Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients
2019; Springer Nature; Volume: 11; Issue: 11 Linguagem: Inglês
10.15252/emmm.201910547
ISSN1757-4684
AutoresFlora Magnotti, Lucie Lefeuvre, Sarah Benezech, Tiphaine Malsot, Louis Waeckel, Amandine Martin, Sébastien Kerever, Daria Chirita, Marine Desjonquères, A. Duquesne, Mathieu Gerfaud‐Valentin, Audrey Laurent, P. Sève, Michel R. Popoff, Thierry Walzer, Alexandre Bélot, Yvan Jamilloux, Thomas Henry,
Tópico(s)Gout, Hyperuricemia, Uric Acid
ResumoArticle7 October 2019Open Access Source DataTransparent process Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients Flora Magnotti Flora Magnotti CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Lucie Lefeuvre Lucie Lefeuvre CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Search for more papers by this author Sarah Benezech Sarah Benezech orcid.org/0000-0002-9587-3896 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Search for more papers by this author Tiphaine Malsot Tiphaine Malsot CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Louis Waeckel Louis Waeckel CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Search for more papers by this author Amandine Martin Amandine Martin CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Sébastien Kerever Sébastien Kerever Department of Anesthesiology and Critical Care, St Louis-Lariboisière University Hospital, AP-HP, ECSTRA Team, Epidemiology and Biostatistics, Sorbonne Paris Cité Research Centre, UMR 1153, Inserm, University Denis Diderot-Paris VII, Paris, France Search for more papers by this author Daria Chirita Daria Chirita CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Marine Desjonqueres Marine Desjonqueres Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Agnès Duquesne Agnès Duquesne Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Mathieu Gerfaud-Valentin Mathieu Gerfaud-Valentin Hospices Civils de Lyon, Lyon, France Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France Search for more papers by this author Audrey Laurent Audrey Laurent Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Pascal Sève Pascal Sève Hospices Civils de Lyon, Lyon, France Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France Search for more papers by this author Michel-Robert Popoff Michel-Robert Popoff Bacterial Toxins, Institut Pasteur, Paris, France Search for more papers by this author Thierry Walzer Thierry Walzer CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Alexandre Belot Alexandre Belot CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Yvan Jamilloux Corresponding Author Yvan Jamilloux [email protected] orcid.org/0000-0001-5249-3650 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France Search for more papers by this author Thomas Henry Corresponding Author Thomas Henry [email protected] orcid.org/0000-0002-0687-8565 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Flora Magnotti Flora Magnotti CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Lucie Lefeuvre Lucie Lefeuvre CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Search for more papers by this author Sarah Benezech Sarah Benezech orcid.org/0000-0002-9587-3896 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Search for more papers by this author Tiphaine Malsot Tiphaine Malsot CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Louis Waeckel Louis Waeckel CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Search for more papers by this author Amandine Martin Amandine Martin CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Sébastien Kerever Sébastien Kerever Department of Anesthesiology and Critical Care, St Louis-Lariboisière University Hospital, AP-HP, ECSTRA Team, Epidemiology and Biostatistics, Sorbonne Paris Cité Research Centre, UMR 1153, Inserm, University Denis Diderot-Paris VII, Paris, France Search for more papers by this author Daria Chirita Daria Chirita CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Marine Desjonqueres Marine Desjonqueres Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Agnès Duquesne Agnès Duquesne Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Mathieu Gerfaud-Valentin Mathieu Gerfaud-Valentin Hospices