Fig 1.
TLR4 LPS and Cobalt-alloy particles induce THP-1 macrophage secretion of IL-1β and TNF-α in a dose dependant manner.
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 p<0.05 respective to control and ** represents significance at p<0.01 respective to 0ng/mL.
Fig 2.
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.
(A) IL-1β (B) TNF-α (C) 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. (D) 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.
Fig 3.
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.
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 [6] 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
Fig 4.
Biological responses to Cobalt-alloy particles in human primary differentiated macrophages induce significantly elevated secretion of both pro and anti-inflammatory cytokines, only when combined with known TLR4 agonist LPS.
(A) IL-1β (B) TNF-α (C) IL-6 and (D) IL-10 cytokine production was assessed after human primary differentiated macrophages (n = 4) were challenged for 20 h with Cobalt-alloy particles (particles:cell = 5:1), Cobalt-alloy/LPS+, and LPS. Note: ** p<0.05 to each treatment groups respective control values.
Fig 5.
Blocking TLR4 does not significantly decrease IL-1β and TNF-α response of human primary monocyte/macrophages to Cobalt-alloy particles.
(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: * p<0.05 to each treatment groups respective control values.
Fig 6.
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.
Fig 7.
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), (B) 2 mg/mouse calvaria of endotoxin-free Cobalt-alloy particles, (C) 5 μg/mL LPS or (D) Cobalt-alloy/LPS+. Measurements represent remaining bone thickness.
Fig 8.
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.
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 in vitro challenge (performed in triplicate). Note: * p<0.05.
Fig 9.
A general schematic showing the effects of Cobalt alloy particulate on macrophages acting directly and indirectly on three major pro-inflammatory innate immune pathways: 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.