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1.
Aquat Toxicol ; 231: 105739, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33434705

RESUMO

Cadmium (Cd) with no known functional role in any life-form has myriad of harmful effects. The present study was designed to elucidate the mechanism of Cd-induced oxystress generation and its impact on antioxidant and apoptosis signaling pathways in head kidney macrophage (HKM) of Channa punctatus Bloch. Fish were sampled and acclimatized with one group treated with cadmium chloride (CdCl2) (1.96 mg/L) and another as untreated control group, both kept under observation for 7 days. Exposure to Cd caused ultrastructural changes along with reduced head kidney somatic index (HKSI). Significantly increased levels of reactive oxygen species (ROS), respiratory burst activity, lipid peroxidation, DNA fragmentation and superoxide dismutase were found in the HKM from the treated group as compared to control. In contrast, antioxidant enzymes like catalase and reduced glutathione activity decreased in the Cd exposed group. The suppressed antioxidant activity was further confirmed and corroborated from the altered expression of Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) genes, the major player of antioxidant pathway. Cd induced alteration in Nrf2-Keap1 signaling pathway was also validated by the diminished levels of Nrf2 dependent expression of protein like heme oxygenase-1 (HO-1). The flow cytometry analysis supported the event of apoptosis in Cd exposed group as compared to control, which was further confirmed by the upregulated expression of caspase-3, caspase-8, caspase-9, TNF-α and p53 genes from the real-time gene expression study. In addition, altered protein level of cytochrome C validates the incidence of apoptosis. Altogether, our results demonstrate that exposure to Cd caused oxidative stress in HKM of Channa punctatus Bloch. by compromising the antioxidant enzyme activities via the down regulation of expression of genes related to antioxidant signaling pathway besides encouraging apoptosis via both mitochondrial and death receptor pathway.


Assuntos
Apoptose , Cádmio/toxicidade , Peixes/metabolismo , Rim Cefálico/citologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/toxicidade
2.
Proc Natl Acad Sci U S A ; 117(52): 33561-33569, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376222

RESUMO

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.


Assuntos
Envelhecimento/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose , Streptococcus pneumoniae/imunologia , Animais , Autofagia , Proteínas de Bactérias/metabolismo , Lipídeos/química , Macrófagos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/ultraestrutura , Estreptolisinas/metabolismo
3.
Int J Nanomedicine ; 15: 6355-6372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922006

RESUMO

Background: Cerium oxide nanoparticles (CeO2NPs) are potent scavengers of cellular reactive oxygen species (ROS). Their antioxidant properties make CeO2NPs promising therapeutic agents for bone diseases and bone tissue engineering. However, the effects of CeO2NPs on intracellular ROS production in osteoclasts (OCs) are still unclear. Numerous studies have reported that intracellular ROS are essential for osteoclastogenesis. The aim of this study was to explore the effects of CeO2NPs on osteoclast differentiation and the potential underlying mechanisms. Methods: The bidirectional modulation of osteoclast differentiation by CeO2NPs was explored by different methods, such as fluorescence microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. The cytotoxic and proapoptotic effects of CeO2NPs were detected by cell counting kit (CCK-8) assay, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and flow cytometry. Results: The results of this study demonstrated that although CeO2NPs were capable of scavenging ROS in acellular environments, they facilitated the production of ROS in the acidic cellular environment during receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent osteoclast differentiation of bone marrow-derived macrophages (BMMs). CeO2NPs at lower concentrations (4.0 µg/mL to 8.0 µg/mL) promoted osteoclast formation, as shown by increased expression of Nfatc1 and C-Fos, F-actin ring formation and bone resorption. However, at higher concentrations (greater than 16.0 µg/mL), CeO2NPs inhibited osteoclast differentiation and promoted apoptosis of BMMs by reducing Bcl2 expression and increasing the expression of cleaved caspase-3, which may be due to the overproduction of ROS. Conclusion: This study demonstrates that CeO2NPs facilitate osteoclast formation at lower concentrations while inhibiting osteoclastogenesis in vitro by inducing the apoptosis of BMMs at higher concentrations by modulating cellular ROS levels.


