Cutting Edge: Reactive Oxygen Species Inhibitors Block Priming, but Not Activation, of the NLRP3 Inflammasome

Unit for Clinical Biochemistry, Institute for Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, 53127 Bonn, Germany.
The Journal of Immunology (Impact Factor: 4.92). 06/2011; 187(2):613-7. DOI: 10.4049/jimmunol.1100613
Source: PubMed

ABSTRACT A common denominator among the multiple damage-inducing agents that ultimately lead to activation of NLRP3 has not yet been identified. Recently, production of reactive oxygen species (ROS) has been suggested to act as a common event upstream of the NLRP3 inflammasome machinery. Because de novo translation of NLRP3 is an essential step in the activation of NLRP3, we investigated the role of substances that inhibit either ROS production or its oxidative activity. Although we observe that NLRP3 inflammasome activation is unique among other known inflammasomes in its sensitivity to ROS inhibition, we have found that this phenomenon is attributable to the fact that NLRP3 strictly requires priming by a proinflammatory signal, a step that is blocked by ROS inhibitors. Although these data do not exclude a general role for ROS production in the process of NLRP3-triggered inflammation, they would put ROS upstream of NLRP3 induction, but not activation.

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Available from: Eva Bartok, Sep 27, 2015
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    • "In line with these findings, we observed that NAC suppressed IL-1b secretion by M. bovis-stimulated THP-1 macrophages. Zhou et al. found that mitochondrial ROS generation is essential for NLRP3 inflammasome activation (Zhou et al. 2011), and Bauernfeind et al. demonstrated that ROS play a part in the priming step, but not activation of the NLRP3 inflammasome (Bauernfeind et al. 2011). However, NAC did not affect priming, the protein levels of Fig. 3 PR-619 decreases IL-1b secretion by M. bovisinfected THP-1 macrophages. "
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    • "Studies have measured an increase in cytoplasmic ROS upon silica treatment and attributed this to NOX2 activation. The interpretation of this measurement is difficult because cells were preactivated by either phorbol myristate acetate or lipopolysaccharide before exposure to silica, which results in generalized and nonphagosomal NOX activation and an increase in cellular ROS even before exposure to silica (Kahlenberg et al., 2005; Li et al., 2009; Bauernfeind et al., 2011; Brass et al., 2012). Our experiments were therefore performed without preactivating macrophages. "
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    ABSTRACT: Chronic inhalation of silica particles causes lung fibrosis and silicosis. Silica taken up by alveolar macrophages causes phagolysosomal membrane damage and leakage of lysosomal material into the cytoplasm to initiate apoptosis. We have investigated the role of reactive oxygen species (ROS) implicated in causing this membrane damage by studying the spatio-temporal generation of ROS. In macrophages, ROS generated by NADPH oxidase 2 (NOX2) was detected in phagolysosomes containing either silica particles or non-toxic latex particles. ROS was only detected in the cytoplasm of cells treated with silica and appeared in parallel with an increase in phagosomal ROS as well as several hours later associated with mitochondrial production of ROS late in apoptosis. Pharmacological inhibition of NOX activity did not prevent silica induced phagolysosomal leakage, but delayed it. In Cos7 cells that do not express NOX2, ROS was detected in silica containing phagolysosomes that leaked. ROS was not detected in phagolysosomes containing latex particles. Leakage of silica containing phagolysosomes in both cell types was transient and after resealing of the membrane, endolysosomal fusion continued. These results demonstrate that silica particles can generate phagosomal ROS independent of NOX activity and we propose that this silica generated ROS can cause phagolysosomal leakage to initiate apoptosis. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 09/2015; 26(18). DOI:10.1091/mbc.E15-03-0126 · 4.47 Impact Factor
    • "However, controversial results were obtained in studies using cells deficient in NADPH protein or cells from patients with defective NADPH oxidase subunits (van de Veerdonk et al., 2010; Meissner et al., 2010; van Bruggen et al., 2010). A previous study using inhibitors of reactive oxygen species implicated the involvement of reactive oxygen species in NLRP3 priming rather than in inflammasome activation (Bauernfeind et al., 2011). Ziram-induced decrease in pro-caspase-1 was inhibited by an oxygen radical scavenger, N-acetylcysteine but not by other oxygen radical scavengers, pyrrolidine dithiocarbamate and ascorbate (data not shown). "
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    ABSTRACT: The NLRP3 inflammasome, composed of caspase-1, NLRP3 and ASC, plays a critical role in the clearance of microbial pathogens. Here, we found that the treatment of mouse macrophages with the zinc-containing dithiocarbamate ziram, a widely used fungicide in agriculture, caused a decrease in pro-caspase-1 and NLRP3 levels while not affecting ASC level. Ziram did not affect levels of pro-caspase-1 and NLRP3 mRNA, and no cleavage products of pro-caspase-1 including p10 subunit, which is an autocleavage product of pro-caspase-1, were detected, indicating that the decrease was associated with degradation of these proteins. The decrease was inhibited by SH-type antioxidants, N-acetyl cysteine, dithiothreitol and 2-mercaptoethanol, or a metal chelator EDTA but not by inhibitors of proteasome, lysosomes, autophagy and matrix metalloproteases. Thiram, a comparator for ziram that does not contain zinc, showed a weaker decrease in protein levels. Furthermore, the zinc-containing dithiocarbamate, zinc diethyldithiocarbamate, efficiently decreased the levels of pro-caspase-1 and NLRP3, whereas dithiocarbamates, dimethyldithiocarbamate and diethyldithiocarbamate without zinc, were less active. The organic zinc compound [3,4-Toluenedithiolato(2-)]zinc hydrate did not induce a decrease in protein levels. Ziram also inhibited IL-1β production by macrophages in response to lipopolysaccharide and bacterial clearance during Salmonella infection of macrophage cells. These results indicate that ziram causes degradation of pro-caspase-1 and NLRP3 in a zinc- and oxidative property-dependent manner and suggest that exposure to ziram may compromise the clearance of microbial pathogens. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Toxicology Letters 04/2015; 235(3). DOI:10.1016/j.toxlet.2015.04.012 · 3.26 Impact Factor
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