RESUMEN
Cells use mitophagy to remove damaged or unwanted mitochondria to maintain homeostasis. Here we report that the intracellular bacterial pathogen Listeria monocytogenes exploits host mitophagy to evade killing. We found that L. monocytogenes induced mitophagy in macrophages through the virulence factor listeriolysin O (LLO). We discovered that NLRX1, the only Nod-like receptor (NLR) family member with a mitochondrial targeting sequence, contains an LC3-interacting region (LIR) and directly associated with LC3 through the LIR. NLRX1 and its LIR motif were essential for L. monocytogenes-induced mitophagy. NLRX1 deficiency and use of a mitophagy inhibitor both increased mitochondrial production of reactive oxygen species and thereby suppressed the survival of L. monocytogenes. Mechanistically, L. monocytogenes and LLO induced oligomerization of NLRX1 to promote binding of its LIR motif to LC3 for induction of mitophagy. Our study identifies NLRX1 as a novel mitophagy receptor and discovers a previously unappreciated strategy used by pathogens to hijack a host cell homeostasis system for their survival.
Asunto(s)
Listeria monocytogenes/fisiología , Proteínas Mitocondriales/fisiología , Mitofagia , Animales , Autofagia , Toxinas Bacterianas/metabolismo , Línea Celular , Femenino , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Listeria monocytogenes/patogenicidad , Listeriosis/metabolismo , Listeriosis/microbiología , Macrófagos/microbiología , Macrófagos/ultraestructura , Masculino , Ratones , Ratones Noqueados , Viabilidad Microbiana , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Dominios Proteicos , Especies Reactivas de Oxígeno/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Metabolic engagement is intrinsic to immune cell function. Prostaglandin E2 (PGE2) has been shown to modulate macrophage activation, yet how PGE2 might affect metabolism is unclear. Here, we show that PGE2 caused mitochondrial membrane potential (Δψm) to dissipate in interleukin-4-activated (M(IL-4)) macrophages. Effects on Δψm were a consequence of PGE2-initiated transcriptional regulation of genes, particularly Got1, in the malate-aspartate shuttle (MAS). Reduced Δψm caused alterations in the expression of 126 voltage-regulated genes (VRGs), including those encoding resistin-like molecule α (RELMα), a key marker of M(IL-4) cells, and genes that regulate the cell cycle. The transcription factor ETS variant 1 (ETV1) played a role in the regulation of 38% of the VRGs. These results reveal ETV1 as a Δψm-sensitive transcription factor and Δψm as a mediator of mitochondrial-directed nuclear gene expression.
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Núcleo Celular/efectos de los fármacos , Dinoprostona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Animales , Núcleo Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Interleucina-4/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.
Asunto(s)
Mucosa Intestinal , Macrófagos , Animales , Macrófagos/ultraestructura , Masculino , Mucosa Intestinal/ultraestructura , Ratas , Ratas Wistar , Intestino Grueso/ultraestructura , Intestino Grueso/inervación , Microscopía Electrónica de Rastreo , Lisosomas/ultraestructura , Lisosomas/metabolismo , Ciego/ultraestructura , Vacuolas/ultraestructuraRESUMEN
Many different types of inorganic materials are processed into nano/microparticles for medical utilization. The impact of selected key characteristics of these particles, including size, shape, and surface chemistries, on biological systems, is frequently studied in clinical contexts. However, one of the most important basic characteristics of these particles, their density, is yet to be investigated. When the particles are designed for drug delivery, highly mobile macrophages are the major participants in cellular levels that process them in vivo. As such, it is essential to understand the impact of particles' densities on the mobility of macrophages. Here, inorganic particles with different densities are applied, and their interactions with macrophages studied. A set of these particles are incubated with the macrophages and the outcomes are explored by optical microscopy. This microscopic view provides the understanding of the mechanistic interactions between particles of different densities and macrophages to conclude that the particles' density can affect the migratory behaviors of macrophages: the higher the density of particles engulfed inside the macrophages, the less mobile the macrophages become. This work is a strong reminder that the density of particles cannot be neglected when they are designed to be utilized in biological applications.
