TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis
2012; Springer Nature; Volume: 32; Issue: 1 Linguagem: Inglês
10.1038/emboj.2012.328
ISSN1460-2075
AutoresSanna Nyström, Daniel J. Antoine, Peter Lundbäck, John G. Lock, Andreia Nita, Kari Högstrand, Alf Grandien, Helena Erlandsson-Harris, Jan Andersson, Steven E. Applequist,
Tópico(s)Gout, Hyperuricemia, Uric Acid
ResumoArticle7 December 2012free access TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis Sanna Nyström Sanna Nyström Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Daniel J Antoine Daniel J Antoine Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, UKThese authors equally contributed to this work Search for more papers by this author Peter Lundbäck Peter Lundbäck Rheumatology Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, SwedenThese authors equally contributed to this work Search for more papers by this author John G Lock John G Lock Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Stockholm, Sweden Search for more papers by this author Andreia F Nita Andreia F Nita Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 'Carol Davila' University of Medicine and Pharmacy, Bucharest, Romania Search for more papers by this author Kari Högstrand Kari Högstrand Department of Medicine, Center for Experimental Hematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Alf Grandien Alf Grandien Department of Medicine, Center for Experimental Hematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Helena Erlandsson-Harris Helena Erlandsson-Harris Rheumatology Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Ulf Andersson Ulf Andersson Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Steven E Applequist Corresponding Author Steven E Applequist Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Sanna Nyström Sanna Nyström Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Daniel J Antoine Daniel J Antoine Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, UKThese authors equally contributed to this work Search for more papers by this author Peter Lundbäck Peter Lundbäck Rheumatology Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, SwedenThese authors equally contributed to this work Search for more papers by this author John G Lock John G Lock Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Stockholm, Sweden Search for more papers by this author Andreia F Nita Andreia F Nita Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 'Carol Davila' University of Medicine and Pharmacy, Bucharest, Romania Search for more papers by this author Kari Högstrand Kari Högstrand Department of Medicine, Center for Experimental Hematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Alf Grandien Alf Grandien Department of Medicine, Center for Experimental Hematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Helena Erlandsson-Harris Helena Erlandsson-Harris Rheumatology Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Ulf Andersson Ulf Andersson Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Steven E Applequist Corresponding Author Steven E Applequist Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden Search for more papers by this author Author Information Sanna Nyström1, Daniel J Antoine2, Peter Lundbäck3, John G Lock4, Andreia F Nita1,5, Kari Högstrand6, Alf Grandien6, Helena Erlandsson-Harris3, Ulf Andersson7 and Steven E Applequist 1 1Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 2Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, UK 3Rheumatology Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden 4Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Stockholm, Sweden 5'Carol Davila' University of Medicine and Pharmacy, Bucharest, Romania 6Department of Medicine, Center for Experimental Hematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 7Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden *Corresponding author. Department of Medicine, Center for Infectious Medicine, F59, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. Tel.:+46 8 585 89688 or +46 73 960 3909; Fax:+46 8 746 76 37; E-mail: [email protected] The EMBO Journal (2013)32:86-99https://doi.org/10.1038/emboj.2012.328 Correction(s) for this article TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis09 January 2013 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Infection of macrophages by bacterial pathogens can trigger Toll-like receptor (TLR) activation as well as