RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) originates from normal pancreatic ducts where digestive juice is regularly produced. It remains unclear how PDAC can escape autodigestion by digestive enzymes. Here we show that human PDAC tumour cells use gasdermin E (GSDME), a pore-forming protein, to mediate digestive resistance. GSDME facilitates the tumour cells to express mucin 1 and mucin 13, which form a barrier to prevent chymotrypsin-mediated destruction. Inoculation of GSDME-/- PDAC cells results in subcutaneous but not orthotopic tumour formation in mice. Inhibition or knockout of mucin 1 or mucin 13 abrogates orthotopic PDAC growth in NOD-SCID mice. Mechanistically, GSDME interacts with and transports YBX1 into the nucleus where YBX1 directly promotes mucin expression. This GSDME-YBX1-mucin axis is also confirmed in patients with PDAC. These findings uncover a unique survival mechanism of PDAC cells in pancreatic microenvironments.
Asunto(s)
Adenocarcinoma , Neoplasias Pancreáticas , Proteínas Citotóxicas Formadoras de Poros , Adenocarcinoma/genética , Animales , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mucina-1 , Mucinas , Neoplasias Pancreáticas/patología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Microambiente Tumoral , Proteína 1 de Unión a la Caja YRESUMEN
OBJECTIVE: Trimetazidine (TMZ) exerts a strong inhibitory effect on ischemia/reperfusion (I/R) injury. Inflammation plays a key role in I/R injury. We hypothesized that TMZ may protect cardiomyocytes from I/R injury by inhibiting inflammation. METHODS: The left anterior descending coronary artery was ligated for 30 min followed by 6 h of reperfusion to establish a model of I/R injury. H9c2 cardiomyocytes were subjected to 2 h of hypoxia and 3 h of normoxic conditions to establish a model of hypoxia/reoxygenation (H/R) injury. We monitored the change in pyroptosis by performing Western blot analysis, microscopy and ELISA. RESULTS: I/R and H/R treatment stimulated gasdermin D-N domain (GSDMD-N) expression in cardiomyocytes (sham onefold vs. I/R 2.5-fold; control onefold vs. H/R 2.0-fold). Moreover, TMZ increased the viability of H9c2 cardiomyocytes subjected to H/R treatment (H/R 65.0% vs. H/R + TMZ 85.3%) and reduced the infarct size in vivo (I/R 47.0% vs. I/R + TMZ 28.3%). H/R and I/R treatment increased the levels of TLR4, MyD88, phospho-NF-κB p65 and the NLRP3 inflammasome; however, TMZ reduced the expression of these proteins. Additionally, TMZ inhibited noncanonical inflammasome signaling induced by I/R injury. CONCLUSIONS: In summary, TMZ alleviated pyroptosis induced by myocardial I/R injury through the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, TMZ represents an alternative treatment for myocardial I/R injury.
Asunto(s)
Daño por Reperfusión Miocárdica/tratamiento farmacológico , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Trimetazidina/farmacología , Animales , Masculino , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , FN-kappa B/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Proteínas de Unión a Fosfato/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/fisiologíaRESUMEN
MATERIALS AND METHODS: Locomotor outcomes in perforin-deficient (Pfp-/-) mice and wild-type littermate controls were measured after severe compression injury of the lower thoracic spinal cord up to six weeks after injury. RESULTS: According to both the Basso mouse scale score and single frame motion analysis, motor recovery of Pfp-/- mice was similar to wild-type controls. Interestingly, immunohistochemical analysis of cell types at six weeks after injury showed enhanced cholinergic reinnervation of spinal motor neurons caudal to the lesion site and neurofilament-positive structures at the injury site in Pfp-/- mice, whereas numbers of microglia/macrophages and astrocytes were decreased compared with controls. CONCLUSIONS: We conclude that, although, loss of perforin does not change the locomotor outcome after injury, it beneficially affects diverse cellular features, such as number of axons, cholinergic synapses, astrocytes and microglia/macrophages at or caudal to the lesion site. Perforin's ability to contribute to Rag2's influence on locomotion was observed in mice doubly deficient in perforin and Rag2 which recovered better than Rag2-/- or Pfp-/- mice, suggesting that natural killer cells can cooperate with T- and B-cells in spinal cord injury.
