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1.
Immunity ; 54(9): 1989-2004.e9, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34363750

RESUMO

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.


Assuntos
Autofagia/fisiologia , Células Endoteliais/fisiologia , Infiltração de Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Animais , Quimiotaxia de Leucócito/fisiologia , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Junções Intercelulares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia
2.
Immunity ; 54(7): 1494-1510.e7, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34033752

RESUMO

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.


Assuntos
Envelhecimento/imunologia , Transporte Biológico/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Animais , Quimiocina CXCL1/imunologia , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Feminino , Junções Intercelulares/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-8B/imunologia , Vênulas/imunologia
3.
Immunity ; 50(2): 273-275, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30784571

RESUMO

Leukocyte trafficking is a key component of steady-state tissue homing and in mounting an inflammatory response. Two recent publications in Immunity by He et al. (2018) and Adrover et al. (2019) report on the diurnal regulation of these responses and the associated pathophysiological implications.


Assuntos
Leucócitos , Movimento Celular , Humanos , Masculino , Transporte Proteico
4.
Immunity ; 49(6): 1062-1076.e6, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30446388

RESUMO

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.


Assuntos
Quimiocina CXCL1 , Quimiocina CXCL2 , Sistema do Grupo Sanguíneo Duffy , Neutrófilos , Receptores de Superfície Celular , Migração Transendotelial e Transepitelial , Animais , Músculos Abdominais/efeitos dos fármacos , Músculos Abdominais/imunologia , Músculos Abdominais/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/imunologia , Quimiocina CXCL2/metabolismo , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/imunologia , Sistema do Grupo Sanguíneo Duffy/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/imunologia , Junções Intercelulares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Migração Transendotelial e Transepitelial/genética , Migração Transendotelial e Transepitelial/imunologia , Fator de Necrose Tumoral alfa/farmacologia
5.
Proc Natl Acad Sci U S A ; 121(18): e2400752121, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38648484

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.


Assuntos
Aterosclerose , Células Endoteliais , Lamina Tipo A , Músculo Liso Vascular , Progéria , Animais , Camundongos , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Progéria/metabolismo , Progéria/genética , Progéria/patologia , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertase 9/genética
6.
EMBO Rep ; 25(9): 3842-3869, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38918502

RESUMO

Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.


Assuntos
Senescência Celular , Quimiocina CXCL1 , Células Endoteliais , Inflamação , Neutrófilos , Neutrófilos/metabolismo , Neutrófilos/imunologia , Animais , Humanos , Camundongos , Inflamação/metabolismo , Inflamação/patologia , Células Endoteliais/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/genética , Adesão Celular
7.
Circulation ; 144(22): 1777-1794, 2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34694158

RESUMO

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. METHODS: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type-specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. RESULTS: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P<0.0001). Ubiquitous progerin suppression and lamin A restoration significantly extended lifespan when induced in 6-month-old mildly symptomatic mice and even in severely ill animals aged 13 months, although the benefit was much more pronounced on early intervention (84.5% lifespan extension in mildly symptomatic mice, P<0.0001, and 6.7% in severely ill mice, P<0.01). It is remarkable that major vascular alterations were prevented and lifespan normalized in HGPSrev mice when progerin suppression and lamin A restoration were restricted to vascular smooth muscle cells and cardiomyocytes. CONCLUSIONS: HGPSrev mice constitute a new experimental model for advancing knowledge of HGPS. Our findings suggest that it is never too late to treat HGPS, although benefit is much more pronounced when progerin is targeted in mice with mild symptoms. Despite the broad expression pattern of progerin and its deleterious effects in many organs, restricting its suppression to vascular smooth muscle cells and cardiomyocytes is sufficient to prevent vascular disease and normalize lifespan.


