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1.
JAMA Netw Open ; 7(4): e244699, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38568695

RESUMO

This cohort study evaluates the role that community-level socioeconomic status plays in hypertension-related hospital readmission within 12 weeks after delivery.


Assuntos
Readmissão do Paciente , Feminino , Humanos , Período Pós-Parto
2.
Cells ; 13(6)2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38534377

RESUMO

The chronic inflammatory component of asthma is propagated by granulocytes, including neutrophils and eosinophils, in the peripheral circulation and airway. Previous studies have suggested that these cells have an altered expression of adhesion-related molecules and a propensity for the release of granule contents that may contribute to tissue damage and enhance inflammatory complications in patients with status asthmaticus. The goal of this prospective cohort study at a tertiary care pediatric hospital with a large population of asthma patients was to assess the role of granulocyte-based inflammation in the development of asthma exacerbation. Subjects were enrolled from two patient populations: those with mild-to-moderate asthma exacerbations seen in the emergency department and those with severe asthma admitted to the intensive care unit (PICU). Clinical data were collected, and blood was drawn. Granulocytes were immediately purified, and the phenotype was assessed, including the expression of cell surface markers, elastase release, and cytokine production. Severe asthmatics admitted to the PICU displayed a significantly higher total neutrophil count when compared with healthy donors. Moreover, little to no eosinophils were found in granulocyte preparations from severe asthmatics. Circulating neutrophils from severe asthmatics admitted to the PICU displayed significantly increased elastase release ex vivo when compared with the PMN from healthy donors. These data suggest that the neutrophil-based activation and release of inflammatory products displayed by severe asthmatics may contribute to the propagation of asthma exacerbations.


Assuntos
Asma , Neutrófilos , Humanos , Criança , Elastase Pancreática , Estudos Prospectivos , Eosinófilos , Inflamação
3.
Lab Chip ; 24(3): 615-628, 2024 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-38189525

RESUMO

Excessive release of neutrophil extracellular traps (NETs) has been reported in various human pathologies, including COVID-19 patients. Elevated NET levels serve as a biomarker, indicating increased coagulopathy and immunothrombosis risks in these patients. Traditional immunoassays employed to quantify NET release focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NET release from primary human neutrophils with single-cell resolution in the presence of the motile bacteria Pseudomonas aeruginosa PAO1 and inflammatory mediators. The device was engineered to have wide chambers and constricted loops to measure NET release in variably confined spaces. Our open NET-array device enabled immunofluorescent labeling of citrullinated histone H3, a NET release marker. We took time-lapse images of primary healthy human neutrophils releasing NETs in clinically relevant infection and inflammation-rich microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α [20 ng mL-1] and interleukin-6 [50 ng mL-1], but not leukotriene B4 [20 nM], compared to the infection alone. We also quantified the temporal dynamics of NET release and differences in the relative areas of NETs, showing a high percentage of variable size NET release with combined PAO1 - inflammatory mediator treatment, in the device chambers. Importantly, we demonstrated reduced NET release in the confined loops of our combined infection-inflammation microsystem. Ultimately, our NET-array device stands as a valuable tool, facilitating experiments that enhance our comprehension of the spatiotemporal dynamics of NET release in response to infection within a defined microenvironment. In the future, our system can be used for high throughput and cost-effective screening of novel immunotherapies on human neutrophils in view of the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.


Assuntos
Armadilhas Extracelulares , Humanos , Neutrófilos/microbiologia , Histonas , Inflamação , Mediadores da Inflamação
4.
JCI Insight ; 8(20)2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37707952

