Prdx4 limits caspase‐1 activation and restricts inflammasome‐mediated signaling by extracellular vesicles
2019; Springer Nature; Volume: 38; Issue: 20 Linguagem: Inglês
10.15252/embj.2018101266
ISSN1460-2075
AutoresSimone Lipinski, Steffen Pfeuffer, Philipp Arnold, Christian Treitz, Konrad Aden, Henriette Ebsen, Maren Falk‐Paulsen, Nicolas Gisch, Antonella Fazio, J. W. Kuiper, Anne Luzius, Susanne Billmann-Born, Stefan Schreiber, Gabriel Núñez, Hans‐Dietmar Beer, Till Strowig, Mohamed Lamkanfi, Andreas Tholey, Philip Rosenstiel,
Tópico(s)interferon and immune responses
ResumoArticle23 September 2019Open Access Source DataTransparent process Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles Simone Lipinski Corresponding Author Simone Lipinski [email protected] orcid.org/0000-0002-9322-7372 Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Steffen Pfeuffer Steffen Pfeuffer Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Philipp Arnold Philipp Arnold Anatomical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Christian Treitz Christian Treitz Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-University, Kiel, Germany Search for more papers by this author Konrad Aden Konrad Aden Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 1st Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Henriette Ebsen Henriette Ebsen Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Maren Falk-Paulsen Maren Falk-Paulsen Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Nicolas Gisch Nicolas Gisch orcid.org/0000-0003-3260-5269 Division of Bioanalytical Chemistry, Priority Area Infections, Research Center Borstel, Leibniz Lung Center, Borstel, Germany Search for more papers by this author Antonella Fazio Antonella Fazio Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Jan Kuiper Jan Kuiper Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Anne Luzius Anne Luzius Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Susanne Billmann-Born Susanne Billmann-Born Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Stefan Schreiber Stefan Schreiber 1st Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Gabriel Nuñez Gabriel Nuñez Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Hans-Dietmar Beer Hans-Dietmar Beer orcid.org/0000-0001-9932-4045 Department of Dermatology, University Hospital Zurich, Zurich, Switzerland Faculty of Medicine, University of Zurich, Zurich, Switzerland Search for more papers by this author Till Strowig Till Strowig Department of Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Mohamed Lamkanfi Mohamed Lamkanfi orcid.org/0000-0002-4898-7663 Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium Search for more papers by this author Andreas Tholey Andreas Tholey Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-University, Kiel, Germany Search for more papers by this author Philip Rosenstiel Corresponding Author Philip Rosenstiel [email protected] orcid.org/0000-0002-9692-8828 Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Simone Lipinski Corresponding Author Simone Lipinski [email protected] orcid.org/0000-0002-9322-7372 Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Steffen Pfeuffer Steffen Pfeuffer Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Philipp Arnold Philipp Arnold Anatomical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Christian Treitz Christian Treitz Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-University, Kiel, Germany Search for more papers by this author Konrad Aden Konrad Aden Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 1st Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Henriette Ebsen Henriette Ebsen Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Maren Falk-Paulsen Maren Falk-Paulsen Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Nicolas Gisch Nicolas Gisch orcid.org/0000-0003-3260-5269 Division of Bioanalytical Chemistry, Priority Area Infections, Research Center Borstel, Leibniz Lung Center, Borstel, Germany Search for more papers by this author Antonella Fazio Antonella Fazio Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Jan Kuiper Jan Kuiper Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Anne Luzius Anne Luzius Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Susanne Billmann-Born Susanne Billmann-Born Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Stefan Schreiber Stefan Schreiber 1st Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Gabriel Nuñez Gabriel Nuñez Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Hans-Dietmar Beer Hans-Dietmar Beer orcid.org/0000-0001-9932-4045 Department of Dermatology, University Hospital Zurich, Zurich, Switzerland Faculty of Medicine, University of Zurich, Zurich, Switzerland Search for more papers by this