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1.
Eur J Immunol ; 54(6): e2350771, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38494423

RESUMEN

Vomocytosis, also known as nonlytic exocytosis, is a process whereby fully phagocytosed microbes are expelled from phagocytes without discernible damage to either the phagocyte or microbe. Although this phenomenon was first described in the opportunistic fungal pathogen Cryptococcus neoformans in 2006, to date, mechanistic studies have been hampered by an inability to reliably stimulate or inhibit vomocytosis. Here we present the fortuitous discovery that macrophages lacking the scavenger receptor MAcrophage Receptor with COllagenous domain (MARCO), exhibit near-total vomocytosis of internalised cryptococci within a few hours of infection. Marco-/- macrophages also showed elevated vomocytosis of a yeast-locked C. albicans strain, suggesting this to be a broadly relevant observation. We go on to show that MARCO's role in modulating vomocytosis is independent of its role as a phagocytic receptor, suggesting that this protein may play an important and hitherto unrecognised role in modulating macrophage behaviour.


Asunto(s)
Cryptococcus neoformans , Macrófagos , Receptores Inmunológicos , Animales , Ratones , Cryptococcus neoformans/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/genética , Candida albicans/inmunología , Fagocitosis/inmunología , Ratones Noqueados , Exocitosis/inmunología , Criptococosis/inmunología
2.
Nat Immunol ; 11(9): 814-9, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20711192

RESUMEN

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.


Asunto(s)
Dermatitis por Contacto , Níquel/inmunología , Receptor Toll-Like 4/inmunología , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Transducción de Señal , Receptor Toll-Like 4/genética
3.
PLoS Pathog ; 14(3): e1006914, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29522575

RESUMEN

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.


Asunto(s)
Infecciones por Adenoviridae/virología , Pulmón/virología , Macrófagos Alveolares/virología , Macrófagos/virología , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Internalización del Virus , Infecciones por Adenoviridae/inmunología , Infecciones por Adenoviridae/metabolismo , Adenovirus Humanos/inmunología , Animales , Humanos , Inmunidad Innata , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Receptores Inmunológicos/genética
4.
Arch Toxicol ; 94(1): 173-186, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31677074

RESUMEN

Macrophages play a major role in the removal of foreign materials, including nano-sized materials, such as nanomedicines and other nanoparticles, which they accumulate very efficiently. Because of this, it is recognized that for a safe development of nanotechnologies and nanomedicine, it is essential to investigate potential effects induced by nano-sized materials on macrophages. To this aim, in this work, a recently established model of primary murine alveolar-like macrophages was used to investigate macrophage responses to two well-known nanoparticle models: 50 nm amino-modified polystyrene, known to induce cell death via lysosomal damage and apoptosis in different cell types, and 50 nm silica nanoparticles, which are generally considered non-toxic. Then, a time-resolved study was performed to characterize in detail the response of the macrophages following exposure to the two nanoparticles. As expected, exposure to the amino-modified polystyrene led to cell death, but surprisingly no lysosomal swelling or apoptosis were detected. On the contrary, a peculiar mitochondrial membrane hyperpolarization was observed, accompanied by endoplasmic reticulum stress (ER stress), increased cellular reactive oxygen species (ROS) and changes of metabolic activity, ultimately leading to cell death. Strong toxic responses were observed also after exposure to silica, which included mitochondrial ROS production, mitochondrial depolarization and cell death by apoptosis. Overall, these results showed that exposure to the two nanoparticles led to a very different series of intracellular events, suggesting that the macrophages responded differently to the two nanoparticle models. Similar time-resolved studies are required to characterize the response of macrophages to nanoparticles, as a key parameter in nanosafety assessment.


Asunto(s)
Macrófagos/efectos de los fármacos , Nanopartículas/toxicidad , Poliestirenos/toxicidad , Dióxido de Silicio/toxicidad , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Células Cultivadas , Dispersión Dinámica de Luz , Estrés del Retículo Endoplásmico/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Lisosomas/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos Alveolares/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nanopartículas/química , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Poliestirenos/química , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo
5.
J Toxicol Environ Health A ; 82(20): 1076-1087, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31797748

