RESUMO
The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis-important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens.
Assuntos
Citotoxinas/toxicidade , Leucócitos/metabolismo , Lipossomos/química , Streptococcus pyogenes/metabolismo , Streptococcus/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Colesterol/metabolismo , Humanos , Leucócitos/efeitos dos fármacos , Testes de NeutralizaçãoRESUMO
BACKGROUND: Streptococcal infections are associated with life-threatening pneumonia and sepsis. The rise in antibiotic resistance calls for novel approaches to treat bacterial diseases. Anti-virulence strategies promote a natural way of pathogen clearance by eliminating the advantage provided to bacteria by their virulence factors. In contrast to antibiotics, anti-virulence agents are less likely to exert selective evolutionary pressure, which is a prerequisite for the development of drug resistance. As part of their virulence mechanism, many bacterial pathogens secrete cytolytic exotoxins (hemolysins) that destroy the host cell by destabilizing their plasma membrane. Liposomal nanotraps, mimicking plasmalemmal structures of host cells that are specifically targeted by bacterial toxins are being developed in order to neutralize-by competitive sequestration-numerous exotoxins. RESULTS: In this study, the liposomal nanotrap technology is further developed to simultaneously neutralize the whole palette of cytolysins produced by Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis-pathogens that can cause life-threatening streptococcal toxic shock syndrome. We show that the mixture of liposomes containing high amounts of cholesterol and liposomes composed exclusively of choline-containing phospholipids is fully protective against the combined action of exotoxins secreted by these pathogens. CONCLUSIONS: Unravelling the universal mechanisms that define targeting of host cells by streptococcal cytolysins paves the way for a broad-spectrum anti-toxin therapy that can be applied without a diagnostic delay for the treatment of bacterial infections including those caused by antibiotic-resistant pathogens.
Assuntos
Lipossomos/farmacologia , Lipossomos/uso terapêutico , Infecções Estreptocócicas/tratamento farmacológico , Antibacterianos/uso terapêutico , Toxinas Bacterianas , Diagnóstico Tardio , Proteínas Hemolisinas , Humanos , Streptococcus , Streptococcus pyogenesRESUMO
Pore-forming toxins (PFTs) form multimeric trans-membrane pores in cell membranes that differ in pore channel diameter (PCD). Cellular resistance to large PFTs (>20 nm PCD) was shown to rely on Ca2+ influx activated membrane repair mechanisms. Small PFTs (<2 nm PCD) were shown to exhibit a high cytotoxic activity, but host cell response and membrane repair mechanisms are less well studied. We used monocytic immune cell lines to investigate the cellular resistance and host membrane repair mechanisms to small PFTs lysenin (Eisenia fetida) and aerolysin (Aeromonas hydrophila). Lysenin, but not aerolysin, is shown to induce Ca2+ influx from the extracellular space and to activate Ca2+ dependent membrane repair mechanisms. Moreover, lysenin binds to U937 cells with higher efficiency as compared to THP-1 cells, which is in line with a high sensitivity of U937 cells to lysenin. In contrast, aerolysin equally binds to U937 or THP-1 cells, but in different plasma membrane areas. Increased aerolysin induced cell death of U937 cells, as compared to THP-1 cells, is suggested to be a consequence of cap-like aerolysin binding. We conclude that host cell resistance to small PFTs attack comprises binding efficiency, pore localization, and capability to induce Ca2+ dependent membrane repair mechanisms.
