526
  25. 25. Caicedo MS, Desai R, McAllister K, Reddy A, Jacobs JJ, Hallab NJ. Soluble and particulate Co-Cr-Mo alloy implant metals activate the inflammasome danger signaling pathway in human macrophages: a novel mechanism for implant debris reactivity. J Orthop Res. 2009;27(7):847–54. pmid:19105226
  26. 26. Fort BP, Dubyak GR, Greenfield EM. Lysosomal disruption by orthopedic wear particles induces activation of the NLRP3 inflammasome and macrophage cell death by distinct mechanisms. J Orthop Res. 2021;39(3):493–505. pmid:32779803
  27. 27. Ferko M-A, Catelas I. Effects of metal ions on caspase-1 activation and interleukin-1β release in murine bone marrow-derived macrophages. PLoS One. 2018;13(8):e0199936. pmid:30138321
  28. 28. Swanson KV, Deng M, Ting JP-Y. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol. 2019;19(8):477–89. pmid:31036962
  29. 29. Sutterwala FS, Haasken S, Cassel SL. Mechanism of NLRP3 inflammasome activation. Ann N Y Acad Sci. 2014;1319(1):82–95. pmid:24840700
  30. 30. Yabal M, Calleja DJ, Simpson DS, Lawlor KE. Stressing out the mitochondria: Mechanistic insights into NLRP3 inflammasome activation. J Leukoc Biol. 2019;105(2):377–99. pmid:30589456
  31. 31. Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL, et al. Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med. 2018;215(3):827–40. pmid:29432122
  32. 32. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol. 2008;9(8):847–56. pmid:18604214
  33. 33. de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974;23(18):2495–531. pmid:4606365
  34. 34. Chevriaux A, Pilot T, Derangère V, Simonin H, Martine P, Chalmin F, et al. Cathepsin B is required for NLRP3 inflammasome activation in macrophages, through NLRP3 interaction. Front Cell Dev Biol. 2020;8:167. pmid:32328491
  35. 35. Orlowski GM, Colbert JD, Sharma S, Bogyo M, Robertson SA, Rock KL. Multiple cathepsins promote pro-IL-1β synthesis and NLRP3-mediated IL-1β activation. J Immunol. 2015;195(4):1685–97. pmid:26195813
  36. 36. Hildebrand D, Bode KA, Rieß D, Cerny D, Waldhuber A, Römmler F, et al. Granzyme A produces bioactive IL-1β through a nonapoptotic inflammasome-independent pathway. Cell Rep. 2014;9(3):910–7. pmid:25437548
  37. 37. Maelfait J, Vercammen E, Janssens S, Schotte P, Haegman M, Magez S, et al. Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8. J Exp Med. 2008;205(9):1967–73. pmid:18725521
  38. 38. VanOs R, Lildhar LL, Lehoux EA, Beaulé PE, Catelas I. In vitro macrophage response to nanometer-size chromium oxide particles. J Biomed Mater Res B Appl Biomater. 2014;102(1):149–59. pmid:23997019
  39. 39. Sadh S, Hajjar S, Ariana A, Phuong MS, Cai D, Thakker P, et al. Coating M-CSF on plastic surface results in the generation of increased numbers of macrophages in vitro. J Immunol Methods. 2020;481–482:112788. pmid:32304707
  40. 40. Aits S. Methods to detect loss of lysosomal membrane integrity. Methods Mol Biol. 2019;1880:315–29. pmid:30610707
  41. 41. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. 2025; Available from: http://www.R-project.org/
    • 42. Landgraeber S, Jäger M, Jacobs JJ, Hallab NJ. The pathology of orthopedic implant failure is mediated by innate immune system cytokines. Mediators Inflamm. 2014;2014:185150. pmid:24891761
    • 43. Caicedo MS, Pennekamp PH, McAllister K, Jacobs JJ, Hallab NJ. Soluble ions more than particulate cobalt-alloy implant debris induce monocyte costimulatory molecule expression and release of proinflammatory cytokines critical to metal-induced lymphocyte reactivity. J Biomed Mater Res A. 2010;93(4):1312–21. pmid:19844976