Assuntos
Diferenciação Celular , Cério/química , Osteoclastos/citologia , Espécies Reativas de Oxigênio/metabolismo , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Nanopartículas/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
4.
PLoS One ; 15(8): e0237795, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833989

RESUMO

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.


Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Buffy Coat/citologia , Diferenciação Celular , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Técnicas de Cocultura , Voluntários Saudáveis , Humanos , Microscopia Intravital , Macrófagos/citologia , Macrófagos/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Monócitos/fisiologia , Cultura Primária de Células
5.
Rev Esp Patol ; 53(3): 188-192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32650970

RESUMO

The new coronavirus SARS-CoV-2, first identified in Wuhan, China in December, 2019, can cause Severe Acute Respiratory Syndrome (SARS) with massive alveolar damage and progressive respiratory failure. We present the relevant autopsy findings of the first patient known to have died from COVID19 pneumonia in Spain, carried out on the 14th of February, 2020, in our hospital (Hospital Arnau de Vilanova-Lliria, Valencia). Histological examination revealed typical changes of diffuse alveolar damage (DAD) in both the exudative and proliferative phase of acute lung injury. Intra-alveolar multinucleated giant cells, smudge cells and vascular thrombosis were present. The diagnosis was confirmed by reverse real-time PCR assay on a throat swab sample taken during the patient's admission. The positive result was reported fifteen days subsequent to autopsy.


Assuntos
Autopsia , Betacoronavirus , Infecções por Coronavirus/patologia , Pulmão/patologia , Pandemias , Pneumonia Viral/patologia , /etiologia , Idoso , Células Epiteliais Alveolares/ultraestrutura , Proteína 1 de Troca de Ânion do Eritrócito/análise , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Betacoronavirus/isolamento & purificação , Carcinoma de Células de Transição/complicações , China , Técnicas de Laboratório Clínico , Infecções Comunitárias Adquiridas/diagnóstico , Infecções por Coronavirus/complicações , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Proteínas de Ligação a DNA/análise , Humanos , Pulmão/virologia , Macrófagos/química , Macrófagos/ultraestrutura , Masculino , Pneumonia/diagnóstico , Pneumonia Viral/complicações , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Espanha/epidemiologia , Fatores de Transcrição/análise , Viagem , Neoplasias da Bexiga Urinária/complicações
6.
Eur J Med Chem ; 200: 112407, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32512480

RESUMO

The photophysical properties of naphthalimide-based fluorophores can be easily tuned by chemical manipulation of the substituents on that privileged scaffold. Replacement of a OMe group at position 6 in 2-(hydroxyl)ethyl-naphthalimide derivatives by diverse amines, including 2-(hydroxyl)ethylamine, trans-(4-acetamido)cyclohexylamine and azetidine increases the solvatochromic (ICT) character, while this replacement in 2-(dimethylamino)ethyl-naphthalimide analogues (PET fluorophores) decrease their solvent polarity sensitivity or even reversed them to solvatochromic fluorophores. These fluorophores resulted macrophage nucleus imaging probes, which bind DNA as intercalants and showed low cytotoxicity in human cancer cells.