Assuntos
Toxinas Bacterianas/toxicidade , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Toxinas Biológicas/toxicidade , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Resistência a Medicamentos , Genes Reporter , Humanos , Monócitos/metabolismo , Monócitos/patologia , Células THP-1 , Células U937RESUMO
Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid specifically enriched in the late endosome-lysosome compartment playing a crucial role for the fate of endocytosed components. Due to its presence in extracellular fluids during diseases associated with endolysosomal dysfunction, it is considered as a possible biomarker of disorders such as genetic lysosomal storage diseases and cationic amphiphilic drug-induced phospholipidosis. However, there is no true validation of this biomarker in human studies, nor a clear identification of the carrier of this endolysosome-specific lipid in biofluids. The present study demonstrates that in absence of any sign of renal failure, BMP, especially all docosahexaenoyl containing species, are significantly increased in the urine of patients treated with the antiarrhythmic drug amiodarone. Such urinary BMP increase could reflect a generalized drug-induced perturbation of the endolysosome compartment as observed in vitro with amiodarone-treated human macrophages. Noteworthy, BMP was associated with extracellular vesicles (EVs) isolated from human urines and extracellular medium of human embryonic kidney HEK293 cells and co-localizing with classical EV protein markers CD63 and ALIX. In the context of drug-induced endolysosomal dysfunction, increased BMP-rich EV release could be useful to remove excess of undigested material. This first human pilot study not only reveals BMP as a urinary biomarker of amiodarone-induced endolysosomal dysfunction, but also highlights its utility to prove the endosomal origin of EVs, also named as exosomes. This peculiar lipid already known as a canonical late endosome-lysosome marker, may be thus considered as a new lipid marker of urinary exosomes.
Assuntos
Endossomos/química , Endossomos/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Idoso , Amiodarona/efeitos adversos , Animais , Antiarrítmicos/efeitos adversos , Biomarcadores/urina , Endossomos/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Nefropatias/induzido quimicamente , Lisofosfolipídeos/química , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Monoglicerídeos/química , Projetos Piloto , Ratos , Células THP-1RESUMO
Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.
Assuntos
Toxinas Bacterianas/metabolismo , Estruturas da Membrana Celular/metabolismo , Estruturas da Membrana Celular/patologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patologia , Cálcio/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Células Jurkat , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Streptococcus pneumoniae/patogenicidade , Células THP-1 , Células U937RESUMO
BACKGROUND: Epidermal hyperplasia represents a morphologic hallmark of psoriatic skin lesions. Langerhans cells (LCs) in the psoriatic epidermis engage with keratinocytes (KCs) in tight physical interactions; moreover, they induce T-cell-mediated immune responses critical to psoriasis. OBJECTIVE: This study sought to improve the understanding of epidermal factors in psoriasis pathogenesis. METHODS: BMP7-LCs versus TGF-ß1-LCs were phenotypically characterized and their functional properties were analyzed using flow cytometry, cell kinetic studies, co-culture with CD4 T cells, and cytokine measurements. Furthermore, immunohistology of healthy and psoriatic skin was performed. Additionally, in vivo experiments with Junf/fJunBf/fK5cre-ERT mice were carried out to assess the role of bone morphogenetic protein (BMP) signaling in psoriatic skin inflammation. RESULTS: This study identified a KC-derived signal (ie, BMP signaling) to promote epidermal changes in psoriasis. Whereas BMP7 is strictly confined to the basal KC layer in the healthy skin, it is expressed at high levels throughout the lesional psoriatic epidermis. BMP7 instructs precursor cells to differentiate into LCs that phenotypically resemble psoriatic LCs. These BMP7-LCs exhibit proliferative activity and increased sensitivity to bacterial stimulation. Moreover, aberrant high BMP signaling in the lesional epidermis is mediated by a KC intrinsic mechanism, as suggested from murine data and clinical outcome after topical antipsoriatic treatment in human patients. CONCLUSIONS: These data indicate that available TGF-ß family members within the lesional psoriatic epidermis preferentially signal through the canonical BMP signaling cascade to instruct inflammatory-type LCs and to promote psoriatic epidermal changes. Targeting BMP signaling might allow to therapeutically interfere with cutaneous psoriatic manifestations.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Linfócitos T CD4-Positivos/imunologia , Epiderme/imunologia , Inflamação/imunologia , Queratinócitos/fisiologia , Células de Langerhans/imunologia , Psoríase/metabolismo , Adulto , Idoso , Animais , Proteína Morfogenética Óssea 7/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Citocinas/metabolismo , Epiderme/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Adulto JovemRESUMO
Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.-Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.