    • 44. Marim FM, Silveira TN, Lima DS Jr, Zamboni DS. A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells. PLoS One. 2010;5(12):e15263. pmid:21179419
    • 45. Assouvie A, Daley-Bauer LP, Rousselet G. Growing murine bone marrow-derived macrophages. Methods Mol Biol. 2018;1784:29–33. pmid:29761385
    • 46. Catelas I, Huk OL, Petit A, Zukor DJ, Marchand R, Yahia L. Flow cytometric analysis of macrophage response to ceramic and polyethylene particles: effects of size, concentration, and composition. J Biomed Mater Res. 1998;41(4):600–7. pmid:9697033
    • 47. Madl AK, Kovochich M, Liong M, Finley BL, Paustenbach DJ, Oberdörster G. Toxicology of wear particles of cobalt-chromium alloy metal-on-metal hip implants Part II: Importance of physicochemical properties and dose in animal and in vitro studies as a basis for risk assessment. Nanomedicine. 2015;11(5):1285–98. pmid:25735266
    • 48. Doorn PF, Campbell PA, Worrall J, Benya PD, McKellop HA, Amstutz HC. Metal wear particle characterization from metal on metal total hip replacements: transmission electron microscopy study of periprosthetic tissues and isolated particles. J Biomed Mater Res. 1998;42(1):103–11. pmid:9740012
    • 49. Catelas I, Campbell PA, Bobyn JD, Medley JB, Huk OL. Wear particles from metal-on-metal total hip replacements: effects of implant design and implantation time. Proc Inst Mech Eng H. 2006;220(2):195–208. pmid:16669387
    • 50. Catelas I, Bobyn JD, Medley JB, Krygier JJ, Zukor DJ, Huk OL. Size, shape, and composition of wear particles from metal-metal hip simulator testing: effects of alloy and number of loading cycles. J Biomed Mater Res A. 2003;67(1):312–27. pmid:14517891
    • 51. Hallab NJ, Jacobs JJ. Biologic effects of implant debris. Bull NYU Hosp Jt Dis. 2009;67(2):182–8. pmid:19583551
    • 52. Zindel J, Kubes P. DAMPs, PAMPs, and LAMPs in immunity and sterile inflammation. Annu Rev Pathol. 2020;15:493–518. pmid:31675482
    • 53. Horie M, Nishio K, Kato H, Endoh S, Fujita K, Nakamura A, et al. The expression of inflammatory cytokine and heme oxygenase-1 genes in THP-1 cells exposed to metal oxide nanoparticles. J Nano Res. 2015;30:116–27.
    • 54. Andujar P, Simon-Deckers A, Galateau-Sallé F, Fayard B, Beaune G, Clin B, et al. Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders. Part Fibre Toxicol. 2014;11:23. pmid:24885771
    • 55. Giampreti A, Lonati D, Ragghianti B, Ronchi A, Petrolini VM, Vecchio S, et al. N-acetyl-cysteine as effective and safe chelating agent in metal-on-metal hip-implanted patients: two cases. Case Rep Orthop. 2016;2016:8682737. pmid:27148463
    • 56. Martell AE. Chelates of ascorbic acid: formation and catalytic properties. In: Seib PA, Tolbert S, editors. Ascorbic acid: chemistry, metabolism, and uses. (ACS Symposium Series; vol. 200). Washington (DC): American Chemical Society; 1982. p. 153–78. https://doi.org/10.1021/ba-1982-0200.ch007
      • 57. Scharf B, Clement CC, Zolla V, Perino G, Yan B, Elci SG, et al. Molecular analysis of chromium and cobalt-related toxicity. Sci Rep. 2014;4:5729. pmid:25034144
      • 58. Lushchak OV, Kubrak OI, Lozinsky OV, Storey JM, Storey KB, Lushchak VI. Chromium(III) induces oxidative stress in goldfish liver and kidney. Aquat Toxicol. 2009;93(1):45–52. pmid:19395069
      • 59. Salloum Z, Lehoux EA, Harper M-E, Catelas I. Effects of cobalt and chromium ions on oxidative stress and energy metabolism in macrophages in vitro. J Orthop Res. 2018;36(12):3178–87. pmid:30144138
      • 60. Stuart JA, Fonseca J, Moradi F, Cunningham C, Seliman B, Worsfold CR, et al. How supraphysiological oxygen levels in standard cell culture affect oxygen-consuming reactions. Oxid Med Cell Longev. 2018;2018:8238459. pmid:30363917
