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1.
Chem Commun (Camb) ; 55(100): 15097-15100, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31782429

RESUMO

We prepared a nanoprobe through self-assembly of gold nanoclusters (AuNCs) and using FITC-modified hyaluronic acid (HA) for ratiometric sensing of highly reactive oxygen species (hROS). Taking advantage of the aggregation-induced emission (AIE) properties of the self-assembled AuNCs, hROS-responsive cleavage of HA, and the ratiometric signal change of dual-emission, the nanoprobe exhibited excellent performance in imaging hROS in living cells.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Espécies Reativas de Oxigênio/análise , Espectrometria de Fluorescência/métodos , Animais , Glutationa/química , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/enzimologia , Macrófagos/metabolismo , Nanopartículas Metálicas/toxicidade , Camundongos , Microscopia de Fluorescência , Células RAW 264.7
2.
Nat Commun ; 10(1): 4904, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659168

RESUMO

Xanthine oxidoreductase has been implicated in cancer. Nonetheless, the role played by its two convertible forms, xanthine dehydrogenase (XDH) and oxidase (XO) during tumorigenesis is not understood. Here we produce XDH-stable and XO-locked knock-in (ki) mice to address this question. After tumor transfer, XO ki mice show strongly increased tumor growth compared to wild type (WT) and XDH ki mice. Hematopoietic XO expression is responsible for this effect. After macrophage depletion, tumor growth is reduced. Adoptive transfer of XO-ki macrophages in WT mice increases tumor growth. In vitro, XO ki macrophages produce higher levels of reactive oxygen species (ROS) responsible for the increased Tregs observed in the tumors. Blocking ROS in vivo slows down tumor growth. Collectively, these results indicate that the balance of XO/XDH plays an important role in immune surveillance of tumor development. Strategies that inhibit the XO form specifically may be valuable in controlling cancer growth.


Assuntos
Neoplasias/enzimologia , Xantina Desidrogenase/genética , Xantina Oxidase/genética , Animais , Proliferação de Células , Feminino , Técnicas de Introdução de Genes , Humanos , Macrófagos/enzimologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo
3.
Int J Mol Sci ; 20(19)2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31546645

RESUMO

Abdominal aortic aneurysm (AAA) is among the top 20 causes of death in the United States. Surgical repair is the gold standard for AAA treatment, therefore, there is a need for non-invasive therapeutic interventions. Aneurysms are more closely associated with the osteoclast-like catabolic degradation of the artery, rather than the osteoblast-like anabolic processes of arterial calcification. We have reported the presence of osteoclast-like cells (OLCs) in human and mouse aneurysmal tissues. The aim of this study was to examine OLCs from aneurysmal tissues as a source of degenerative proteases. Aneurysmal and control tissues from humans, and from the mouse CaPO4 and angiotensin II (AngII) disease models, were analyzed via flow cytometry and immunofluorescence for the expression of osteoclast markers. We found higher expression of the osteoclast markers tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), and cathepsin K, and the signaling molecule, hypoxia-inducible factor-1α (HIF-1α), in aneurysmal tissue compared to controls. Aneurysmal tissues also contained more OLCs than controls. Additionally, more OLCs from aneurysms express HIF-1α, and produce more MMP-9 and cathepsin K, than myeloid cells from the same tissue. These data indicate that OLCs are a significant source of proteases known to be involved in aortic degradation, in which the HIF-1α signaling pathway may play an important role. Our findings suggest that OLCs may be an attractive target for non-surgical suppression of aneurysm formation due to their expression of degradative proteases.


