RESUMEN
Cellular senescence is a response to many stressful insults. DNA damage is a consistent feature of senescent cells, but in many cases its source remains unknown. Here, we identify the cellular endonuclease caspase-activated DNase (CAD) as a critical factor in the initiation of senescence. During apoptosis, CAD is activated by caspases and cleaves the genomic DNA of the dying cell. The CAD DNase is also activated by sub-lethal signals in the apoptotic pathway, causing DNA damage in the absence of cell death. We show that sub-lethal signals in the mitochondrial apoptotic pathway induce CAD-dependent senescence. Inducers of cellular senescence, such as oncogenic RAS, type-I interferon, and doxorubicin treatment, also depend on CAD presence for senescence induction. By directly activating CAD experimentally, we demonstrate that its activity is sufficient to induce senescence in human cells. We further investigate the contribution of CAD to senescence in vivo and find substantially reduced signs of senescence in organs of ageing CAD-deficient mice. Our results show that CAD-induced DNA damage in response to various stimuli is an essential contributor to cellular senescence.
Asunto(s)
Senescencia Celular , Daño del ADN , Humanos , Animales , Ratones , Apoptosis , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/genética , Ratones Noqueados , Doxorrubicina/farmacologíaRESUMEN
The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.
Asunto(s)
Apoptosis/genética , Proteína 11 Similar a Bcl2/metabolismo , Dineínas/metabolismo , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Proteína 11 Similar a Bcl2/genética , Células CACO-2 , Línea Celular Tumoral , Regulación de la Expresión Génica , Células HeLa , Humanos , Células MCF-7 , Ratones , Unión Proteica , Multimerización de Proteína/genética , Estabilidad Proteica , Interferencia de ARN , Proteína X Asociada a bcl-2/genéticaRESUMEN
Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever. In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex. Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Coxiella burnetii/metabolismo , ARN Helicasas DEAD-box/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Fiebre Q/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Apoptosis , Supervivencia Celular , Coxiella burnetii/genética , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Mutación , Fiebre Q/microbiología , Células THP-1RESUMEN
GM-CSF is a potent inflammatory cytokine regulating myeloid cell differentiation, hematopoiesis, and various other functions. It is functionally associated with a number of inflammatory pathologies including rheumatoid arthritis and inflammatory bowel disease. GM-CSF has been found to promote NLRP3-dependent IL-1ß secretion, which may have a significant role in driving inflammatory pathologies. However, the molecular mechanisms remain unknown. Here, we show that GM-CSF induces IL-1ß secretion through a ROS-dependent pathway. TNF is required for reactive oxygen species (ROS) generation that strikingly does not promote NLRP3 activation, but instead drives ubiquitylation of IL-1ß, promoting its cleavage through basal NRLP3 activity. GM-CSF regulates this pathway through suppression of antioxidant responses via preventing upregulation of NRF2. Thus, the pro-inflammatory effect of GM-CSF on IL-1ß is through suppression of antioxidant responses, which leads to ubiquitylation of IL-1ß and enhanced processing. This study highlights the role of metabolic regulation of inflammatory signaling and reveals a novel mechanism for GM-CSF to promote inflammation.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Antioxidantes/farmacología , Células Cultivadas , Regulación hacia Abajo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Inflamasomas/metabolismo , Interleucina-1beta/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
PURPOSE: To analyse recent epidemiological trends of bloodstream infections (BSI) caused by Enterococcus spp. In adult patients admitted to tertiary care centres in Germany. METHODS: Epidemiological data from the multicentre R-NET study was analysed. Patients presenting with E. faecium or E. faecalis in blood cultures in six German tertiary care university hospitals between October 2016 and June 2020 were prospectively evaluated. In vancomycin-resistant enterococci (VRE), the presence of vanA/vanB was confirmed via molecular methods. RESULTS: In the 4-year study period, 3001 patients with BSI due to Enterococcus spp. were identified. E. faecium was detected in 1830 patients (61%) and E. faecalis in 1229 patients (41%). Most BSI occurred in (sub-) specialties of internal medicine. The pooled incidence density of enterococcal BSI increased significantly (4.0-4.5 cases per 10,000 patient days), which was primarily driven by VRE BSI (0.5 to 1.0 cases per 10,000 patient days). In 2020, the proportion of VRE BSI was > 12% in all study sites (range, 12.8-32.2%). Molecular detection of resistance in 363 VRE isolates showed a predominance of the vanB gene (77.1%). CONCLUSION: This large multicentre study highlights an increase of BSI due to E. faecium, which was primarily driven by VRE. The high rates of hospital- and ICU-acquired VRE BSI point towards an important role of prior antibiotic exposure and invasive procedures as risk factors. Due to limited treatment options and high mortality rates of VRE BSI, the increasing incidence of VRE BSI is of major concern.