Nod-like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase-1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming-free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage-associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re-introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4-agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system. Introduction Activation of innate immune receptors in cells of monocytic lineage promotes anti-microbial defenses. Macrophages in particular are suited to recognize the microbe-associated molecular pattern molecules through germ-line expression of pattern recognition receptors (PRRs). Two primary families of PRRs are the Toll-like receptors (TLRs) and Nod-like receptors (NLRs), which are key in activating innate immune responses to protect the host from infection. TLRs and NLRs are thought to activate distinct signalling pathways which promote diverse outcomes defending against infection. Activation of TLRs triggers the production of anti-microbial effector molecules nitrous oxide (NO) and mitochondria-dependent reactive oxygen species (mROS) (Kawai and Akira, 2011; West et al, 2011). TLRs also activate the transcription factors NF-κB and IRF3 to induce inflammatory cytokines, the inactive pro-forms of inflammatory cytokines such as IL-1β, and type I interferons and chemokines (Kawai et al, 2001; Kawai and Akira, 2011). Despite this arsenal of anti-microbial effectors, it is often observed that bacterial pathogens activate macrophage cell death (Ashida et al, 2011). Bacterial virulence factors can trigger cell death upon infection and some of these have now been found to be NLR agonists. For example, the mouse NLR Naip5 directly interacts with bacterial flagellin and forms a complex with NLRC4 (Kofoed and Vance, 2011; Zhao et al, 2011). This detection initiates the formation of a multi-protein complex called an inflammasome able to promote caspase-1 and caspase-11 catalytic activity (Schroder and Tschopp, 2010; Akhter et al, 2012). Flagellin activates the NLRC4 inflammasome by the canonical pathway leading to regulated cell death called pyroptosis (defined by a requirement for an 'activated' but unprocessed procaspase-1) independently of caspase-11 (Broz et al, 2010; Kayagaki et al, 2011; Motani et al, 2011; Galluzzi et al, 2012). NLRC4 inflammasome activation also induces lysosome exocytosis, phagosome-lysozome fusion, as well as the processing of the pro-form of the inflammatory cytokines IL-1β and IL-18 by a catalytic caspase-1 (Broz et al, 2010; Bergsbaken et al, 2011; Motani et al, 2011; Akhter et al, 2012). In vivo NLRC4 inflammasome activation, and in some cases pyroptosis, leads to anti-microbial activity (Miao et al, 2011) as well as sepsis associated with multi-antibiotic-resistant commensal pathogens in a damaged gut (Ayres et al, 2012). Confounding the uninfluenced study of inflammasome activation and associated cell death is the presence of 'signal-1' or priming. The NLRC4 inflammasome, assisted by Naip5, is activated by the carboxy-terminal tail of flagellin produced by a variety of enteric pathogens (Lightfield et al, 2008, 2011). However, infection of macrophages with flagellated bacteria activates multiple innate immune receptors as well as allows for modulation of NF-κB, inflammasome, and mitochondrial activity via microbe-derived factors (Ashida et al, 2011). This makes the use of infection models unsuited for the study of uninfluenced NLRC4/Naip5 activation. Transduction of macrophages with retroviral vectors expressing flagellin or flagellin COOH tails activates NLRC4/Naip5-dependent pyroptosis (Lightfield et al, 2008; Miao et al, 2010b; Lightfield et al, 2011; Ayres et al, 2012). However, these techniques deliver viral dsRNA activating the viral infection sensor Tank Binding Kinase 1, a key signalling adaptor downstream of RIG-I-like receptors (Sutlu et al, 2012). Transfection of recombinant flagellin protein using cationic lipids activates the primed NLRC4/Naip5 inflammasome and IL-1β processing (Miao et al, 2006; Buzzo et al, 2010; Miao et al, 2010b; Bauernfeind et al, 2011; Lightfield et al, 2011; Zhao et al, 2011) and release of IL-18 without overt priming (Bauernfeind et al, 2011). However, cationic lipids induce autophagy (Man et al, 2010) that affects pro-IL-1β levels, processing, and release (Dupont et al, 2011; Harris et al, 2011). Direct interactions observed between the central autophagy regulator Beclin1 and NLRC4 also suggest that autophagy influences NLRC4/Naip5 responses (Jounai