RESUMEN

Contaminated marine bathing water has been reported to adversely affect human health. Our data demonstrated a correlation between total endotoxin (lipopolysaccharide; LPS) levels and degree of contamination of marine bathing waters. To assess the potential health implications of LPS present in marine bathing waters, the inflammation-inducing potency of water samples collected at different time points at multiple sampling sites were assessed using a cell culture-based assay. The numbers of fecal indicator bacteria (FIB) were also examined in the same samples. Water samples were used to stimulate two cell culture models: (1) a novel non-transformed continuously growing murine cell line Max Plank Institute (MPI) characteristic of alveolar macrophages and (2) human MonoMac 6 monocyte cell line. The inflammatory potential of the samples was assessed by measuring the release of inflammatory cytokines. The presence of high levels of LPS in contaminated bathing water led to induction of inflammatory response from our in vitro cell-based bioassays suggesting its potential health impact. This finding introduces an in vitro culture assay that reflects the level of LPS in water samples. These observations further promote previous finding that LPS is a reliable surrogate biomarker for fecal contamination of bathing water.


Asunto(s)
Citocinas/inmunología , Lipopolisacáridos/efectos adversos , Macrófagos/microbiología , Agua de Mar/microbiología , Contaminación del Agua/efectos adversos , Animales , Playas , Línea Celular , Inglaterra , Monitoreo del Ambiente , Humanos , Ratones , Microbiología del Agua
6.
Emerg Infect Dis ; 24(5): 935-937, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29664378

RESUMEN

We report identification of an Oropouche virus strain in a febrile patient from Ecuador by using metagenomic sequencing and real-time reverse transcription PCR. Virus was isolated from patient serum by using Vero cells. Phylogenetic analysis of the whole-genome sequence showed the virus to be similar to a strain from Peru.


Asunto(s)
Infecciones por Bunyaviridae/virología , Orthobunyavirus/aislamiento & purificación , Adulto , Animales , Infecciones por Bunyaviridae/epidemiología , Chlorocebus aethiops , Ecuador/epidemiología , Humanos , Masculino , Orthobunyavirus/genética , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Vero
7.
Proc Natl Acad Sci U S A ; 110(24): E2191-8, 2013 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-23708119

RESUMEN

Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.


Asunto(s)
Células de la Médula Ósea/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Macrófagos/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/inmunología , Interleucina-1alfa/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos Alveolares/citología , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mycobacterium tuberculosis/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fagocitosis/inmunología , Propionibacterium acnes/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Factor de Transcripción STAT5/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Transcriptoma/inmunología
8.
Biology (Basel) ; 13(5)2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38785798

RESUMEN

Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor's cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7-a commonly used experimental murine macrophage model-to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts.

9.
Cell Rep ; 43(3): 113935, 2024 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-38460129

RESUMEN

Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity.


Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína Sequestosoma-1 , Cuerpos de Procesamiento , Inflamación , Autofagia/fisiología
10.
J Innate Immun ; 16(1): 226-247, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38527452

RESUMEN

INTRODUCTION: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αß) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients. METHODS: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-ß was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αß, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αßR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction. RESULTS: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αß by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αß-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αß-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-ß pretreatment enhances the subsequent induction of IFN-αß in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αß overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice. CONCLUSION: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects.


Asunto(s)
Adenoviridae , Citocinas , Factor 3 Regulador del Interferón , Lipopolisacáridos , Macrófagos , Ratones Noqueados , Animales , Ratones , Lipopolisacáridos/inmunología , Humanos , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Macrófagos/inmunología , Citocinas/metabolismo , Ratones Endogámicos C57BL , Factor 7 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Vectores Genéticos , Infecciones por Adenoviridae/inmunología , Interferón Tipo I/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Interferón beta/metabolismo
11.
J Immunol ; 186(9): 5478-88, 2011 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-21441453

RESUMEN

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-ß (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-ß production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-ß responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αß responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/inmunología , Lipopolisacáridos/inmunología , Mastocitos/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Western Blotting , Separación Celular , Inmunoprecipitación de Cromatina , Citocinas/biosíntesis , Citocinas/inmunología , Citometría de Flujo , Antígeno 96 de los Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4 , Transfección
12.
Nat Commun ; 14(1): 4895, 2023 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-37580395

RESUMEN

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.