Assuntos
Aneurisma da Aorta Abdominal/enzimologia , Osteoclastos/enzimologia , Animais , Aneurisma da Aorta Abdominal/metabolismo , Catepsina K/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Osteoclastos/metabolismo , Proteólise , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/metabolismo
4.
Nutrients ; 11(9)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500218

RESUMO

Omega-3 polyunsaturated fatty acids (ω3-PUFAs) have potential protective activity in a variety of infectious diseases, but their actions and underlying mechanisms in Toxoplasma gondii infection remain poorly understood. Here, we report that docosahexaenoic acid (DHA) robustly induced autophagy in murine bone marrow-derived macrophages (BMDMs). Treatment of T. gondii-infected macrophages with DHA resulted in colocalization of Toxoplasma parasitophorous vacuoles with autophagosomes and reduced intracellular survival of T. gondii. The autophagic and anti-Toxoplasma effects induced by DHA were mediated by AMP-activated protein kinase (AMPK) signaling. Importantly, BMDMs isolated from Fat-1 transgenic mice, a well-known animal model capable of synthesizing ω3-PUFAs from ω6-PUFAs, showed increased activation of autophagy and AMPK, leading to reduced intracellular survival of T. gondii when compared with wild-type BMDMs. Moreover, Fat-1 transgenic mice exhibited lower cyst burden in the brain following infection with the avirulent strain ME49 than wild-type mice. Collectively, our results revealed mechanisms by which endogenous ω3-PUFAs and DHA control T. gondii infection and suggest that ω3-PUFAs might serve as therapeutic candidate to prevent toxoplasmosis and infection with other intracellular protozoan parasites.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antiparasitários/farmacologia , Autofagia/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Macrófagos/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/prevenção & controle , Toxoplasmose Cerebral/prevenção & controle , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/parasitologia , Encéfalo/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Macrófagos/enzimologia , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/parasitologia , Transdução de Sinais , Toxoplasma/patogenicidade , Toxoplasmose Animal/enzimologia , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia , Toxoplasmose Cerebral/enzimologia , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologia
5.
Free Radic Res ; 53(8): 875-881, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257950

RESUMO

Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that, when secreted, can act as a proinflammatory signal. Here we studied the regulation of intracellular PRDX1 by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in the RAW 264.7 mouse macrophage cell line. While LPS or IFN-γ alone did not affect PRDX1 protein levels, their combination led to an almost complete loss of the PRDX1 dimer. This was likely mediated by the increased production of nitric oxide (NO) as it was reversed by the NO synthase inhibitor L-N-methylarginine (L-NMMA), while a NO-releasing agent decreased PRDX1 levels. Inhibition of the proteasome with MG132 also prevented the loss of the PRDX1 dimer, suggesting that the decrease is due to a NO-activated proteasomal degradation pathway. By contrast with the decrease in protein levels, LPS increased PRDX1 mRNA and this effect was amplified by IFN-γ. Two other Nrf2 target genes, thioredoxin reductase (TXNRD1) and haem oxygenase (HMOX1), were also induced by LPS but IFN-γ did not increase their expression further. This study shows that inflammation differentially regulates PRDX1 at the levels of protein stability and gene expression, and that NO plays a key role in this mechanism.


Assuntos
Inflamação , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Peroxirredoxinas/metabolismo , Animais , Regulação da Expressão Gênica , Interferon gama , Lipopolissacarídeos , Macrófagos/enzimologia , Camundongos , Peroxirredoxinas/genética , Proteólise , Células RAW 264.7 , Espécies Reativas de Nitrogênio/metabolismo
6.
Nucleic Acids Res ; 47(16): 8746-8754, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31287870

RESUMO

RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5' leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. We demonstrate here that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5') and the RNA exosome (3'). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.


Assuntos
Processamento Alternativo , Proteínas de Drosophila/genética , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribonuclease P/genética , Animais , Evolução Biológica , Linhagem Celular , Biologia Computacional/métodos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Embrião não Mamífero , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Íntrons , Macrófagos/citologia , Macrófagos/enzimologia , Masculino , Conformação de Ácido Nucleico , Subunidades Proteicas/metabolismo , Proteólise , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo
7.
Eur J Pharmacol ; 860: 172559, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31325435