author Till Strowig Till Strowig Department of Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Mohamed Lamkanfi Mohamed Lamkanfi orcid.org/0000-0002-4898-7663 Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium Search for more papers by this author Andreas Tholey Andreas Tholey Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-University, Kiel, Germany Search for more papers by this author Philip Rosenstiel Corresponding Author Philip Rosenstiel [email protected] orcid.org/0000-0002-9692-8828 Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Author Information Simone Lipinski *,1,‡, Steffen Pfeuffer1,‡, Philipp Arnold2, Christian Treitz3, Konrad Aden1,4, Henriette Ebsen1, Maren Falk-Paulsen1, Nicolas Gisch5, Antonella Fazio1, Jan Kuiper1, Anne Luzius1, Susanne Billmann-Born1, Stefan Schreiber4, Gabriel Nuñez6, Hans-Dietmar Beer7,8, Till Strowig9, Mohamed Lamkanfi10,11, Andreas Tholey3 and Philip Rosenstiel *,1 1Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 2Anatomical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany 3Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-University, Kiel, Germany 41st Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 5Division of Bioanalytical Chemistry, Priority Area Infections, Research Center Borstel, Leibniz Lung Center, Borstel, Germany 6Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, MI, USA 7Department of Dermatology, University Hospital Zurich, Zurich, Switzerland 8Faculty of Medicine, University of Zurich, Zurich, Switzerland 9Department of Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany 10Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium 11VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium ‡These authors contributed equally to this work *Corresponding author. Tel: +49 431 500 15111; E-mail: [email protected] *Corresponding author. Tel: +49 4341 500 15111; E-mail: [email protected] The EMBO Journal (2019)38:e101266https://doi.org/10.15252/embj.2018101266 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 Abstract Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1β by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1β generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1β-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs. Synopsis This study shows that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1β generation by interfering with inflammasome activity. Prdx4 forms a redox-sensitive regulatory complex with the caspase-1 at the cysteine in position 397 that leads to caspase-1 sequestration and inactivation. Lack of Prdx4 in myeloid cells leads to elevated IL-1β levels and increased clinical symptoms in a murine septic shock model. Prdx4 interacts with caspase-1 in the cytosolic compartment and in extracellular vesicles (EVs) of activated macrophages. Purified EVs from macrophages contain all components of the NLRP3 inflammasome as well as Prdx4 and are able to convey a robust IL-1β-dependent inflammatory response in vitro and in vivo. Loss of Prdx4 leads to an increased pro-inflammatory potential of EVs, indicating a potential role of Prdx4 in the regulation of cell-to-cell communication via macrophage-derived EVs. Introduction Inflammation is the physiologic response to infection or injury and aims to restore cellular and tissue integrity. Multimeric protein complexes termed "inflammasomes" are key mediators of acute and chronic inflammatory responses. They assemble in response to cellular stress and regulate the maturation and secretion of IL-1-like cytokines, which induce a potent pro-inflammatory host response (Schroder & Tschopp, 2010). Pathologic conditions that lead to loss of control of IL-1β processing and secretion are associated with various inflammatory diseases including hereditary periodic fever syndromes, gout, atherosclerosis (Ridker et al, 2017), and type 2 diabetes (Neven et al, 2004; Martinon & Tschopp, 2005; Dinarello et al, 2010; Duewell et al, 2010). The NLRP3 (NOD-like receptor pyrin domain containing 3) inflammasome is the prototypical and best-studied inflammasome and is strongly expressed in myeloid cells (Manji et al, 2002). The sensor and scaffolding protein NLRP3 and pro-IL-1β are induced in the presence of LPS (lipopolysaccharide), other TLR or NLR agonists, or certain cytokines such as TNF-α or IL-1β (Bauernfeind et al, 2009; Franchi et al, 2009). Following this priming step, NLRP3 is activated by a drop in intracellular K+ concentrations (Munoz-Planillo et al, 2013) or by reactive oxygen species (Gaidt et al, 2016; Gross et al, 2016) commonly caused by various endogenous and exogenous danger signals like extracellular ATP-induced purinergic receptor P2X7 (P2X7R) activation (Ferrari et al, 1997), monosodium urate, bacterial-derived