et al, 2011). For these reasons, it is not known if pyroptosis is dependent on, or is affected by, priming and/or activation of other signalling pathways. Furthermore, it is also unknown if NLRC4/Naip5 inflammasome activation leads to the de novo production of NO, ROS, or inflammatory cytokines. Pyroptosis is associated with anti-microbial activity (Miao et al, 2010a), pathology (Kovarova et al, 2012), and coincides with the release of damage-associated molecular patterns (DAMPs). The DAMP high mobility group protein box 1 (HMGB1) is a highly conserved nuclear protein capable of promoting both sterile and infectious inflammation by numerous mechanisms after release from activated or dying cells (Andersson and Tracey, 2011). HMGB1 contains three cysteine residues (Cys23, Cys45, and Cys106) and the degree of reduction and oxidation (redox), and/or sulphonation of these residues directly affects its structure-to-function relationship. These well-defined, mutually exclusive forms exhibit either chemotactic activity with Cxcl12 (completely reduced 'all-thiol'), TLR4 agonist activity (oxidized at Cys23-Cys46, reduced Cys106), or an apparently 'inactive' form (sulphonated at Cys106) (Yang et al, 2010, 2012; Schiraldi et al, 2012; Venereau et al, 2012). These structural isoforms have a direct relationship to in vivo inflammation where the all thiol form exhibits chemotactic activity and the in vivo oxidation of injected HMGB1 reduces this activity (Venereau et al, 2012). However, it is not known if changes in HMGB1 cysteine redox isoforms are only passive processes in response to environmental oxidation or if specific cellular pathways can effect these changes. The role of cysteine redox isoforms in other functions attributed to HMGB1 is unknown. Various HMGB1 cysteine redox isoforms have been studied during apoptotic events associated with irradiation (Kazama et al, 2008). Apoptosis generally involves mitochondrial dysfunction and manipulation of mitochondrial ROS production affects HMGB1 cysteine isoforms (Kazama et al, 2008). However, compared to apoptosis, it is not known if pyroptosis involves the mitochondria or ROS production. Priming inflammasomes with LPS induces mitochondrial ROS but it is unknown if these non-lethal changes affect changes in HMGB1 isoforms or if fulminant cell death could suffice. The release of inflammatory molecules during cell death is likely an important factor in pathology associated with inflammasome activation. Priming, however, complicates our understanding of what responses are specific to inflammasome activation or to the priming event. Understanding these dynamics will help clarify the roles these factors play in the outcomes of cell death and pyroptotic-associated pathology. Here, we develop a priming-free inflammasome activation system to address questions regarding the necessity and role of priming in inflammasome activation, pyroptosis, and DAMP production. Results NLRC4/Naip5 agonist expression induces rapid cell death without priming NLRC4 gene expression is independent of priming (Bauernfeind et al, 2011). To test if NLRC4/Naip5 inflammasome activation is independent of priming, we developed a single-construct retroviral Tet-ON vector to express the C-terminal 34 or 19 amino acids of S. typhimurium flagellin (FliC) fused to Enhanced GFP (EGFP) in macrophages (Figure 1A). Constructs expressing this NLRC4/Naip5 agonist (EGFP-C34) and control (EGFP-C19) were stably transduced into immortalized mouse BMDMs (B10R) expressing macrophage surface markers (Supplementary Figure 1). B10R and inducible-retroviral transductants (called C34 or C19) were tested for the expression of transcripts of NLRC4/Naip5 inflammasome members, associated molecules, and NF-κB and IRF responsive genes. B10R, C34, and C19 cells expressed NLRC4, Naip5, caspase-1, PKR, and ASC, but not the NF-κB-responsive gene IL-1β or the IRF-responsive gene Cxcl10 demonstrating a lack of active TLR and anti-viral signalling (Figure 1B). C34 and C19 cells were then treated with Doxacyclin (Dox) and monitored for inducible gene expression (EGFP signal) as well as cell viability. We observed a rapid increase in fluorescence in C34 and C19 cells, however, only C34 cells underwent cell death (Figure 1C and D). Cell death, as determined by propidium iodide (PI) staining, occurred concurrently with LDH release indicating that PI staining was not likely a function of pore formation in viable cells but stained truly non-viable cells (Figure 1D and E). Figure 1.Macrophage pyroptosis by NLRC4/Naip5 agonists expression is priming independent and is largely unaffected by priming. (A) Self-inactivating retroviral vector driving expression of the EGFP-C34 or EGFP-C19 fusions from the tetracycline-regulated promoter (tetR) by the reverse tetracycline-controlled transactivator rtTA2S-M2 (rtTA) by Dox. See Supplementary data for a detailed description of domains. (B) Expression of gene transcripts by B10R, C34, and C19 cells key to NLRC4/Naip5 inflammasome activation and representative of NF-κB, and IRF activation. Transcript expression after 3 h LPS treatment (100 ng/ml) is shown. (C, D) Kinetic analysis of induced C34 and C19 EGFP fusion proteins (C) and cell death (D) determined by flow cytometry±LPS priming (100 ng/ml, 3 h) followed by±Dox (500 ng/ml). (E) Kinetic analysis of cell death in C34 and C19 cells determined by LDH release±LPS priming (100 ng/ml, 3 h) followed by±Dox (500 ng/ml). (F) Determination of cell death by flow cytometry in C34, C34/CrmA, C34/cFLIPS, or C34/cFLIPL cells±Dox (500 ng/ml) for 2 h. (G) Kinetic analysis of IL-1β detection in the cell supernatant as detected by ELISA±LPS priming (100 ng/ml, 3 h) followed by±Dox addition (500 ng/ml) (P-value=0.0023). (H) IL-1β processing in supernatant determined by immunoblot in C34 cells±LPS priming (100 ng/ml, 3 h)±Dox (500 ng/ml) 8 h post Dox addition. (I–K) Induction of C34 and C19 EGFP fusion proteins (I) cell death (J) and TNFα production following±3 or 18 h LPS priming (100 ng/ml) followed by±Dox (0–500 ng/ml) determined by flow cytometry and ELISA 24 h post Dox addition. Data are mean±s.e.m. of triplicate samples representative of two (C, D, E, G), three (I, J), or four (F) independent experiments. (H) Representative of four independent experiments is shown. Significant differences are indicated *P⩽0.05, ***P⩽0.001. Download figure Download PowerPoint Inflammasome activity and pyroptosis is dependent on caspase-1 (Schroder and Tschopp, 2010). To determine if cell death was dependent on caspase-1, we co-expressed the serpin family caspase-1 inhibitor CrmA in C34 cells. Upon Dox addition, we observed decreased cell death (Figure 1F) but no changes in EGFP expression. CrmA has also been shown to inhibit caspase-8 activity (Smith et al, 1996; Oberst et al, 2011). However, caspase-8 can be specifically inhibited by FLIP-short (cFLIPS) and FLIP-long (cFLIPL) (Weinlich et al, 2011). To determine if cell death involved caspase-8, we overexpressed cFLIPS or cFLIPL in C34 cells. Overexpression of either form of cFLIP was unable to inhibit cell death after Dox addition (Figure 1F) and did not affect EGFP expression despite robust FLIP overexpression (Supplementary Figure 3). Together, these results demonstrate that the presence of a defined inflammasome agonist promotes pyroptotic cell death in the absence of priming and manipulation of signalling pathways. Priming allows IL-1β production but does not affect pyroptosis after NLRC4/Naip5 agonist expression Our results indicate that expression of an NLRC4/Naip5 agonist alone induces pyroptosis. However, regulated cell death pathways can be influenced by signalling pathways such as NF-κB supplied by priming (e.g., LPS; Ashida et al, 2011). As inflammasome activation routinely occurs in the presence of priming we assessed the ability of priming to affect cell death. Treatment of B10R, C34, or C19 cells with LPS lead to no discernable changes in NLRC4, Naip5, PKR, or Caspase-1 expression but did upregulate IL-1β and Cxcl10 transcripts demonstrating functional signalling pathways (Figure 1B). However, 3 h pre-treatment with LPS prior to Dox treatment did not lead to changes in the degree of cell death (Figure 1C–E) but did lead to the expression of pro-IL-1β, which underwent processing and release upon Dox treatment in C34 cells but not in C19 cells (Figure 1G and H). In C34 cells, the detection of IL-1β coincided with the degree of death observed (Figure 1G). As the effects of LPS priming may take longer to affect pyroptosis we repeated experiments with both 3 and 18 h LPS priming. Primings were followed by induction of the system with various levels of Dox to allow for variations in NLRC4/Naip5 agonist expression. We observed only a slight trend for priming to effect pyroptosis (∼5–7% inhibition) with 3 or 18 h LPS priming at intermediate levels of agonist expression (Figure 1I and J). Responses to LPS were confirmed by TNFα production (Figure 1K). These results