Assuntos
Toxinas Bacterianas/farmacologia , Membrana Celular/metabolismo , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Estreptolisinas/farmacologia , Proteínas de Bactérias/farmacologia , Transporte Biológico , Sistemas CRISPR-Cas , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Humanos , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bacterial infectious diseases are a leading cause of death. Pore-forming toxins (PFTs) are important virulence factors of Gram-positive pathogens, which disrupt the plasma membrane of host cells and can lead to cell death. Yet, host defense and cell membrane repair mechanisms have been identified: i.e., PFTs can be eliminated from membranes as microvesicles, thus limiting the extent of cell damage. Released into an inflammatory environment, these host-derived PFTs-carrying microvesicles encounter innate immune cells as first-line defenders. This study investigated the impact of microvesicle- or liposome-sequestered PFTs on human macrophage polarization in vitro. We show that microvesicle-sequestered PFTs are phagocytosed by macrophages and induce their polarization into a novel CD14+MHCIIlowCD86low phenotype. Macrophages polarized in this way exhibit an enhanced response to Gram-positive bacterial ligands and a blunted response to Gram-negative ligands. Liposomes, which were recently shown to sequester PFTs and so protect mice from lethal bacterial infections, show the same effect on macrophage polarization in analogy to host-derived microvesicles. This novel type of polarized macrophage exhibits an enhanced response to Gram-positive bacterial ligands. The specific recognition of their cargo might be of advantage in the efficiency of targeted bacterial clearance.
Assuntos
Toxinas Bacterianas/imunologia , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/imunologia , Transdução de Sinais , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade , Imunomodulação , Imunofenotipagem , Modelos Biológicos , Monócitos/imunologia , Monócitos/metabolismo , FenótipoRESUMO
Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca2+-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores. Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Bicamadas Lipídicas/metabolismo , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/fisiologia , Proteínas de Bactérias/metabolismo , Derrame de Bactérias/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Colesterol/metabolismo , Células HEK293 , Humanos , Microscopia Intravital , Bicamadas Lipídicas/imunologia , Microfluídica , Infecções Pneumocócicas/microbiologia , Estreptolisinas/metabolismoRESUMO
BACKGROUND: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency. OBJECTIVE: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. METHODS: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. RESULTS: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-ß1, thereby allowing LC differentiation marked by a low cytokine expression profile. CONCLUSION: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.
Assuntos
Células Dendríticas/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Monócitos/citologia , Pele/citologia , Adulto , Diferenciação Celular/fisiologia , Células Cultivadas , Células Dendríticas/metabolismo , Embrião de Mamíferos , Sangue Fetal/citologia , Humanos , Inflamação/metabolismo , Fator 4 Semelhante a Kruppel , Monócitos/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.
Assuntos
Diferenciação Celular/imunologia , Inflamação/imunologia , MAP Quinase Quinase 6/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Animais , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/imunologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HL-60 , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/farmacologia , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/imunologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-ß1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-ß1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-ß1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-ß1-ALK5 signaling. Conversely, TGF-ß1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-ß1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-ß1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-ß1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-ß1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Células de Langerhans/citologia , Células de Langerhans/metabolismo , Animais , Proteína Morfogenética Óssea 7/deficiência , Proteína Morfogenética Óssea 7/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/biossíntese , Células Epidérmicas , Epiderme/embriologia , Epiderme/metabolismo , Expressão Gênica , Humanos , Células de Langerhans/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
BACKGROUND AND AIM: Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in myeloid leukemia. We therefore studied its expression and function in cluster of differentiation 34-positive (CD34(+)) primary human hematopoietic progenitor cells. MATERIALS AND METHODS: CD34(+) cells were differentiated into various myeloid lineages using the appropriate cytokines. EVI1 expression was measured by quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR) and intranuclear fluorescence-activated cell sorting (FACS). Experimental manipulation of EVI1 levels was achieved using retroviral infection. RESULTS: EVI1 mRNA and its variant myelodysplastic syndrome 1 (MDS1)/EVI1, which gives rise to a partially antagonistic protein, were detectable in CD34(+) cells, but their levels declined rapidly during differentiation into the granulocyte, monocyte, dendritic, erythroid, and megakaryocyte lineages. Similarly, EVI1 protein levels decreased during myeloid differentiation. Attempts to experimentally express EVI1 in CD34(+) and U937 cells indicated that ectopic expression of EVI1 may cause growth arrest, apoptosis and/or senescence of human hematopoietic cells. CONCLUSION: EVI1 is expressed in human hematopoietic progenitor cells, but is down-regulated during differentiation. Ectopic expression of EVI1 may activate cellular safeguards against oncogene activation.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Proteínas de Ligação a DNA/biossíntese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/biossíntese , Antígenos CD34/biossíntese , Citometria de Fluxo , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Proto-Oncogenes , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937RESUMO
