Fig 1.
Inflammasome deficient mice are resistant to developing DTH responses to implant metals as determined by paw inflammation to metal challenge.
(A) Schematic model for generating metal-DTH to implant metal(s) and particle-induced osteolysis in vivo over 21 days. Metal-DTH responses were determined by measuring paw thickness/inflammation 48 h post challenge on day 14 in both (B) male and (C) female C57BL/6 and Caspase-1-/- mice aged 12–16 weeks that were either non-sensitized (vehicle group) or metal-sensitized (DTH group). Data represent two independent experiments with n = 4–5 mice/group in each experiment. Paw inflammation is represented as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test and asterisk (*) denote significant differences at p < 0.05 compared to respective vehicle group.
Fig 2.
Female metal-sensitized (metal-DTH) BL/6 mice exhibit higher levels of lymphocyte proliferation reactivity.
(A) Schematic model demonstrating experimental design. Lymphocytes were purified from mouse spleens at Day 21 and co-cultured with or without Ni or Co challenge for four days to measure amount of lymphocyte proliferation responses via LTT from either (B) male or (C) female metal-DTH BL/6 and Caspase-1 -/- mice, and cell proliferation was measured by 3H-thymidine incorporation. (D) Comparison of lymphocyte metal sensitivity responses in male vs. female BL/6 mice, and (E) percentage increase of lymphocyte proliferation to metal challenge; calculated using cell proliferation to metal(s) vs. respective cell proliferation to media. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test and asterisk (**) denote significant differences at p < 0.005 compared to respective in vitro media challenged cell group.
Fig 3.
Female vs male BL/6 metal-DTH mice exhibit preferential IL-17A/F producing T-cells to implant metal-challenge.
(A) Schematic model demonstrating experimental design. At day 21, spleens were harvested. Lymphocytes were purified from mouse spleens and co-cultured with or without Ni or Co challenge for four days and supernatants were collected and analyzed for cytokine production. Lymphocytic (B) IFN-gamma and (C) IL-17A/F production was measured from metal-DTH treated male BL/6 and Caspase-1 -/-. Lymphocytic (D) IFN-gamma and (E) IL-17A/F production was also measured from metal-DTH treated female BL/6 and Caspase-1 -/- mice. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test and asterisk (**) denote significant differences at p < 0.005 and (*) p < 0.05 compared to respective in vitro media challenged cell group.
Fig 4.
Metal-sensitized caspase-1 knockout female (DTH: Female caspase-1 -/-) mice produce IFN-gamma dominated T-cell responses to implant metal-challenge.
Percentage increase of lymphocyte cytokine production of IFN-gamma or IL-17A/F in response to (A) Ni challenge and (B) Co challenge; calculated using cytokine production to metal(s) vs. respective negative control media cell cytokine production from female metal-DTH BL/6 and Caspase-1 -/- mice. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test.
Fig 5.
Female vs male BL/6 lymphocyte cytokine responses to CoCrMo-alloy particle challenge.
Percentage increase of lymphocyte cytokine production of IFN-gamma or IL-17A/F in response to CoCrMo-alloy particle challenge from either vehicle or metal-DTH (A) male BL/6 or (B) female BL/6 mice; calculated using cytokine production to CoCrMo-alloy particle vs. respective negative control media cell cytokine production. (C) Comparison of male vs. female metal-DTH lymphocytic cytokine responses to CoCrMo-alloy particle challenge. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test.
Fig 6.
A pre-existing condition of metal-DTH augments metal particle-induced osteolysis.
(A) Schematic model demonstrating experimental design where both vehicle and metal-DTH treated male and female C57BL/6 and Caspase-1 -/- mice received 2 mg/mouse calvaria of endotoxin-free CoCrMo-alloy particles. 7 days post-particle implantation (on day 21), calvaria were retrieved and analyzed by microCT. (B) Representative images and graphical representation of the percentage of particle-induced osteolysis from vehicle and metal-DTH treated (C) male and (D) female BL/6 and Caspase-1 -/- mice. (E) Comparison of percentage of bone loss from metal-DTH treated male vs. female BL/6 mice. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test.
Fig 7.
Age and sex effects lymphocyte metal-DTH responses.
Post metal-DTH induction, lymphocytes were purified from mouse spleens and co-cultured with or without Ni or Co challenge. Comparison of lymphocyte proliferation to Ni and Co from metal-DTH younger and aged: (A) males, (B) females. And comparison of LTT responses among (C) aged male vs. female metal-DTH treated BL/6 mice. Cell proliferation was measured by 3H-thymidine incorporation. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test and asterisk (**) denote significant differences at p < 0.005 and (*) p < 0.05 compared to respective in vitro media challenged cell group.
Fig 8.
IL-17 production is reduced in aged (vs young) metal-DTH female mice.
Lymphocytes were restimulated in vitro with soluble metal ions for 4 days and (A) IFN-gamma and (B) IL-17A/F production were measured from supernatants by ELISA. Percentage increase of lymphocyte cytokine production of IFN-gamma or IL-17A/F in response to (C) Ni or (C) Co metal ion challenge; calculated using cytokine production to metal ions vs. respective negative control media cell cytokine production. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test.
Fig 9.
Young female mice are more susceptible to experiencing metal-induced PIO.
Metal-DTH treated young and aged male and female BL/6 mice received 2 mg/mouse calvaria of endotoxin-free CoCrMo-alloy particles. 7 days post-particle implantation (on day 21), calvaria were retrieved and analyzed by microCT. (A) Representative images and (B) graphical representation of the percentage of particle-induced osteolysis. Data represents two independent experiments with n = 4–5 mice/group in each experiment. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Mann-Whitney test.