RESUMO

Modulation of the immune response to initiate and halt the inflammatory process occurs both at the site of injury as well as systemically. Due to the evolving role of cellular metabolism in regulating cell fate and function, tendon injuries that undergo normal and aberrant repair were evaluated by metabolic profiling to determine its impact on healing outcomes. Metabolomics revealed an increasing abundance of the immunomodulatory metabolite itaconate within the injury site. Subsequent single-cell RNA-Seq and molecular and metabolomic validation identified a highly mature neutrophil subtype, not macrophages, as the primary producers of itaconate following trauma. These mature itaconate-producing neutrophils were highly inflammatory, producing cytokines that promote local injury fibrosis before cycling back to the bone marrow. In the bone marrow, itaconate was shown to alter hematopoiesis, skewing progenitor cells down myeloid lineages, thereby regulating systemic inflammation. Therapeutically, exogenous itaconate was found to reduce injury-site inflammation, promoting tenogenic differentiation and impairing aberrant vascularization with disease-ameliorating effects. These results present an intriguing role for cycling neutrophils as a sensor of inflammation induced by injury - potentially regulating immune cell production in the bone marrow through delivery of endogenously produced itaconate - and demonstrate a therapeutic potential for exogenous itaconate following tendon injury.


Assuntos
Neutrófilos , Succinatos , Humanos , Neutrófilos/metabolismo , Succinatos/farmacologia , Succinatos/metabolismo , Succinatos/uso terapêutico , Macrófagos/metabolismo , Inflamação/metabolismo
5.
Nat Commun ; 14(1): 2610, 2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37147288

RESUMO

Severe COVID-19 is characterized by an increase in the number and changes in the function of innate immune cells including neutrophils. However, it is not known how the metabolome of immune cells changes in patients with COVID-19. To address these questions, we analyzed the metabolome of neutrophils from patients with severe or mild COVID-19 and healthy controls. We identified widespread dysregulation of neutrophil metabolism with disease progression including in amino acid, redox, and central carbon metabolism. Metabolic changes in neutrophils from patients with severe COVID-19 were consistent with reduced activity of the glycolytic enzyme GAPDH. Inhibition of GAPDH blocked glycolysis and promoted pentose phosphate pathway activity but blunted the neutrophil respiratory burst. Inhibition of GAPDH was sufficient to cause neutrophil extracellular trap (NET) formation which required neutrophil elastase activity. GAPDH inhibition increased neutrophil pH, and blocking this increase prevented cell death and NET formation. These findings indicate that neutrophils in severe COVID-19 have an aberrant metabolism which can contribute to their dysfunction. Our work also shows that NET formation, a pathogenic feature of many inflammatory diseases, is actively suppressed in neutrophils by a cell-intrinsic mechanism controlled by GAPDH.


Assuntos
COVID-19 , Armadilhas Extracelulares , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora) , Humanos , COVID-19/metabolismo , Armadilhas Extracelulares/metabolismo , Metaboloma , Metabolômica , Neutrófilos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo
6.
J Cyst Fibros ; 22(1): 140-145, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36041886

RESUMO

BACKGROUND: As people with Cystic Fibrosis (CF) live longer, extra-pulmonary complications such as CF-related bone disease (CFBD) are becoming increasingly important. The etiology of CFBD is poorly understood but is likely multifactorial. Bones undergo continuous remodeling via pathways including RANK (receptor activator of NF-κB)/sRANKL (soluble ligand)/OPG (osteoprotegerin). We sought to examine the association between sRANKL (stimulant of osteoclastogenesis) and OPG levels (inhibitor of osteoclast formation) and CFBD to investigate their potential utility as biomarkers of bone turnover in people with CF. METHODS: We evaluated sRANKL and OPG in plasma from people with CF and healthy controls (HC) and compared levels in those with CF to bone mineral density results. We used univariable and multivariable analysis to account for factors that may impact sRANKL and OPG. RESULTS: We found a higher median [IQR] sRANKL 10,896pg/mL [5,781-24,243] CF; 2,406pg.mL [659.50-5,042] HC; p= 0.0009), lower OPG 56.68pg/mL [36.28-124.70] CF; 583.20pg/mL [421.30-675.10] HC; p < 0.0001), and higher RANKL/OPG in people with CF no BD than in HC (p < 0.0001). Furthermore, we found a higher RANKL/OPG ratio 407.50pg/mL [214.40-602.60] CFBD; 177.70pg/mL [131.50-239.70] CF no BD; p = 0.007) in people with CFBD versus CF without bone disease. This difference persisted after adjusting for variables thought to impact bone health. CONCLUSIONS: The current screening recommendations of imaging for CFBD may miss important markers of bone turnover such as the RANKL/OPG ratio. These findings support the investigation of therapies that modulate the RANK/RANKL/OPG pathway as potential therapeutic targets for bone disease in CF.