indicate that NLRC4/Naip5 agonist induced pyroptosis is generally refractory to TLR activation. NLRC4/Naip5 agonist expression induces rapid 'explosive' pyroptosis To understand the physical behaviour of cells undergoing unprimed pyroptosis, we subjected C34 and C19 cells to live-cell confocal microscopy. Cells stained with plasma membrane (PM) dye were imaged after Dox addition and data from PM, EGFP, YFP, and transmission (Trans) images were collected. C34 and C19 cells were observed to be slightly motile and YFP positive at the experimental outset (Figure 2A and B; Supplementary Movies 1 and 2). Induced cells gain EGFP signal that forms a slight, dispersed, punctate staining pattern in both C34 and C19 cells (Figure 2A and B). However, at various time points after induction C34 cells rapidly rupture with little noticeable change in previous behaviour. This rupture leads to an ejection of intracellular contents (Figure 2A, arrow) followed by a collapse in the size of the remaining cell body (defined by PM staining) and loss of EGFP and YFP signals (Figure 2A; Supplementary Movie 1). In contrast, Dox-treated C19 cells remained slightly motile and became progressively EGFP bright without undergoing cell death (Figure 2B; Supplementary Movie 2). Analysis of Dox-treated C34 and C19 cells for the full time course indicated that observations made with individual cells are representative of larger populations. These data demonstrate that pyroptosis is mechanistically different from classic apoptosis and directly suggests a mechanism for inflammatory cytokine and DAMP release. Figure 2.Sudden cell rupture occurs following cytoplasmic NLRC4/Naip5 agonist expression. Upper left: plasma membrane (PM) staining, upper right: EGFP signal, lower left: YFP signal, lower right: transmitted white light (Trans). (A) PM stained C34 cells gain EGFP signal with time then rupture releasing cellular contents. Arrows highlight the release of cellular contents detected by transmission microscopy and PM staining. (B) PM surface stained C19 cells gain EGFP signal with time but fail to rupture and die. Data are representative of ⩾100 independent cell events. Scale bar 10 μm. Download figure Download PowerPoint NLRC4/Naip5 agonist expression does not lead to inflammatory cytokine expression, NO, or ROS production Priming of inflammasome systems make it difficult to test whether inflammasome activation leads to the de novo expression of inflammatory cytokines and anti-microbial chemical production typical of innate immune receptor agonism. To study responses typical of TLR agonism during NLRC4/Naip5 activation, we activated C34 and C19 cells for 3 h with Dox or LPS. LPS activation of C34 and C19 cells resulted in the induction of inflammatory transcripts (Figure 3A). However, activation of C34 and C19 cells with Dox revealed no inflammasome-specific increases in gene transcripts for IL-6, Cxcl10, Caspase-11, IL-1β, CSF2, or TNFα despite being activated as indicated by EGFP expression (Figure 3A; Supplementary Figure 4). Figure 3.NLRC4/Naip5 agonist expression does not lead to inflammatory cytokine, NO, or ROS production. (A) mRNA and EGFP expression in Dox stimulated (500 ng/ml) or LPS stimulated (100 ng/ml) C34 or C19 cells at 3 h Hprt1 direct transcript values and Hprt1-relative expression data for IL-6, Cxcl10, and Caspse-11. (B) Nitrite levels in culture supernatants of C34 cells 24 h post Dox (500 ng/ml) or LPS (100 ng/ml) addition. (C) ROS levels in living C34 cells 8 h post Dox (500 ng/ml) or LPS (100 ng/ml) addition. First panel: representative histogram of unstimulated (grey fill), LPS-stimulated (dotted line), or Dox-stimulated (solid line) cells. Second panel: quantitative CellROX staining data. Data are mean±s.e.m. of triplicate samples representative of three (A, C) or four (B) independent experiments. Significant differences are indicated **P⩽0.01. Download figure Download PowerPoint Expression of iNOS is induced by TLR activation leading to the production of the anti-microbial chemical NO that affects inflammatory responses. To see if NLRC4/Naip5-activated cells produce NO, we activated C34 cells with Dox or LPS for 24 h and assayed supernatants for NO (assayed as nitrite). LPS stimulated cells produced NO but activation with Dox did not despite EGFP expression (Figure 3B). Activation of outer-membrane expressed TLRs in macrophages leads to the production of mitochondrial-derived ROS, which contributes to inflammatory and anti-microbial