      • 61. Reinheckel T, Tholen M. Low-level lysosomal membrane permeabilization for limited release and sublethal functions of cathepsin proteases in the cytosol and nucleus. FEBS Open Bio. 2022;12(4):694–707. pmid:35203107
      • 62. Katsnelson MA, Lozada-Soto KM, Russo HM, Miller BA, Dubyak GR. NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx. Am J Physiol Cell Physiol. 2016;311(1):C83–100. pmid:27170638
      • 63. Furuoka M, Ozaki K-I, Sadatomi D, Mamiya S, Yonezawa T, Tanimura S, et al. TNF-α induces caspase-1 activation independently of simultaneously induced NLRP3 in 3T3-L1 cells. J Cell Physiol. 2016;231(12):2761–7. pmid:26989816
      • 64. Chuang J-P, Kao C-Y, Lee J-C, Ling P, Maa M-C, Leu T-H. EPS8 regulates an NLRP3 inflammasome-independent caspase-1 activation pathway in monosodium urate crystal-treated RAW264.7 macrophages. Biochem Biophys Res Commun. 2020;530(3):487–93. pmid:32595041
      • 65. Feltham R, Vince JE, Lawlor KE. Caspase-8: not so silently deadly. Clin Transl Immunology. 2017;6(1):e124. pmid:28197335
      • 66. Antonopoulos C, Russo HM, El Sanadi C, Martin BN, Li X, Kaiser WJ, et al. Caspase-8 as an effector and regulator of NLRP3 inflammasome signaling. J Biol Chem. 2015;290(33):20167–84. pmid:26100631
      • 67. Petit A, Mwale F, Zukor DJ, Catelas I, Antoniou J, Huk OL. Effect of cobalt and chromium ions on bcl-2, bax, caspase-3, and caspase-8 expression in human U937 macrophages. Biomaterials. 2004;25(11):2013–8. pmid:14741615
      • 68. Liu G, Guo T, Zhang Y, Liu N, Chen J, Chen J, et al. Apoptotic pathways of macrophages within osteolytic interface membrane in periprosthestic osteolysis after total hip replacement. APMIS. 2017;125(6):565–78. pmid:28345781
      • 69. Petit A, Zukor DJ, Antoniou J, Ralston W, Huk OL. Expression of caspase-8 and caspase-3 proteins in interface membranes from aseptically loose total hip arthroplasty. J Mater Sci Mater Med. 2002;13(11):1001–5. pmid:15348168
      • 70. Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL, et al. Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med. 2018;215(3):827–40. pmid:29432122
      • 71. Chauhan D, Bartok E, Gaidt MM, Bock FJ, Herrmann J, Seeger JM, et al. BAX/BAK-induced apoptosis results in caspase-8-dependent IL-1β maturation in macrophages. Cell Rep. 2018;25(9):2354-2368.e5. pmid:30485805
      • 72. Vince JE, Wong WW-L, Gentle I, Lawlor KE, Allam R, O’Reilly L, et al. Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation. Immunity. 2012;36(2):215–27. pmid:22365665
      • 73. Yabal M, Müller N, Adler H, Knies N, Groß CJ, Damgaard RB, et al. XIAP restricts TNF- and RIP3-dependent cell death and inflammasome activation. Cell Rep. 2014;7(6):1796–808. pmid:24882010
      • 74. Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D’Cruz AA, et al. RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun. 2015;6:6282. pmid:25693118
      • 75. Gaidt MM, Ebert TS, Chauhan D, Schmidt T, Schmid-Burgk JL, Rapino F, et al. Human monocytes engage an alternative inflammasome pathway. Immunity. 2016;44(4):833–46. pmid:27037191
      • 76. Netea MG, Nold-Petry CA, Nold MF, Joosten LAB, Opitz B, van der Meer JHM, et al. Differential requirement for the activation of the inflammasome for processing and release of IL-1β in monocytes and macrophages. Blood. 2009;113(10):2324–35. pmid:19104081
      • 77. Nelson CL, McLaren AC, McLaren SG, Johnson JW, Smeltzer MS. Is aseptic loosening truly aseptic? Clin Orthop Relat Res. 2005;437:25–30.
      • 78. Pajarinen J, Jamsen E, Konttinen YT, Goodman SB. Innate immune reactions in septic and aseptic osteolysis around hip implants. J Long Term Eff Med Implants. 2014;24(4):283–96. pmid:25747031