RESUMO
Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma.
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Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.
Assuntos
Cílios , Ciliopatias , Humanos , Animais , Camundongos , Cílios/genética , Cílios/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas/genética , Aminoácidos/metabolismo , Mamíferos/metabolismo , Proteínas do Citoesqueleto/genéticaRESUMO
Mycobacterium abscessus infections are emerging in cystic fibrosis patients, and treatment success rate in these patients is only 33% due to extreme antibiotic resistance. Thus, new treatment options are essential. An interesting target could be Lsr2, a nucleoid-associated protein involved in mycobacterial virulence. Zafirlukast is a Food and Drug Administration (FDA)-approved drug against asthma that was shown to bind Lsr2. In this study, zafirlukast treatment is shown to reduce M. abscessus growth, with a minimal inhibitory concentration of 16 µM and a bactericidal concentration of 64 µM in replicating bacteria only. As an initial response, DNA condensation, a known stress response of mycobacteria, occurs after 1 h of treatment with zafirlukast. During continued zafirlukast treatment, the morphology of the bacteria alters and the structural integrity of the bacteria is lost. After 4 days of treatment, reduced viability is measured in different culture media, and growth of M. abscessus is reduced in a dose-dependent manner. Using transmission electron microscopy, we demonstrated that the hydrophobic multilayered cell wall and periplasm are disorganized and ribosomes are reduced in size and relocalized. In summary, our data demonstrate that zafirlukast alters the morphology of M. abscessus and is bactericidal at 64 µM. The bactericidal concentration of zafirlukast is relatively high, and it is only effective on replicating bacteria but as zafirlukast is an FDA-approved drug, and currently used as an anti-asthma treatment, it could be an interesting drug to further study in in vivo experiments to determine whether it could be used as an antibiotic for M. abscessus infections.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Mycobacterium abscessus , Fenilcarbamatos , Sulfonamidas , Compostos de Tosil , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Antibacterianos/farmacologia , Compostos de Tosil/farmacologia , Sulfonamidas/farmacologia , Fenilcarbamatos/farmacologia , Indóis/farmacologia , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genéticaRESUMO
PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.
Assuntos
Proteínas Ferro-Enxofre , Peixe-Zebra , Animais , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Masculino , Feminino , Fenótipo , Fibroblastos/metabolismo , Fibroblastos/patologia , Citosol/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Microcefalia/genética , Microcefalia/patologia , Lactente , MetalochaperonasRESUMO
Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and/or SepF. We have isolated SepF homologs from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 44 nm. Interestingly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF regulates ring diameter. Importantly, when SepF chimeras with different diameters were expressed in the bacterial host Bacillus subtilis, the thickness of its septa changed accordingly. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mold to shape the cell wall.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Divisão Celular , Parede Celular/metabolismo , PolimerizaçãoRESUMO
AIMS: Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce. METHODS AND RESULTS: Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with <10. Mitochondrial dysfunction was not explained by reduced abundance or fragmentation of mitochondria, as evaluated by transmission electron microscopy. Rather, improper organization of mitochondria relative to myofibrils (expressed as a percentage of disorganized mitochondria) was strongly associated with mitochondrial dysfunction. Pre-incubation with the cardiolipin-stabilizing drug elamipretide and raising mitochondrial NAD+ levels both boosted NADH-linked respiration. CONCLUSION: Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.
Assuntos
Cardiomiopatia Hipertrófica , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/patologia , NAD/genética , Cardiomiopatia Hipertrófica/genética , Mutação , Mitocôndrias Cardíacas/patologia , RespiraçãoRESUMO
The most prevalent route of HIV-1 infection is across mucosal tissues after sexual contact. Langerhans cells (LCs) belong to the subset of dendritic cells (DCs) that line the mucosal epithelia of vagina and foreskin and have the ability to sense and induce immunity to invading pathogens. Anatomical and functional characteristics make LCs one of the primary targets of HIV-1 infection. Notably, LCs form a protective barrier against HIV-1 infection and transmission. LCs restrict HIV-1 infection through the capture of HIV-1 by the C-type lectin receptor Langerin and subsequent internalization into Birbeck granules. However, the underlying molecular mechanism of HIV-1 restriction in LCs remains unknown. Here we show that human E3-ubiquitin ligase tri-partite-containing motif 5α (TRIM5α) potently restricts HIV-1 infection of LCs but not of subepithelial DC-SIGN+ DCs. HIV-1 restriction by TRIM5α was thus far considered to be reserved to non-human primate TRIM5α orthologues, but our data strongly suggest that human TRIM5α is a cell-specific restriction factor dependent on C-type lectin receptor function. Our findings highlight the importance of HIV-1 binding to Langerin for the routeing of HIV-1 into the human TRIM5α-mediated restriction pathway. TRIM5α mediates the assembly of an autophagy-activating scaffold to Langerin, which targets HIV-1 for autophagic degradation and prevents infection of LCs. By contrast, HIV-1 binding to DC-SIGN+ DCs leads to disassociation of TRIM5α from DC-SIGN, which abrogates TRIM5α restriction. Thus, our data strongly suggest that restriction by human TRIM5α is controlled by C-type-lectin-receptor-dependent uptake of HIV-1, dictating protection or infection of human DC subsets. Therapeutic interventions that incorporate C-type lectin receptors and autophagy-targeting strategies could thus provide cell-mediated resistance to HIV-1 in humans.