Asunto(s)
Locomoción/fisiología , Regeneración Nerviosa/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Traumatismos de la Médula Espinal/enzimología , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Citotóxicas Formadoras de Poros/deficienciaRESUMEN
Pyroptosis is a highly inflammatory and lytic type of programmed cell death (PCD) commenced by inflammasomes, which sense perturbations in the cytosolic environment. Recently, several ground-breaking studies have linked a family of pore-forming proteins known as gasdermins (GSDMs) to pyroptosis. The human genome encodes six GSDM proteins which have a characteristic feature of forming pores in the plasma membrane resulting in the disruption of cellular homeostasis and subsequent induction of cell death. GSDMs have an N-terminal cytotoxic domain and an auto-inhibitory C-terminal domain linked together through a flexible hinge region whose proteolytic cleavage by various enzymes releases the N-terminal fragment that can insert itself into the inner leaflet of the plasma membrane by binding to acidic lipids leading to pore formation. Emerging studies have disclosed the involvement of GSDMs in various modalities of PCD highlighting their role in diverse cellular and pathological processes. Recently, the cryo-EM structures of the GSDMA3 and GSDMD pores were resolved which have provided valuable insights into the pore formation process of GSDMs. Here, we discuss the current knowledge regarding the role of GSDMs in PCD, structural and molecular aspects of autoinhibition, and pore formation mechanism followed by a summary of functional consequences of gasdermin-induced membrane permeabilization.
Asunto(s)
Apoptosis/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Animales , Humanos , Proteínas Citotóxicas Formadoras de Poros/química , Conformación ProteicaRESUMEN
BACKGROUND: End stage renal disease (ESRD) has caused public health problem with high prevalence worldwide. Peritoneum from peritoneal dialysis patients with ESRD can induce pathological changes of the peritoneum, including fibrosis. The trans-differentiation of pericytes has been found to be closely associated with inflammatory diseases, such as organ fibrosis. However, the function of macrophages in regulating the transition of pericyte to peritoneal fibrosis is unclear. METHODS: Histological examination was conducted using Hematoxylin and eosin (HE) staining and Masson's trichrome staining. The protein levels were determined via western blot. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-1ß concentrations. Gasdermin D (GSDMD) was knocked out in mice by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9). RESULTS: Mice receiving dextrose peritoneal dialysate displayed mesothelial cell monolayer loss and thickness of submesothelial compact zone increase. Moreover, dextrose peritoneal dialysate treatment up-regulated GSDMD expression. GSDMD knockdown inhibited IL-1ß production in macrophages. Further, pericytes were treated with cultural supernatant from macrophages. We found that GSDMD knockdown suppressed fibrosis and vascular endothelial growth factor (VEGF)/phosphoinositide 3-kinase (PI3K) pathway in pericytes. In addition, GSDMD were knocked out in mice using CRISPR/Cas9. The histological examinations revealed that GSDMD-/- alleviated the damage of peritoneal tissue and thickness of submesothelial compact zone. GSDMD-/- attenuated interleukin-1beta (IL-1ß) level and peritoneal fibrosis induced by dextrose peritoneal dialysate treatment in pericytes in vivo. CONCLUSION: These results demonstrated that macrophages can regulate the transition of pericyte to peritoneal fibrosis via the GSDMD/IL-1ß axis, which provides a new therapeutic target.
Asunto(s)
Transición Epitelial-Mesenquimal , Interleucina-1beta/metabolismo , Macrófagos/fisiología , Pericitos/fisiología , Fibrosis Peritoneal/etiología , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animales , Western Blotting , Transición Epitelial-Mesenquimal/fisiología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Interleucina-1beta/fisiología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Pericitos/metabolismo , Fibrosis Peritoneal/metabolismo , Proteínas de Unión a Fosfato/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiologíaRESUMEN
Bacterial infections are predominantly treated with antibiotics, and resistance to antibiotics is becoming an increasing threat to our health. Pore-forming toxins (PFTs) are virulence factors secreted by many pathogenic bacterial strains, both in acute and chronic infections. They are special membrane-targeting proteins that exert toxic effects by forming pores in the cell membrane. Recent studies have elucidated the structure of PFTs and the detailed molecular mechanisms of their pathogenicity. Here, we discuss recent findings that highlight the regulatory mechanisms and important roles of two types of PFTs, α-PFTs and ß-PFTs, in mediating the virulence of bacteria, and the therapeutic potential of targeting PFTs for antibacterial treatment. Therapeutic strategies based on PFTs are highly specific and may alleviate the issue of increasing resistance to antibiotics.
