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1.
Nat Commun ; 11(1): 4071, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792491

RESUMO

Arrest of oligodendrocyte (OL) differentiation and remyelination following myelin damage in multiple sclerosis (MS) is associated with neurodegeneration and clinical worsening. We show that Glutathione S-transferase 4α (Gsta4) is highly expressed during adult OL differentiation and that Gsta4 loss impairs differentiation into myelinating OLs in vitro. In addition, we identify Gsta4 as a target of both dimethyl fumarate, an existing MS therapy, and clemastine fumarate, a candidate remyelinating agent in MS. Overexpression of Gsta4 reduces expression of Fas and activity of the mitochondria-associated Casp8-Bid-axis in adult oligodendrocyte precursor cells, leading to improved OL survival during differentiation. The Gsta4 effect on apoptosis during adult OL differentiation was corroborated in vivo in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis models, where Casp8 activity was reduced in Gsta4-overexpressing OLs. Our results identify Gsta4 as an intrinsic regulator of OL differentiation, survival and remyelination, as well as a potential target for future reparative MS therapies.


Assuntos
Glutationa Transferase/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Caspase 8/genética , Caspase 8/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glutationa Transferase/genética , Homeostase/genética , Homeostase/fisiologia , Imuno-Histoquímica , Masculino , Microglia/citologia , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Fagocitose/genética , Fagocitose/fisiologia , Processamento de Proteína Pós-Traducional , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Remielinização/genética , Remielinização/fisiologia
2.
Proc Natl Acad Sci U S A ; 117(27): 15818-15826, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541024

RESUMO

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Clonagem Molecular , Ativação do Complemento , Miócitos de Músculo Liso/metabolismo , Fagocitose/fisiologia , Animais , Antígeno CD47/metabolismo , Linhagem da Célula , Proliferação de Células , Complemento C3/genética , Complemento C3/metabolismo , Feminino , Humanos , Inflamação , Macrófagos/metabolismo , Masculino , Camundongos Knockout para ApoE , Miócitos de Músculo Liso/citologia , Placa Aterosclerótica/metabolismo , Análise de Sequência de RNA , Regulação para Cima
3.
Braz J Med Biol Res ; 53(7): e9207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520207

RESUMO

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.


Assuntos
Citoesqueleto/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Inativação Gênica , Vetores Genéticos , Humanos , Camundongos , Células RAW 264.7 , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Transfecção
4.
PLoS One ; 15(5): e0232432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365067

RESUMO

CR3 and CR4, the leukocyte specific ß2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of ß2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells.


Assuntos
Células Dendríticas/imunologia , Inflamação/imunologia , Integrina alfaXbeta2/imunologia , Antígeno de Macrófago 1/imunologia , Macrófagos/imunologia , Podossomos/imunologia , Anticorpos Bloqueadores/imunologia , Antígenos CD18/imunologia , Adesão Celular/imunologia , Adesão Celular/fisiologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Movimento Celular/fisiologia , Células Dendríticas/patologia , Células Dendríticas/fisiologia , Fibrinogênio/imunologia , Humanos , Técnicas In Vitro , Inflamação/patologia , Lipopolissacarídeos/imunologia , Macrófagos/patologia , Macrófagos/fisiologia , Fagocitose/imunologia , Fagocitose/fisiologia , Podossomos/patologia
5.
Nat Cell Biol ; 22(5): 546-558, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341550

RESUMO

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.


Assuntos
Células Epiteliais/fisiologia , Epitélio/fisiologia , Animais , Proliferação de Células/fisiologia , Feminino , Lactação/fisiologia , Macrófagos/fisiologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Fagocitose/fisiologia , Gravidez
6.
Am J Physiol Heart Circ Physiol ; 318(6): H1447-H1460, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32330087