Assuntos
Antígenos CD/metabolismo , Autofagia , Proteínas de Transporte/metabolismo , HIV-1/fisiologia , Células de Langerhans/metabolismo , Células de Langerhans/virologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de HIV/metabolismo , Fatores de Restrição Antivirais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade nas Mucosas , Células de Langerhans/citologia , Células de Langerhans/imunologia , Receptores de Superfície Celular/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.
Assuntos
Adenosina Trifosfatases , Fosfolipídeos , Fosfolipídeos/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Membrana/metabolismo , LisossomosRESUMO
Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.
Assuntos
Epilepsia do Lobo Temporal/complicações , Hipocampo/metabolismo , Distúrbios do Metabolismo do Ferro/etiologia , Ferro/metabolismo , Estado Epiléptico/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Feminino , Humanos , Distúrbios do Metabolismo do Ferro/patologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Ratos , Estado Epiléptico/metabolismo , Estado Epiléptico/patologiaRESUMO
Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.
Assuntos
Antiácidos/metabolismo , Lipídeos/biossíntese , Lipídeos/química , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Animais , Regulação Bacteriana da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Lisossomos , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Mycobacterium kansasii/genética , PrevalênciaRESUMO
The lysosomal storage disease Niemann-Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1-/- mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1-/- liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1-/- liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1-/- liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1-/- hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.
Assuntos
Glucosilceramidase/metabolismo , Fígado/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Receptores Depuradores/metabolismo , Animais , Transporte Biológico/fisiologia , Catepsina D/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Doença de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Lisossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Esfingomielinas/metabolismoRESUMO
Disarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram-negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane-based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence-based high-throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen-43. VUF15259 also impairs ß-barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam-complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug-like compounds.
Assuntos
Sistemas de Secreção Tipo V/antagonistas & inibidores , Sistemas de Secreção Tipo V/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Endopeptidases/metabolismo , Bactérias Gram-Negativas , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/metabolismo , Fatores de Virulência/metabolismoRESUMO
Insight into the molecular and cellular processes in blood-retinal barrier (BRB) development, including the contribution of paracellular and transcellular pathways, is still incomplete but may help to understand the inverse process of BRB loss in pathologic eye conditions. In this comprehensive observational study, we describe in detail the formation of the BRB at the molecular level in physiologic conditions, using mice from postnatal day (P)3 to P25. Our data indicate that immature blood vessels already have tight junctions at P5, before the formation of a functional BRB. Expression of the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), which is known to be involved in transcellular transport and associated with BRB permeability, decreased during development and was absent when a functional barrier was formed. Moreover, we show that PLVAP deficiency causes a transient delay in retinal vascular development and changes in mRNA expression levels of endothelial permeability pathway proteins.-Van der Wijk, A.-E., Wisniewska-Kruk, J., Vogels, I. M. C., van Veen, H. A., Ip, W. F., van der Wel, N. N., van Noorden, C. J. F., Schlingemann, R. O., Klaassen, I. Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.
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Barreira Hematorretiniana/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Animais , Barreira Hematorretiniana/embriologia , Barreira Hematorretiniana/ultraestrutura , Western Blotting , Éxons/genética , Genótipo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Transmissão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by Klebsiella pneumoniae in PAD4+/+ and PAD4-/- mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4+/+ but not of PAD4-/- mice. Combined light and electron microscopy showed NET-like structures surrounding Klebsiella in areas of CitH3 staining in the lung; however, these were also seen in PAD4-/- mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, Klebsiella and LPS could still induce NETosis in PAD4-/- neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.
Assuntos
Armadilhas Extracelulares/imunologia , Infecções por Klebsiella/imunologia , Desiminases de Arginina em Proteínas/imunologia , Sepse/imunologia , Animais , Armadilhas Extracelulares/enzimologia , Humanos , Infecções por Klebsiella/enzimologia , Klebsiella pneumoniae/imunologia , Camundongos , Camundongos Knockout , Proteína-Arginina Desiminase do Tipo 4 , Sepse/enzimologiaRESUMO
Background and Purpose- We developed a rat model of silent brain infarcts based on microsphere infusion and investigated their impact on perfusion and tissue damage. Second, we studied the extent and mechanisms of perfusion recovery. Methods- At day 0, 15 µm fluorescent microspheres were injected into the right common carotid artery of F344 rats. At days 1, 7, or 28, the brain was removed, cut in 100-µm cryosections, and processed for immunofluorescent staining and analysis. Results- Injection of microspheres caused mild and transient damage to the treated hemisphere, with a decrease in perfused capillary volume at day 1, as compared with the untreated hemisphere. At day 1 but not at days 7 and 28, we observed IgG staining outside of the vessels, indicating vessel leakage. All microspheres were located inside the lumen of the vessels at day 1, whereas the vast majority (≈80%) of the microspheres were extravascular at day 7, and 100% at day 28. This was accompanied by restoration of perfused capillary volume. Conclusions- Microspheres cause mild and transient damage, and effective extravasation mechanisms exist in the brain to clear microsized emboli from the vessels.
Assuntos
Infarto Encefálico , Microesferas , Animais , Infarto Encefálico/induzido quimicamente , Infarto Encefálico/metabolismo , Infarto Encefálico/patologia , Modelos Animais de Doenças , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
BACKGROUND: Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils. METHODS: To follow eosinophil-virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti-IL-5 (mepolizumab), followed by a challenge with rhinovirus-16 (RV16). RESULTS: DiD-RSV and DiD-influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus-induced loss of asthma control. CONCLUSIONS: This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations.
Assuntos
Asma/etiologia , Eosinófilos/metabolismo , Viroses/complicações , Viroses/virologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Asma/diagnóstico , Asma/metabolismo , Modelos Animais de Doenças , Eosinófilos/patologia , Eosinófilos/ultraestrutura , Humanos , Vírus da Influenza A/fisiologia , Lectinas Tipo C/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Testes de Função RespiratóriaRESUMO
Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle.
Assuntos
Barreira Hematoencefálica/microbiologia , Infecções do Sistema Nervoso Central/patologia , Interações Hospedeiro-Patógeno , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/crescimento & desenvolvimento , Animais , Encéfalo/microbiologia , Modelos Animais de Doenças , Macrófagos/microbiologia , Peixe-ZebraRESUMO
Objective- Nbeal2-/- mice, a model of human gray platelet syndrome, have reduced neutrophil granularity and impaired host defense against systemic Staphylococcus aureus infection. We here aimed to study the role of Nbeal2 deficiency in both leukocytes and platelets during gram-negative pneumonia and sepsis. Approach and Results- We studied the role of Nbeal2 in platelets and leukocytes during murine pneumonia and sepsis by Klebsiella pneumoniae. Apart from platelet α-granule deficiency and reduced neutrophil granularity, also monocyte granularity was reduced in Nbeal2-/- mice, whereas plasma levels of MPO (myeloperoxidase), elastase, NGAL (neutrophil gelatinase-associated lipocalin), and MMP-9 (matrix metalloproteinase 9), and leukocyte CD11b expression were increased. Nbeal2-/- leukocytes showed unaltered in vitro antibacterial response and phagocytosis capacity against Klebsiella, and unchanged reactive nitrogen species and cytokine production. Also during Klebsiella pneumonia and sepsis, Nbeal2-/- mice had similar bacterial growth in lung and distant body sites, with enhanced leukocyte migration to the bronchoalveolar space. Despite similar infection-induced inflammation, organ damage was increased in Nbeal2-/- mice, which was also seen during endotoxemia. Platelet-specific Nbeal2 deficiency did not influence leukocyte functions, indicating that Nbeal2 directly modifies leukocytes. Transfusion of Nbeal2-/- but not of Nbeal2+/+ platelets into thrombocytopenic mice was associated with bleeding in the lung but similar host defense, pointing at a role for platelet α-granules in maintaining vascular integrity but not host defense during Klebsiella pneumosepsis. Conclusions- These data show that Nbeal2 deficiency-resulting in gray platelet syndrome-affects platelets, neutrophils, and monocytes, with intact host defense but increased organ damage during gram-negative pneumosepsis.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/deficiência , Síndrome da Plaqueta Cinza/metabolismo , Infecções por Klebsiella/metabolismo , Klebsiella pneumoniae/patogenicidade , Insuficiência de Múltiplos Órgãos/metabolismo , Pneumonia Bacteriana/metabolismo , Sepse/metabolismo , Animais , Plaquetas/microbiologia , Proteínas Sanguíneas/genética , Antígeno CD11b/sangue , Modelos Animais de Doenças , Feminino , Síndrome da Plaqueta Cinza/sangue , Síndrome da Plaqueta Cinza/genética , Interações Hospedeiro-Patógeno , Infecções por Klebsiella/sangue , Infecções por Klebsiella/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Lipocalina-2/sangue , Masculino , Metaloproteinase 9 da Matriz/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/microbiologia , Insuficiência de Múltiplos Órgãos/sangue , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/microbiologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Elastase Pancreática/sangue , Peroxidase/sangue , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Transfusão de Plaquetas , Pneumonia Bacteriana/sangue , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/microbiologia , Sepse/sangue , Sepse/genética , Sepse/microbiologiaRESUMO
Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.
Assuntos
Cápsulas Bacterianas/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium marinum/patogenicidade , Sistemas de Secreção Tipo VII/metabolismo , Virulência/fisiologia , Animais , Linhagem Celular , Cromatografia em Camada Fina , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Immunoblotting , Microscopia Eletrônica , Mycobacterium marinum/metabolismo , Fatores de Virulência/metabolismo , Peixe-ZebraRESUMO
The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics-based membrane protein predictions, ca. one-third could be predicted to be membrane-localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were identified. In addition to previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology, unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.