activity (West et al, 2011). To see if NLRC4/Naip5-activated cells produce ROS, we treated C34 cells with Dox or LPS and assayed living cells 8 h later for the production of whole-cell ROS. LPS stimulated cells produced ROS but activation with Dox did not despite EGFP expression (Figure 3C). Together these results show that during pyroptosis, activation of the inflammasome does not lead to de novo synthesis of inflammatory cytokines or reactive chemical species associated with inflammation and anti-microbial activity. NLRC4/Naip5 agonist expression leads to pyroptosis independent of overt mitochondrial dysfunction Primed apoptosis leading to NLRP3 activation involves a collapse of inner mitochondrial membrane potential and ROS production (Zhou et al, 2011; Shimada et al, 2012). Our results indicating that pyroptosis occurs without ROS production led us to investigate mitochondrial responses in response to NLRP4/Naip5 activation. To do this, we overexpressed the anti-apoptotic Bcl-2-like protein Bcl-XL in C34 cells to inhibit mitochondrial outer membrane permeabilization (MOMP), which occurs during activation of the intrinsic pathway of apoptosis (Kroemer et al, 2007) and inhibits apoptosis associated with NLRP3 activation (Shimada et al, 2012). Overexpression of Bcl-XL in C34 cells inhibited etoposide-induced apoptosis but not pyroptosis (Figure 4A). Bcl-XL expression did not affect EGFP expression after Dox treatment (Figure 4B). These results indicate that standard MOMP is not a response initiated during pyroptosis. Figure 4.Pyroptosis due to NLRC4/Naip5 agonist expression is unaffected by Bcl-XL and independent of mitochondrial dysfunction. (A, B) Determination of cell death (A) and EGFP expression (B) by flow cytometry in C34 or C34/Bcl-XL cells±Dox (500 ng/ml)±Etoposide (50 μM) after 24 h. (C–E) Changes in ΔΨm (C) viability (D) and EGFP expression (E)±Dox treatment (500 ng/ml)±CsA (10 μM)±CCCP (3 μM) after TMRE loading in C34 or C19 cells measured by flow cytometry. (F, G) Determination of cell death (F) and EGFP expression (G) in C34 cells by flow cytometry during inhibition of state 3 mitochondrial respiration. Data are mean±s.e.m. of triplicate samples representative of three (A–E) or two (F, G) independent experiments. Significant differences are indicated ***P⩽0.001. NS, not significant. Download figure Download PowerPoint Decreases in the inner mitochondrial transmembrane potential (ΔΨm) can occur following MOMP (Kroemer et al, 2007). Dissipation of ΔΨm leads to reductions in ATP synthesis, increased ROS production, and correlates with the mitochondria release of the NLRP3 agonist oxidized mitochondrial DNA (Shimada et al, 2012). To determine if ΔΨm changes with pyroptosis, we loaded cells with the cationic dye TMRE that accumulates in active mitochondria. Treatment of C34 cells with the mitochondrial decoupler CCCP resulted in a rapid loss of ΔΨm while treatment of cells with Dox only resulted in a slight but significant decrease (Figure 4C). This decrease was dependent on NLRC4/Naip5 agonist expression as it occurred in Dox-treated C34 but not in C19 cells. To determine if this decrease in ΔΨm was functionally correlated with pyroptosis, we treated C34 cells with the mitochondrial permeability transition pore inhibitor Cyclosporin A (CsA) followed by Dox. CsA treatment inhibited the NLRC4/Naip5 inflammasome-dependent ΔΨm decrease (Figure 4C). However, this was unable to prevent pyroptosis (Figure 4D). Neither TMRE loading nor CsA treatment affected EGFP expression after Dox treatment (Figure 4E). These observations show that ΔΨm decreases seen during pyroptosis do not correlate with cell death. This together with observations that C34 cells overexpressing Bcl-XL inhibit MOMP-associated apoptosis but have no effect on pyroptosis suggest that pyroptosis does not involve mitochondria in a manner similar to 'type II extrinsic' or 'intrinsic apoptosis' demonstrating clear phenotypic differences (Galluzzi et al, 2012). Programmed and regulated cell death are energy-dependent processes. We reasoned if pyroptosis is a form of regulated cell death it may require a functional mitochondria. To test the energy requirement of pyroptosis, we inhibited the state three respiration step of mitochondria (ADP to ATP phosphorylization) using high-dose TMRE treatment (Scaduto and Grotyohann, 1999). Following inhibition, we treated C34 cells with Dox. Activation led to cell death while pre-treatment with high-dose TMRE inhib
Referência(s)