Asunto(s)
Criptococosis , Fagocitosis , Receptores Depuradores de Clase A , Receptor Toll-Like 4 , Animales , Humanos , Ratones , Cryptococcus neoformans , Macrófagos/microbiología , Receptor Toll-Like 4/genética , Receptores Depuradores de Clase A/metabolismo
13.
Emerg Top Life Sci ; 6(4): 411-422, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36453919

RESUMEN

Micro and nanosize plastic polymers degrading from large plastic compounds are accumulating in the natural environment and expose potential biological threats to human health. These particles are largely persistent and consequently accumulate in the exposed individuals. The presence of microplastics has already been demonstrated in various human organs including the lung, the gastrointestinal system and the blood raising concerns about their possible harmful effects. The chemical composition, size and shape of microplastics as well as their weathering status represent important factors influencing the potential impact of microplastics on tissues. In addition, microplastics can function as vectors for adsorbed chemical compounds and may harbour and deliver live microbial pathogens or their ligands. In vitro and in vivo animal studies demonstrated that microplastics are taken up to cells in a size and cell type dependent manner. Once inside the targeted cell they activate oxidative processes, mitochondrial dysfunction and ER-stress. These molecular processes result in the activation or repression of cell type specific functions and potentially in the induction of cytotoxicity. The microplastic elicited events may result in inflammation, organ damage and fibrosis of the targeted organs as well as in systemic immunological and metabolic conditions. In addition, microplastics may impact on the gut microbiota which may exert further gastrointestinal and systemic metabolic and immunological effects. In this minireview, we evaluate the factors and mechanisms that influence potential microplastic induced cellular and organ pathologies in humans and discuss limitations of current understanding regarding microplastic elicited conditions as well as future perspectives for research.


Asunto(s)
Microbioma Gastrointestinal , Microplásticos , Animales , Humanos , Microplásticos/toxicidad , Plásticos , Tracto Gastrointestinal , Ambiente
14.
Biosci Rep ; 42(7)2022 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-35735109

RESUMEN

Inflammation is central to several diseases. TLR4 mediates inflammation by recognising and binding to bacterial lipopolysaccharides and interacting with other proteins in the TLR4 signalling pathway. Although there is extensive research on TLR4-mediated inflammation, there are gaps in understanding its mechanisms. Recently, TLR4 co-localised with LPCAT2, a lysophospholipid acetyltransferase. LPCAT2 is already known to influence lipopolysaccharide-induced inflammation; however, the mechanism of LPCAT2 influencing lipopolysaccharide-mediated inflammation is not understood. The present study combined computational analysis with biochemical analysis to investigate the influence of LPCAT2 on lysine acetylation in LPS-treated RAW264.7 cells. The results suggest for the first time that LPCAT2 influences lysine acetylation in LPS-treated RAW264.7 cells. Moreover, we detected acetylated lysine residues on TLR4. The present study lays a foundation for further research on the role of lysine acetylation on TLR4 signalling. Moreover, further research is required to characterise LPCAT2 as a protein acetyltransferase.


Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Lipopolisacáridos , Receptor Toll-Like 4/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Inflamación/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Lisina/metabolismo , Ratones , Células RAW 264.7 , Receptor Toll-Like 4/genética
15.
J Virol ; 83(18): 9411-22, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19570869

RESUMEN

Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus Parapoxvirus. It displays only limited homology with orthopoxviruses and has some molecular features such as an unusual high GC content distinct from orthopoxviruses. Inactivated PPVO (iPPVO) displays strong immunostimulatory capacities mediating antiviral activity in vivo. The role of dendritic cells (DC) and the pattern recognition receptors and signaling requirements responsible for immunostimulation by iPPVO are unknown. We demonstrate here that bone marrow-derived plasmacytoid DC (BM-pDC) and bone marrow-derived conventional DC (BM-cDC) secrete alpha/beta interferon (IFN-alpha/beta) in response to iPPVO. Furthermore, iPPVO induces tumor necrosis factor alpha (TNF-alpha) and interleukin-12/23p40 (IL-12/23p40) release and major histocompatibility complex class II (MHC-II), MHC-I, and CD86 upregulation by bone marrow-derived DC (BMDC). After engulfment, iPPVO is located in endosomal compartments and in the cytosol of BMDC. iPPVO elicits IFN-alpha/beta by Toll-like receptor (TLR)-independent pathways in BM-cDC, since IFN-alpha/beta release does not require myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-alpha release and enhanced expression of MHC-I and CD86 but not of MHC-II by BMDC chiefly requires MyD88 but not TLR2 or TLR4. Induction of IFN-alpha by iPPVO in BM-cDC occurred in the absence of IFN regulatory factor 3 (IRF3) but required the presence of IRF7, whereas iPPVO-triggered IFN-beta production required the presence of either IRF7 or IRF3. These results provide the first evidence that iPPVO mediates its immunostimulatory properties by TLR-independent and TLR-dependent pathways and demonstrate an important role of cDC for IFN-alpha/beta production.


Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Parapoxvirus/inmunología , Receptores Toll-Like/metabolismo , Animales , Células de la Médula Ósea , Ratones , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Inactivación de Virus
16.
PLoS Pathog ; 4(11): e1000208, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19008951

RESUMEN

The early systemic production of interferon (IFN)-alphabeta is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-alphabeta response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-alphabeta mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-alphabeta production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-alphabeta response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-alphabeta and IL-6 in vivo by distinct pathways and confirm that IFN-alphabeta positively regulates the IL-6 response. Finally, by measuring TNF-alpha responses to LPS in Ad-infected wild type and IFN-alphabetaR(-/-) mice, we show that IFN-alphabeta is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-alphabeta response, which is responsible for the bulk of IFN-alphabeta production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-alphabeta induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.


Asunto(s)
Adenoviridae/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Bazo/citología , Animales , Endosomas , Humanos , Interferón Tipo I/genética , Interleucina-6/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos , Proteínas Quinasas Activadas por Mitógenos , ARN Mensajero/análisis , Transducción de Señal , Bazo/inmunología , Distribución Tisular , Regulación hacia Arriba
17.
PLoS Negl Trop Dis ; 14(1): e0007897, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31961856

RESUMEN

Oropouche virus (OROV) is responsible for outbreaks of Oropouche fever in parts of South America. We recently identified and isolated OROV from a febrile Ecuadorian patient, however, a previously published qRT-PCR assay did not detect OROV in the patient sample. A primer mismatch to the Ecuadorian OROV lineage was identified from metagenomic sequencing data. We report the optimisation of an qRT-PCR assay for the Ecuadorian OROV lineage, which subsequently identified a further five cases in a cohort of 196 febrile patients. We isolated OROV via cell culture and developed an algorithmically-designed primer set for whole-genome amplification of the virus. Metagenomic sequencing of the patient samples provided OROV genome coverage ranging from 68-99%. The additional cases formed a single phylogenetic cluster together with the initial case. OROV should be considered as a differential diagnosis for Ecuadorian patients with febrile illness to avoid mis-diagnosis with other circulating pathogens.


Asunto(s)
Infecciones por Bunyaviridae/virología , Orthobunyavirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Infecciones por Bunyaviridae/diagnóstico , Estudios de Cohortes , Ecuador , Genoma Viral , Humanos , Metagenoma , Orthobunyavirus/clasificación , Orthobunyavirus/genética , Filogenia , ARN Viral/genética
18.
J Virol ; 81(23): 13242-7, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17898065

RESUMEN

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.


Asunto(s)
ADN Viral/química , Herpesvirus Humano 4/fisiología , Histonas/análisis , Linfocitos/virología , Regiones Promotoras Genéticas , Proteínas de la Matriz Viral/biosíntesis , Acetilación , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Herpesvirus Humano 4/química , Humanos , Linfocitos/química , Metilación , Datos de Secuencia Molecular , Unión Proteica , Proteínas de la Matriz Viral/genética
19.
Immunobiology ; 213(3-4): 193-203, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18406367

RESUMEN

In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.


Asunto(s)
Infecciones por Bacterias Gramnegativas/metabolismo , Lipopolisacáridos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Humanos , Sistema Inmunológico , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Antígeno 96 de los Linfocitos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Transducción de Señal , Receptor Toll-Like 4/metabolismo
20.
Front Immunol ; 9: 438, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29593716

RESUMEN

Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis (Mtb) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hamper the detailed molecular understanding of host-Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung AMs, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular, and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell lines. MPI cells were also suitable for the determination of anti-tuberculosis (TB) drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, interleukin 6 (IL-6), IL-1α, and IL-1ß, as compared to stimulation with heat-killed (HK) bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including HK Mtb. By contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using light chain 3B immunostaining, as well as LysoTracker labeling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for TB research, allowing a deeper understanding of AMs functions in this pathology.


Asunto(s)
Macrófagos Alveolares/fisiología , Mycobacterium tuberculosis/fisiología , Tuberculosis/inmunología , Animales , Autofagia , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Pulmón/patología , Macrófagos Alveolares/microbiología , Ratones , Ratones Endogámicos BALB C , Fagosomas/metabolismo , Células THP-1
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