pore-forming toxins, or nigericin (Kanneganti et al, 2006; Mariathasan et al, 2006). Upon activation, NLRP3 oligomerizes and forms a molecular platform by recruiting the adapter protein ASC (apoptosis-associated speck-like protein containing a CARD) and pro-caspase-1 (Martinon et al, 2009). Clustering of pro-caspase-1 molecules leads to proximity-induced auto-proteolysis into p20 and p10 subunits, which in turn cleave pro-IL-1β to generate active IL-1β (Dinarello, 1998). Mature IL-1β is released into the extracellular space alongside active caspase-1 and oligomeric particles of the NLRP3 inflammasome (Baroja-Mazo et al, 2014). Ever since an alternative secretory pathway for the leaderless IL-1β has been reported (Rubartelli et al, 1990), the exact manner of release remains matter of debate. Suggested mechanisms include exocytosis via secretory lysosomes (Andrei et al, 1999, 2004), secretion by microvesicle shedding (MacKenzie et al, 2001), release of multivesicular bodies that may contain exosomes (Qu et al, 2007), an autophagy-based secretory pathway (Dupont et al, 2011), gasdermin D-dependent secretion via pores (Evavold et al, 2018) and a loss of membrane integrity leading to passive IL-1β release that occurs in parallel with pyroptotic death of the secreting cell (Shirasaki et al, 2014; Martin-Sanchez et al, 2016). We have previously shown that the 2-Cys oxidoreductase peroxiredoxin-4 (Prdx4) is induced in response to microbial danger signals, particularly downstream of the innate immune receptor NOD2 and that Prdx4 negatively regulates NF-κB signaling (Weichart et al, 2006). Here, we report that Prdx4 limits inflammasome activity by thiol-mediated inactivation of caspase-1. Mechanistically, we provide evidence that Prdx4 and caspase-1 interact in the cytosol and form a redox-sensitive regulatory complex via caspase-1 cysteine 397 and a high-molecular-weight (HMW) complex of Prdx4. Furthermore, we show that Prdx4 is co-localized with components of the inflammasome in extracellular vesicles (EVs). Within EVs, loss of Prdx4 resulted in increased levels of cleaved caspase-1 and IL-1β maturation. Importantly, EVs, derived from inflammasome-activated macrophages, were able to transmit an IL-1β-dependent immune response to recipient cells, whereby Prdx4 deficiency boosted the pro-inflammatory potential of EVs. We thus define a critical role for Prdx4 in the post-translational and post-secretional regulation of inflammasome activation and induction of inflammatory responses. Results Prdx4 protects from LPS-induced septic shock To determine how Prdx4 influences inflammatory responses in vivo, we generated Prdx4-knockout (KO) mice (Appendix Fig S1). Mice were fertile and showed no spontaneous phenotype. To investigate the role of Prdx4 during inflammation, we challenged mice with sub-lethal doses of LPS. We found that Prdx4-deficient mice had increased body weight loss and delayed restoration of weight compared to their wild-type (WT) littermates (Fig 1A). Consistent with the increased body weight loss, Prdx4 KO mice had significant higher Cxcl1, TNF-α and IL-1β levels in serum and peritoneal lavages at 24 h post-LPS injection (Fig 1B–D). As IL-1β is a major mediator of LPS-induced systemic immune responses, we next blocked IL-1β-mediated signaling using the interleukin-1-receptor antagonist (IL-1RA) Anakinra. In all IL-1RA-treated animals, weight loss was attenuated in response to LPS administration and no differences were found between Prdx4 KO and WT littermates (Fig 2A). Also, excessive serum Cxcl1, TNF-α, and IL-1β levels in LPS-treated Prdx4 KO mice were significantly lowered upon the injection of IL-1RA (Fig 2B). Thus, we concluded that loss of Prdx4 results in an aggravated inflammatory response, which involves increased IL-1β signaling. Figure 1. Prdx4 protects from LPS-induced septic shock A. Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n = 7 mice; vertical lines indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (two-way-ANOVA, Bonferroni post-test). B–D. Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot represents an individual mouse. Horizontal lines indicate mean. **P < 0.01; ***P < 0.001; n.s. not significant (two-tailed t-test). Data are representative of two independent experiments. Download figure Download PowerPoint Figure 2. Role of IL-1 receptor blockade and myeloid-specific ablation of Prdx4 in the endotoxin-shock model Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 μg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n = 5 mice; vertical lines indicate SEM. ***P < 0.001 (two-way-ANOVA, Bonferroni post-test). Serum concentration of Cxcl1, TNF-α, and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS, and IL-1RA or control. Each dot represents an individual mouse. Horizontal lines indicate mean. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant (two-tailed t-test). Percent body weight of male Prdx4-flox and Prdx4-ΔLysMCre mice over the 48 h course of 4.5 mg/kg BW LPS (i.p.). Each circle represents a mean of n = 7 mice; vertical lines indicate SEM. *P < 0.05; ***P < 0.001 (two-way-ANOVA, Bonferroni post-test). Serum concentration of Cxcl1, TNF-α, and IL-1β in Prdx4-flox and Prdx4-ΔLysMCre mice injected with LPS. Each dot represents an individual mouse. Horizontal lines indicate mean. *P < 0.05; ***P < 0.001; n.s. not significant (two-tailed t-test). Data are representative of two independent experiments. Download figure Download PowerPoint Prdx4-deficient macrophages display elevated cytokine responses and inflammasome activation We next sought to determine the major cellular source of the increased IL-1β generation. As myeloid cells have been described as critical producers of pro-inflammatory cytokines in LPS-induced septic responses (Dinarello et al, 1974; Baracos et al, 1983), we crossed floxed Prdx4 mice to a LysMCre deleter strain in order to obtain mice that specifically lack Prdx4 in cells of myeloid origin, hereafter referred to as Prdx4-ΔLysMCre (Appendix Fig S2A). Knockout of Prdx4 was confirmed by Western blot analysis of bone marrow-derived macrophages (BMDMs) with antibodies against Prdx4 (Appendix Fig S2B). Since the results from the whole-body knockout mice showed the largest difference in body weight loss between 40 and 60 h after LPS injection, Prdx4-ΔLysMCre and floxed littermates were monitored for 48 h post-LPS injection. Comparable to Prdx4 KO mice, Prdx4-ΔLysMCre mice showed a significantly increased body weight loss starting from 36 h after LPS injection until the end point (Fig 2C). Also, we found higher Cxcl1, TNF-α, and IL-1β levels in the serum of Prdx4-ΔLysMCre mice compared to floxed littermates (Fig 2D). Collectively, these results suggest a critical role of the myeloid compartment for the Prdx4-mediated protection during endotoxin shock. Because Prdx4 deficiency led to increased cytokine responses following LPS challenge in vivo, we used BMDMs from Prdx4 WT and KO mice to characterize the altered responses to LPS in more detail. In a time course of LPS stimulation, we confirmed that LPS-induced release of Cxcl1 and TNF-α was significantly increased in Prdx4-deficient BMDMs (Fig 3A). Importantly, we found that the absence of Prdx4 also led to a time-dependent release of IL-1β. This is of interest since LPS stimulation alone is usually not sufficient to trigger significant IL-1β release in WT BMDMs (Hagar et al, 2013; Kayagaki et al, 2013). Thus, we next induced IL-1β release by activation of the inflammasome. We confirmed that loss of Prdx4 leads to excessive release of IL-1β in BMDMs that were primed with LPS to induce expression of inflammasome components (Bauernfeind et al, 2009) followed by a time course of ATP treatment (Fig 3B). Accordingly, we detected increased levels of mature IL-1β in the supernatant of Prdx4-deficient BMDMs (Fig 3C). Next, we used HEK293 cells that were forced to secrete IL-1β by pro-IL-1β/caspase-1 overexpression. In line with our previous findings, co-expression of Prdx4 decreased levels of mature IL-1β (Appendix Fig S3). Since previous reports demonstrated a role for Prdx4 in the redox-dependent regulation of NF-κB activation (Jin et al, 1997; Weichart et al, 2006; Yu et al, 2010) and reactive oxygen species (ROS) also contribute to NF-κB-dependent NLRP3 priming (Bauernfeind et al, 2011), we hypothesized that Prdx4 deficiency might affect inflammasome priming leading to the observed differences in IL-1β levels. Unexpectedly, we did neither find genotype-dependent differences on Nlrp3 protein levels or stability, nor on Nlrp3 or Il1b mRNA levels in response to LPS-induced priming or on other inflammasome components or redox proteins related to inflammasome activation (Fig EV1). To investigate whether the formation of ASC specks downstream of inflammasome activation is affected by Prdx4, BMDMs were stimulated with nigericin after LPS priming or left untreated. We did not find differences in ASC speck formation (Fig 3D), indicating that increased IL-1β levels in Prdx4 KO BMDMs do not result from increased ASC speck formation. However, we detected increased levels of cleaved caspase-1 in the supernatant of Prdx4 KO BMDMs after nigericin-induced inflammasome activation (Fig 3E), indicating that Prdx4 negatively influences caspase-1 activation. In order to validate whether unrestrained caspase-1 activity accounts for the IL-1β hypersecretion in Prdx4-deficient BMDMs, we used the selective caspase-1 inhibitor YVAD. We found that YVAD