RESUMO

Abdominal aortic aneurysm (AAA) is characterized with progressive weakening and considerable dilation of the aortic wall. Despite the high risk of mortality in the elderly population, there are still no clinical pharmacological therapies to alleviate AAA progression. Macrophage-derived MMP9 acts as a key factor in extracellular matrix degradation and is crucial for aortic aneurysm development and aortic rupture. Here, we demonstrated that the transcription level of MMP9 was suppressed with a concentration-dependent manner in macrophages after Imatinib treatment, which was accompanied by the down-regulation of MMP9 protein expression and reduced MMP9 secretion in vitro. Imatinib administration (50 mg/kg/d, i.g.) was carried out one week after the establishment of elastase-induced AAA in rats, stabilizing aneurysm progression and improving survival rate via decreasing the aortic diameter and preventing elastin degradation. Expression and activity of MMP9 in the artery tissues were significantly suppressed after Imatinib treatment via in situ assessment like immunohistochemistry and zymography, although macrophage infiltration was not affected. Furthermore, we found that Imatinib inhibited MMP9 transcription through reduction of STAT3 phosphorylation and translocation from nucleus to cytoplasm. These observations indicated that Imatinib prevents aneurysm progression by inhibiting STAT3-mediated MMP9 expression and activation, suggesting a new application of Imatinib on AAA clinical therapy.


Assuntos
Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/prevenção & controle , Progressão da Doença , Mesilato de Imatinib/farmacologia , Macrófagos/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Elastase Pancreática/efeitos adversos , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Macrófagos/enzimologia , Masculino , Metaloproteinase 9 da Matriz/genética , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo
8.
Zhonghua Gan Zang Bing Za Zhi ; 27(7): 516-520, 2019 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357777

RESUMO

Objective: To investigate the effects of different expression of monoacylglycerol lipase (MAGL) in tumor-associated macrophages (TAMs) with the proliferation of MHCC97H human liver cancer cells in vivo and its mechanism. Methods: Human peripheral blood-derived monocyte was induced to differentiate into M2-type TAMs and was identified by flow cytometry. The co-culture model of TAMs and MHCC97H human liver cancer cells was established, and the expression of MAGL in TAMs cells was detected by qRT-PCR. The expression of MAGL in TAMs cells was detected by plasmid transfection. ELISA and qRT-PCR was used to detect the mRNA expression levels and secretion levels of inflammatory factors in TAMs cells. The subcutaneous tumor model of MHCC97H mice was constructed to observe the effect of different expression of MAGL in TAMs cells with the proliferation of MHCC97H human liver cancer cells in vivo. F-test was used for the measurement of homogeneity of variance between two independent samples. A t-test was used for homogeneity of variance, and the corrected t-test was used for non-homogeneity of variance. Results: Human peripheral blood-derived monocytes were successfully induced to differentiate into M2-type TAMs. An in vitro co-culture model was established. qRT-PCR showed that MHCC97H human liver cancer cells significantly down-regulated the expressional level of MAGL in TAMs cells. The constructed subcutaneous tumor model of mice demonstrated that up-regulation up-regulation of MAGL expression in M2-type TAMs inhibited the proliferation of MHCC97H human liver cancer cells in vivo. Furthermore, the mechanistic study illustrated that the high expression of MAGL promoted the transcription and secretion of inflammatory factors such as interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha in M2-type TAMs cells. Conclusion: The overexpression of MAGL inhibits the proliferation of MHCC97H hepatocellular carcinoma cells in vivo, and its mechanism may be associated to the release of inflammatory factors that from TAMs cells.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Macrófagos/enzimologia , Monoacilglicerol Lipases/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Humanos , Camundongos
9.
Oxid Med Cell Longev ; 2019: 3264858, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178956

RESUMO

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.


Assuntos
Macrófagos/metabolismo , NADPH Oxidase 4/metabolismo , NF-kappa B/metabolismo , Animais , Polaridade Celular/fisiologia , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Humanos , Interferon gama/farmacologia , Interleucinas/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/deficiência , Espécies Reativas de Oxigênio/metabolismo
10.
mSphere ; 4(3)2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31167948

RESUMO

Epidemiological evidence correlates low serum vitamin A (retinol) levels with increased susceptibility to active tuberculosis (TB); however, retinol is biologically inactive and must be converted into its bioactive form, all-trans retinoic acid (ATRA). Given that ATRA triggers a Niemann-Pick type C2 (NPC2)-dependent antimicrobial response against Mycobacterium tuberculosis, we investigated the mechanism by which the immune system converts retinol into ATRA at the site of infection. We demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived dendritic cells (DCs), but not macrophages, express enzymes in the vitamin A metabolic pathway, including aldehyde dehydrogenase 1 family, member a2 (ALDH1A2) and short-chain dehydrogenase/reductase family, member 9 (DHRS9), enzymes capable of the two-step conversion of retinol into ATRA, which is subsequently released from the cell. Additionally, mRNA and protein expression levels of ALDH1A2 and DC marker CD1B were lower in tuberculosis lung tissues than in normal lung. The conditioned medium from DCs cultured with retinol stimulated antimicrobial activity from M. tuberculosis-infected macrophages, as well as the expression of NPC2 in monocytes, which was blocked by specific inhibitors, including retinoic acid receptor inhibitor (RARi) or N,N-diethylaminobenzaldehyde (DEAB), an ALDH1A2 inhibitor. These results indicate that metabolism of vitamin A by DCs transactivates macrophage antimicrobial responses.IMPORTANCE Tuberculosis (TB) is the leading cause of death by a single infectious agent worldwide. One factor that contributes to the success of the microbe is the deficiency in immunomodulatory nutrients, such as vitamin A (retinol), which are prevalent in areas where TB is endemic. Clinical trials show that restoration of systemic retinol levels in active TB patients is ineffective in mitigating the disease; however, laboratory studies demonstrate that activation of the vitamin A pathway in Mycobacterium tuberculosis-infected macrophages triggers an antimicrobial response. Therefore, the goal of this study was to determine the link between host retinol levels and retinoic acid-mediated antimicrobial responses against M. tuberculosis By combining established in vitro models with in situ studies of lung tissue from TB patients, this study demonstrates that the innate immune system utilizes transcellular metabolism leading to activation between dendritic cells and macrophages as a means to combat the pathogen.


Assuntos
Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Mycobacterium tuberculosis/imunologia , Vitamina A/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/imunologia , Adulto , /imunologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Células Dendríticas/microbiologia , Humanos , Pulmão/microbiologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/microbiologia , Retinal Desidrogenase/genética , Retinal Desidrogenase/imunologia , Tuberculose/microbiologia
11.
Gen Physiol Biophys ; 38(4): 315-324, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31241043

RESUMO

Diosmin is an unsaturated flavonoid glycoside, presents in citrus fruits. The aim of this study is to investigate the molecular mechanism of diosmin with respect to the NF-κB and MAPKs signaling pathways. Firstly, 10, 20, 30, 40 and 50 µM diosmin were treated to lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The anti-inflammatory effects of diosmin was displayed via measuring prostaglandin E2 (PGE2), nitric oxide (NO), interleukines (IL-6, IL-12), tumor necrosis factor α (TNF-α) production, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6, IL-12, TNF-α mRNA levels, and phosphorylation levels of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκB-α) and mitogen-activated protein kinases (MAPKs); JNK, ERK, and p38 in LPS induced RAW264.7 macrophages. Our study showed that especially high concentrations of diosmin decreased NO, PGE2, IL-6, IL-12, TNF-α production and mRNA levels of these mediators (p < 0.05). The expression of phosphorylated-JNK was significantly suppressed by diosmin at 40 and 50 µM concentrations. Furthermore, diosmin significantly inhibited the expression of phosphorylated-ERK, p38, and p-IκB-α in a dose-dependent manner. Our results suggest that diosmin is a potent anti-inflammatory agent and has potential for development into a therapeutic agent for inflammation-associated disorders.


Assuntos
Diosmina/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos
12.
PLoS One ; 14(5): e0216405, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071151

RESUMO

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.