RESUMEN
Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.
Asunto(s)
Apoptosis/fisiología , Inmunidad/fisiología , Mitocondrias/fisiología , Animales , Muerte Celular/inmunología , Células Cultivadas , Células Epiteliales/fisiología , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Serina Endopeptidasas/fisiología , Transducción de Señal/fisiología , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
OBJECTIVES: To analyse the influence of antibiotic consumption on healthcare-associated healthcare onset (HAHO) Clostridioides difficile infection (CDI) in a German university hospital setting. METHODS: Monthly ward-level antibiotic consumption measured in DDD/100 patient days (pd) and CDI surveillance data from five university hospitals in the period 2017 through 2019 were analysed. Uni- and multivariable analyses were performed with generalized estimating equation models. RESULTS: A total of 225 wards with 7347 surveillance months and 4â036â602 pd participated. With 1184 HAHO-CDI cases, there was a median incidence density of 0.17/1000 pd (IQR 0.03-0.43) across all specialties, with substantial differences among specialties. Haematology-oncology wards showed the highest median incidence density (0.67/1000 pd, IQR 0.44-1.01), followed by medical ICUs (0.45/1000 pd, IQR 0.27-0.73) and medical general wards (0.32/1000 pd, IQR 0.18-0.53). Multivariable analysis revealed carbapenem (mostly meropenem) consumption to be the only antibiotic class associated with increased HAHO-CDI incidence density. Each carbapenem DDD/100 pd administered increased the HAHO-CDI incidence density by 1.3% [incidence rate ratio (IRR) 1.013; 95% CI 1.006-1.019]. Specialty-specific analyses showed this influence only to be valid for haematological-oncological wards. Overall, factors like ward specialty (e.g. haematology-oncology ward IRR 2.961, 95% CI 2.203-3.980) or other CDI cases on ward had a stronger influence on HAHO-CDI incidence density (e.g. community-associated CDI or unknown association case in same month IRR 1.476, 95% CI 1.242-1.755) than antibiotic consumption. CONCLUSIONS: In the German university hospital setting, monthly ward-level carbapenem consumption seems to increase the HAHO-CDI incidence density predominantly on haematological-oncological wards. Furthermore, other patient-specific factors seem to be equally important to control HAHO-CDI.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium , Infección Hospitalaria , Humanos , Antibacterianos/uso terapéutico , Hospitales Universitarios , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Carbapenémicos , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/epidemiología , Incidencia , Estudios RetrospectivosRESUMEN
The mitochondrial apoptosis pathway has the function to kill the cell, but recent work shows that this pathway can also be activated to a sublethal level, where signal transduction can be observed but the cell survives. Intriguingly, this signaling has been shown to contribute to inflammatory activity of epithelial cells upon infection with numerous agents. This suggests that microbial recognition can generate sublethal activity in the mitochondrial apoptosis pathway. Because this recognition is achieved by pattern recognition receptors (PRRs), it also implies that PRR signals are linked to the mitochondrial apoptosis apparatus. We here test this hypothesis during infection of epithelial cells with modified vaccinia virus Ankara (MVA). MVA recognition is achieved through receptors specific for nucleic acids, and we present evidence that the three receptors, Toll-like receptor 3 (TLR3), RIG-I/MDA5, and cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING), are involved in this signaling. When stimulated directly by specific ligands, all three receptors could trigger sublethal apoptosis signals. During infection with MVA, sublethal apoptosis signals were unmasked in X-linked IAP (XIAP)-deficient cells, where apoptosis induction was observed. Deletion of any of the three signaling adapters, TRIF, MAVS, and STING, reduced the DNA damage response, a sensitive measure of sublethal apoptosis signals. Our results suggest that PRRs signal via mitochondria, where they generate sublethal signals through the BCL-2-family, which may contribute to the response to infectious agents. IMPORTANCE A contribution of the mitochondrial apoptosis apparatus, in the absence of cell death, to the reaction of nonprofessional immune cells to viruses is suggested to play a role as a broad alert system of an infected cell: the apoptosis system can be activated by many upstream signals and could therefore act as a central coordinator of viral recognition. The proapoptotic activity of PRRs has been documented in multiple situations, but this activity seems too low to be meaningful, and a physiological significance of such activity is not immediately obvious. This work suggests the alternative interpretation that PRRs do not have the primary function to induce apoptosis but to trigger sublethal signals in the apoptosis system. A number of lines of recent research suggest that mitochondria contribute to cellular reactions, and this pathway may be a way of triggering an early host response.