Assuntos
Núcleo Celular , Corantes Fluorescentes/química , Macrófagos/ultraestrutura , Imagem Molecular/métodos , Naftalimidas/química , Linhagem Celular Tumoral , DNA/metabolismo , Humanos , Substâncias Intercalantes/química , Relação Estrutura-Atividade
7.
Int J Nanomedicine ; 15: 3483-3496, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32523344

RESUMO

Background: The use of polycaprolactone (PCL) for bone defects in a clinical setting is limited due to a lack of bioactivity. Exosomes derived from mesenchymal stem cells (MSCs) have an important immunoregulatory potential and together with S-nitrosoglutathione (GSNO) they possess therapeutic potential for bone regeneration. Materials and Methods: In this study, PCL was modified with GSNO and MSC-derived exosomes and the impact on macrophages and osteogenes is evaluated. Results: MSC-derived exosomes exhibited a cup-shaped morphology and were internalized by macrophages and human bone marrow-derived mesenchymal stromal cells (hBMSCs). The pattern of internalization of scaffold-immobilized exosomes was similar in RAW264.7 cells and hBMSCs after 4h and 24h of co-culture. Assessment of macrophage morphology under inflammatory conditions by scanning electronic microscopy (SEM) and confocal microscopy demonstrated macrophages were significantly elongated and expression of pro-inflammatory genes markedly decreased when co-cultured with PCL/PDA + GSNO + exosome scaffolds. Furthermore, this scaffold modification significantly enhanced osteogenic differentiation of hBMSCs. Discussion: This study demonstrated the possibility of using a GSNO- and exosome-based strategy to adapt barrier membrane scaffolds. PCL/PDA + GSNO + exosome scaffolds may serve as an important barrier membrane for osteogenesis and tissue regeneration.


Assuntos
Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , S-Nitrosoglutationa/metabolismo , Animais , Diferenciação Celular , Endocitose , Exossomos/ultraestrutura , Humanos , Inflamação/patologia , Macrófagos/patologia , Macrófagos/ultraestrutura , Camundongos , Células RAW 264.7 , Tecidos Suporte/química
9.
J Neurol Neurosurg Psychiatry ; 91(6): 650-659, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32245766

RESUMO

OBJECTIVE: To describe the pathological features of Guillain-Barré syndrome focusing on macrophage-associated myelin lesions. METHODS: Longitudinal sections of sural nerve biopsy specimens from 11 patients with acute inflammatory demyelinating polyneuropathy (AIDP) exhibiting macrophage-associated demyelinating lesions were examined using electron microscopy. A total of 1205 nodes of Ranvier were examined to determine the relationship of the macrophage-associated demyelinating lesions with the nodal regions. Additionally, immunohistochemical and immunofluorescent studies were performed to elucidate the sites of complement deposition. RESULTS: Overall, 252 macrophage-associated myelin lesions were identified in longitudinal sections. Of these, 40 lesions exhibited complete demyelination with no association with the lamellar structures of myelin. In 183 lesions, macrophage cytoplasm was located at internodes without association with the nodes of Ranvier or paranodes. In particular, these internodal lesions were more frequent in one patient (152 lesions). In the remaining 29 lesions, the involvement of nodal regions was obvious. Lesions involving nodal regions were more frequently observed than those involving internodes in four patients. Invasion of the macrophage cytoplasmic processes into the space between the paranodal myelin terminal loops and the axolemma from the nodes of Ranvier was observed in three of these patients. Immunostaining suggested complement deposition corresponding to putative initial macrophage-associated demyelinating lesions. CONCLUSIONS: The initial macrophage-associated demyelinating lesions appeared to be located at internodes and at nodal regions. The sites at which the macrophages initiated phagocytosis of myelin might be associated with the location of complement deposition in certain patients with AIDP.


Assuntos
Doenças Desmielinizantes/patologia , Síndrome de Guillain-Barré/patologia , Macrófagos/ultraestrutura , Bainha de Mielina/ultraestrutura , Neurônios/ultraestrutura , Idoso , Axônios/patologia , Axônios/ultraestrutura , Feminino , Humanos , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Bainha de Mielina/patologia , Neurônios/patologia , Nós Neurofibrosos/patologia , Nós Neurofibrosos/ultraestrutura
10.
J Leukoc Biol ; 108(1): 253-266, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272506

RESUMO

Berberine (BBR) is an isoquinoline alkaloid extracted from several commonly used Chinese herbs. Our previous studies demonstrated BBR-mediated alleviation of lung injury due to inflammation and decrease in the mortality of mice with influenza viral pneumonia. The recent argument of autophagy against inflammatory responses has aroused wide concerns. This study focuses on the reactive oxygen species-Nod-like receptor protein 3 (ROS-NLRP3) pathway to investigate whether BBR inhibits NLRP3 inflammasome activation by inducing mitophagy. Our results demonstrate that BBR and mitochondrion-targeted superoxide dismutase mimetic (Mito-TEMPO; a specific mitochondrial ROS scavenger) significantly restricted NLRP3 inflammasome activation, increased mitochondrial membrane potential (MMP), and decreased mitochondrial ROS (mtROS) generation in J774A.1 macrophages infected with PR8 influenza virus. These observations suggest that the inhibitory effects of BBR on NLRP3 inflammasome activation were associated with the amelioration of mtROS generation. BBR treatment induced regular mitophagy, as evident from the increase in microtubule-associated protein 1 light chain 3 II, decrease in p62, colocalization of LC3 and mitochondria, and formation of autophagosomes. However, 3-methyladenine, an autophagy inhibitor, reversed the inhibitory effects of BBR on mitochondrial damage and NLRP3 inflammasome activation in influenza virus-infected macrophages, indicating the involvement of mitophagy in mediating the inhibitory effects of BBR on NLRP3 inflammasome activation. Furthermore, the knockdown of Bcl-2/adenovirus E18-19-kDa interacting protein 3 (BNIP3) expression attenuated the effects of BBR on mitophagy induction to some extent, suggesting that the BBR-induced mitophagy may be, at least in part, mediated in a BNIP3-dependent manner. Similar results were obtained in vivo using a mouse model of influenza viral pneumonia that was administered with BBR. Taken together, these findings suggest that restricting NLRP3 inflammasome activation by decreasing ROS generation through mitophagy induction may be crucial for the BBR-mediated alleviation of influenza virus-induced inflammatory lesions.


Assuntos
Berberina/farmacologia , Inflamassomos/metabolismo , Macrófagos/virologia , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Orthomyxoviridae/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Macrófagos/ultraestrutura , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Orthomyxoviridae/efeitos dos fármacos , Pneumonia/patologia , Pneumonia/virologia , Regulação para Cima/efeitos dos fármacos
11.
Micron ; 132: 102851, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32092694

RESUMO

Kupffer cells are liver-resident macrophages that play an important role in mediating immune-related functions in mammals and humans. They are well-known for their capacity to phagocytose large amounts of waste complexes, cell debris, microbial particles and even malignant cells. Location, appearance and functional aspects are important features used to identify these characteristic cells of the liver sinusoid. To-date, there is limited information on the occurrence of macrophages in zebrafish liver. Therefore, we aimed to characterise the ultrastructural and functional aspects of liver-associated macrophages in the zebrafish model by taking advantage of the latest advances in zebrafish genetics and multimodal correlative imaging. Herein, we report on the occurrence of macrophages within the zebrafish liver exhibiting conventional ultrastructural features (e.g. presence of pseudopodia, extensive lysosomal apparatus, a phagolysosome and making up ∼3% of the liver volume). Intriguingly, these cells were not located within the sinusoidal vascular bed of hepatic tissue but instead resided between hepatocytes and lacked phagocytic function. While our results demonstrated the presence and structural similarities with liver macrophages from other experimental models, their functional characteristics were distinctly different from Kupffer cells that have been described in rodents and humans. These findings illustrate that the innate immune system of the zebrafish liver has some distinctly different characteristics compared to other animal experimental models. This conclusion underpins our call for future studies in order to have a better understanding of the physiological role of macrophages residing between the parenchymal cells of the zebrafish liver.