Assuntos
Doenças Ósseas , Fibrose Cística , Humanos , Fibrose Cística/complicações , Densidade Óssea , Osteoprotegerina/metabolismo , Remodelação Óssea , Biomarcadores
7.
J Leukoc Biol ; 111(4): 849-856, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34342036

RESUMO

Type 1 diabetes (T1D) is a chronic inflammatory condition sometimes complicated by acute diabetic ketoacidosis (DKA). A subset of patients with T1D develop DKA independent of known risk factors. This study tested the hypothesis that circulating polymorphonuclear leukocytes (PMN) from children with T1D and DKA would exhibit a primed phenotype and that the signature would be unique in patients predisposed to have DKA. Using a prospective cohort study design, neutrophil phenotype was assessed in 30 patients with T1D seen in endocrinology clinic for routine care, 30 patients with acute DKA, and 36 healthy donors. Circulating PMN from patients with DKA display a primed phenotype with increased basal cell-surface CD11b, L-selectin shedding, and enhanced fMLF-elicited reactive oxygen species (ROS) production. Moreover, PMN from T1D patients both with and without DKA lack the capacity to be further primed by incubation with TNF-α, a classic priming stimulus. Primed PMN phenotypic signatures demonstrated are independent of hemoglobin A1c, the premier biological marker for DKA risk, and are consistent with a hyperinflammatory state. A single nucleotide polymorphism in TLR-1 (1805G>T), known to be associated with a hyperinflammatory PMN phenotype, correlated with DKA. This study elucidated a novel phenotypic signature in circulating PMN from children with T1D with and without DKA, and suggests the possibility of a previously unrecognized PMN phenotype with potential clinical implications. Immunophenotype and genotype may be applicable as biomarkers for DKA risk stratification in patients with T1D.


Assuntos
Diabetes Mellitus Tipo 1 , Cetoacidose Diabética , Biomarcadores , Criança , Diabetes Mellitus Tipo 1/complicações , Cetoacidose Diabética/complicações , Humanos , Neutrófilos , Fenótipo , Estudos Prospectivos
8.
Inflammation ; 45(2): 800-811, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34718927

RESUMO

Severe lung inflammation is common in life-threatening coronavirus disease 2019 (COVID-19). This study tested the hypothesis that polymorphonuclear (PMN, neutrophil) phenotype early in the course of disease progression would predict peak lung disease severity in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is increasingly evident that PMN activation contributes to tissue injury resulting from extracellular reactive oxygen species generation, granule exocytosis with release of proteases, neutrophil extracellular trap (NET) formation, and release of cytokines. The current study focuses on PMN activation in response to SARS-CoV-2 infection, specifically, the association between NETs and lung disease. This is a prospective cohort study at an academic medical center with patients enrolled within 4 days of admission at 3 tertiary hospitals: Clements University Hospital, Parkland Memorial Hospital, and Children's Health in Dallas, TX. Patients were categorized as having minimal or moderate to severe lung disease based on peak respiratory support. Healthy donor controls matched for age, sex, race, and ethnicity were also enrolled. Neutrophils from COVID-19 patients displayed greater IL-8 expression, elastase release, and NET formation as compared with neutrophils from healthy donors. Importantly, neutrophils from COVID-19 patients had enhanced NET formation in the absence of any additional stimulus, not seen in PMN from healthy donors. Moreover, PMA-elicited NET formation by circulating PMN correlated with severity of lung disease. We speculate that neutrophil immuno-phenotyping can be used to predict lung disease severity in COVID-19 patients.