Civils de Lyon, Lyon, France Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France Search for more papers by this author Audrey Laurent Audrey Laurent Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Pascal Sève Pascal Sève Hospices Civils de Lyon, Lyon, France Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France Search for more papers by this author Michel-Robert Popoff Michel-Robert Popoff Bacterial Toxins, Institut Pasteur, Paris, France Search for more papers by this author Thierry Walzer Thierry Walzer CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Alexandre Belot Alexandre Belot CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France Search for more papers by this author Yvan Jamilloux Corresponding Author Yvan Jamilloux [email protected] orcid.org/0000-0001-5249-3650 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Hospices Civils de Lyon, Lyon, France Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France Search for more papers by this author Thomas Henry Corresponding Author Thomas Henry [email protected] orcid.org/0000-0002-0687-8565 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France Search for more papers by this author Author Information Flora Magnotti1, Lucie Lefeuvre1,2, Sarah Benezech1,2,‡, Tiphaine Malsot1, Louis Waeckel1,2, Amandine Martin1, Sébastien Kerever3, Daria Chirita1, Marine Desjonqueres2,4, Agnès Duquesne2,4, Mathieu Gerfaud-Valentin2,5, Audrey Laurent2,4, Pascal Sève2,5, Michel-Robert Popoff6, Thierry Walzer1, Alexandre Belot1,2,4, Yvan Jamilloux *,1,2,5,‡ and Thomas Henry *,1,‡ 1CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ. Lyon, Lyon, France 2Hospices Civils de Lyon, Lyon, France 3Department of Anesthesiology and Critical Care, St Louis-Lariboisière University Hospital, AP-HP, ECSTRA Team, Epidemiology and Biostatistics, Sorbonne Paris Cité Research Centre, UMR 1153, Inserm, University Denis Diderot-Paris VII, Paris, France 4Service de Néphrologie, Rhumatologie, Dermatologie pédiatriques, HFME, Bron, France 5Service de Médecine Interne, Hôpital de la Croix-Rousse, Lyon, France 6Bacterial Toxins, Institut Pasteur, Paris, France ‡These authors contributed equally to this work as senior authors ‡Present address: Institut d'Hématologie et Oncologie Pédiatrique, Lyon, France *Corresponding author. Tel: +33 4 37 28 23 72; E-mail: [email protected] *Corresponding author. Tel: +33 4 37 28 23 72; E-mail: [email protected] EMBO Mol Med (2019)11:e10547https://doi.org/10.15252/emmm.201910547 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 Familial Mediterranean fever (FMF) is the most frequent hereditary systemic autoinflammatory syndrome. FMF is usually caused by biallelic mutations in the MEFV gene, encoding Pyrin. Conclusive genetic evidence lacks for about 30% of patients diagnosed with clinical FMF. Pyrin is an inflammasome sensor maintained inactive by two kinases (PKN1/2). The consequences of MEFV mutations on inflammasome activation are still poorly understood. Here, we demonstrate that PKC superfamily inhibitors trigger inflammasome activation in monocytes from FMF patients while they trigger a delayed apoptosis in monocytes from healthy donors. The expression of the pathogenic p.M694V MEFV allele is necessary and sufficient for PKC inhibitors (or mutations precluding Pyrin phosphorylation) to trigger caspase-1- and gasdermin D-mediated pyroptosis. In line with colchicine efficacy in patients, colchicine fully blocks this response in FMF patients' monocytes. These results indicate that Pyrin inflammasome activation is solely controlled by Pyrin (de)phosphorylation in FMF patients while a second control mechanism restricts its activation in healthy donors/non-FMF patients. This study paves the way toward a functional characterization of MEFV variants and a functional test to diagnose FMF. Synopsis Familial Mediterranean fever (FMF) is a systemic auto-inflammatory disease associated with MEFV mutations. MEFV encodes Pyrin, an inflammasome sensor. The link between MEFV mutations and the dysregulated activation of the Pyrin inflammasome observed in FMF patients is unclear. Pyrin dephosphorylation is insufficient to trigger full inflammasome activation in healthy donors'monocytes while it is sufficient in FMF patients monocytes. The pathogenic MEFV mutation most frequently observed in FMF patients triggers constitutive inflammasome activation only when combined to a phosphonull MEFV mutation. UCN-01-induced dephosphorylation of Pyrin triggers inflammasome activation in FMF patients' monocytes but not in monocytes from other patients paving the way to a functional diagnosis of FMF. Introduction Familial Mediterranean fever (FMF) is the most frequent hereditary systemic autoinflammatory disorder characterized by recurrent episodes of fever, serositis, and abdominal pain (Sonmez et al, 2016). Its feared complication is secondary AA amyloidosis, which can lead to end-stage kidney disease. Colchicine, an inhibitor of microtubule polymerization, decreases chronic inflammation and represents the cornerstone of FMF treatment (Goldfinger, 1972). Daily and lifelong administration of colchicine is currently recommended for FMF patients. Familial Mediterranean fever diagnosis relies first on clinical criteria. Due to the absence of pathognomonic clinical signs and to heterogeneity in clinical presentations (Mor et al, 2003; Padeh et al, 2010), FMF diagnosis can be challenging (Giancane et al, 2015). Familial Mediterranean fever is associated with mutations in the MEFV gene. Mendelian transmission of the disease occurs mostly in an autosomal recessive mode. As of today, genetic screening confirms the FMF diagnosis upon identification of biallelic mutations in clearly pathogenic MEFV variants (Shinar et al, 2012). Nine sequence variants of MEFV are considered clearly pathogenic (Shinar et al, 2012). Yet, there are 365 MEFV variants listed in the Infevers database (Sarrauste de Menthiere et al, 2003), most of them of uncertain significance, which can result in misdiagnosis or diagnosis delay (Lidar et al, 2005). Furthermore, a substantial proportion of clinically diagnosed FMF patients (up to 30%) presents only a single MEFV pathogenic variant (Dode et al, 2000; Lachmann et al, 2006; Jeru et al, 2013). Finally, no MEFV variant is found in 5–14% of clinically diagnosed FMF patients (Lachmann et al, 2006; Toplak et al, 2012). Due to all these situations, genetic testing has a 70–80% positive predictive value (Soriano & Manna, 2012) and the median delay between disease onset and diagnosis remains long (1.4 years for patients born in the 21st century; Toplak et al, 2012). Furthermore, the generalization of next-generation sequencing leads to the identification of novel rare variants of unknown impact. Functional assays robustly discriminating pathogenic MEFV variants from non-pathogenic MEFV polymorphisms are needed to sustain diagnosis and the development of personalized medicine (Van Gorp et al, 2016). MEFV encodes Pyrin, an inflammasome sensor detecting Rho A GTPase inhibition (Xu et al, 2014). Inactivation of Rho A by various bacterial toxins triggers activation of the Pyrin inflammasome, i.e., oligomerization of the inflammasome adaptor ASC, caspase-1 activation, secretion of the pro-inflammatory cytokines IL-1β and IL-18, and an inflammatory cell death termed pyroptosis (Cookson & Brennan, 2001; Martinon et al, 2002; Xu et al, 2014). At steady state, Pyrin is maintained inactive by phosphorylation of its serine residues S208 and S242. Two kinases (PKN1/2) from the PKC superfamily phosphorylate Pyrin, leading to its sequestration by 14-3-3 chaperone proteins (Gao et al, 2016; Masters et al, 2016; Park et al, 2016; Van Gorp et al, 2016). Rho A inhibition leads to dephosphorylation of Pyrin, its release from the 14-3-3 proteins and the assembly/activation of the Pyrin inflammasome. Of note, in healthy individuals, the transition from 14-3-3-free Pyrin to ASC oligomerization and Pyrin inflammasome activation requires microtubule dynamics (Gao et al, 2016). Colchicine specifically blocks the Pyrin inflammasome downstream of Pyrin release from the 14-3-3 proteins and upstream of ASC oligomerization (Gao et al, 2016). In FMF patients, the microtubule-dependent mechanism might be deficient since a recent report indicated that colchicine is inefficient to block Pyrin inflammasome activation in PBMCs from FMF patients (Van Gorp et al, 2016). This in vitro result, at odds with the clinical efficacy of colchicine in FMF patients, is still poorly understood. A two-step activation model is emerging with (i) dephosphorylation of Pyrin following inhibition of PKN1/2 and (ii) Pyrin inflammasome maturation involving a colchicine-targetable microtubule dynamics event (Gao et al, 2016). The link between the two steps remains unclear. Particularly, it is unknown whether dephosphorylation of Pyrin automatically leads to Pyrin inflammasome activation in cells with intact microtubule dynamics. Finally, the impact of MEFV mutations on each step is controversial (Gao et al, 2016; Masters et al, 2016; Park et al, 2016; Van Gorp et al, 2016). In this work, we demonstrate that PKC superfamily inhibitors trigger inflammasome activation, IL-1β secretion, and pyroptosis in monocytes from FMF patients while they fail to do so in monocytes from healthy donors (HD) in which they trigger a delayed apoptosis. PKC superfamily inhibitor-mediated inflammasome activation was blocked by colchicine in FMF patients' monocytes in line with the efficacy of this drug in patients. The mechanism of the differential control of the Pyrin inflammasome was pinpointed to specific MEFV mutations in human monocyte cell lines expressing either one of three common clearly pathogenic MEFV variants, p.M694V, p.M694I, or p.M680I. Importantly, the cytotoxic effect of PKC superfamily inhibitors on the p.M694V allele-expressing cells could be recapitulated genetically by mutating the Pyrin Serine 242 or S208 residues. These results suggest that, while Pyrin inflammasome is controlled by two independent mechanisms in healthy donors, in FMF patients, the Pyrin inflammasome lacks one safeguard mechanism and is only regulated by Pyrin phosphorylation. Finally, our results indicate that these differences could be exploited to develop a functional diagnostic test. Results PKC inhibitors trigger IL-1β release in monocytes from FMF patients The current model for Pyrin inflammasome activation indicates that activation results from the dephosphorylation of Pyrin following the lack of sustained activation of PKN1/2, two kinases from the PKC superfamily (Park et al, 2016). To explore the mechanisms underlying deregulation of the Pyrin inflammasome in FMF patients, we decided to assess the efficacy of staurosporine (a potent PKC superfamily inhibitor targeting PKN1/2; Davis et al, 2011) to trigger IL-1β release in primary monocytes from HD or FMF patients. We observed no to very low IL-1β release from monocytes isolated from HD in response to LPS + staurosporine (Fig 1A). In our experimental conditions, monocytes from 31 out of the 33 HD (94%) released < 50 pg/ml of IL-1β (Fig 1A). In sharp contrast, monocytes from FMF patients released moderate to high levels of IL-1β, leading to an average level 17-fold higher (422 pg/ml, P < 0.0001) than the average level in the supernatant of HD monocytes (25 pg/ml) (Fig 1A and Appendix Fig S1A for a detailed version including patients' genotype). These differences were conserved over several staurosporine concentrations and at several times post-treatment (Appendix Fig S2A and B). This result indicates strongly differing inflammasome responses to PKC superfamily inhibition between FMF patients and HD. To confirm this result, we used UCN-01, a hydroxylated derivative of staurosporine, which displays a better selectivity for PKC superfamily kinases (Tamaoki, 1991). Similar findings were observed (Fig 1B and Appendix Fig S1B) with monocytes from FMF patients releasing > 10-fold higher IL-1β levels than HD monocytes did. The same trend (Appendix Fig S2C and D) was observed using the bisindolylmaleimide RO 31–8220, another PKC superfamily inhibitor of different chemical structure (Davis et al, 1992). IL-1β levels following treatment with UCN-01 and staurosporine were significantly correlated in the different patients (Appendix Fig S2E). As seen with staurosporine, the difference in IL-1β response between monocytes from HD and FMF patients was conserved over a large range of concentrations of UCN-01 (Appendix Fig S2F). The hyper-responsiveness of FMF monocytes to PKC superfamily inhibitors thus differs from their hyper-responsiveness to Clostridioides difficile toxin TcdB, which was observed only at low doses of TcdB (Jamilloux et al, 2018). Figure 1. PKC inhibitors specifically trigger IL-1β release and a fast cell death in monocytes from FMF patients A–I . Monocytes from healthy donors (HD) or FMF patients were either primed with LPS (A–C, G–I) or not (D–F) and treated with (A, C) 1.25 μM staurosporine (Stauro), (B, D–F) 12.5 μM UCN-01, or (G–I) 5 μM nigericin (Nig). (A, B) IL-1β and (C) TNF level were quantified by ELISA at 90 min post stimulation. (D, G) Cell death was monitored in real time by measuring propidium iodide influx/fluorescence every 5 min. (E, H) The time required to reach 20% cell death and (F, I) the area under the curve (AUC) were computed for each HD or FMF patients. Data information: (A–C, E–F, H, I) Each dot represents the mean value from three biological replicates for one HD or patient. The bar represents the median ± interquartile range. a.u.: arbitrary units. (D, G) Each point of the curve corresponds to the average cell death values from the indicated number of HD or FMF patients (for each individual, the value is the mean of a biological triplicate). The dotted line indicates the 20% cell death value. (A, B, F) ***P < 0.0001 by Wilcoxon rank-sum test. (C) One-way ANOVA with Sidak's multiple comparison tests was performed. LPS: N. S. Not significant P = 0.99; LPS + Staurosporine N.S.: P = 0.77. (E, H) Unpaired t-tests were performed, and two-tailed P-values are shown. (E) ***P < 0.0001, (H) N.S.: P = 0.79. (I) N.S. P = 0.9 by Wilcoxon rank-sum test. Source data are available online for this figure. Source Data for Figure 1 [emmm201910547-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint IL-1β levels were substantially decreased upon addition, 30 min before UCN-01, of the caspase-1 inhibitors VX-765 or YVAD-FMK (Appendix Fig S2G). Neither the caspase-3 inhibitor (DEVD-FMK) nor the caspase-8 inhibitor (IETD-FMK) demonstrated a robust inhibition of IL-1β release. This result suggests that UCN-01 triggers inflammasome activation in FMF patient monocytes. As previously described (Van Gorp et al, 2016), we did not observe any difference in IL-1β release in response to engagement of the NLRP3 inflammasome by LPS + ATP (Appendix Fig S2H) or of the NLRC4 inflammasome (Jamilloux et al, 2018). Furthermore, LPS + staurosporine treatment did not lead to differential TNF secretion between monocytes from HD and FMF patients (Fig 1C), indicating that the differing response to PKC inhibitors between HD and FMF patients is specific to inflammasome activation. These results suggest that dephosphorylation of Pyrin is sufficient to trigger inflammasome activation in monocytes from FMF patients while a PKC-independent mechanism limits IL-1β release in HD monocytes. PKC inhibitors trigger fast cell death in monocytes from FMF patients Inflammasome activation is often associated with a fast cell death process termed pyroptosis. We thus investigated whether PKC inhibitors, in the absence of LPS priming, trigger cell death in monocytes from HD and FMF patients. Indeed, UCN-01 triggered a very rapid influx of propidium iodide in monocytes from FMF patients while it was much delayed in monocytes from HD (Fig 1D). These kinetics were determined to be significantly different by quantifying the time post-UCN-01 addition leading to 20% cell death (dotted line in Fig 1D and E, P < 0.0001) and the area under the curve (AUC, Fig 1F, P < 0.0001). The difference in cell death was specific to PKC inhibitors since NLRP3 inflammasome activation by LPS + nigericin (Mariathasan et al, 2006) triggered propidium iodide influx with similar kinetics in monocytes from HD and FMF patients (Fig 1G–I and Appendix Fig S2I). Importantly, the UCN-01-mediated fast cell death was observed in the absence of LPS treatment, indicating that the Pyrin inflammasome does not require TLR-mediated priming, as previously demonstrated following C. difficile toxin treatment (Van Gorp et al, 2016; Jamilloux et al, 2018). PKC inhibitors differentially trigger pyroptosis or apoptosis in monocytes from FMF patients and HD The kinetics of monocytes death and