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Protective Effect of Inflammasome Activation by Hydrogen Peroxide in a Mouse Model of Septic Shock

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Protective Effect of Inflammasome Activation by Hydrogen Peroxide in a Mouse Model of Septic Shock Protective Effect of Inflammasome Activation by Hydrogen Peroxide in a Mouse Model of Septic Shock Olivier Huet, MD, PhD1,2,3; Raelene J. Pickering, PhD1; Chris Tikellis, PhD1; Celine Latouche, PhD1; Fenella Long, PhD1; Bronwyn Kingwell, PhD1; Bryan Dickinson, PhD5; Chris J. Chang, PhD5; Seth Masters, PhD4; Fabienne Mackay, PhD3; Mark E. Cooper, MD, PhD1; Judy B. de Haan, PhD1 Objectives: To study the effect of a lack of antioxidant defenses Klebsiella pneumonia, compared to ­wild-type mice. Furthermore, this during lethal pneumonia induced by Klebsiella pneumonia, com- protective effect was suppressed when antioxidant defenses were pared to wild-type mice. restored. Infected glutathione peroxidase 1 mice showed an early Setting: Laboratory experiments. and significant, albeit transient, increase in the activity of the NOD-like Subjects: C57Bl6 and glutathione peroxidase 1 knockout mice. receptor family, pyrin domain containing 3 inflammasome when com- Intervention: Murine acute pneumonia model induced by Klebsi- pared with wild-type mice. The key role of the NOD-like receptor fam- ella pneumonia. ily, pyrin domain containing 3 inflammasome during acute pneumonia Measurements and Main Results: We show here that despite a lack of was confirmed in vivo when the protective effect was suppressed by one of the major antioxidant defense enzymes, glutathione peroxidase treating glutathione peroxidase 1 mice with an interleukin-1 recep- 1 knockout mice are protected during lethal pneumonia induced by tor antagonist. Additionally we report, in vitro, that increased con- centrations of active caspase-1 and interleukin-1β are related to an 1Diabetes Complications, BakerIDI Heart and Diabetes Institute, Mel- increased concentration of hydrogen peroxide in bacterially infected bourne, VIC, Australia. glutathione peroxidase 1 macrophages and that restoring hydrogen 2 Department of Anaesthesia and Intensive Care, CHRU La Cavale peroxide antioxidant defenses suppressed this effect. Blanche, Université de Bretagne Ouest, Brest, France. Conclusions: Our findings demonstrate that, contrary to current 3Australian and New Zealand Intensive Care Research Centre, School of Public Health and Preventive Medicine, Monash University, Melbourne, thinking, an early intervention targeting NOD-like receptor family, VIC, Australia. pyrin domain containing 3 inflammasome activity induces a timely 4Inflammation Division, The Walter and Eliza Hall Institute, Parkville, VIC, and efficient activation of the innate immune response during Australia. acute infection. Our findings also demonstrate a role for hydrogen 5 Departments of Chemistry and Molecular and Cell Biology and the How- peroxide in the mechanisms tightly regulating NOD-like receptor ard Hughes Medical Institute, University of California, Berkeley, CA. family, pyrin domain containing 3 activation. (Crit Care Med 2017; Drs. Huet and de Haan designed experiments, critically evaluated the results, and wrote the article. Drs. Huet, Pickering, Tikellis, Latouche, 45:e184–e194) and Long performed experiments. Drs. Dickinson and Chang designed Key Words: antioxidant defense; caspase-1; hydrogen peroxide; and synthesized the PF6-AM fluorescent probe. Drs. Kingwell, Masters, glutathione peroxidase; inflammasome; innate immune response; Mackay, and Cooper were involved in study design and article editing. interleukin-1β; sepsis; septic shock Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ccmjournal). Supported in part by the Victorian Government’s Operational Infrastruc- ture Support Program. recent worldwide survey reported that 29.5% of Dr. Huet was supported in part by an SFAR fellowship. Dr. Tikellis received patients admitted to ICU had sepsis at admission or support for this article research from the National Health and Medical during their ICU stay, with a mortality rate among Research, Canberra, Australia. Dr. Kingwell’s institution received funding A from the Victorian state government. Dr. Chang received support for this those affected of 25.8% (1). article research from the National Institutes of Health and Howard Hughes A prolonged and increased nonspecific systemic inflam- Medical Institute. The remaining authors have disclosed that they do not have any potential conflicts of interest. matory response (2) and enhanced oxidative stress (3) have For information regarding this article, E-mail: [email protected] been considered to be contributors to severe sepsis. This ratio- Copyright © 2017 by the Society of Critical Care Medicine and Wolters