Asunto(s)
Macrófagos , Humanos , Tamaño de la Partícula , Macrófagos/ultraestructuraRESUMEN
Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.
Asunto(s)
Proteínas del Helminto/farmacología , Factor 88 de Diferenciación Mieloide/metabolismo , Fagosomas/efectos de los fármacos , Choque Séptico/prevención & control , Receptor Toll-Like 4/metabolismo , Animales , Autofagia/efectos de los fármacos , Autofagia/inmunología , Células Cultivadas , Femenino , Humanos , Immunoblotting , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Factor 88 de Diferenciación Mieloide/inmunología , Fagosomas/inmunología , Fagosomas/metabolismo , Choque Séptico/genética , Choque Séptico/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.
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Envejecimiento/inmunología , Interacciones Huésped-Patógeno/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas Asociadas a Microtúbulos/metabolismo , Fagocitosis , Streptococcus pneumoniae/inmunología , Animales , Autofagia , Proteínas Bacterianas/metabolismo , Lípidos/química , Macrófagos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Streptococcus pneumoniae/ultraestructura , Estreptolisinas/metabolismoRESUMEN
Crohn's disease is an inflammatory disease of the gut caused by a complex interplay among genetic, microbial, and environmental factors. The intestinal tract is constantly exposed to metals and other trace elements ingested as food. Synchrotron radiation-induced X-ray fluorescence spectroscopy and X-ray absorption fine structure analysis revealed the deposition of nickel particles within Crohn's disease tissue specimens. After nickel particle stimulation, THP-1 cells showed filopodia formation and autophagic vacuoles containing lipid bodies. Nickel particles precipitated colitis in mice bearing mutations of the IBD susceptibility protein A20/TNFAIP3. Nickel particles also exacerbated dextran sulfate sodium-induced colitis in mice harboring myeloid cell-specific Atg5 deficiency. These findings illustrate that nickel particle ingestion may worsen Crohn's disease by perturbing autophagic processes in the intestine, providing new insights into environmental factors in Crohn's disease pathogenesis.
Asunto(s)
Enfermedad de Crohn/patología , Progresión de la Enfermedad , Inflamación/patología , Intestinos/patología , Níquel/toxicidad , Animales , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia/metabolismo , Sulfato de Dextran , Susceptibilidad a Enfermedades , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Macrófagos/ultraestructura , Ratones Endogámicos C57BL , Células THP-1 , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Despite aquaporin water channels (AQPs) play a critical role in maintaining water homeostasis in female reproductive tract and prompt a gradual increase in water content in cervical edema as pregnancy progressed, their relationship with macrophage infiltration and collagen content in human cervical remodeling need to be further investigated. This is the first study to examine the expression and localization of AQP3, AQP4, AQP5, AQP8, and macrophages simultaneously in human cervical ripening. The immunoreactivity of these AQPs was 2.6 to 6-fold higher on gestational weeks 26 (GD26W) than that on GD6W and GD15W, but AQP4 expression on GD39W dropped a similar extent on GD15W, other AQPs continued to rise on GD39W. The AQP3, AQP4, and AQP5 intensity seemed more abundant in cervical stroma than in the perivascular area on GD26W; the distribution of AQP3, AQP5, and AQP8 in cervical stroma was equivalent to that in the perivascular area on GD39W. Macrophage numbers were 1.7-fold higher in subepithelium region and 3.0-fold higher in center area on GD26W than that on GD15W; such numbers remained elevated on GD39W. The electron micrographs showed that cervical extensibility increased significantly on GD26W and GD39W accompanied with increased macrophage infiltration, cervical water content, and much more space among collagen fibers. These findings suggest that the upregulation of AQPs expression in human cervix is closely related to enhanced macrophage infiltration during pregnancy; there may be a positive feedback mechanism between them to lead the increase of water content and the degradation of collagen.
Asunto(s)
Acuaporinas/análisis , Cuello del Útero/fisiología , Macrófagos/fisiología , Adolescente , Adulto , Acuaporina 3/análisis , Acuaporina 4/análisis , Acuaporina 5/análisis , Acuaporinas/fisiología , Recuento de Células , Maduración Cervical/fisiología , Cuello del Útero/química , Cuello del Útero/citología , Colágeno/análisis , Colágeno/metabolismo , Femenino , Edad Gestacional , Humanos , Macrófagos/ultraestructura , Microscopía Electrónica , Embarazo , Adulto JovenRESUMEN
Characterized by a dismal survival rate and limited response to therapy, glioblastoma (GBM) remains one of the most aggressive human malignancies. Recent studies of the role of tumor-associated macrophages (TAMs) in the progression of GBMs have demonstrated that TAMs are significant contributors to tumor growth, invasion, and therapeutic resistance. TAMs, which include brain-resident microglia and circulating bone marrow derived-monocytes (BMDMs), are typically grouped together in histopathological and molecular analyses due to the lack of reliable markers of distinction. To develop more effective therapies aimed at specific TAM populations, we must first understand how these cells differ both morphologically and behaviorally. Furthermore, we must develop a deeper understanding of the mechanisms encouraging their infiltration and how these mechanisms can be therapeutically exploited. In this study, we combined immunocompetent lineage tracing mouse models of GBM with high-resolution open-skull 2-photon microscopy to investigate the phenotypical and functional characteristics of TAMs. We demonstrate that TAMs are composed of 2 morphologically distinct cell types that have differential migratory propensities. We show that BMDMs are smaller, minimally branched cells that are highly migratory compared with microglia, which are larger, highly branched stationary cells. In addition, 2 populations of monocytic macrophages were observed that differed in terms of CX3CR1 expression and migratory capacity. Finally, we demonstrate the efficacy of anti-vascular endothelial growth factor A blockade for prohibiting TAM infiltration, especially against BMDMs. Taken together, our data clearly define characteristics of individual TAM populations and suggest that combination therapy with antivascular and antichemotaxis therapy may be an attractive option for treating these tumors.
Asunto(s)
Linaje de la Célula/genética , Glioblastoma/genética , Macrófagos/ultraestructura , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Receptor 1 de Quimiocinas CX3C/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/diagnóstico por imagen , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Microglía/patología , Microglía/ultraestructura , Monocitos/patología , Monocitos/ultraestructura , Microambiente Tumoral/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Glucocorticoids delay fracture healing and induce osteoporosis. However, the mechanisms by which glucocorticoids delay bone repair have yet to be clarified. Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of plasminogen activators and an adipocytokine that regulates metabolism. We herein investigated the roles of macrophages in glucocorticoid-induced delays in bone repair after femoral bone injury using PAI-1-deficient female mice intraperitoneally administered with dexamethasone (Dex). Dex significantly decreased the number of F4/80-positive macrophages at the damaged site two days after femoral bone injury. It also attenuated bone injury-induced decreases in the number of hematopoietic stem cells in bone marrow in wild-type and PAI-1-deficient mice. PAI-1 deficiency significantly weakened Dex-induced decreases in macrophage number and macrophage colony-stimulating factor (M-CSF) mRNA levels at the damaged site two days after bone injury. It also significantly ameliorated the Dex-induced inhibition of macrophage phagocytosis at the damaged site. In conclusion, we herein demonstrated that Dex decreased the number of macrophages at the damaged site during early bone repair after femoral bone injury partly through PAI-1 and M-CSF in mice.
Asunto(s)
Regeneración Ósea , Glucocorticoides/farmacología , Macrófagos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Animales , Médula Ósea/patología , Regeneración Ósea/efectos de los fármacos , Recuento de Células , Dexametasona/farmacología , Femenino , Fémur/efectos de los fármacos , Fémur/lesiones , Fémur/patología , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Trastornos Hemorrágicos/patología , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/deficienciaRESUMEN
BACKGROUND: Interferon alfa (IFN-α) has been proved effective in treating chronic hepatitis B (CHB), owing to its ability to suppress hepatitis B surface antigen and hepatitis B virus (HBV) covalently closed circular DNA. However, the underlying mechanisms are unclear. METHODS: We investigated the antiviral activities of exosomes from responders and nonresponders to pegylated IFN-α (PegIFN-α) as well as the supernatants of IFN-α-treated macrophages derived from THP-1 (the human leukemia monocyte cell line). Then the expression profiles of exosomal microRNAs (miRNAs) were analyzed using miRNA sequencing. The luciferase reporter assay was used to locate the binding position of HBV genomic sequence targeted by the identified miRNA. RESULTS: Exosomes from PegIFN-α-treated patients, particularly responders, as well as the supernatants of IFN-α-treated macrophages exhibited anti-HBV activities, as manifested by the suppression of hepatitis B surface antigen, hepatitis B e antigen, HBV DNA, and covalently closed circular DNA levels in HBV-related cell lines. PegIFN-α treatment up-regulated exosomal hsa-miR-193a-5p, hsa-miR-25-5p, and hsa-miR-574-5p, which could partially inhibit HBV replication and transcription, and hsa-miR-574-5p reduced pregenomic RNA and polymerase messenger RNA levels by binding to the 2750-2757 position of the HBV genomic sequence. CONCLUSIONS: Exosomes can transfer IFN-α-related miRNAs from macrophages to HBV-infected hepatocytes, and they exhibit antiviral activities against HBV replication and expression.
Asunto(s)
Exosomas/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Macrófagos/metabolismo , MicroARNs/metabolismo , Adolescente , Adulto , Anciano , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Quimioterapia Combinada , Guanina/análogos & derivados , Guanina/uso terapéutico , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Interferón-alfa/farmacología , Macrófagos/inmunología , Macrófagos/ultraestructura , Persona de Mediana Edad , Células THP-1 , Replicación Viral/efectos de los fármacos , Adulto JovenRESUMEN
Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients and experimentally infected animals indicate a critical role for augmented expression of proinflammatory chemokines and cytokines in severe disease. Here, we demonstrate that SARS-CoV-2 infection of human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells was abortive, but induced the production of multiple antiviral and proinflammatory cytokines (interferon-α, interferon-ß, tumor necrosis factor, and interleukins 1ß, 6, and 10) and a chemokine (CXCL10). Despite the lack of efficient replication in MDMs, SARS-CoV-2 induced profound interferon-mediated cell death of host cells. Macrophage activation and death were not enhanced by exposure to low levels of convalescent plasma, suggesting that antibody-dependent enhancement of infection does not contribute to cell death. Together, these results indicate that infection of macrophages and dendritic cells potentially plays a major role in coronavirus disease 2019 pathogenesis, even in the absence of productive infection.
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COVID-19/terapia , Células Dendríticas/virología , Macrófagos/virología , SARS-CoV-2/inmunología , COVID-19/inmunología , Muerte Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/ultraestructura , Humanos , Inmunización Pasiva , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Macrófagos/inmunología , Macrófagos/ultraestructura , Microscopía Electrónica de Transmisión , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Sueroterapia para COVID-19RESUMEN
The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study, we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs), that occurs between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long-distance tumor cell streaming in vitro Using an in vivo zebrafish model that recreates macrophage-mediated tumor cell invasion, we observed TNT-mediated macrophage-dependent tumor cell invasion, distant metastatic foci and areas of metastatic spread. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that leads to tumor progression and metastasis.
Asunto(s)
Neoplasias de la Mama/genética , Comunicación Celular/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Neoplasias Mamarias Animales/genética , Animales , Transporte Biológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Embrión no Mamífero , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Xenoinjertos , Humanos , Macrófagos/ultraestructura , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Cultivo Primario de Células , Células RAW 264.7 , Ratas , Transducción de Señal , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismo , Pez CebraRESUMEN
BACKGROUND: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. METHODS: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. RESULTS: In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. CONCLUSION: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7's neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7's primary role in neuronal tissues is not related to antiviral immunity.
Asunto(s)
Ganglios Espinales/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Neuroglía/metabolismo , Células Receptoras Sensoriales/metabolismo , Receptor Toll-Like 7/biosíntesis , Animales , Femenino , Ganglios Espinales/ultraestructura , Expresión Génica , Cobayas , Macrófagos/ultraestructura , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/ultraestructura , Células Receptoras Sensoriales/ultraestructura , Receptor Toll-Like 7/genéticaRESUMEN
While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Regulación hacia Abajo , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Pseudomonas aeruginosa , Sistemas de Secreción Tipo III/metabolismo , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Línea Celular , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Mutación , Fagosomas/microbiología , Fagosomas/ultraestructura , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Sistemas de Secreción Tipo III/genéticaRESUMEN
Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163-CD4+, CD163+CD4+, and CD163-CD4- cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68+CD4+CD163- cells seemed to be preferentially localized in the intestinal villus, whereas CD68+CD163+CD4+ cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I-III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs.
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Íleon/ultraestructura , Macrófagos/ultraestructura , Animales , Ratas , Ratas WistarRESUMEN
Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.
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Citocinas/metabolismo , Eugenol/uso terapéutico , Inmunidad , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/inmunología , Óxido Nítrico/biosíntesis , Fosforilcolina/análogos & derivados , Administración Oral , Animales , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Interacciones Farmacológicas , Quimioterapia Combinada , Eugenol/administración & dosificación , Eugenol/farmacología , Femenino , Inmunidad/efectos de los fármacos , Concentración 50 Inhibidora , Leishmania donovani/efectos de los fármacos , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/inmunología , Leishmania donovani/ultraestructura , Leishmaniasis Visceral/parasitología , Estadios del Ciclo de Vida/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/parasitología , Macrófagos/ultraestructura , Masculino , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo II/metabolismo , Parásitos/efectos de los fármacos , Parásitos/crecimiento & desarrollo , Parásitos/inmunología , Parásitos/ultraestructura , Fosforilación/efectos de los fármacos , Fosforilcolina/administración & dosificación , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates â¼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.
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Proteínas de Unión al GTP/inmunología , Inmunidad Innata/inmunología , Interferón gamma/inmunología , Macrófagos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Cromosomas de los Mamíferos/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Inmunidad Celular/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Listeria monocytogenes/inmunología , Mediciones Luminiscentes , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Óxido Nítrico/metabolismo , Toxoplasma/metabolismoRESUMEN
BACKGROUND: Ischemia is the partial or complete blockage of blood supply to tissues. Extracellular vesicles (EVs) are emerging as a therapeutic tool for ischemic diseases. Most EV-based ischemia therapies are based on various stem cells. Here, we propose an alternative cell source for the isolation of pro-angiogenic EVs. METHODS: EVs were isolated from a mouse macrophage cell line (Raw 264.7). The characteristic features of the macrophage-derived EVs (MAC-EVs) were assessed using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting (WB) analysis. WB and qRT-PCR were performed to identify the pro-angiogenic VEGF and Wnt3a proteins and microRNAs (miR-210, miR-126, and miR-130a) in the MAC-EVs. In vitro and in vivo Matrigel plug assays were performed to investigate the capacity of the MAC-EVs for tube (blood vessel-like) formation and new blood vessel formation and assessed by histology. RESULTS: The MAC-EVs was positive for ALIX and negative for calnexin, with a round shape and an average size of 189 ± 65.1 nm. WB and qRT-PCR results revealed that VEGF, Wnt3a and miR-130a were more abundant in the MAC-EVs than cells. MAC-EVs treatment resulted in increased endothelial cellular proliferation, migration, and tube formation in vitro. In vivo assay results revealed that MAC-EVs increased the formation of new and larger blood vessels in the Matrigel plug of mice compared to the formation in the control group. CONCLUSION: Our results suggest that MAC-EVs have the potential to induce angiogenesis in vitro and in vivo, could serve as a pro-angiogenic alternative for ischemic diseases.
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Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Neovascularización Fisiológica , Inductores de la Angiogénesis/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Colágeno , Modelos Animales de Enfermedad , Combinación de Medicamentos , Células Endoteliales/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Fluorescencia , Laminina , Macrófagos/ultraestructura , Ratones , Ratones Desnudos , Proteoglicanos , Células RAW 264.7RESUMEN
The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rod-like crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.