RESUMO

Macrophages play a pivotal role in tissue repair following myocardial infarction (MI). In response to injury, they exist along a spectrum of activation states tightly regulated by their microenvironment. Cardiosphere-derived cells (CDCs) have been shown to mediate cardioprotection via modulation of the macrophage response. Our study was designed to gain mechanistic insight into the role of CDC-derived extracellular vesicles (EVs) in modulating macrophage phenotypes and operant signaling pathways to better understand their potential contribution to immunomodulatory cardioprotection. We found that CDC-derived EVs alter the functional phenotype of macrophages, modifying levels of phagocytosis and efferocytosis without changing viability or proliferation. Interestingly, extracellular vesicles differentially regulate several M1/M2 genes dependent on macrophage activation before EV treatment but consistently upregulate arginase 1 regardless of macrophage origin or polarization state. CDC-derived EVs polarize M1 macrophages to a proangiogenic phenotype dependent on arginase 1 upregulation and independent of VEGF-A. In addition, EV-dependent arginase 1 upregulation downregulates nitric oxide (NO) secretion in activated macrophages. These data suggest a novel urea-cycle-dependent mechanism in macrophages that promotes angiogenesis and provides additional mechanistic insight into the potential contribution of CDC-derived extracellular vesicles in immunomodulatory cardioprotection.NEW & NOTEWORTHY We hypothesized that in the window of therapeutic extracellular vesicle (EV) administration, inflammatory M1 macrophages are likely the primary target of cardiosphere-derived cell (CDC)-derived EVs. The effect of CDC-EVs on this population, however, is currently unknown. In this study, we demonstrate that CDC-derived EVs polarize M1 macrophages to a proangiogenic phenotype dependent on arginase 1 upregulation. These results provide insight into an immunomodulatory mechanism of CDC-EVs in a more physiologically relevant model of post-myocardial infarction (post-MI) macrophage polarization.


Assuntos
Arginase/metabolismo , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Animais , Proliferação de Células/fisiologia , Sobrevivência Celular , Humanos , Camundongos , Fagocitose/fisiologia , Fenótipo
7.
Invest Ophthalmol Vis Sci ; 61(3): 9, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32176260

RESUMO

Purpose: To investigate diurnal variation in the length of mouse rod outer segments in vivo. Methods: The lengths of rod inner and outer segments (RIS, ROS) of dark-adapted albino mice maintained on a 12-hour dark:12-hour light cycle with light onset 7 AM were measured at prescribed times (6:30 AM, 11 AM, 3:30 PM) during the diurnal cycle with optical coherence tomography (OCT), taking advantage of increased visibility, after a brief bleaching exposure, of the bands corresponding to RIS/ROS boundaries and ROS tips (ROST). Results: Deconvolution of OCT depth profiles resolved two backscatter bands located 7.4 ± 0.1 and 10.8 ± 0.2 µm (mean ± SEM) proximal to Bruch's membrane (BrM). These bands were identified with histology as arising from the apical surface of RPE and ROST, respectively. The average length of dark-adapted ROS at 6:30 AM was 17.7 ± 0.8 µm. By 11 AM, the average ROS length had decreased by 10% to 15.9 ± 0.7 µm. After 11 AM, the ROS length increased steadily at an average rate of 0.12 µm/h, returning to baseline length by 23.5 hours in the cycle. Conclusions: The diurnal variation in ROS length measured in these experiments is consistent with prior histological investigations showing that rodent rod discs are phagocytosed by the RPE maximally over several hours around the time of normal light onset. The rate of recovery of ROS to baseline length before normal light onset is consistent with the hypothesis that disc membrane synthesis is fairly constant over the diurnal cycle.


Assuntos
Ritmo Circadiano/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Albinismo Ocular/patologia , Animais , Lâmina Basilar da Corioide/ultraestrutura , Adaptação à Escuridão/fisiologia , Camundongos Endogâmicos BALB C , Microscopia Confocal , Fagocitose/fisiologia , Retina/anatomia & histologia , Retina/diagnóstico por imagem , Segmento Interno das Células Fotorreceptoras da Retina/fisiologia , Segmento Interno das Células Fotorreceptoras da Retina/ultraestrutura , Segmento Externo da Célula Bastonete/ultraestrutura , Espalhamento de Radiação , Tomografia de Coerência Óptica/métodos
8.
Am J Pathol ; 190(6): 1224-1235, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32201264

RESUMO

Intratracheal instillation of apoptotic cells enhances resolution of experimental lung inflammation by incompletely understood mechanisms. We report that this intervention induces functional regulatory T lymphocytes (Tregs) in mouse lung experimentally inflamed by intratracheal administration of lipopolysaccharide. Selective depletion demonstrated that Tregs were necessary for maximal apoptotic cell-directed enhancement of resolution, and adoptive transfer of additional Tregs was sufficient to promote resolution without administering apoptotic cells. After intratracheal instillation, labeled apoptotic cells were observed in most CD11c+CD103+ myeloid dendritic cells migrating to mediastinal draining lymph nodes and bearing migratory and immunoregulatory markers, including increased CCR7 and ß8 integrin (ITGB8) expression. In mice deleted for αv integrin in the myeloid line to reduce phagocytosis of dying cells by CD103+ dendritic cells, exogenous apoptotic cells failed to induce transforming growth factor-ß1 expression or Treg accumulation and failed to enhance resolution of lipopolysaccharide-induced lung inflammation. We conclude that in murine lung, myeloid phagocytes encountering apoptotic cells can deploy αv integrin-mediated mechanisms to induce Tregs and enhance resolution of acute inflammation.


Assuntos
Apoptose/fisiologia , Integrina alfaV/metabolismo , Pneumonia/metabolismo , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Fatores de Transcrição Forkhead/metabolismo , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Fagocitose/fisiologia , Pneumonia/patologia
9.
Invest Ophthalmol Vis Sci ; 61(2): 4, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031576

RESUMO

Purpose: Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. Methods: Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O2) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. Results: Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. Conclusions: VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult. These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.


Assuntos
Colecalciferol/farmacologia , Hiperóxia/fisiopatologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vitaminas/farmacologia , Adolescente , Adulto , Cadáver , Células Cultivadas , Criança , Pré-Escolar , Células Endoteliais da Veia Umbilical Humana , Humanos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Receptores Notch/metabolismo , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
10.
J Neurosci ; 40(10): 2154-2165, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31980585

RESUMO

Astrocytes are an integral component of the neurovascular unit where they act as homeostatic regulators, especially after brain injuries, such as stroke. One process by which astrocytes modulate homeostasis is the release of functional mitochondria (Mt) that are taken up by other cells to improve their function. However, the mechanisms underlying the beneficial effect of Mt transfer are unclear and likely multifactorial. Using a cell culture system, we established that astrocytes release both intact Mt and humanin (HN), a small bioactive peptide normally transcribed from the Mt genome. Further experiments revealed that astrocyte-secreted Mt enter microglia, where they induce HN expression. Similar to the effect of HN alone, incorporation of Mt by microglia (1) upregulated expression of the transcription factor peroxisome proliferator-activated receptor gamma and its target genes (including mitochondrial superoxide dismutase), (2) enhanced phagocytic activity toward red blood cells (an in vitro model of hematoma clearance after intracerebral hemorrhage [ICH]), and (3) reduced proinflammatory responses. ICH induction in male mice caused profound HN loss in the affected hemisphere. Intravenously administered HN penetrated perihematoma brain tissue, reduced neurological deficits, and improved hematoma clearance, a function that normally requires microglia/macrophages. This study suggests that astrocytic Mt-derived HN could act as a beneficial secretory factor, including when transported within Mt to microglia, where it promotes a phagocytic/reparative phenotype. These findings also indicate that restoring HN levels in the injured brain could represent a translational target for ICH. These favorable biological responses to HN warrant studies on HN as therapeutic target for ICH.SIGNIFICANCE STATEMENT Astrocytes are critical for maintaining brain homeostasis. Here, we demonstrate that astrocytes secrete mitochondria (Mt) and the Mt-genome-encoded, small bioactive peptide humanin (HN). Mt incorporate into microglia, and both Mt and HN promote a "reparative" microglia phenotype characterized by enhanced phagocytosis and reduced proinflammatory responses. Treatment with HN improved outcomes in an animal model of intracerebral hemorrhage, suggesting that this process could have biological relevance to stroke pathogenesis.