Assuntos
Anti-Inflamatórios/farmacologia , Atorvastatina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Proteínas de Membrana/biossíntese , Zimosan/toxicidade , Animais , Movimento Celular/efeitos dos fármacos , Feminino , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/epidemiologia , Inflamação/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Metaloporfirinas/farmacologia , Camundongos , Monócitos/enzimologia , Monócitos/patologia , Neutrófilos/enzimologia , Neutrófilos/patologia , Protoporfirinas/farmacologia
13.
J Vasc Res ; 56(3): 139-151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31064000

RESUMO

BACKGROUND: It has been reported that smoking is one of the strongest positive risk factors for abdominal aortic aneurysms (AAAs). Although many studies have been directed to decipher the effect of smoking on AAA, its effect on macrophage activation has not yet been explored. OBJECTIVES: We have reported the importance of osteoclastogenesis (OCG) in aneurysm formation. Therefore, we examined the effect of cigarette smoking on OCG and arterial aneurysmal formation by using cigarette smoke extract (CSE) in this study. METHODS: Macrophage cell lines were stimulated with CSE, and their activation and differentiation were examined in vitro. Since macrophages activated through the OCG pathway are identified by tartrate-resistant acid phosphatase (TRAP) expression, these cells are referred to as TRAP-positive macrophages (TPMs) in this study. We also applied CSE-contained PBS in the calcium chloride-induced mouse carotid aneurysm model in vivo. RESULTS: Macrophages stimulated with CSE expressed significantly higher levels of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, cathepsin K, matrix metalloproteinase-9 and membrane-type metalloproteinase (MT1-MMP). CSE-treated mouse aneurysms showed increased aneurysm size with increased TPM infiltration and protease expression compared to non-CSE-treated mouse aneurysms. CONCLUSIONS: These results suggest that CSE intensifies OCG in macrophages and promotes arterial aneurysmal progression.


Assuntos
Aneurisma/induzido quimicamente , Doenças das Artérias Carótidas/induzido quimicamente , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fumaça/efeitos adversos , Fosfatase Ácida Resistente a Tartarato/metabolismo , Produtos do Tabaco/efeitos adversos , Aneurisma/enzimologia , Aneurisma/patologia , Animais , Cloreto de Cálcio , Doenças das Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/patologia , Catepsina K/metabolismo , Modelos Animais de Doenças , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/enzimologia , Osteoclastos/patologia , Células RAW 264.7 , Transdução de Sinais
14.
Ecotoxicol Environ Saf ; 174: 574-583, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30870658

RESUMO

Endocrine disruptors interfere with normal sexual and reproductive development of numerous organisms. Widely used in several chemical and manufacturing industries, nonylphenol (NP), a potent xenoestrogen, has the potential to perturb immune system. Using rat splenic macrophages (SMΦ) as the model system, NP-modulation of lipopolysaccharide (LPS)-induced inflammatory response has been investigated. Our results demonstrate that NP (0.1-10 µM) attenuates catalase activity, reactive oxygen species (ROS) generation and nitric oxide (NO) synthesis in LPS-treated SMΦ in vitro. NP inhibition of LPS-induced nuclear factor kappa B (NF-κB) activation and pro-inflammatory cytokine gene expression corroborate well with attenuation of suppressor of cytokine signalling 3 (SOCS3). Besides, elevated expression of anti-inflammatory factors reveals inverse correlation with suppression of endotoxin-induced M1 polarization in NP pre-incubated cells. While LPS promotes, NP prevents ERK1/2 (extracellular-signa1-regulated kinase 1/2) phosphorylation and MEK-inhibitor abrogates SOCS3 expression and NO production suggesting involvement of ERK1/2 in NP inhibition of SOCS3 expression. Further, translational inhibitor cycloheximide prevents LPS-induced NF-κB activation indicating functional importance of de novo synthesis of SOCS3, at least in part, in toll-like receptor 4 (TLR4)-mediated inflammatory response. Collectively, present study provides evidence favouring participation of SOCS3 in NP modulation of inflammatory response in rat SMΦ.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Fenóis/farmacologia , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Catalase/metabolismo , Citocinas/genética , Citocinas/metabolismo , Interações de Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Baço/citologia , Receptor 4 Toll-Like/metabolismo
15.
PLoS One ; 14(3): e0214150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30889221

RESUMO

Myeloperoxidase (MPO) is a highly abundant protein within the neutrophil that is associated with lipoprotein oxidation, and increased plasma MPO levels are correlated with poor prognosis after myocardial infarct. Thus, MPO inhibitors have been developed for the treatment of heart failure and acute coronary syndrome in humans. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide PF-06282999 is a recently described selective small molecule mechanism-based inactivator of MPO. Here, utilizing PF-06282999, we investigated the role of MPO to regulate atherosclerotic lesion formation and composition in the Ldlr-/- mouse model of atherosclerosis. Though MPO inhibition did not affect lesion area in Ldlr-/- mice fed a Western diet, reduced necrotic core area was observed in aortic root sections after MPO inhibitor treatment. MPO inhibition did not alter macrophage content in and leukocyte homing to atherosclerotic plaques. To assess non-invasive monitoring of plaque inflammation, [18F]-Fluoro-deoxy-glucose (FDG) was administered to Ldlr-/- mice with established atherosclerosis that had been treated with clinically relevant doses of PF-06282999, and reduced FDG signal was observed in animals treated with a dose of PF-06282999 that corresponded with reduced necrotic core area. These data suggest that MPO inhibition does not alter atherosclerotic plaque area or leukocyte homing, but rather alters the inflammatory tone of atherosclerotic lesions; thus, MPO inhibition could have utility to promote atherosclerotic lesion stabilization and prevent atherosclerotic plaque rupture.


Assuntos
Acetamidas/farmacologia , Aterosclerose/tratamento farmacológico , Macrófagos/enzimologia , Peroxidase/antagonistas & inibidores , Placa Aterosclerótica/tratamento farmacológico , Pirimidinonas/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Peroxidase/genética , Peroxidase/metabolismo , Placa Aterosclerótica/enzimologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Receptores de LDL/deficiência , Receptores de LDL/metabolismo
16.
Biochimie ; 166: 84-93, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30914255

RESUMO

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2-8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M-1 s-1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.


Assuntos
Catepsinas/química , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Corantes Fluorescentes/química , Humanos , Macrófagos/enzimologia , Camundongos , Oligopeptídeos/química , Baço/enzimologia , Especificidade por Substrato , Células THP-1
17.
Nat Immunol ; 20(4): 420-432, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858618

RESUMO

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.


Assuntos
Dano ao DNA , Macrófagos/metabolismo , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acrilamidas/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Piperidinas/farmacologia
18.
Mol Immunol ; 109: 27-37, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30851634

RESUMO

Small Ras GTPases are key molecules that regulate a variety of cellular responses in different cell types. Rap1 plays important functions in the regulation of macrophage biology during inflammation triggered by toll-like receptors (TLRs). However, despite sharing a relatively high degree of similarity with Rap1, no studies concerning Rap2 in macrophages and innate immunity have been reported yet. In this work, we show that either way alterations in the levels of Rap2a hampers proper macrophages response to TLR stimulation. Rap2a is activated by LPS in macrophages, and although putative activator TLR-inducible Ras guanine exchange factor RasGEF1b was sufficient to induce, it was not fully required for Rap2a activation. Silencing of Rap2a impaired LPS-induced production of IL-6 cytokine and KC/Cxcl1 chemokine, and also NF-κB activity as measured by reporter gene studies. Surprisingly, overexpression of Rap2a did also lead to marked inhibition of NF-κB activation induced by LPS, Pam3CSK4 and downstream TLR signaling molecules. We also found that Rap2a can inhibit the LPS-induced phosphorylation of the NF-κB subunit p65 at serine 536. Collectively, our data suggest that expression levels of Rap2a in macrophages might be tightly regulated to avoid unbalanced immune response. Our results implicate Rap2a in TLR-mediated responses by contributing to balanced NF-κB activity status in macrophages.


Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Macrófagos/enzimologia , NF-kappa B/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Macrófagos/patologia , Camundongos , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Proteínas rap de Ligação ao GTP/genética , Fatores ras de Troca de Nucleotídeo Guanina
19.
J Vasc Surg ; 70(2): 588-598.e2, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30792060

RESUMO

OBJECTIVE: Macrophages play a critical role in the initiation and progression of abdominal aortic aneurysm (AAA) and are classically distinguished into M1 "proinflammatory" and M2 "anti-inflammatory" macrophages. Topical application of elastase associated with transforming growth factor ß (TGF-ß) systemic neutralization reproduces the main pathologic features of human AAA, offering a new model to investigate their role. The aim of this study was to investigate whether macrophages contribute to the expression of canonical M1/M2 markers in the aorta in the AAA model induced by elastase and systemic blockade of TGF-ß and whether blocking of TGF-ß activity affects macrophage phenotype and the expression of the M2 marker arginase 1 (ARG1). METHODS: C57Bl/6J male mice (6-8 weeks old) were randomly assigned to three experimental groups: mice that had local application of heat-inactivated elastase or elastase and mice that had elastase application and received injection of anti-TGF-ß (elastase + anti-TGF-ß group). Monocyte-macrophage depletion was achieved in the elastase + anti-TGF-ß group using liposome clodronate. Macrophage phenotype was characterized by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. Human infrarenal AAA tissues (n = 10) were obtained to analyze ARG1 expression. RESULTS: Analysis of gene expression in the infrarenal aortic wall revealed that after 14 days, no significant difference for the expression of CCL2, NOS2, and Ym1/2 was observed in the elastase group compared with the elastase + anti-TGF-ß group, whereas the expression of ARG1, interleukin (IL) 1ß, and IL-6 was significantly increased. Macrophage depletion in the elastase + anti-TGF-ß group led to a significant decrease of IL-1ß, IL-6, ARG1, and Ym1/2 gene expression. Immunofluorescent staining confirmed that TGF-ß neutralization significantly enhanced ARG1 protein expression in the aneurysmal tissue. Flow cytometry analysis revealed an increase of macrophages expressing ARG1 in the aorta of mice treated with elastase + anti-TGF-ß compared with the elastase group, and their proportion increased with aneurysmal dilation. In humans, ARG1 protein expression was increased in aneurysmal tissues compared with controls, and positive cells were mainly found in the adventitia. CONCLUSIONS: TGF-ß neutralization finely tunes macrophage phenotype in elastase-induced AAA and leads to an increase in ARG1 gene and protein expression in the aortic wall. Even if further studies are required to elucidate its role in AAA development, ARG1 could represent a new prognostic or therapeutic target in aneurysmal disease.


Assuntos
Anticorpos Neutralizantes , Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/enzimologia , Arginase/metabolismo , Macrófagos/enzimologia , Elastase Pancreática , Fator de Crescimento Transformador beta/metabolismo , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/patologia , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Transdução de Sinais , Fator de Crescimento Transformador beta/imunologia , Regulação para Cima
20.
BMC Vet Res ; 15(1): 45, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30704453

RESUMO

BACKGROUND: Bacterial lipopolysaccharide and interferon-γ stimulation of rodent macrophages in vitro induces up-regulation of inducible nitric oxide synthase, whereas interleukin-4 stimulation results in increased activity of arginase-1. Thus different stimulants result in differing macrophage phenotypes, appropriate for responses to a range of pathogens. The current study was conducted in order to determine whether bovine macrophages derived from monocytes and spleen respond similarly. RESULTS: Lipopolysaccharide and interferon-γ did not induce detectable increases in nitric oxide production by bovine monocyte-derived or splenic macrophages in vitro. Similarly, interleukin-4 and interleukin-13 did not affect arginase activity. However, changes in transcription of genes coding for these products were detected. CONCLUSION: Differences between macrophage activation patterns exist between cattle and other species and these differences may occur during the post-transcription phase.


Assuntos
Ativação de Macrófagos , Macrófagos/imunologia , Baço/citologia , Animais , Arginase/metabolismo , Biomarcadores/metabolismo , Bovinos , Polaridade Celular , Células Cultivadas , Feminino , Interferon gama/imunologia , Interleucina-13/imunologia , Interleucina-4/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/enzimologia , Masculino , Óxido Nítrico/metabolismo , Baço/imunologia , Transcrição Genética
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