Assuntos
Fígado/citologia , Fígado/ultraestrutura , Macrófagos/ultraestrutura , Peixe-Zebra/anatomia & histologia , Animais , Macrófagos do Fígado/ultraestrutura , Contagem de Leucócitos , Microscopia Eletrônica , Fagocitose , Fagossomos , Coloração e Rotulagem
12.
Sci Rep ; 10(1): 3061, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080266

RESUMO

Macrophages are central cells both in the immune response and in iron homeostasis. Iron is both essential and potentially toxic. Therefore, iron acquisition, transport, storage, and release are tightly regulated, by several important proteins. Cytosolic ferritin is an iron storage protein composed of 24 subunits of either the L- or the H-type chains. H-ferritin differs from L-ferritin in the capacity to oxidize Fe2+ to Fe3+. In this work, we investigated the role played by H-ferritin in the macrophages' ability to respond to immune stimuli and to deal with exogenously added iron. We used mice with a conditional deletion of the H-ferritin gene in the myeloid lineage to obtain bone marrow-derived macrophages. These macrophages had normal viability and gene expression under basal culture conditions. However, when treated with interferon-gamma and lipopolysaccharide they had a lower activation of Nitric Oxide Synthase 2. Furthermore, H-ferritin-deficient macrophages had a higher sensitivity to iron-induced toxicity. This sensitivity was associated with a lower intracellular iron accumulation but a higher production of reactive oxygen species. These data indicate that H-ferritin modulates macrophage response to immune stimuli and that it plays an essential role in protection against iron-induced oxidative stress and cell death.


Assuntos
Ferritinas/metabolismo , Ferro/metabolismo , Macrófagos/metabolismo , Oxirredutases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Compostos Férricos/farmacologia , Ferritinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hemina/farmacologia , Ferro/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genética , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/ultraestrutura , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Oxirredutases/genética , Compostos de Amônio Quaternário/farmacologia
13.
PLoS One ; 15(1): e0227030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910224

RESUMO

Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.


Assuntos
Vírus da Dengue , Células Endoteliais/metabolismo , Vesículas Extracelulares/virologia , Macrófagos/ultraestrutura , Antígenos CD/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Citocinas/metabolismo , Dengue/imunologia , Vírus da Dengue/imunologia , Células Endoteliais/imunologia , Vesículas Extracelulares/fisiologia , Humanos , Macrófagos/virologia , Permeabilidade , Células U937
14.
Mol Pharm ; 17(1): 155-166, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31742407

RESUMO

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.


Assuntos
Darunavir/administração & dosagem , Inibidores da Protease de HIV/administração & dosagem , Pró-Fármacos/administração & dosagem , Pró-Fármacos/síntese química , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Darunavir/síntese química , Darunavir/química , Darunavir/farmacocinética , Farmacorresistência Viral/efeitos dos fármacos , Inibidores da Protease de HIV/farmacocinética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Ratos , Espectrometria de Massas em Tandem
15.
J Morphol ; 281(1): 135-152, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774588

RESUMO

Rapid activation of resident glia occurs after spinal cord injury. Somewhat later, innate and adaptive immune responses occur with the invasion of peripheral immune cells into the wound site. The activation of resident and peripheral immune cells has been postulated to play harmful as well as beneficial roles in the regenerative process. Mauthner cells, large identifiable neurons located in the hindbrain of most fish and amphibians, provided the opportunity to study the morphological relationship between reactive cells and Mauthner axons (M-axons) severed by spinal cord crush or by selective axotomy. After crossing in the hindbrain, the M-axons of adult goldfish, Carassius auratus, extend the length of the spinal cord. Following injury, the M-axon undergoes retrograde degeneration within its myelin sheath creating an axon-free zone (proximal dieback zone). Reactive cells invade the wound site, enter the axon-free dieback zone and are observed in the vicinity of the retracted M-axon tip as early as 3 hr postinjury. Transmission electron microscopy allowed the detection of microglia/macrophages and granulocytes, some of which appear to be neutrophil-like, at each of these locations. We believe that this is the first report of the invasion of such cells within the myelin sheath of an identifiable axon in the vertebrate central nervous system (CNS). We speculate that microglia/macrophages and granulocytes that are attracted within a few hours to the damaged M-axon are part of an inflammatory response that allows phagocytosis of debris and plays a role in the regenerative process. Our results provide the baseline from which to utilize immunohistochemical and genetic approaches to elucidate the role of non-neuronal cells in the regenerative process of a single axon in the vertebrate CNS.