Assuntos
Astrócitos/metabolismo , Hemorragia Cerebral , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microglia/metabolismo , Mitocôndrias/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologia , Fenótipo , Ratos , Ratos Sprague-Dawley
11.
J Neurosci ; 40(7): 1453-1482, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31896673

RESUMO

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.


Assuntos
Hipocampo/citologia , Neurogênese/fisiologia , Neurônios/citologia , Fagocitose/fisiologia , Animais , Apoptose , Sinalização do Cálcio , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Meios de Cultivo Condicionados , Retroalimentação Fisiológica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Hipocampo/crescimento & desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Purinérgicos P2Y12/fisiologia , Transcriptoma , c-Mer Tirosina Quinase/fisiologia
12.
Nat Rev Immunol ; 20(4): 254-267, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31822793

RESUMO

The clearance of apoptotic cells by professional and non-professional phagocytes - a process termed 'efferocytosis' - is essential for the maintenance of tissue homeostasis. Accordingly, defective efferocytosis underlies a growing list of chronic inflammatory diseases. Although much has been learnt about the mechanisms of apoptotic cell recognition and uptake, several key areas remain incompletely understood. This Review focuses on new discoveries related to how phagocytes process the metabolic cargo they receive during apoptotic cell uptake; the links between efferocytosis and the resolution of inflammation in health and disease; and the roles of efferocytosis in host defence. Understanding these aspects of efferocytosis sheds light on key physiological and pathophysiological processes and suggests novel therapeutic strategies for diseases driven by defective efferocytosis and impaired inflammation resolution.


Assuntos
Inflamação/fisiopatologia , Fagócitos/fisiologia , Fagocitose/fisiologia , Apoptose/fisiologia , Homeostase/fisiologia , Humanos
13.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31685547

RESUMO

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1 +) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1 + strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.


Assuntos
Fermentação/fisiologia , Mucor/metabolismo , Mucor/patogenicidade , Virulência/fisiologia , Álcool Desidrogenase/metabolismo , Animais , Linhagem Celular , Fermentação/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/farmacologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucor/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Células RAW 264.7 , Virulência/efeitos dos fármacos
14.
Glia ; 68(1): 44-59, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31429116

RESUMO

Synaptic strength reduces during sleep, but the underlying mechanisms of this process are unclear. This study showed reduction of synaptic proteins in rat prefrontal cortex (PFC) at AM7 or Zeitgeber Time (ZT0), when the light phase or sleeping period for rats started. At this time point, microglia were weakly activated, displaying larger and more granular somata with increased CD11b expression compared with those at ZT12, as revealed by flow cytometry. Expression of opsonins, such as complements or MFG-E8, matrix metalloproteinases, and microglial markers at ZT0 were increased compared with that at ZT12. Microglia at ZT0 phagocytosed synapses, as revealed by immunohistochemical staining. Immunoblotting detected more synapsin I in the isolated microglia at ZT0 than at ZT12. Complement C3- or MFG-E8-bound synapses were the most abundant at ZT0, some of which were phagocytosed by microglia. Systemic administration of synthetic glucocorticoid dexamethasone reduced microglial size, granularity and CD11b expression at ZT0, resembling microglia at ZT12, and increased synaptic proteins and decreased the sleeping period. Noradrenaline (NA) suppressed glutamate-induced phagocytosis in primary cultured microglia. Systemic administration of the brain monoamine-depleting agent reserpine decreased NA content and synapsin I expression in PFC, and increased expression of microglia markers, C3 and MFG-E8, while increasing the sleeping period. A NA precursor l-threo-dihydroxyphenylserine abolished the reserpine-induced changes. These results suggest that microglia may eliminate presumably weak synapses during every sleep phase. The circadian changes in concentrations of circulating glucocorticoids and brain NA might be correlated with the circadian changes of microglial phenotypes and synaptic strength.


Assuntos
Microglia/metabolismo , Fagócitos/metabolismo , Fagocitose/fisiologia , Córtex Pré-Frontal/metabolismo , Fases do Sono/fisiologia , Sinapses/metabolismo , Animais , Células Cultivadas , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Dexametasona/farmacologia , Masculino , Microglia/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos Wistar , Fases do Sono/efeitos dos fármacos , Sinapses/efeitos dos fármacos
15.
Int J Parasitol ; 50(1): 75-83, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31857073

RESUMO

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.


Assuntos
Dreissena/imunologia , Hemócitos/parasitologia , Mytilus edulis/imunologia , Fagocitose/fisiologia , Espécies Sentinelas , Animais , Cryptosporidium , Transmissão de Doença Infecciosa , Dreissena/citologia , Água Doce/parasitologia , Hemócitos/imunologia , Humanos , Imunidade Celular/fisiologia , Mytilus edulis/citologia , Água do Mar/parasitologia , Toxoplasma
16.
Crit Care Med ; 48(1): e58-e65, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31634237

RESUMO

OBJECTIVES: Interleukin-17D has been shown to participate in the control of viral infections and cancer. Here we hypothesized that interleukin-17D may play a potential role in sepsis. DESIGN: Prospective randomized animal investigation and in vitro human blood studies. SETTING: Research laboratory from a university hospital. SUBJECTS: Female C57BL/6J mice, sepsis patients by Sepsis-3 definitions, ICU patient controls, and healthy individuals. INTERVENTIONS: Serum concentrations of interleukin-17D were measured and analyzed in human sepsis patients, patient controls, and healthy individuals. The contribution of interleukin-17D to sepsis-related survival, bacterial burden, and organ injury was assessed in a murine model of cecal ligation and puncture-induced polymicrobial sepsis by the use of anti-interleukin-17D antibody and recombinant interleukin-17D protein. The effects of interleukin-17D on bacterial phagocytosis by macrophages were also investigated using in vitro cell models. MEASUREMENTS AND MAIN RESULTS: On the day of ICU admission (day 0), septic patients had significantly higher serum concentrations of interleukin-17D than patient controls and healthy individuals. Serum interleukin-17D levels remained significantly elevated in septic patients from ICU admission to day 3 and correlated with Sequential (Sepsis-related) Organ Failure Assessment scores and documented bacteremia on day 0. Furthermore, nonsurvivors of septic patients displayed significantly higher interleukin-17D levels compared with survivors of septic patients on days 0 and 1 of ICU admission. In animal models of sepsis, treatment with anti-interleukin-17D antibody protected mice from cecal ligation and puncture-induced severe sepsis, which was associated with improved bacterial clearance and organ injury. Conversely, administration of recombinant interleukin-17D protein aggravated cecal ligation and puncture-induced nonsevere sepsis. Furthermore, we found that interleukin-17D impaired bacterial phagocytosis by macrophages. Phagocytosis inhibition by interleukin-17D involved its ability to down-regulate the activation of nuclear factor-κB signaling pathway in macrophages upon bacterial infection. CONCLUSIONS: This study indicates a previously undescribed role of interleukin-17D in sepsis and identifies a new target for antisepsis treatment.


Assuntos
Interleucina-27/sangue , Interleucina-27/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Sepse/sangue , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Índice de Gravidade de Doença
17.
Nat Cell Biol ; 21(12): 1532-1543, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792382

RESUMO

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.


Assuntos
Apoptose/fisiologia , Cloretos/metabolismo , Inflamação/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Apoptose/genética , Transporte Biológico/genética , Transporte Biológico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Inflamação/genética , Inflamação/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Fagócitos/fisiologia , Fagocitose/genética , Fagocitose/fisiologia , Transdução de Sinais/genética , Simportadores de Cloreto de Sódio-Potássio/genética , Transcrição Genética/genética , Transcrição Genética/fisiologia
18.
Lipids Health Dis ; 18(1): 215, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31823799