Assuntos
Lamina Tipo A/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Progéria , Animais , Modelos Animais de Doenças , Humanos , Lamina Tipo A/genética , Camundongos , Camundongos Transgênicos , Progéria/genética , Progéria/metabolismo
8.
J Immunol ; 202(5): 1549-1558, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30665935

RESUMO

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Fagócitos/metabolismo , Superóxidos/metabolismo , Simportadores/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Ânions/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Simportadores/genética
9.
Kidney Int ; 97(3): 516-527, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31866111

RESUMO

Here we investigated the role of murine mast cell protease 4 (MCPT4), the functional counterpart of human mast cell chymase, in an experimental model of renal ischemia reperfusion injury, a major cause of acute kidney injury. MCPT4-deficient mice had worsened kidney function compared to wildtype mice. MCPT4 absence exacerbated pathologic neutrophil infiltration in the kidney and increased kidney myeloperoxidase expression, cell death and necrosis. In kidneys with ischemia reperfusion injury, when compared to wildtype mice, MCPT4-deficient mice showed increased surface expression of adhesion molecules necessary for leukocyte extravasation including neutrophil CD162 and endothelial cell CD54. In vitro, human chymase mediated the cleavage of neutrophil expressed CD162 and also CD54, P- and E-Selectin expressed on human glomerular endothelial cells. MCPT4 also dampened systemic neutrophil activation after renal ischemia reperfusion injury as neutrophils expressed more CD11b integrin and produced more reactive oxygen species in MCPT4-deficient mice. Accordingly, after renal injury, neutrophil migration to an inflammatory site distal from the kidney was increased in MCPT4-deficient versus wildtype mice. Thus, contrary to the described overall aggravating role of mast cells, one granule-released mediator, the MCPT4 chymase, exhibits a potent anti-inflammatory function in renal ischemia reperfusion injury by controlling neutrophil extravasation and activation thereby limiting associated damage.


Assuntos
Injúria Renal Aguda , Quimases , Mastócitos/enzimologia , Traumatismo por Reperfusão , Injúria Renal Aguda/prevenção & controle , Animais , Células Endoteliais , Rim , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , Traumatismo por Reperfusão/prevenção & controle
10.
Gut ; 67(8): 1505-1516, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-28601846

RESUMO

OBJECTIVE: Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients' susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients' neutrophils and blood. DESIGN: Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR. RESULTS: The impaired superoxide production by patients' neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 phox /NOX2 and p22 phox ), its cytosolic partner p47 phox but not p67 phox . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients' plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients' neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients' neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production. CONCLUSION: Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.


Assuntos
Cirrose Hepática Alcoólica/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Cirrose Hepática Alcoólica/patologia , Masculino , Pessoa de Meia-Idade
11.
Eur J Clin Invest ; 48 Suppl 2: e12951, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29757466

RESUMO

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.


Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Proteínas de Bactérias/farmacocinética , Ativação Enzimática/fisiologia , Ativadores de Enzimas/farmacologia , Humanos , NADPH Oxidase 2/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/fisiologia , Acetato de Tetradecanoilforbol/farmacocinética
12.
J Hepatol ; 64(5): 1041-1048, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26719020

RESUMO

BACKGROUND & AIMS: Myeloperoxidase exocytosis and production of hydrogen peroxide via the neutrophil superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contribute to efficient elimination of bacteria. Cirrhosis impairs immune functions and increases susceptibility to bacterial infection. We recently showed that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a severe impairment of formylpeptide receptor (fPR)-mediated intracellular signaling and superoxide production. Here, we performed ex vivo studies with these patients' neutrophils to further investigate myeloperoxidase release, bactericidal capacity and signaling events following fPR stimulation by the formylpeptide formyl-met-leu-phe (fMLP). METHODS: Myeloperoxidase release was studied by measuring extracellular myeloperoxidase activity. Activation of signaling effectors was studied by Western blot and their respective contribution to myeloperoxidase release studied using pharmacological antagonists. RESULTS: fMLP-induced myeloperoxidase release was strongly impaired in patients' neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced phosphorylation of major signaling effectors, AKT, ERK1/2 and p38-MAP-Kinases, was also strongly deficient despite a similar expression of signaling effectors or fPR. However, based on effector inhibition in healthy neutrophils, AKT and p38-MAPK but not ERK1/2 upregulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients' neutrophils exhibited a defective bactericidal capacity that was reversed ex vivo by the TLR7/8 agonist CL097, through potentiation of the fMLP-induced AKT/p38-MAPK signaling axis and myeloperoxidase release. CONCLUSIONS: We provide first evidence that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity, which can be reversed via TLR7/8 activation. These defects, together with the previously described severe deficient superoxide production, may increase cirrhotic patients' susceptibility to bacterial infections.