Assuntos
Axônios/patologia , Carpa Dourada/fisiologia , Granulócitos/patologia , Macrófagos/patologia , Microglia/patologia , Bainha de Mielina/fisiologia , Traumatismos da Medula Espinal/patologia , Animais , Axônios/ultraestrutura , Axotomia , Granulócitos/ultraestrutura , Macrófagos/ultraestrutura , Microglia/ultraestrutura , Bainha de Mielina/ultraestrutura , Neutrófilos/patologia , Neutrófilos/ultraestrutura
16.
Arterioscler Thromb Vasc Biol ; 39(12): 2492-2504, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597449

RESUMO

OBJECTIVE: Understanding message delivery among vascular cells is essential for deciphering the intercellular communications in cardiovascular diseases. MicroRNA (miR)-92a is enriched in endothelial cells (ECs) and circulation under atheroprone conditions. Macrophages are the primary immune cells in atherosclerotic lesions that modulate lesion development. Therefore, we hypothesize that, in response to atheroprone stimuli, ECs export miR-92a to macrophages to regulate their functions and enhance atherosclerotic progression. Approach and Results: We investigated the macrophage functions that are regulated by EC miR-92a under atheroprone microenvironments. We first determined the distributions of functional extracellular miR-92a by fractionating the intravesicular and extravesicular compartments from endothelial conditioned media and mice serum. The results indicate that extracellular vesicles are the primary vehicles for EC miR-92a transportation. Overexpression of miR-92a in ECs enhanced the proinflammatory responses and low-density lipoprotein uptake, while impaired the migration, of cocultured macrophage. Opposite effects were found in macrophages cocultured with ECs with miR-92a knockdown. Further analyses demonstrated that intravesicular miR-92a suppressed the expression of target gene KLF4 (Krüppel-like factor 4) in macrophages, suggesting a mechanism by which intravesicular miR-92a regulates recipient cell functions. Indeed, the overexpression of KLF4 rescued the EC miR-92a-induced macrophage atheroprone phenotypes. Furthermore, an inverse correlation of intravesicular miR-92a in blood serum and KLF4 expression in lesions was observed in atherosclerotic animals, indicating the potential function of extracellular miR-92a in regulating vascular diseases. CONCLUSIONS: EC miR-92a can be transported to macrophages through extracellular vesicles to regulate KLF4 levels, thus leading to the atheroprone phenotypes of macrophage and, hence, atherosclerotic lesion formation.


Assuntos
Aterosclerose/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , MicroRNAs/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Comunicação Celular , Células Cultivadas , Líquido Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Macrófagos/ultraestrutura , Camundongos , MicroRNAs/biossíntese , Microscopia Eletrônica de Transmissão
17.
Cell Tissue Res ; 377(3): 527-547, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31485720