Asunto(s)
Antibacterianos/farmacología , Infecciones Bacterianas/microbiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Animales , Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Infecciones Bacterianas/tratamiento farmacológico , Farmacorresistencia Bacteriana/fisiología , Humanos , Factores de VirulenciaRESUMEN
Pyroptosis is a new form of programmed cell death generated by some inflammasomes, piloting the cleavage of gasdermin (GSDM) and stimulation of dormant cytokines like IL-18 and IL-1ß; these reactions are narrowly linked to certain diseases like diabetic nephropathy and atherosclerosis. Doxorubicin, a typical anthracycline, and famous anticancer drug has emerged as a prominent medication in several cancer chemotherapies, although its application is accompanied with expending of dose-dependent, increasing, irreversible and continuing cardiotoxic side effects. However, the exact path that links the induced pyroptosis to the mechanism by which Doxorubicin (DOX) acts against breast cancer cells is still puzzling. The present study seeks to elucidate the potential link between DOX-induced cell death and pyroptosis in two human breast cancer cell lines (MDA-MB-231 and T47D). We proved that treatment with DOX reduced the cell viability in a dose-dependent way and induced pyroptosis morphology in MDA-MB-231 and T47D cells. Also, protein expression analyses revealed GSDME as a key regulator in DOX-induced pyroptosis and highlighted the related role of Caspase-3 activation. Furthermore, DOX treatments induced intracellular accumulation of ROS, stimulated the phosphorylation of JNK, and Caspase-3 activation, subsequently. In conclusion, the study suggests that GSDME triggered DOX-induced pyroptosis in the caspase-3 dependent reactions through the ROS/JNK signalling pathway. Additionally, it showed that the DOX-induced cardiotoxicity and pyroptosis in breast cancer cells can be minimized by reducing the protein level of GSDME; thus, these outcomes provide a new research target and implications for the anticancer investigations and therapeutic applications.
Asunto(s)
Neoplasias de la Mama , Caspasa 3/fisiología , Doxorrubicina/farmacología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Piroptosis/efectos de los fármacos , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Antimicrobial peptides (AMPs) have been regarded as a potential weapon to fight against drug-resistant bacteria, which is threating the globe. Thus, more and more AMPs had been designed or identified. There is a need to integrate them into a platform for researchers to facilitate investigation and analyze existing AMPs. The AMP database has become an important tool for the discovery and transformation of AMPs as agents. A database linking antimicrobial peptides (LAMPs), launched in 2013, serves as a comprehensive tool to supply exhaustive information of AMP on a single platform. LAMP2, an updated version of LAMP, holds 23 253 unique AMP sequences and expands to link 16 public AMP databases. In the current version, there are more than 50% (12 236) sequences only linking a single database and more than 45% of AMPs linking two or more database links. Additionally, updated categories based on primary structure, collection, composition, source and function have been integrated into LAMP2. Peptides in LAMP2 have been integrated in 8 major functional classes and 38 functional activities. More than 89% (20 909) of the peptides are experimentally validated peptides. A total of 1924 references were extracted and regarded as the evidence for supporting AMP activity and cytotoxicity. The updated version will be helpful to the scientific community.