Assuntos
COVID-19 , Armadilhas Extracelulares , Humanos , Pulmão , Neutrófilos , Estudos Prospectivos , SARS-CoV-2 , Índice de Gravidade de Doença
9.
J Immunol ; 207(3): 966-973, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290104

RESUMO

Neutrophils, polymorphonuclear leukocytes (PMN), play a critical role in the innate immune response to Staphylococcus aureus, a pathogen that continues to be associated with significant morbidity and mortality. Neutrophil extracellular trap (NET) formation is involved in ensnaring and killing of S. aureus, but this host-pathogen interaction also leads to host tissue damage. Importantly, NET components including neutrophil proteases are under consideration as therapeutic targets in a variety of disease processes. Although S. aureus lipoproteins are recognized to activate cells via TLRs, specific mechanisms of interaction with neutrophils are poorly delineated. We hypothesized that a lipoprotein-containing cell membrane preparation from methicillin-resistant S. aureus (MRSA-CMP) would elicit PMN activation, including NET formation. We investigated MRSA-CMP-elicited NET formation, regulated elastase release, and IL-8 production in human neutrophils. We studied PMN from healthy donors with or without a common single-nucleotide polymorphism in TLR1, previously demonstrated to impact TLR2/1 signaling, and used cell membrane preparation from both wild-type methicillin-resistant S. aureus and a mutant lacking palmitoylated lipoproteins (lgt). MRSA-CMP elicited NET formation, elastase release, and IL-8 production in a lipoprotein-dependent manner. TLR2/1 signaling was involved in NET formation and IL-8 production, but not elastase release, suggesting that MRSA-CMP-elicited elastase release is not mediated by triacylated lipoproteins. MRSA-CMP also primed neutrophils for enhanced NET formation in response to a subsequent stimulus. MRSA-CMP-elicited NET formation did not require Nox2-derived reactive oxygen species and was partially dependent on the activity of peptidyl arginine deiminase (PAD). In conclusion, lipoproteins from S. aureus mediate NET formation via TLR2/1 with clear implications for patients with sepsis.


Assuntos
Membrana Celular/metabolismo , Armadilhas Extracelulares/metabolismo , Lipoproteínas/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Neutrófilos/imunologia , Proteína-Arginina Desiminase do Tipo 1/metabolismo , Infecções Estafilocócicas/imunologia , Células Cultivadas , Humanos , Interleucina-8/metabolismo , Lipoproteínas/genética , Lipoilação , Staphylococcus aureus Resistente à Meticilina/genética , Mutação/genética , Elastase Pancreática/metabolismo , Polimorfismo de Nucleotídeo Único , Transdução de Sinais/genética , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo
10.
J Immunol ; 204(3): 671-681, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31871022

RESUMO

Neutrophils, polymorphonuclear (PMN) leukocytes, play an important role in the early innate immune response to Mycobacterium tuberculosis infection in the lung. Interactions between PMN and mycobacterial lipids impact the activation state of these migrated cells with consequences for the surrounding tissue in terms of resolution versus ongoing inflammation. We hypothesized that lipoarabinomannan from M. tuberculosis (Mtb LAM) would prime human PMN in a TLR2-dependent manner and investigated this with specific comparison with the purified synthetic TLR2 agonists, Pam3CSK4 and FSL-1. In contrast to Pam3CSK4 and FSL-1, we found Mtb LAM did not induce any of the classical PMN priming phenotypes, including enhancement of NADPH oxidase activity, shedding of l-selectin, or mobilization of CD11b. However, exposure of PMN to Mtb LAM did elicit pro- and anti-inflammatory cytokine production and release in a TLR2/1-dependent manner, using the TLR1 single-nucleotide polymorphism rs5743618 (1805G/T) as a marker for TLR2/1 specificity. Moreover, Mtb LAM did not elicit p38 MAPK phosphorylation or endocytosis, although these processes occurred with Pam3CSK4 stimulation, and were necessary for the early priming events to occur. Interestingly, Mtb LAM did not abrogate priming responses elicited by Pam3CSK4 Notably, subfractionation of light membranes from Pam3CSK4 versus Mtb LAM-stimulated cells demonstrated differential patterns of exocytosis. In summary, Mtb LAM activates PMN via TLR2/1, resulting in the production of cytokines but does not elicit early PMN priming responses, as seen with Pam3CSK4 We speculate that the inability of Mtb LAM to prime PMN may be due to differential localization of TLR2/1 signaling.