RESUMO

BACKGROUND: Macrophage are specialized cells that contributes to the removal of detrimental contents via phagocytosis. Lipid accumulation in macrophages, whether from phagocytosis of dying cells or from circulating oxidized low-density lipoproteins, alters macrophage biology and functionality. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. However, the potential of CPT1a to activate macrophage phagocytic function have not been elucidated. METHODS: Using a murine macrophage cell line, RAW264.7, we determine if intracellular accumulation of 7-ketocholesterol (7-KC) modulates macrophage phagocytic function through CPT1a gene expression. In addition, the effects of CPT1a genetic modification on macrophage phenotype and phagocytosis has been studied. RESULTS: Our results revealed that CPT1a gene expression decreased by the accumulation of 7-KC at the higher dose of 7-KC. This was concomitant with an impair ability to phagocytize bioparticles and an inflammatory phenotype. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC-laden macrophages, increased the gene expression of CPT1a, diminished the gene expression of the inflammatory marker iNOS and restored macrophage phagocytosis. Furthermore, CPT1a Knockdown per se was detrimental for macrophage phagocytosis whereas transcriptional activation of CPT1a heightened the uptake of bioparticles. CONCLUSIONS: Altogether, our findings indicate that downregulation of CPT1a by lipid content modulates macrophage phagocytosis and inflammatory phenotype.


Assuntos
Carnitina O-Palmitoiltransferase/genética , Expressão Gênica/fisiologia , Inflamação , Cetocolesteróis/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Animais , Carnitina O-Palmitoiltransferase/fisiologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Cetocolesteróis/farmacologia , Ativação de Macrófagos/fisiologia , Camundongos , Células RAW 264.7 , Transfecção
19.
Nat Commun ; 10(1): 5606, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811209

RESUMO

Feeding on unicellular photosynthetic organisms by unicellular eukaryotes is the base of the aquatic food chain and evolutionarily led to the establishment of photosynthetic endosymbionts/organelles. Photosynthesis generates reactive oxygen species and damages cells; thus, photosynthetic organisms possess several mechanisms to cope with the stress. Here, we demonstrate that photosynthetic prey also exposes unicellular amoebozoan and excavates predators to photosynthetic oxidative stress. Upon illumination, there is a commonality in transcriptomic changes among evolutionarily distant organisms feeding on photosynthetic prey. One of the genes commonly upregulated is a horizontally transferred homolog of algal and plant genes for chlorophyll degradation/detoxification. In addition, the predators reduce their phagocytic uptake while accelerating digestion of photosynthetic prey upon illumination, reducing the number of photosynthetic cells inside the predator cells, as this also occurs in facultative endosymbiotic associations upon certain stresses. Thus, some mechanisms in predators observed here probably have been necessary for evolution of endosymbiotic associations.


Assuntos
Cadeia Alimentar , Fotossíntese/fisiologia , Comportamento Predatório/fisiologia , Simbiose/fisiologia , Amebozoários/fisiologia , Amebozoários/efeitos da radiação , Animais , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Evolução Biológica , Clorofila , Técnicas de Cocultura , Eucariotos , Evolução Molecular , Luz/efeitos adversos , Naegleria/crescimento & desenvolvimento , Naegleria/fisiologia , Organelas/fisiologia , Estresse Oxidativo , Fagocitose/fisiologia , Comportamento Predatório/efeitos da radiação , Domínios Proteicos , Espécies Reativas de Oxigênio , Simbiose/efeitos da radiação , Transcriptoma
20.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868579

RESUMO

Autoreactive thymocytes are eliminated during negative selection in the thymus, a process important for establishing self-tolerance. Thymic phagocytes serve to remove dead thymocytes, but whether they play additional roles during negative selection remains unclear. Here, using a murine thymic slice model in which thymocytes undergo negative selection in situ, we demonstrate that phagocytosis promotes negative selection, and provide evidence for the escape of autoreactive CD8 T cells to the periphery when phagocytosis in the thymus is impaired. We also show that negative selection is more efficient when the phagocyte also presents the negative selecting peptide. Our findings support a model for negative selection in which the death process initiated following strong TCR signaling is facilitated by phagocytosis. Thus, the phagocytic capability of cells that present self-peptides is a key determinant of thymocyte fate.


Assuntos
Morte Celular , Ativação Linfocitária , Fagocitose/fisiologia , Timócitos/metabolismo , Animais , Apresentação do Antígeno , Células da Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Tolerância a Antígenos Próprios , Transdução de Sinais , Timo/imunologia
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