Asunto(s)
Sistemas de Administración de Bases de Datos , Bases de Datos de Proteínas , Proteínas Citotóxicas Formadoras de Poros , Internet , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiologíaRESUMEN
The aggregation and accumulation of amyloid-ß plaques and tau proteins in the brain have been central characteristics in the pathophysiology of Alzheimer's disease (AD), making them the focus of most of the research exploring potential therapeutics for this neurodegenerative disease. With success in interventions aimed at depleting amyloid-ß peptides being limited at best, a greater understanding of the physiological role of amyloid-ß peptides is needed. The development of amyloid-ß plaques has been determined to occur 10-20 years prior to AD symptom manifestation, hence earlier interventions might be necessary to address presymptomatic AD. Furthermore, recent studies have suggested that amyloid-ß peptides may play a role in innate immunity as an antimicrobial peptide. These findings, coupled with the evidence of pathogens such as viruses and bacteria in AD brains, suggests that the buildup of amyloid-ß plaques could be a response to the presence of viruses and bacteria. This has led to the foundation of the antimicrobial hypothesis for AD. The present review will highlight the current understanding of amyloid-ß, and the role of bacteria and viruses in AD, and will also explore the therapeutic potential of antimicrobial and antiviral drugs in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Antiinfecciosos/uso terapéutico , Antivirales/uso terapéutico , Encéfalo/microbiología , Encéfalo/virología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/microbiología , Enfermedad de Alzheimer/virología , Péptidos beta-Amiloides/fisiología , Animales , Antiinfecciosos/farmacología , Antivirales/farmacología , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/tratamiento farmacológico , Barrera Hematoencefálica , Ensayos Clínicos como Asunto , Citocinas/metabolismo , Flavonoides/farmacología , Flavonoides/uso terapéutico , Humanos , Inmunidad Innata , Inflamación , Ratones , Ratones Noqueados , Neuroglía/metabolismo , Proteínas Citotóxicas Formadoras de Poros/fisiología , Terapias en Investigación , Virosis/complicaciones , Virosis/tratamiento farmacológicoRESUMEN
Perforin-2 (P2) is a pore-forming protein with cytotoxic activity against intracellular bacterial pathogens. P2 knockout (P2KO) mice are unable to control infections and die from normally non-lethal bacterial infections. Here we show that P2KO mice as compared to WT mice show significantly higher levels of systemic inflammation, measured by inflammatory markers in serum, due to continuous microbial translocation from the gut which cannot be controlled as these mice lack P2. Systemic inflammation in young and old P2KO mice induces intrinsic B cell inflammation. Systemic and B cell intrinsic inflammation are negatively associated with in vivo and in vitro antibody responses. Chronic inflammation leads to class switch recombination defects, which are at least in part responsible for the reduced in vivo and in vitro antibody responses in young and old P2KO vs. WT mice. These defects include the reduced expression of activation-induced cytidine deaminase (AID), the enzyme for class switch recombination, somatic hypermutation and IgG production and of its transcriptional activators E47 and Pax5. Of note, the response of young P2KO mice is not different from the one observed in old WT mice, suggesting that the chronic inflammatory status of mice lacking P2 may accelerate, or be equivalent, to that seen in old mice. The inflammatory status of the splenic B cells is associated with increased frequencies and numbers of the pro-inflammatory B cell subset called Age-associated B Cells (ABCs) in the spleen and the visceral adipose tissue (VAT) of P2KO old mice. We show that B cells differentiate into ABCs in the VAT following interaction with the adipocytes and their products, and this occurs more in the VAT of P2KO mice as compared to WT controls. This is to our knowledge the first study on B cell function and antibody responses in mice lacking P2.
Asunto(s)
Linfocitos B/inmunología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Adipocitos/patología , Animales , Formación de Anticuerpos , Traslocación Bacteriana , Diferenciación Celular , Cambio de Clase de Inmunoglobulina , Vacunas contra la Influenza/inmunología , Masculino , Ratones , Bazo/inmunologíaRESUMEN
Apicomplexan parasites cause diseases, including malaria and toxoplasmosis, in a range of hosts, including humans. These intracellular parasites utilize pore-forming proteins that disrupt host cell membranes to either traverse host cells while migrating through tissues or egress from the parasite-containing vacuole after replication. This review highlights recent insight gained from the newly available three-dimensional structures of several known or putative apicomplexan pore-forming proteins that contribute to cell traversal or egress. These new structural advances suggest that parasite pore-forming proteins use distinct mechanisms to disrupt host cell membranes at multiple steps in parasite life cycles. How proteolytic processing, secretion, environment, and the accessibility of lipid receptors regulate the membranolytic activities of such proteins is also discussed.
Asunto(s)
Apicomplexa/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Proteínas Protozoarias/fisiología , Animales , Interacciones Huésped-Parásitos , Humanos , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Protozoarias/químicaRESUMEN
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disease. Inherited forms of HLH are caused by biallelic mutations in several effectors of granule-dependent lymphocyte-mediated cytotoxicity. A small proportion of patients with a so-called "secondary" form of HLH, which develops in the aftermath of infection, autoimmunity, or cancer, carry a monoallelic mutation in one or more HLH-associated genes. Although this observation suggests that HLH may have a polygenic mode of inheritance, the latter is very difficult to prove in humans. In order to determine whether the accumulation of partial genetic defects in lymphocyte-mediated cytotoxicity can contribute to the development of HLH, we generated mice that were doubly or triply heterozygous for mutations in HLH-associated genes, those coding for perforin, Rab27a, and syntaxin-11. We found that the accumulation of monoallelic mutations did indeed increase the risk of developing HLH immunopathology after lymphocytic choriomeningitis virus infection. In mechanistic terms, the accumulation of heterozygous mutations in the two degranulation genes Rab27a and syntaxin-11, impaired the dynamics and secretion of cytotoxic granules at the immune synapse of T lymphocytes. In addition, the accumulation of heterozygous mutations within the three genes impaired natural killer lymphocyte cytotoxicity in vivo. The genetic defects can be ranked in terms of the severity of the resulting HLH manifestations. Our results form the basis of a polygenic model of the occurrence of secondary HLH.