Assuntos
Cirrose Hepática Alcoólica/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neutrófilos/metabolismo , Peroxidase/metabolismo , Western Blotting , Feminino , Humanos , Cirrose Hepática Alcoólica/patologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais
13.
Hepatology ; 57(3): 1163-71, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23080369

RESUMO

UNLABELLED: Cirrhosis is commonly accompanied by impaired defense functions of polymorphonuclear leucocytes (PMNs), increased patient susceptibility to infections, and hepatocellular carcinoma (HCC). PMN antimicrobial activity is dependent on a massive production of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) 2 (NADPH oxidase 2; NOX2), termed respiratory burst (RB). Rapamycin, an antagonist of mammalian target of rapamycin (mTOR), may be used in the treatment of HCC and in transplanted patients. However, the effect of mTOR inhibition on the PMN RB of patients with cirrhosis remains unexplored and was studied here using the bacterial peptide, formyl-Met-Leu-Phe (fMLP), as an RB inducer. fMLP-induced RB of PMN from patients with decompensated alcoholic cirrhosis was strongly impaired (30%-35% of control) as a result of intracellular signaling alterations. Blocking mTOR activation (phospho-S2448-mTOR) with rapamycin further aggravated the RB defect. Rapamycin also inhibited the RB of healthy PMNs, which was associated with impaired phosphorylation of the NOX2 component, p47phox (phox: phagocyte oxidase), on its mitogen-activated protein kinase (MAPK) site (S345) as well as a preferential inhibition of p38-MAPK relative to p44/42-MAPK. However, rapamycin did not alter the fMLP-induced membrane association of p47phox and p38-MAPK in patients' PMNs, but did prevent their phosphorylation at the membranes. The mTOR contribution to fMLP-induced RB, phosphorylation of p47phox and p38-MAPK was further confirmed by mTOR knockdown in HL-60 cells. Finally, rapamycin impaired PMN bactericidal activity, but not bacterial uptake. CONCLUSION: mTOR significantly up-regulates the PMN RB of patients with cirrhosis by p38-MAPK activation. Consequently, mTOR inhibition by rapamycin dramatically aggravates their PMN RB defect, which may increase patients' susceptibility to infection. Thus, concerns should be raised about the use of rapamycin in immuno-depressed patients.


Assuntos
Cirrose Hepática Alcoólica/metabolismo , Neutrófilos/metabolismo , Explosão Respiratória/imunologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Antibióticos Antineoplásicos/farmacologia , Citosol/metabolismo , Interações Medicamentosas , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Feminino , Células HL-60 , Humanos , Cirrose Hepática Alcoólica/tratamento farmacológico , Cirrose Hepática Alcoólica/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fagocitose/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Explosão Respiratória/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
ACS Cent Sci ; 7(8): 1300-1310, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34471675