Assuntos
Lipopolissacarídeos/metabolismo , Mycobacterium tuberculosis/fisiologia , Neutrófilos/imunologia , Receptor 2 Toll-Like/metabolismo , Tuberculose Pulmonar/imunologia , Células Cultivadas , Citocinas/metabolismo , Exocitose , Humanos , Imunidade Inata , Lipopeptídeos/metabolismo , NADPH Oxidases/metabolismo , Ativação de Neutrófilo , Polimorfismo de Nucleotídeo Único , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo
11.
Front Immunol ; 10: 1472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338092

RESUMO

The mortality rate of patients with critical illness has decreased significantly over the past two decades, but the rate of decline has slowed recently, with organ dysfunction as a major driver of morbidity and mortality. Among patients with the systemic inflammatory response syndrome (SIRS), acute lung injury is a common component with serious morbidity. Previous studies in our laboratory using a murine model of SIRS demonstrated a key role for NADPH oxidase 2 (Nox2)-derived reactive oxygen species in the resolution of inflammation. Nox2-deficient (gp91phox-/y) mice develop profound lung injury secondary to SIRS and fail to resolve inflammation. Alveolar macrophages from gp91phox-/y mice express greater levels of chemotactic and pro-inflammatory factors at baseline providing evidence that Nox2 in alveolar macrophages is critical for homeostasis. Based on the lung pathology with increased thrombosis in gp91phox-/y mice, and the known role of platelets in the inflammatory process, we hypothesized that Nox2 represses platelet activation. In the mouse model, we found that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) and CXCL7 were increased in the bronchoalveolar fluid of gp91phox-/y mice at baseline and 24 h post intraperitoneal zymosan-induced SIRS consistent with platelet activation. Activated platelets interact with leukocytes via P-selectin glycoprotein ligand 1 (PSGL-1). Within 2 h of SIRS induction, alveolar neutrophil PSGL-1 expression was higher in gp91phox-/y mice. Platelet-neutrophil interactions were decreased in the peripheral blood of gp91phox-/y mice consistent with movement of activated platelets to the lung of mice lacking Nox2. Based on the severe lung pathology and the role of platelets in the formation of neutrophil extracellular traps (NETs), we evaluated NET production. In contrast to previous studies demonstrating Nox2-dependent NET formation, staining of lung sections from mice 24 h post zymosan injection revealed a large number of citrullinated histone 3 (H3CIT) and myeloperoxidase positive cells consistent with NET formation in gp91phox-/y mice that was virtually absent in WT mice. In addition, H3CIT protein expression and PAD4 activity were higher in the lung of gp91phox-/y mice post SIRS induction. These results suggest that Nox2 plays a critical role in maintaining homeostasis by regulating platelet activation and NET formation in the lung.


Assuntos
Lesão Pulmonar Aguda/patologia , Armadilhas Extracelulares/imunologia , NADPH Oxidase 2/metabolismo , Ativação Plaquetária/imunologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Animais , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Pulmão/patologia , Macrófagos Alveolares/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Fator Plaquetário 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/genética
12.
Inflammation ; 42(1): 185-198, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30288635

RESUMO

Acute lung injury (ALI), developing as a component of the systemic inflammatory response syndrome (SIRS), leads to significant morbidity and mortality. Reactive oxygen species (ROS), produced in part by the neutrophil NADPH oxidase 2 (Nox2), have been implicated in the pathogenesis of ALI. Previous studies in our laboratory demonstrated the development of pulmonary inflammation in Nox2-deficient (gp91phox-/y) mice that was absent in WT mice in a murine model of SIRS. Given this finding, we hypothesized that Nox2 in a resident cell in the lung, specifically the alveolar macrophage, has an essential anti-inflammatory role. Using a murine model of SIRS, we examined whole-lung digests and bronchoalveolar lavage fluid (BALf) from WT and gp91phox-/y mice. Both genotypes demonstrated neutrophil sequestration in the lung during SIRS, but neutrophil migration into the alveolar space was only present in the gp91phox-/y mice. