Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis

Surveillance of cell wall diffusion barrier integrity modulates water and solute transport in plants

We acknowledge support from the ERA-NET Coordinating Action in Plant Sciences program project ERACAPS13.089_RootBarriers, with support from Biotechnology and Biological Sciences Research Council (grant no. BB/N023927/1 to D.E.S.), the German Research Foundation (DFG; grant no. FR 1721/2-1 to R.B.F. and the AgreenSkills+ fellowship programme to MC-P which has received funding from the EU’s Seventh Framework Programme under grant agreement N° FP7-609398 (AgreenSkills+ contract). This work was also funded by the Ministry of Education, Youth and Sports of the Czech Republic (National Program for Sustainability I, grant no. LO1204), the Swedish Governmental Agency for Innovation Systems (Vinnova) and the Swedish Research Council (VR). We thank Kevin Mackenzie (University of Aberdeen–Microscopy Histology Facility) and Carine Alcon (BPMP-PHIV microscopy platform) for assistance using the confocal microscope and stereo microscope for observing the root samples, and the Swedish Metabolomics Centre (http://www.swedishmetabolomicscentre.se/) for access to instrumentation.Peer reviewedPublisher PD

Neutrophils in cancer: neutral no more

Neutrophils are indispensable antagonists of microbial infection and facilitators of wound healing. In the cancer setting, a newfound appreciation for neutrophils has come into view. The traditionally held belief that neutrophils are inert bystanders is being challenged by the recent literature. Emerging evidence indicates that tumours manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumour behaviour. In this Review, we discuss the involvement of neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and therapeutic targets

Agent-Based Modeling of Endotoxin-Induced Acute Inflammatory Response in Human Blood Leukocytes

Inflammation is a highly complex biological response evoked by many stimuli. A persistent challenge in modeling this dynamic process has been the (nonlinear) nature of the response that precludes the single-variable assumption. Systems-based approaches offer a promising possibility for understanding inflammation in its homeostatic context. In order to study the underlying complexity of the acute inflammatory response, an agent-based framework is developed that models the emerging host response as the outcome of orchestrated interactions associated with intricate signaling cascades and intercellular immune system interactions.An agent-based modeling (ABM) framework is proposed to study the nonlinear dynamics of acute human inflammation. The model is implemented using NetLogo software. Interacting agents involve either inflammation-specific molecules or cells essential for the propagation of the inflammatory reaction across the system. Spatial orientation of molecule interactions involved in signaling cascades coupled with the cellular heterogeneity are further taken into account. The proposed in silico model is evaluated through its ability to successfully reproduce a self-limited inflammatory response as well as a series of scenarios indicative of the nonlinear dynamics of the response. Such scenarios involve either a persistent (non)infectious response or innate immune tolerance and potentiation effects followed by perturbations in intracellular signaling molecules and cascades.The ABM framework developed in this study provides insight on the stochastic interactions of the mediators involved in the propagation of endotoxin signaling at the cellular response level. The simulation results are in accordance with our prior research effort associated with the development of deterministic human inflammation models that include transcriptional dynamics, signaling, and physiological components. The hypothetical scenarios explored in this study would potentially improve our understanding of how manipulating the behavior of the molecular species could manifest into emergent behavior of the overall system

To die or not to die : study of the mechanisms of neutrophil survival and death in infectious context

Au cours des 30 dernières années, les formes inflammatoires de mort cellulaire sont apparues comme des processus cruciaux conduisant à l'inflammation et à la défense de l'hôte contre les infections. Le système immunitaire inné de l'hôte joue un rôle important dans la reconnaissance et l'élimination rapides des pathogènes envahissants. La reconnaissance est obtenue par plusieurs classes de récepteurs appelés "Pattern Recognition Receptors" (PRR), qui détectent des molécules microbiennes conservées appelées "Pattern-Associated Molecular Patterns" (PAMPs). Bien que la plupart de ces voies déclenchent la sécrétion de cytokines, les protéines de type récepteur de Nod, appelés "NOD-like Receptors" (NLR), une classe de PRR cytosoliques, déclenchent une réponse plus forte, caractérisée par l'induction de la mort des cellules hôtes et la sécrétion de cytokines inflammatoires via l'assemblage d'inflammasomes. Les inflammasomes sont des complexes multiprotéiques qui activent la caspase-1/-11 inflammatoire, qui à son tour favorisent la libération de cytokines (interleukine (IL)-1ß/-18) et la pyroptose, une forme inflammatoire de mort cellulaire. Les neutrophiles jouent un rôle essentiel contre les infections. Bien que plusieurs types de mort du neutrophile contribuent à la défense de l'hôte contre les infections, comme la NETose, les neutrophiles sont considérés comme incapables d'induire la pyroptose dépendante de la Caspase-1, médiée par l'activation de l'inflammasome. Mon principal travail en thèse consistait à déterminer les mécanismes de régulation de la pyroptose du neutrophile ainsi que les conséquences en immunopathologie. Nous avons identifié la bactérie Pseudomonas aeruginosa comme capable de déclencher une mort Caspase-1-dépendante chez les neutrophiles humains et murins. En particulier, la délétion des Exotoxines U ou S chez P. aeruginosa redirige entièrement la mort des neutrophiles vers la pyroptose dépendante de la Caspase-1. Mécaniquement, P. aeruginosa active l'inflammasome NLRC4 du neutrophile, induisant le clivage de la gasdermine D (GSDMD) par la Caspase-1 et l'induction de pyroptose chez le neutrophile. De plus, nous montrons que le clivage de GSDMD favorise l'activation de la "Peptidyl arginine Deaminase 4" (PAD4) dépendante du calcium. PAD4 induit la citrullination des histones, conduisant à la décondensation et à la translocation de l'ADN dans le cytosol de la cellule hôte, sans qu'il soit expulsé des cellules dans l'environnement extracellulaire. Enfin, nous montrons par microscopie intravitale que la pyroptose du neutrophile se produit in vivo dans les poumons de souris MRP8- GFP infectées par P. aeruginosa, et que la suppression de la caspase-1 spécifiquement dans les neutrophiles (chez les souris MRP8-CreCasp1flox) réduit la production d'IL-1ß et la sensibilité à l'infection à P. aeruginosa in vivo. Ces résultats suggèrent que les neutrophiles ne sont pas résistants à la pyroptose dépendante de Caspase-1, mais sont plutôt finement régulés. En étudiant les neutrophiles défectueux pour la NADPH oxydase chez les patients atteints de Maladies Granulomateuses Chroniques (MGC), nous avons dévoilé une fonction clé de la NADPH oxydase qui limite spécifiquement la pyroptose du neutrophile. Par conséquent, les pathogènes bactériens associés à la MGC déclenchent une pyroptose inflammatoire dépendante des caspases (-1/-4/-11) chez les patients MGC, mais pas dans les neutrophiles de patients sains. Mécaniquement, le manque d'oxydation des Caspases inflammatoires médié par la NADPH oxydase dans divers Cystéines déclenche une pyroptose chez les neutrophiles, inefficace contre tous les pathogènes CGD testés. Ensemble, nos données démontrent que les neutrophiles ne sont pas universellement résistants à la pyroptose dépendante de Caspase-1 chez le neutrophile et avons identifié la NADPH oxydase comme un élément clé, essentiel contre les dommages inflammatoires induits par la pyroptose neutrophile.Over the last 30 years, inflammatory forms of cell death have emerged as crucial processes driving inflammation and host defense against infections. The host innate immune system plays an important role in the rapid recognition and elimination of invading pathogens. Recognition is achieved by several classes of germ-line encoded pattern-recognition receptors (PRRs), which detect conserved microbial molecules called pathogen-associated molecular patterns (PAMPs). While most of these pathways initiate the secretion of cytokines, Nod-like Receptors (NLRs) proteins, one class of cytosolic PRRs, trigger a stronger response, characterised by the induction of host cell death and secretion of inflammatory cytokines via the assembly of inflammasomes. Inflammasomes are multiprotein complexes that activate inflammatory caspase-1/-11, which in turn promote cytokine release (interleukin (IL)-1ß/-18) and pyroptosis, an inflammatory form of host cell death, that kills the infected cells, remove the replicative niche of intracellular pathogens and re-exposes the pathogen to extracellular immune responses. Neutrophils play an essential role against infections. Although multiple neutrophil death programs contribute to host defense against infections, such as NETosis, neutrophils are thought to be defective in Caspase-1-dependent pyroptosis, mediated by the activation of the pro-inflammatory complex inflammasome. My main PhD work consisted in deciphering the mechanisms of regulation of neutrophil pyroptosis and its consequences in immunopathology. For this purpose, we screened several bacterial species for their ability to bypass neutrophil resistance to canonical inflammasome-induced pyroptosis and found that the bacterial pathogen Pseudomonas aeruginosa triggers a Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of the Exotoxins U or S in P. aeruginosa entirely rewires neutrophil death towards Caspase-1-driven pyroptosis, suggesting that these bacterial Exotoxins somehow suppress caspase-1-mediated neutrophil pyroptosis. Mechanistically, P. aeruginosa-induced pyroptosis requires the expression of a functional Type-3 Secretion System (T3SS) and Flagellin. Consequently, P. aeruginosa selectively activates the neutrophil NLRC4 inflammasome, which ensures Caspase1-driven Gasdermin D (GSDMD) cleavage and the induction of neutrophil pyroptosis. Furthermore, we show that GSDMD activation promotes Calcium-dependent Peptidyl Arginine Deaminase 4 (PAD4) activation. PAD4 citrullinates histones, which leads to DNA decondensation and translocation in the host cell cytosol without it being expulsed from the cells into the extracellular environment. Finally, we show by intravital microscopy that neutrophil pyroptosis occurs in lungs of P. aeruginosa-infected MRP8- GFP mice, and that neutrophil-targeted deletion of caspase-1 (in MRP8-CreCasp1flox mice) reduces both IL-1ß production and susceptibility to P. aeruginosa infection in vivo. These results suggested that neutrophils are not resistant to Caspase-1-dependent pyroptosis but are rather tightly regulated. Studying NADPH oxidase-defective neutrophils from Chronic Granulomatous Diseases (CGDs) patients, we unveiled a key function of the NADPH oxidase at specifically restraining neutrophil pyroptosis. Hence, CGD-associated bacterial pathogens triggered inflammatory Caspases (-1/-4/-11)-dependent pyroptosis in CGD but not in healthy neutrophils. Mechanistically, lack of NADPH-dependent oxidation of inflammatory Caspases in various Cysteines unleashed inflammasome-driven pyroptosis in neutrophils, inefficient against all CGD pathogen tested. Together, our data demonstrated that neutrophils are not universally resistant for Caspase- 1-dependent pyroptosis and identified the NADPH oxidase as an essential safe-keeper against neutrophil pyroptosis-driven inflammatory damages

Vivre ou mourir : étude des mécanismes de survie et de mort des neutrophiles dans le contexte infectieux

Over the last 30 years, inflammatory forms of cell death have emerged as crucial processes driving inflammation and host defense against infections. The host innate immune system plays an important role in the rapid recognition and elimination of invading pathogens. Recognition is achieved by several classes of germ-line encoded pattern-recognition receptors (PRRs), which detect conserved microbial molecules called pathogen-associated molecular patterns (PAMPs). While most of these pathways initiate the secretion of cytokines, Nod-like Receptors (NLRs) proteins, one class of cytosolic PRRs, trigger a stronger response, characterised by the induction of host cell death and secretion of inflammatory cytokines via the assembly of inflammasomes. Inflammasomes are multiprotein complexes that activate inflammatory caspase-1/-11, which in turn promote cytokine release (interleukin (IL)-1ß/-18) and pyroptosis, an inflammatory form of host cell death, that kills the infected cells, remove the replicative niche of intracellular pathogens and re-exposes the pathogen to extracellular immune responses. Neutrophils play an essential role against infections. Although multiple neutrophil death programs contribute to host defense against infections, such as NETosis, neutrophils are thought to be defective in Caspase-1-dependent pyroptosis, mediated by the activation of the pro-inflammatory complex inflammasome. My main PhD work consisted in deciphering the mechanisms of regulation of neutrophil pyroptosis and its consequences in immunopathology. For this purpose, we screened several bacterial species for their ability to bypass neutrophil resistance to canonical inflammasome-induced pyroptosis and found that the bacterial pathogen Pseudomonas aeruginosa triggers a Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of the Exotoxins U or S in P. aeruginosa entirely rewires neutrophil death towards Caspase-1-driven pyroptosis, suggesting that these bacterial Exotoxins somehow suppress caspase-1-mediated neutrophil pyroptosis. Mechanistically, P. aeruginosa-induced pyroptosis requires the expression of a functional Type-3 Secretion System (T3SS) and Flagellin. Consequently, P. aeruginosa selectively activates the neutrophil NLRC4 inflammasome, which ensures Caspase1-driven Gasdermin D (GSDMD) cleavage and the induction of neutrophil pyroptosis. Furthermore, we show that GSDMD activation promotes Calcium-dependent Peptidyl Arginine Deaminase 4 (PAD4) activation. PAD4 citrullinates histones, which leads to DNA decondensation and translocation in the host cell cytosol without it being expulsed from the cells into the extracellular environment. Finally, we show by intravital microscopy that neutrophil pyroptosis occurs in lungs of P. aeruginosa-infected MRP8- GFP mice, and that neutrophil-targeted deletion of caspase-1 (in MRP8-CreCasp1flox mice) reduces both IL-1ß production and susceptibility to P. aeruginosa infection in vivo. These results suggested that neutrophils are not resistant to Caspase-1-dependent pyroptosis but are rather tightly regulated. Studying NADPH oxidase-defective neutrophils from Chronic Granulomatous Diseases (CGDs) patients, we unveiled a key function of the NADPH oxidase at specifically restraining neutrophil pyroptosis. Hence, CGD-associated bacterial pathogens triggered inflammatory Caspases (-1/-4/-11)-dependent pyroptosis in CGD but not in healthy neutrophils. Mechanistically, lack of NADPH-dependent oxidation of inflammatory Caspases in various Cysteines unleashed inflammasome-driven pyroptosis in neutrophils, inefficient against all CGD pathogen tested. Together, our data demonstrated that neutrophils are not universally resistant for Caspase- 1-dependent pyroptosis and identified the NADPH oxidase as an essential safe-keeper against neutrophil pyroptosis-driven inflammatory damages.Au cours des 30 dernières années, les formes inflammatoires de mort cellulaire sont apparues comme des processus cruciaux conduisant à l'inflammation et à la défense de l'hôte contre les infections. Le système immunitaire inné de l'hôte joue un rôle important dans la reconnaissance et l'élimination rapides des pathogènes envahissants. La reconnaissance est obtenue par plusieurs classes de récepteurs appelés "Pattern Recognition Receptors" (PRR), qui détectent des molécules microbiennes conservées appelées "Pattern-Associated Molecular Patterns" (PAMPs). Bien que la plupart de ces voies déclenchent la sécrétion de cytokines, les protéines de type récepteur de Nod, appelés "NOD-like Receptors" (NLR), une classe de PRR cytosoliques, déclenchent une réponse plus forte, caractérisée par l'induction de la mort des cellules hôtes et la sécrétion de cytokines inflammatoires via l'assemblage d'inflammasomes. Les inflammasomes sont des complexes multiprotéiques qui activent la caspase-1/-11 inflammatoire, qui à son tour favorisent la libération de cytokines (interleukine (IL)-1ß/-18) et la pyroptose, une forme inflammatoire de mort cellulaire. Les neutrophiles jouent un rôle essentiel contre les infections. Bien que plusieurs types de mort du neutrophile contribuent à la défense de l'hôte contre les infections, comme la NETose, les neutrophiles sont considérés comme incapables d'induire la pyroptose dépendante de la Caspase-1, médiée par l'activation de l'inflammasome. Mon principal travail en thèse consistait à déterminer les mécanismes de régulation de la pyroptose du neutrophile ainsi que les conséquences en immunopathologie. Nous avons identifié la bactérie Pseudomonas aeruginosa comme capable de déclencher une mort Caspase-1-dépendante chez les neutrophiles humains et murins. En particulier, la délétion des Exotoxines U ou S chez P. aeruginosa redirige entièrement la mort des neutrophiles vers la pyroptose dépendante de la Caspase-1. Mécaniquement, P. aeruginosa active l'inflammasome NLRC4 du neutrophile, induisant le clivage de la gasdermine D (GSDMD) par la Caspase-1 et l'induction de pyroptose chez le neutrophile. De plus, nous montrons que le clivage de GSDMD favorise l'activation de la "Peptidyl arginine Deaminase 4" (PAD4) dépendante du calcium. PAD4 induit la citrullination des histones, conduisant à la décondensation et à la translocation de l'ADN dans le cytosol de la cellule hôte, sans qu'il soit expulsé des cellules dans l'environnement extracellulaire. Enfin, nous montrons par microscopie intravitale que la pyroptose du neutrophile se produit in vivo dans les poumons de souris MRP8- GFP infectées par P. aeruginosa, et que la suppression de la caspase-1 spécifiquement dans les neutrophiles (chez les souris MRP8-CreCasp1flox) réduit la production d'IL-1ß et la sensibilité à l'infection à P. aeruginosa in vivo. Ces résultats suggèrent que les neutrophiles ne sont pas résistants à la pyroptose dépendante de Caspase-1, mais sont plutôt finement régulés. En étudiant les neutrophiles défectueux pour la NADPH oxydase chez les patients atteints de Maladies Granulomateuses Chroniques (MGC), nous avons dévoilé une fonction clé de la NADPH oxydase qui limite spécifiquement la pyroptose du neutrophile. Par conséquent, les pathogènes bactériens associés à la MGC déclenchent une pyroptose inflammatoire dépendante des caspases (-1/-4/-11) chez les patients MGC, mais pas dans les neutrophiles de patients sains. Mécaniquement, le manque d'oxydation des Caspases inflammatoires médié par la NADPH oxydase dans divers Cystéines déclenche une pyroptose chez les neutrophiles, inefficace contre tous les pathogènes CGD testés. Ensemble, nos données démontrent que les neutrophiles ne sont pas universellement résistants à la pyroptose dépendante de Caspase-1 chez le neutrophile et avons identifié la NADPH oxydase comme un élément clé, essentiel contre les dommages inflammatoires induits par la pyroptose neutrophile

Regulated cell death in neutrophils : from apoptosis to NETosis and pyroptosis

Neutrophils are among the most abundant immune cells, representing about 50%-70% of all circulating leu-kocytes in humans. Neutrophils rapidly infiltrate inflamed tissues and play an essential role in host defense against infections. They exert microbicidal activity through a variety of specialized effector mechanisms, including phagocytosis, production of reactive oxygen species, degranulation and release of secretory vesicles containing broad-spectrum antimicrobial factors. In addition to their homeostatic turnover by apoptosis, recent studies have revealed the mechanisms by which neutrophils undergo various forms of regulated cell death. In this review, we will discuss the different modes of regulated cell death that have been described in neutrophils, with a particular emphasis on the current understanding of neutrophil pyroptosis and its role in infections and autoinflammation

Age-related variation in health status after age 60.

Disability, functionality, and morbidity are often used to describe the health of the elderly. Although particularly important when planning health and social services, knowledge about their distribution and aggregation at different ages is limited. We aim to characterize the variation of health status in a 60+ old population using five indicators of health separately and in combination.3080 adults 60+ living in Sweden between 2001 and 2004 and participating at the SNAC-K population-based cohort study. Health indicators: number of chronic diseases, gait speed, Mini Mental State Examination (MMSE), disability in instrumental-activities of daily living (I-ADL), and in personal-ADL (P-ADL).Probability of multimorbidity and probability of slow gait speed were already above 60% and 20% among sexagenarians. Median MMSE and median I-ADL showed good performance range until age 84; median P-ADL was close to zero up to age 90. Thirty% of sexagenarians and 11% of septuagenarians had no morbidity and no impairment, 92% and 80% of them had no disability. Twenty-eight% of octogenarians had multimorbidity but only 27% had some I-ADL disability. Among nonagenarians, 13% had severe disability and impaired functioning while 12% had multimorbidity and slow gait speed.Age 80-85 is a transitional period when major health changes take place. Until age 80, most people do not have functional impairment or disability, despite the presence of chronic disorders. Disability becomes common only after age 90. This implies an increasing need of medical care after age 70, whereas social care, including institutionalization, becomes a necessity only in nonagenarians