RESUMO

The uptake of macromolecules and larger energy-rich particles into the cell is known as phagocytosis. Phagocytosed material is enzymatically degraded in membrane-bound vesicles of the endosome/lysosome system (intracellular digestion). Whereas most, if not all, cells of the animal body are equipped with the molecular apparatus for phagocytosis and intracellular digestion, a few cell types are specialized for a highly efficient mode of phagocytosis. These are the ("professional") macrophages, motile cells that seek out and eliminate pathogenic invaders or damaged cells. Macrophages form the backbone of the innate immune system. Developmentally, they derive from specialized compartments within the embryonic mesoderm and early vasculature as part of the process of hematopoiesis. Intensive research has revealed in detail molecular and cellular mechanisms of phagocytosis and intracellular digestion in macrophages. In contrast, little is known about a second type of cell that is "professionally" involved in phagocytosis, namely the "enteric phagocyte." Next to secretory (zymogenic) cells, enteric phagocytes form one of the two major cell types of the intestine of most invertebrate animals. Unlike vertebrates, these invertebrates only partially digest food material in the intestinal lumen. The resulting food particles are absorbed by phagocytosis or pinocytosis and digested intracellularly. In this review, we provide a brief overview of the enteric phagocytes described electron microscopically for diverse invertebrate clades, to then to compare these cells with the "canonical" phagocyte ultrastructure established for macrophages. In addition, we will review observations and speculations associated with the hypothesis that macrophages are evolutionarily derived from enteric phagocytes. This idea was already proposed in the late nineteenth century by Elias Metschnikoff who pioneered the research of phagocytosis for both macrophages and enteric phagocytes. We presume that modern approaches to better understand phagocytosis will be helped by considering the deep evolutionary relationship between the two cell types.


Assuntos
Macrófagos/ultraestrutura , Fagocitose/fisiologia , Animais , Evolução Biológica
18.
Toxicol Lett ; 316: 10-19, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476341

RESUMO

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 µg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.


Assuntos
Células Epiteliais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Tabaco/efeitos adversos , Traqueia/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Masculino , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos Sprague-Dawley , Medição de Risco , Fatores de Tempo , Técnicas de Cultura de Tecidos , Traqueia/metabolismo , Traqueia/ultraestrutura
20.
Int J Pediatr Otorhinolaryngol ; 127: 109656, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31470202

RESUMO

OBJECTIVE: To determine the distribution of perivascularresident macrophages (PVMs) in BLB and their relationship with capillaries, and to explore the possible mechanisms responsible for lipopolysaccharide (LPS)-induced activation of PVMs and the breakdown of BLB. METHODS: Adult Balb/c mice were either trans-tympanically injected with LPS, or mock-treated. Auditory brainstem response was tested before and 48 h after treatments. Distribution of pericytes, PVMs and capillaries was analyzed by immunohistochemical staining, and BLB permeability was estimated by FITC-dextran leakage assay. Ultrastructure of stria vascularis was examined by transmission electron microscope. Protein and mRNA level of matrix metallopeptidase 9 (MMP-9), zona occludens-1 (ZO-1), interleukin-33 (IL-33) and its receptor suppression of tumorigenicity 2 (ST2) was measured by IHC and qRT-PCR. RESULTS: Unlike pericytes that surround one capillary, PVMs branched to connect with more than one capillary. LPS caused hearing loss in mice. Following LPS challenge, cochleae showed vascular leakage in stria vascularis, and PVMs presented morphological changes including reduced contact with capillaries. TEM revealed a reduced number of tight junction contact points between endothelial cells and a wider space between PVMs, pericytes and endothelial cells. The mRNA and protein levels of MMP-9 and ST2 in stria vascularis were up-regulated, while ZO-1 were down-regulated after exposure to LPS. CONCLUSIONS: Our results suggest that PVMs may play a more significant role than pericytes in maintaining the integrity of BLB. Our findings also reveal a possible mechanism contributing to LPS-induced activation of PVMs, breakdown of BLB and hearing loss.


Assuntos
Perda Auditiva/metabolismo , Macrófagos/patologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Estria Vascular/ultraestrutura , Vestíbulo do Labirinto , Animais , Capilares/patologia , Cóclea/irrigação sanguínea , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Perda Auditiva/induzido quimicamente , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Lipopolissacarídeos , Macrófagos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Pericitos/ultraestrutura , Estria Vascular/metabolismo , Estria Vascular/patologia , Junções Íntimas/ultraestrutura , Regulação para Cima , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
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