Asunto(s)
Linfohistiocitosis Hemofagocítica/genética , Herencia Multifactorial , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Qa-SNARE/genética , Proteínas de Unión al GTP rab/genética , Animales , Degranulación de la Célula/genética , Cruzamientos Genéticos , Citotoxicidad Inmunológica/genética , Dosificación de Gen , Predisposición Genética a la Enfermedad , Heterocigoto , Sinapsis Inmunológicas/inmunología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Mutación , Proteínas Citotóxicas Formadoras de Poros/fisiología , Proteínas Qa-SNARE/fisiología , Organismos Libres de Patógenos Específicos , Linfocitos T Citotóxicos/inmunología , Proteínas de Unión al GTP rab/fisiología , Proteínas rab27 de Unión a GTPRESUMEN
Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-γ pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Vigilancia Inmunológica/inmunología , Mieloma Múltiple/inmunología , Proteínas de Neoplasias/fisiología , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Antineoplásicos/uso terapéutico , Ácidos Borónicos/uso terapéutico , Bortezomib , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Cruzamientos Genéticos , Ciclofosfamida/uso terapéutico , Progresión de la Enfermedad , Genes myc , Predisposición Genética a la Enfermedad , Inmunoterapia , Interferón gamma/deficiencia , Interferón gamma/genética , Interferón gamma/fisiología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Pirazinas/uso terapéutico , Receptores Virales/deficiencia , Receptores Virales/genética , Receptores Virales/fisiología , Carga Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Celular/genética , Interferón gamma/fisiología , Peste/inmunología , Neumonía Bacteriana/inmunología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Yersinia pestis/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peste/complicaciones , Peste/genética , Neumonía Bacteriana/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Membranes are essential in defining the border and ensuring function of all living cells. As such they are vulnerable and have been a preferred target of attack throughout evolution. The most powerful way of damaging a membrane is through the insertion of pore-forming proteins. Research over the last decades shows that such proteins are produced by bacteria to attack bacterial or eukaryotic cells, vertebrates to kill invading organisms or infected cells, and by eukaryotic cells to "kill" mitochondria and trigger apoptosis. The breadth of effect of these proteins is bringing together, in a very exciting way, research communities that used to be unaware of each other.
Asunto(s)
Toxinas Bacterianas/metabolismo , Membrana Celular/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Animales , Bacterias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacología , Humanos , Perforina/química , Perforina/farmacología , Perforina/fisiología , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/farmacologíaRESUMEN
Perforin (PFN) is the key pore-forming molecule in the cytotoxic granules of immune killer cells. Expressed only in killer cells, PFN is the rate-limiting molecule for cytotoxic function, delivering the death-inducing granule serine proteases (granzymes) into target cells marked for immune elimination. In this chapter we describe our current understanding of how PFN accomplishes this task. We discuss where PFN is expressed and how its expression is regulated, the biogenesis and storage of PFN in killer cells and how they are protected from potential damage, how it is released, how it delivers Granzymes into target cells and the consequences of PFN deficiency.