completely reduced the elevated IL-1β levels in the supernatant of Prdx4-deficient BMDMs (Appendix Fig S4), confirming that Prdx4-dependent IL-1β hypersecretion is dependent on caspase-1. Next, we investigated the impact of Prdx4 on canonical caspase-1 inflammasome activation and IL-1β release. We found that loss of Prdx4 led to increased IL-1β release compared to WT BMDMs in response to canonical inflammasome activation induced by either ATP and nigericin (NLRP3 inflammasome), double-stranded DNA (AIM2 inflammasome), or flagellin (NLRC4 inflammasome), although the highest fold change was found for ATP and nigericin stimulation (Fig 3F). Interestingly, the ATP-, nigericin-, and flagellin-induced LDH release was affected by Prdx4 as well (Fig 3G). We therefore concluded that Prdx4 negatively regulates caspase-1-dependent inflammasome responses in myeloid cells. Figure 3. Prdx4-deficient macrophages display elevated cytokine responses and inflammasome activation Concentration of Cxcl1, TNF-α, and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated). IL-1β release of Prdx4 WT and KO BMDMs, untreated, or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM). Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml), and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice. Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to nigericin (10 μg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC, and nuclei were counterstained using DAPI. Scale bar indicates 20 μm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n = 4 mice; vertical lines indicate SD. n.s. not significant (two-tailed t-test). Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to nigericin (10 μg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS priming alone or without stimulation. Whole-cell lysates were analyzed for pro-caspase-1 and Gapdh levels. IL-1β release in Prdx4 WT and KO BMDMs, untreated, or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or nigericin (10 μg/ml) or transfected for 3 h with poly(dA:dT) or flagellin (1 μg/ml each) or treated with transfection agent only. Quantification of cell death by LDH release in Prdx4 WT and KO BMDMs, untreated, or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or nigericin (10 μg/ml) or transfected for 3 h with poly(dA:dT) or flagellin (1 μg/ml each) or treated with transfection agent only. Data information: (A, B) Each dot represents a mean of n = 3 mice; vertical lines indicate SD. **P < 0.01; ***P < 0.001 (two-way-ANOVA, Bonferroni post-test). (F, G) Bars represent a mean of n = 3 mice; vertical lines indicate SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant (two-tailed t-test). All data are representative of two independent experiments. Source data are available online for this figure. Source Data for Figure 3 [embj2018101266-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Prdx4 does not impact priming of inflammasome components or associated factors (relates to Fig 3) qRT–PCR analysis of Nlrp3, Il1b, Il18, ASC, caspase-1, Nlrp1, Trxnip, and Nos2 relative to Gapdh mRNA in Prdx4 WT and KO BMDMs, primed for 6 h with LPS or left untreated. Western blot analysis of NLRP3, pro-caspase-1, ASC, pro-IL-1β, Prdx4, and β-actin (loading control) in Prdx4 WT and KO BMDMs at 6 h after LPS stimulation. Western blot analysis of NLRP3, Prdx4, and β-actin (loading control) in Prdx4 WT and KO BMDMs at 6 h after LPS stimulation and CHX treatment for the time points indicated. Data information: (A) Each dot represents a biological replicate; horizontal lines indicate mean. Vertical lines indicate SD (C). n.s. not significant (two-tailed t-test). Data are representative of one experiment with n = 4 mice per genotype with n = 2 technical replicates (A) or two (B, C) independent experiments with n = 3 mice per genotype. Source data are available online for this figure. Download figure Download PowerPoint Prdx4 interacts with C397 of caspase-1 to block its function In order to investigate the molecular mechanism by which Prdx4 negatively regulates caspase-1-dependent inflammasome activation, we hypothesized that Prdx4 directly interacts with caspase-1 to limit its downstream cleavage and activation. To test this hypothesis, we assessed whether Prdx4 and caspase-1 interact in vitro using active forms of recombinant human PRDX4 (rPRDX4) and human caspase-1 (rCASP-1). Under physiologic conditions and depending on the redox environment, Prdx4 is known to form oligomeric high-molecular-weight (≥ 250-kDa) structures, with a high abundance of decamers consisting of five disulfide-linked dimers (Tavender et al, 2008). We therefore co-incubated rPRDX4 with rCASP-1 and analyzed the proteins under non-red
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