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Ions released from metal implants have been associated with adverse tissue reactions and are therefore a major concern. Studies with macrophages have shown that cobalt, chromium, and nickel ions can activate the NLRP3 inflammasome, a multiprotein complex responsible for the activation of caspase-1 (a proteolytic enzyme converting pro-interleukin [IL]-1β to mature IL-1β). However, the mechanism(s) of inflammasome activation by metal ions remain largely unknown. The objectives of the present study were to determine if, in macrophages: 1. caspase-1 activation and IL-1β release induced by metal ions are oxidative stress-dependent; and 2. IL-1β release induced by metal ions is NF-κB signaling pathway-dependent. Lipopolysaccharide (LPS)-primed murine bone marrow-derived macrophages (BMDM) were exposed to Co2+ (6-48 ppm), Cr3+ (100-500 ppm), or Ni2+ (12-96 ppm), in the presence or absence of a caspase-1 inhibitor (Z-WEHD-FMK), an antioxidant (L-ascorbic acid [L-AA]), or an NF-κB inhibitor (JSH-23). Culture supernatants were analyzed for caspase-1 by western blotting and/or IL-1β release by ELISA. Immunoblotting revealed the presence of caspase-1 (p20 subunit) in supernatants of BMDM incubated with Cr3+, but not with Ni2+ or Co2+. When L-AA (2 mM) was present with Cr3+, the caspase-1 p20 subunit was undetectable and IL-1β release decreased down to the level of the negative control, thereby demonstrating that caspase-1 activation and IL-1β release induced by Cr3+ was oxidative stress-dependent. ELISA demonstrated that Cr3+ induced the highest release of IL-1β, while Co2+ had no or limited effects. In the presence of Ni2+, the addition of L-AA (2 mM) also decreased IL-1β release, below the level of the negative control, suggesting that IL-1β release induced by Ni2+ was also oxidative stress-dependent. Finally, when present during both priming with LPS and activation with Cr3+, JSH-23 blocked IL-1β release, demonstrating NF-κB involvement. Overall, this study showed that while both Cr3+ and Ni2+ may be inducing inflammasome activation, Cr3+ is likely a more potent activator, acting through oxidative stress and the NF-κB signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Iron acquisition by bacteria is well studied, but iron export from bacteria is less understood. Herein, we identified dr1440 with a P-type ATPase motif as a potential exporter of iron from Deinococcus radiodurans, a bacterium known for its extreme resistance to radiation and oxidants. The DR1440 was located in cell membrane as demonstrated by fluorescence labelling analysis. Mutation of dr1440 resulted in cellular accumulation of iron ions, and expression level of dr1440 was up-regulated significantly under iron ion or hydrogen peroxide stress in the wild-type strain, implicating DR1440 as a potential iron efflux protein. The dr1440 mutant displayed higher sensitivity to iron ions and oxidative stresses including hydrogen peroxide, hypochlorous acid, and gamma-ray irradiation compared with the wild-type strain. The high amount of iron in the mutant strain resulted in severe protein carbonylation, suggesting that DR1440 might contribute to intracellular protein protection against reactive oxygen species (ROS) generated from ferrous ion-mediated Fenton-reaction. Mutations of S297A and C299A led to intracellular accumulation of iron, indicating that S297 and C299 might be important functional residues of DR1440. Thus, DR1440 is a potential iron efflux protein involved in iron homeostasis and oxidative stress-resistance of D. radiodurans., Competing Interests: The authors have declared that no competing interests exist.