nale has been used to justify several large randomized clini- Kluwer Health, Inc. All Rights Reserved. cal trials intended to blunt the inflammatory response, none DOI: 10.1097/CCM.0000000000002070 of which have succeeded (4). These failures highlight our e184 www.ccmjournal.org February 2017 • Volume 45 • Number 2 Copyright © 2016 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved. Copyright © 2016 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved. Online Laboratory Investigations limited knowledge of the mechanisms leading to severe sepsis, Animal Experiments and current studies suggest that a more complex coexistence Experiments were approved by the Alfred Medical Research of pro- and anti-inflammatory responses is involved, with a and Education Precinct Animal Ethics Committee, Mel- specific temporal relationship (5–7). Recent clinical reports bourne, VIC, Australia. The mouse model of acute pneumo- focusing on the early stages of infection stress the critical need nia has been validated and published previously (18). Equal for a strong and transient proinflammatory response to gen- numbers of male and female mice were used (n = 17 to 20 for erate a better clinical outcome during acute infections (8, 9). survival experiments and 6 to 10 for biochemistry and pathol- Furthermore, it has been shown that the ratio of pro- to anti- ogy experiments). Mice were 10–12 weeks old. inflammatory factors changes during disease progression, and this ratio seems to be key to the avoidance of septic shock (9). Tissue Collection 3 The NOD-like receptor family, pyrin domain containing 3 Klebsiella pneumoniae (5 × 10 colony-formatting units)– (NLRP3) inflammasome controls the activation of two proin- infected mice were euthanized on days 1 and 2 post infection. flammatory cytokines: interleukin (IL)-1β and IL-18. IL-1β, a Lungs and plasma were collected. Lungs were processed for key proinflammatory cytokine involved in the innate immune immunohistochemical analysis or stored at −80°C for RNA response, is synthesized as an inactive precursor that becomes and protein extraction. activated after cleavage by caspase-1. When activated, both caspase-1 and IL-1β are secreted out of the cell, which is a Quantitative Real-Time Polymerase Chain Reaction hallmark feature of the activated inflammasome. The NLRP3 Adhesion molecules, proinflammatory cytokines, and oxida- tive stress markers were assessed in lung homogenates as previ- inflammasome plays a key role during infection, as recently ously described (19). shown for a bacterial model of septic shock where its inactiva- tion induced higher mortality (10, 11). Lung Tissue and Circulating Inflammatory Markers Cellular mechanisms controlling the activation of the IL-6, IL-1β, IL-10, and interferon-γ (IFN-γ) were measured in inflammasome are not fully understood (12). Recent evidence lung homogenates and plasma by using commercially available suggests a role for reactive oxygen species (ROS), but this enzyme-linked immunosorbent assay (ELISA) kits (R&D sys- remains to be fully delineated. It has been hypothesized that tems, Minneapolis, MN) as per manufacturer’s instructions. the NLRP3 inflammasome is a general sensor of cellular stress through its activation by ROS that have been generated in the Western Blots proximity of the inflammasome (13–16). However, the specific Caspase-1 and IL-1β protein were measured by Western blots. ROS and the mechanism by which the NLRP3 inflammasome Anti-mouse IL-1β primary antibody (number, 8689; Cell Sig- senses ROS are currently unclear and remain controversial. naling Technology, Danvers, MA) or an anti-mouse caspase-1 Using an endotoxemic murine model, it has been reported primary antibody (number, sc-1218R; SantaCruz Biotechnol- that the absence of the cytosolic Cu/Zn isoform, superoxide ogy, Dallas, TX) and α-tubulin (number, 2144; Cell Signaling dismutase (SOD)1, is followed by inactivation of the NLRP3 Technology) were used. ECL Advance Western Blotting detec- inflammasome in vitro and in vivo (16). The absence of SOD1 tion and quantitation by densitometry revealed protein levels. .− activity significantly increases the concentration of O2 by sup- Data are expressed relative to α-tubulin. pressing its dismutation into hydrogen peroxide (H2O2). Indeed, .− oxidation of key inflammasome proteins mediated by O2 was Histology shown to prevent inflammasome activation (16). SOD1 was, Formalin-fixed, paraffin-embedded 4-µm-thick lung sections therefore, hypothesized to play a key role in the activation of were stained with hematoxylin and eosin and analyzed for inflam- the NLRP3 inflammasome. However, a role for H2O2 cannot be matory infiltrate (assigned a score of 0–5) according to Yatmaz ruled out given its rapid formation due to dismutation
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