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS, progeria) is a rare genetic disease characterized by premature aging and death in childhood for which there were no approved drugs for its treatment until last November, when lonafarnib obtained long-sought FDA approval. However, the benefits of lonafarnib in patients are limited, highlighting the need for new therapeutic strategies. Here, we validate the enzyme isoprenylcysteine carboxylmethyltransferase (ICMT) as a new therapeutic target for progeria with the development of a new series of potent inhibitors of this enzyme that exhibit an excellent antiprogeroid profile. Among them, compound UCM-13207 significantly improved the main hallmarks of progeria. Specifically, treatment of fibroblasts from progeroid mice with UCM-13207 delocalized progerin from the nuclear membrane, diminished its total protein levels, resulting in decreased DNA damage, and increased cellular viability. Importantly, these effects were also observed in patient-derived cells. Using the Lmna G609G/G609G progeroid mouse model, UCM-13207 showed an excellent in vivo efficacy by increasing body weight, enhancing grip strength, extending lifespan by 20%, and decreasing tissue senescence in multiple organs. Furthermore, UCM-13207 treatment led to an improvement of key cardiovascular hallmarks such as reduced progerin levels in aortic and endocardial tissue and increased number of vascular smooth muscle cells (VSMCs). The beneficial effects go well beyond the effects induced by other therapeutic strategies previously reported in the field, thus supporting the use of UCM-13207 as a new treatment for progeria.

15.
Antioxid Redox Signal ; 33(5): 354-373, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-31968991

RESUMO

Significance: Despite their intrinsic cytotoxic properties, mounting evidence indicates that reactive oxygen species (ROS) physiologically produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) of epithelial cells (NOX1, dual oxidase [DUOX]2) and phagocytes (NOX2) are critical for innate immune response and homeostasis of the intestinal mucosa. However, dysregulated ROS production could be a driving factor in inflammatory bowel diseases (IBDs). Recent Advances: In addition to NOX2, recent studies have demonstrated that NOX1- and DUOX2-derived ROS can regulate intestinal innate immune defense and homeostasis by impacting many processes, including bacterial virulence, expression of bacteriostatic proteins, epithelial renewal and restitution, and microbiota composition. Moreover, the antibacterial role of DUOX2 is a function conserved in evolution as it has been described in invertebrates, and lower and higher vertebrates. In humans, variants of the NOX2, NOX1, and DUOX2 genes, which are associated with impaired ROS production, have been identified in very early onset IBD, but overexpression of NOX/DUOX, especially DUOX2, has also been described in IBD, suggesting that loss-of-function or excessive activity of the ROS-generating enzymes could contribute to disease progression. Critical Issues: Therapeutic perspectives aiming at targeting NOX/DUOX in IBD should take into account the two sides of NOX/DUOX-derived ROS in intestinal inflammation. Hence, NOX/DUOX inhibitors or ROS inducers should be considered as a function of the disease context. Future Directions: A thorough understanding of the physiological and pathological regulation of NOX/DUOX in the gastrointestinal tract is an absolute pre-requisite for the development of therapeutic strategies that can modulate ROS levels in space and time.


Assuntos
Gastroenterite/etiologia , Gastroenterite/metabolismo , Trato Gastrointestinal/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Biomarcadores , Gerenciamento Clínico , Suscetibilidade a Doenças , Oxidases Duais/genética , Oxidases Duais/metabolismo , Gastroenterite/patologia , Gastroenterite/terapia , Trato Gastrointestinal/patologia , Expressão Gênica , Humanos , Imunidade Inata , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia
16.
J Clin Invest ; 130(5): 2301-2318, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31971917

RESUMO

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability-enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.


Assuntos
Permeabilidade Capilar/imunologia , Microvasos/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Migração Transendotelial e Transepitelial/imunologia , Animais , Permeabilidade Capilar/genética , Masculino , Camundongos , Camundongos Transgênicos , Microvasos/patologia , Neutrófilos/patologia , Migração Transendotelial e Transepitelial/genética
17.
Nat Med ; 25(3): 423-426, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30778239

RESUMO

CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.


Assuntos
Sistemas CRISPR-Cas , Terapia Genética/métodos , Lamina Tipo A/genética , Progéria/terapia , Animais , Células HEK293 , Humanos , Lamina Tipo A/metabolismo , Camundongos , Mutação Puntual , Progéria/genética
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