Asunto(s)
Apoptosis , Mitocondrias , Ácidos Nucleicos , Receptores de Reconocimiento de Patrones , Virosis , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Humanos , Inmunidad Innata , Mitocondrias/inmunología , Nucleotidiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Receptor Toll-Like 3/metabolismo , Virus Vaccinia , Virosis/inmunologíaRESUMEN
The SARS-CoV-2 pandemic has highlighted the importance of viable infection surveillance and the relevant infrastructure. From a German perspective, an integral part of this infrastructure, genomic pathogen sequencing, was at best fragmentary and stretched to its limits due to the lack or inefficient use of equipment, human resources, data management and coordination. The experience in other countries has shown that the rate of sequenced positive samples and linkage of genomic and epidemiological data (person, place, time) represent important factors for a successful application of genomic pathogen surveillance. Planning, establishing and consistently supporting adequate structures for genomic pathogen surveillance will be crucial to identify and combat future pandemics as well as other challenges in infectious diseases such as multi-drug resistant bacteria and healthcare-associated infections. Therefore, the authors propose a multifaceted and coordinated process for the definition of procedural, legal and technical standards for comprehensive genomic pathogen surveillance in Germany, covering the areas of genomic sequencing, data collection and data linkage, as well as target pathogens. A comparative analysis of the structures established in Germany and in other countries is applied. This proposal aims to better tackle epi- and pandemics to come and take action from the "lessons learned" from the SARS-CoV-2 pandemic.
Asunto(s)
COVID-19 , Infección Hospitalaria , Humanos , Pandemias/prevención & control , COVID-19/epidemiología , COVID-19/prevención & control , SARS-CoV-2/genética , GenómicaRESUMEN
The SARS-CoV2 pandemic has shown a deficit of essential epidemiological infrastructure, especially with regard to genomic pathogen surveillance in Germany. In order to prepare for future pandemics, the authors consider it urgently necessary to remedy this existing deficit by establishing an efficient infrastructure for genomic pathogen surveillance. Such a network can build on structures, processes, and interactions that have already been initiated regionally and further optimize them. It will be able to respond to current and future challenges with a high degree of adaptability.The aim of this paper is to address the urgency and to outline proposed measures for establishing an efficient, adaptable, and responsive genomic pathogen surveillance network, taking into account external framework conditions and internal standards. The proposed measures are based on global and country-specific best practices and strategy papers. Specific next steps to achieve an integrated genomic pathogen surveillance include linking epidemiological data with pathogen genomic data; sharing and coordinating existing resources; making surveillance data available to relevant decision-makers, the public health service, and the scientific community; and engaging all stakeholders. The establishment of a genomic pathogen surveillance network is essential for the continuous, stable, active surveillance of the infection situation in Germany, both during pandemic phases and beyond.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/prevención & control , Pandemias/prevención & control , Alemania/epidemiología , GenómicaRESUMEN
Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.
Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteasas Ubiquitina-Específicas , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , UbiquitinaciónRESUMEN
Structure and integrity of the mitochondrial network play important roles in many cellular processes. Loss of integrity can lead to the activation of a variety of signalling pathways and affect the cell's response to infections. The activation of such mitochondria-mediated cellular responses has implications for infection recognition, signal transduction and pathogen control. Although we have a basic understanding of mitochondrial factors such as mitochondrial DNA or RNA that may be involved in processes like pro-inflammatory signalling, the diverse roles of mitochondria in host defence remain unclear. Here we will first summarise the functions of mitochondria in the host cell and provide an overview of the major known mitochondrial stress responses. We will then present recent studies that have contributed to the understanding of the role of mitochondria in infectious diseases and highlight a number of recently investigated models of bacterial and viral infections.
Asunto(s)
Mitocondrias , Virosis , Humanos , Inmunidad Innata , Transducción de SeñalRESUMEN
Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysMcreMcl1fl/fl mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Bacterias/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Inmunización Pasiva/métodos , Intestinos/inmunología , Activación Neutrófila/inmunología , Polisacáridos Bacterianos/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Bacterias/clasificación , Bacterias/efectos de los fármacos , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Intestinos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Polisacáridos Bacterianos/inmunologíaRESUMEN
Innate lymphoid cells (ILCs) comprise five distinct subsets. ILCs are found at mucosal barriers and may fight invading pathogens. Chlamydia is an intracellular bacterium that infects the mucosa of the genital tract and can cause severe tissue damage. Here, we used a mouse infection model with Chlamydia muridarum to measure the reaction of genital tract ILCs to the infection. Tissue-resident natural killer (NK) cells were the largest group in the uninfected female genital tract, and their number did not substantially change. Conventional NK cells were present in the greatest numbers during acute infection, while ILC1s continuously increased to high numbers. ILC2 and ILC3s were found at lower numbers that oscillated by a factor of 2 to 4. The majority of ILC3s transdifferentiated into ILC1s. NK cells and ILC1s produced gamma interferon (IFN-γ) and, rarely, tumor necrosis factor (TNF), but only early in the infection. Lack of B and T cells increased ILC numbers, while the loss of myeloid cells decreased them. ILCs accumulated to a high density in the oviduct, a main site of tissue destruction. ILC subsets are part of the inflammatory and immune reaction during infection with C. muridarum and may contribute to tissue damage during chlamydial infection.
Asunto(s)
Infecciones por Chlamydia/inmunología , Genitales Femeninos/inmunología , Linfocitos/inmunología , Animales , Femenino , Inmunidad Innata , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Interleukin-1ß (IL-1ß) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1ß activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1ß that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1ß by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1ß and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Interleucina-1beta/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Proteínas Portadoras/agonistas , Proteínas Portadoras/metabolismo , Caspasa 1/metabolismo , Inflamasomas/inmunología , Inflamasomas/metabolismo , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/deficiencia , Proteínas Inhibidoras de la Apoptosis/genética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Proteínas Mitocondriales/agonistas , Imitación Molecular , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina-Proteína Ligasas , Proteína Inhibidora de la Apoptosis Ligada a X/deficiencia , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Checkpoint kinase 1 (CHK1) is critical for S-phase fidelity and preventing premature mitotic entry in the presence of DNA damage. Tumor cells have developed a strong dependence on CHK1 for survival, and hence, this kinase has developed into a promising drug target. Chk1 deficiency in mice results in blastocyst death due to G2/M checkpoint failure showing that it is an essential gene and may be difficult to target therapeutically. Here, we show that chemical inhibition of CHK1 kills murine and human hematopoietic stem and progenitor cells (HSPCs) by the induction of BCL2-regulated apoptosis. Cell death in HSPCs is independent of p53 but requires the BH3-only proteins BIM, PUMA, and NOXA. Moreover, Chk1 is essential for definitive hematopoiesis in the embryo. Noteworthy, cell death inhibition in HSPCs cannot restore blood cell formation as HSPCs lacking CHK1 accumulate DNA damage and stop dividing. Moreover, conditional deletion of Chk1 in hematopoietic cells of adult mice selects for blood cells retaining CHK1, suggesting an essential role in maintaining functional hematopoiesis. Our findings establish a previously unrecognized role for CHK1 in establishing and maintaining hematopoiesis.
Asunto(s)
Apoptosis/genética , Células de la Médula Ósea/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Benzodiazepinonas/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/deficiencia , Embrión de Mamíferos , Feto , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazoles/farmacología , Quinolinas/farmacología , Quinuclidinas/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All Chlamydiales are obligate intracellular bacteria, and different species infect vastly different hosts. Chlamydia trachomatis infects human epithelial cells; Parachlamydia acanthamoebae replicates in amoebae. We here report that apoptosis impedes growth of P. acanthamoebae in mammalian cells. In HeLa human epithelial cells, P. acanthamoebae infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-XL or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of P. acanthamoebae in HeLa cells, as measured by fluorescence in situ hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with C. trachomatis inhibited P. acanthamoebae-induced apoptosis, suggesting that the known antiapoptotic activity of C. trachomatis can also block P. acanthamoebae-induced apoptosis. C. trachomatis coinfection could not rescue P. acanthamoebae growth in HeLa; in coinfected cells, C. trachomatis even suppressed the growth of P. acanthamoebae independently of apoptosis, while P. acanthamoebae surprisingly enhanced the growth of C. trachomatis Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.