Asunto(s)
Citotoxicidad Inmunológica , Neoplasias/inmunología , Perforina/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Virus/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Humanos , Inmunidad Celular/fisiología , Perforina/química , Proteínas Citotóxicas Formadoras de Poros/químicaRESUMEN
The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by numerous Gram-positive bacterial pathogens. These toxins are released in the extracellular environment as water-soluble monomers or dimers that bind to cholesterol-rich membranes and assemble into large pore complexes. Depending upon their concentration, the nature of the host cell and membrane (cytoplasmic or intracellular) they target, the CDCs can elicit many different cellular responses. Among the CDCs, listeriolysin O (LLO), which is a major virulence factor of the facultative intracellular pathogen Listeria monocytogenes, is involved in several stages of the intracellular lifecycle of the bacterium and displays unique characteristics. It has long been known that following L. monocytogenes internalization into host cells, LLO disrupts the internalization vacuole, enabling the bacterium to replicate into the host cell cytosol. LLO is then used by cytosolic bacteria to spread from cell to cell, avoiding bacterial exposure to the extracellular environment. Although LLO is continuously produced during the intracellular lifecycle of L. monocytogenes, several processes limit its toxicity to ensure the survival of infected cells. It was previously thought that LLO activity was limited to mediating vacuolar escape during bacterial entry and cell to cell spreading. This concept has been challenged by compelling evidence suggesting that LLO secreted by extracellular L. monocytogenes perforates the host cell plasma membrane, triggering important host cell responses. This chapter provides an overview of the well-established intracellular activity of LLO and the multiple roles attributed to LLO secreted by extracellular L. monocytogenes.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Proteínas Hemolisinas/fisiología , Listeria monocytogenes/patogenicidad , Animales , Toxinas Bacterianas/química , Colesterol/metabolismo , Citotoxinas/química , Citotoxinas/metabolismo , Citotoxinas/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/química , Proteínas Hemolisinas/química , Humanos , Listeria monocytogenes/genética , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/fisiología , Factores de Virulencia/química , Factores de Virulencia/fisiologíaRESUMEN
Proteins with membrane-attack complex/perforin (MACPF) domains are found in almost all kingdoms of life, and they have a variety of biological roles, including defence and attack, organism development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with another group of proteins with unknown biological function, known as aegerolysins. These two protein groups coincide in only a few species, and they might operate in concert as cytolytic bi-component pore-forming agents. Representative proteins here include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These have been shown to act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin content. The aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style is discussed.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/fisiología , Proteínas Fúngicas/fisiología , Proteínas Hemolisinas/fisiología , Perforina/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Secuencia de Aminoácidos , Animales , Complejo de Ataque a Membrana del Sistema Complemento/química , Proteínas Fúngicas/química , Proteínas Hemolisinas/química , Humanos , Datos de Secuencia Molecular , Perforina/química , Filogenia , Pleurotus/genética , Pleurotus/patogenicidad , Proteínas Citotóxicas Formadoras de Poros/química , Multimerización de Proteína/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Infection with murine gammaherpesvirus 68 has become an accepted model for studying the virus/host interactions with regard to gammaherpesvirus infections. Previous studies using gene-deficient mice have revealed that neither IFNγ nor perforin is essential in controlling the outcome of infection or the virus load during chronic infection in C57BL/6 mice. However, pronounced multiorgan fibrosis and splenic atrophy are observed in mice lacking IFNγ or the IFNγ receptor. To study the interplay between perforin and IFNγ in controlling the virus-induced pathology and the viral load during chronic gammaherpesvirus infection, we infected IFNγ/perforin double-deficient C57BL/6 mice and followed the course of infection. While absence of perforin prevented the splenic atrophy in IFNγ-deficient mice, fibrosis did not disappear. Moreover, double-deficient mice developed extreme splenomegaly, were unable to control the viral load and displayed chronic immune activation. Thus, IFNγ and perforin act in concert to minimize pathology and control the viral load in mice chronically infected with MHV68. Furthermore, while certain aspect of the virus-induced pathology in IFNγ-deficient mice may be alleviated in double-deficient mice, other aspects are exaggerated, and the normal architecture of the spleen is completely destroyed. We believe that these findings add to the understanding of the virus/host interaction during chronic gammaherpes virus infection.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Interferón gamma/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Rhadinovirus , Animales , Quimiocina CXCL1/sangre , Citocinas/sangre , Femenino , Infecciones por Herpesviridae/patología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Receptores de Interferón/fisiología , Receptor de Interferón gammaRESUMEN
Pore-forming toxins (PFTs) form a large group of bacterial virulence factors that play an important role in various infectious diseases. These include infections with problematic pathogens such as Streptococcus pneumoniae, Staphylococcus aureus, group A and B streptococci, Escherichia coli and Mycobacterium tuberculosis. PFTs perforate host cell membranes, which contributes to the establishment or exacerbation of an infection mainly in two ways: first, by disrupting the host immune response, and second, by helping bacteria to cross epithelial and endothelial barriers, thus allowing them to spread to other parts of the host. Although perforation of the plasma membrane can lead to host cell death, cells possess molecular defence mechanisms and under certain conditions can successfully defend themselves against PFTs. PFTs, as well as the immune response against PFTs, form a potential target for novel prophylactics and therapeutics against bacterial infectious disease, including against antibiotic-resistant strains.