Transforming growth factor-ß1 (TGF-ß1) is a fundamental regulator of immune cell development and function. In this study, we investigated the effects of TGF-ß1 on the differentiation of human Langerhans cells (LCs) and identified Axl as a key TGF-ß1 effector. Axl belongs to the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, whose members function as inhibitors of innate inflammatory responses in dendritic cells and are essential to the prevention of lupus-like autoimmunity. We found that Axl expression is induced by TGF-ß1 during LC differentiation and that LC precursors acquire Axl early during differentiation. We also describe prominent steady-state expression as well as inflammation-induced activation of Axl in human epidermal keratinocytes and LCs. TGF-ß1-induced Axl enhances apoptotic cell (AC) uptake and blocks proinflammatory cytokine production. The antiinflammatory role of Axl in the skin is reflected in a marked impairment of the LC network preceding spontaneous skin inflammation in mutant mice that lack all three TAM receptors. Our findings highlight the importance of constitutive Axl expression to tolerogenic barrier immunity in the epidermis and define a mechanism by which TGF-ß1 enables silent homeostatic clearing of ACs to maintain long-term self-tolerance.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Células de Langerhans/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Diferenciação Celular/genética , Células Cultivadas , Dermatite de Contato/genética , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Homeostase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Células de Langerhans/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved.
Assuntos
Acetiltransferases/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteroides/metabolismo , Esteróis/metabolismo , Acetilação , Acetiltransferases/genética , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico/fisiologia , Hidrolases de Éster Carboxílico/genética , Homeostase , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Pregnenolona/metabolismo , Pregnenolona/toxicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Esteroides/química , Esterol Esterase , Esteróis/química , Especificidade por SubstratoRESUMO
Steryl esters are stored in intracellular lipid droplets from which they are mobilized upon demand and hydrolyzed to yield free sterols and fatty acids. The mechanisms that control steryl ester mobilization are not well understood. We have previously identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester hydrolysis, Yeh1, Yeh2, and Tgl1 (R. Köffel, R. Tiwari, L. Falquet, and R. Schneiter, Mol. Cell. Biol. 25:1655-1668, 2005). Both Yeh1 and Tgl1 localize to lipid droplets, whereas Yeh2 is localized to the plasma membrane. To characterize the precise function of these three partially redundant lipases, we examined steryl ester mobilization under heme-deficient conditions. S. cerevisiae is a facultative anaerobic organism that becomes auxotrophic for sterols and unsaturated fatty acids in the absence of molecular oxygen. Anaerobic conditions can be mimicked in cells that are deficient for heme synthesis. We here report that Yeh1 is the sole active steryl ester hydrolase under such heme-deficient conditions, indicating that Yeh1 is activated whereas Yeh2 and Tgl1 are inactivated by the lack of heme. The heme-dependent activation of Yeh1 is mediated at least in part by an increase in steady-state levels of Yeh1 at the expense of Yeh2 and Tgl1 in exponentially growing cells. This increase in steady-state levels of Yeh1 requires Rox3, a component of the mediator complex that regulates transcription by RNA polymerase II. These data thus provide the first link between fat degradation and the transcriptional control of lipase activity in yeast.
Assuntos
Carboxilesterase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/fisiologia , Heme/deficiência , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Ácidos Graxos , Complexo Mediador , RNA Polimerase II/fisiologia , Esterol Esterase , Especificidade por Substrato , Fatores de Transcrição/fisiologia , Transfecção , Regulação para CimaRESUMO
Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization.
Assuntos
Hidrolases de Éster Carboxílico/fisiologia , Lipídeos de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Esteróis/metabolismo , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/análise , Hidrolases de Éster Carboxílico/genética , Membrana Celular/química , Membrana Celular/enzimologia , Ésteres/metabolismo , Genes Fúngicos/fisiologia , Humanos , Hidrólise , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutação/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Esterol Esterase , Triglicerídeos/metabolismoRESUMO
Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3'-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.