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Apoptosis , Chlamydiales/fisiología , Resistencia a la Enfermedad , Microbiología Ambiental , Infecciones por Bacterias Gramnegativas/microbiología , Interacciones Huésped-Patógeno , Animales , Biomarcadores , Línea Celular , Chlamydiales/aislamiento & purificación , Células Epiteliales , Células HeLa , Humanos , Mitocondrias/metabolismoRESUMEN
OBJECTIVES: To analyse the rectal carriage rate and the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREfm) recovered from patients upon hospital admission. METHODS: Adult patients were screened at six German university hospitals from five different federal states upon hospital admission for rectal colonization with VREfm between 2014 and 2018. Molecular characterization of VREfm was performed by WGS followed by MLST and core-genome MLST analysis. RESULTS: Of 16350 patients recruited, 263 were colonized with VREfm, with increasing prevalence rates during the 5 year study period (from 0.8% to 2.6%). In total, 78.5% of the VREfm were vanB positive and 20.2% vanA positive, while 1.2% harboured both vanA and vanB. The predominant ST was ST117 (56.7%) followed by ST80 (15%), ST203 (10.9%), ST78 (5.7%) and ST17 (3.2%). ST117/vanB VREfm isolates formed a large cluster of 96 closely related isolates extending across all six study centres and four smaller clusters comprising 13, 5, 4 and 3 isolates each. In contrast, among the other STs inter-regional clonal relatedness was rarely observed. CONCLUSIONS: To our knowledge, this is the largest admission prevalence and molecular epidemiology study of VREfm. These data provide insight into the epidemiology of VREfm at six German university hospitals and demonstrate the remarkable inter-regional clonal expansion of the ST117/vanB VREfm clone.
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Infección Hospitalaria , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Enterococos Resistentes a la Vancomicina , Adulto , Infección Hospitalaria/epidemiología , Enterococcus faecium/genética , Genotipo , Alemania/epidemiología , Infecciones por Bacterias Grampositivas/epidemiología , Hospitales , Humanos , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Prevalencia , Vancomicina , Enterococos Resistentes a la Vancomicina/genéticaRESUMEN
Conditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.
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Comunicación Celular/inmunología , Enfermedad Injerto contra Huésped/inmunología , Íleon/inmunología , Ganglios Linfáticos/inmunología , Mesenterio/inmunología , Neutrófilos/inmunología , Enfermedad Aguda , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/genética , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Íleon/patología , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/genética , Janus Quinasa 1/inmunología , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Ganglios Linfáticos/patología , Mesenterio/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/genética , Infiltración Neutrófila/inmunología , Neutrófilos/patología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The obligate intracellular bacterium Chlamydia trachomatis replicates in a cytosolic vacuole in human epithelial cells. Infection of human cells with C. trachomatis causes substantial changes to many host cell-signalling pathways, but the molecular basis of such influence is not well understood. Studies of gene transcription of the infected cell have shown altered transcription of many host cell genes, indicating a transcriptional response of the host cell to the infection. We here describe that infection of HeLa cells with C. trachomatis as well as infection of murine cells with Chlamydia muridarum substantially inhibits protein synthesis of the infected host cell. This inhibition was accompanied by changes to the ribosomal profile of the infected cell indicative of a block of translation initiation, most likely as part of a stress response. The Chlamydia protease-like activity factor (CPAF) also reduced protein synthesis in uninfected cells, although CPAF-deficient C. trachomatis showed no defect in this respect. Analysis of polysomal mRNA as a proxy of actively transcribed mRNA identified a number of biological processes differentially affected by chlamydial infection. Mapping of differentially regulated genes onto a protein interaction network identified nodes of up- and down-regulated networks during chlamydial infection. Proteomic analysis of protein synthesis further suggested translational regulation of host cell functions by chlamydial infection. These results demonstrate reprogramming of the host cell during chlamydial infection through the alteration of protein synthesis.