Metal-induced delayed type hypersensitivity responses potentiate particle induced osteolysis in a sex and age dependent manner.

It is widely recognized that innate macrophage immune reactions to implant debris are central to the inflammatory responses that drive biologic implant failure over the long term. Less common, adaptive lymphocyte immune reactions to implant debris, such as delayed type hypersensitivity (DTH), can also affect implant performance. It is unknown which key patient factors, if any, mediate these adaptive immune responses that potentiate particle/macrophage mediated osteolysis. The objective of this investigation was to determine to what degree known adaptive immune responses to metal implant debris can affect particle-induced osteolysis (PIO); and if this pathomechanism is dependent on: 1) innate immune danger signaling, i.e., NLRP3 inflammasome activity, 2) sex, and/or 3) age. We used an established murine calvaria model of PIO using male and female wild-type C57BL/6 vs. Caspase-1 deficient mice as well as young (12-16 weeks old) vs. aged (18-24 months old) female and male C57BL/6 mice. After induction of metal-DTH, and Cobalt-alloy particle (ASTM F-75, 0.4um median diameter) calvaria challenge, bone resorption was assessed using quantitative micro-computed tomography (micro-CT) analysis and immune responses were assessed by measuring paw inflammation, lymphocyte transformation test (LTT) reactivity and adaptive immune cytokines IFN-gamma and IL-17 (ELISA). Younger aged C57BL/6 female mice exhibited the highest rate and severity of metal sensitivity lymphocyte responses that also translated into higher PIO compared to any other experimental group. The absence of inflammasome/caspase-1 activity significantly suppressed DTH metal-reactivity and osteolysis in both male and female Caspase-1 deficient mice. These murine model results indicate that young female mice are more predisposed to metal-DTH augmented inflammatory responses to wear debris, which is highly influenced by active NLRP3 inflammasome/caspase-1 danger signaling. If these results are clinically meaningful for orthopedic patients, then younger female individuals should be appropriately assessed and followed for DTH derived peri-implant complications

Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation - Fig 6

<p>Hematoxylin and eosin staining of C57BL/6 mouse calvarial tissue and bone thickness 10 d post-op that either received (n = 1/5 represented per group): (A) sham-surgery (sterile PBS), showing no signs of inflammation or significant osteolysis with remaining bone thickness at 193.8 um or (B) 2 mg/mouse calvaria of endotoxin-free Cobalt-alloy particles, with inflammatory infiltrate into the calvarial bone identified by arrows and osteolysis with remaining bone thickness at 101.8 um.</p

An in vivo murine calvarial model of particle-induced osteolysis demonstrates Cobalt-alloy debris co-challenge with TLR4 induces osteolysis but not more than Cobalt alloy alone.

<p>C57BL/6 12 wk old male mice either received (n = 5 per group): (1) sham-surgery (sterile PBS), (2) 2 mg/mouse calvaria of endotoxin-free Cobalt-alloy particles, (3) 5 μg/mL LPS or (4) Cobalt-alloy/LPS+. 10 days later, calvaria were retrieved and analyzed by microCT. (A) Representative images (n = 1/5) and (B) Graphical representation of the percentage decrease in bone thickness relative to sham controls (average of n = 5 per group). (C) IL-1β and TNF-α cytokine production by isolated peritoneal male C57BL/6 macrophages after 20 h of <i>in vitro</i> challenge (performed in triplicate). Note: * <i>p<0</i>.<i>05</i>.</p

TLR4 LPS and Cobalt-alloy particles induce THP-1 macrophage secretion of IL-1β and TNF-α in a dose dependant manner.

<p>IL-1β and TNF-α cytokine production was assessed after THP-1 differentiated macrophages were challenged with (A-B) increasing concentrations of TLR4 agonist LPS and (C-D) increasing dose of Cobalt-alloy (particles to cells ratio) for 20 h and was quantified by ELISA. Cobalt induced significantly less IL-1β and TNF-α than TLR agonist LPS. Note: * indicates <i>p</i><0.05 respective to control and ** represents significance at p<0.01 respective to 0ng/mL.</p

Blocking TLR4 does not significantly decrease IL-1β and TNF-α response of human primary monocyte/macrophages to Cobalt-alloy particles.

<p>(A) IL-1β and (B) TNF-α secreted by human primary monocytes/macrophages (n = 5) challenged with Cobalt-alloy particles (particles:cell = 5:1), LPS or Alum (NLRP3 inflammasome activator) for 20 h, with or without ZVAD-FMK (caspase-1 inhibitor) or PAb TLR4 antibody and was quantified by ELISA. Cytokine levels with use of PAb TLR4 are represented as percent increase as compared to respective control cells and averaged as a group. Note: * <i>p<0</i>.<i>05</i> to each treatment groups respective control values.</p

Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation - Fig 9

<p><b>A general schematic showing the effects of Cobalt alloy particulate on macrophages acting directly and indirectly on three major pro-inflammatory innate immune pathways</b>: 1) general toxins (such as inducing hypoxia like cell responses), 2) as danger associated molecular patterns, DAMPs (inflammasome induced activation), and 3) interacting with the pathogen associated molecular pattern (PAMP) pathway of TLR4.</p

Blocking inflammasome danger signaling was more effective than attempts to block TLR4 in both THP-1 human monocytes and differentiated macrophages challenged with Cobalt-alloy particles.

<p>IL-1β and TNF-α was assessed after (A-B) THP-1 differentiated macrophages and (C-D) THP-1 monocytes were challenged with Cobalt-alloy particles (particles:cell = 5:1), Cobalt-alloy/LPS+, and LPS in the presence of either PAb isotype matched control antibody (4μg/mL), PAb-hTLR4 (4μg/mL), or Cathepsin-B inhibitor (10uM) for 20 hours. Cathepsin B danger signal (inflammasome) blocking for Cobalt alloy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160141#pone.0160141.ref006" target="_blank">6</a>] significantly decreased IL-1β responses to all challenges, more than TLR blocking in both macrophages (A-B) and monocytes (C-D). Note: */** p<0.05 to each treatment groups respective control values</p

TLR4 agonist LPS and Cobalt-alloy particles in THP-1 Macrophages induce significantly elevated secretion of IL-1β, TNF-α and IL-10, while only Cobalt particles significantly increase IL-1β without concomitant IL-10 increase.

<p>(A) IL-1β (B) TNF-α <b>(C)</b> and IL-10 cytokine production was assessed after THP-1 differentiated macrophages were challenged with Cobalt-alloy particles (particles:cell = 5:1), Cobalt-alloy/LPS+, and LPS (100ng/mL) for 20 h and was quantified by ELISA. <b>(D)</b> THP-1 macrophages were challenged with Co-alloy particles and incubated with 15μg/ml DQ ovalbumin simultaneously for 4 hrs and subsequently were fixed and evaluated for the presence of large pools of dispersed DQ ovalbumin fluorescence co-localized with particles, indicative of lysosomal destabilization. Note: * p<0.05 compared to control macrophages.</p