its association with IL-1β release suggest that PKC inhibitors trigger pyroptosis in monocytes from FMF patients. To strengthen this finding, we directly evaluated the ability of the inflammasome adaptor ASC to form specks as a readout of inflammasome complex formation. At 40 min post-UCN-01 treatment, monocytes were fixed and immuno-stained for ASC. More than 35% of monocytes from FMF patients displayed ASC specks while UCN-01 treatment did not substantially increase the frequency of speck-containing cells in HD monocytes (Fig 2A and B and control experiment in Appendix Fig S3). This result indicates that the Pyrin inflammasome activation is controlled by a phosphorylation-independent mechanism upstream of ASC oligomerization in monocytes from HD and that this control mechanism is defective in FMF patients. Figure 2. The PKC inhibitor, UCN-01, triggers pyroptosis or apoptosis in monocytes from FMF patients and HD, respectively A, B. Monocytes from HD or FMF patients were treated with 12.5 μM UCN-01 for 40 min or primed with LPS (3 h) and treated with 5 μM nigericin (Nig) for 90 min. (A) Cells were immuno-stained for ASC. ASC specks are indicated by red arrowheads. Representative confocal microscopy images from one HD (top panels) and one FMF patient (bottom panels) are shown. Scale bars: 10 μm and 2.5 μm in the main figures and onsets, respectively. (B) Quantification of ASC specks in HD and FMF patients' monocytes by immunofluorescence. C. The frequency of cells positive for active caspase-1 was quantified by flow cytometry, using FLICA-Caspase-1 in HD and FMF patients. D, E. Cell death was assessed at 90 min post-UCN-01 or post-LPS + nigericin treatment by determining the percentage of Annexin-V+/PI− cells and of PI+ cells among dead cells (Annexin-V+ and/or PI + cells) using flow cytometry. (D) Representative FACS plots from one healthy donor (HD) and one FMF patient are shown. Percentage are indicated for the two right gates. (E) Cell death modality was assessed by determining the percentage of treatment-induced Annexin-V+/PI− cells and of PI+ cells among dead cells, using flow cytometry. Data information: (B, C, E) Kruskal–Wallis with Dunn's multiple comparison tests were performed to compare HD and FMF responses. Adjusted P-values are detailed below. (B) Each dot (HD)/symbol (FMF) represents the percentage of cells containing an ASC speck for one individual. Symbol to FMF patient #: square #2 (M694I/M694I), round #26 (V726A/V726A), triangle #13 (M694V/R761H), diamond #18 (M694I/M694I). UCN-01 **P = 0.0095; LPS + nigericin N.S. P = 0.75. (C) Each dot (HD)/symbol(FMF) represents the percentage of cells stained with FLICA-Casp1 for one individual, the bar represents the median (±interquartile range). Symbol to FMF patient #: round #26 (V726A/V726A), triangle #13 (M694V/R761H), triangle pointing down #23 (M694V/M694V), hexagon #24 (M694V/M694V), star #10 (M694V/M694V). Untreated: N.S. P = 1; UCN-01: **P = 0.0069; LPS + nigericin: N.S. P = 1. (E) Each dot (HD)/triangle (FMF) represents the value for one individual, and the bar represents the median (± interquartile range). UCN-01: ***P < 0.0001; LPS + nigericin N.S. P = 0.51. Source data are available online for this figure. Source Data for Figure 2 [emmm201910547-sup-0004-SDataFig2.zip] Download figure Download PowerPoint The UCN-01-mediated induction of inflammasome activation in monocytes from FMF patients was further confirmed by quantifying cells containing active caspase-1, using the fluorescent inhibitor probe FAM-YVAD-FMK, referred to as FLICA-Casp1. As quantified by flow cytometry, 33% of monocytes from FMF patients stained positive for FLICA-Casp1 at 40 min post-UCN-01 addition, while only 6% of monocytes from HD did, a proportion similar to the one observed in the untreated samples (Fig 2C). These results establish that PKC inhibitors specifically trigger pyroptosis in monocytes from FMF patients. Yet, PKC inhibitors also lead to a late cell death in HD monocytes (Fig 1D). Based on the well-known activity of PKC inhibitors to trigger apoptosis (Nie et al, 2014), and on the absence of signs of inflammasome activation (Figs 1B
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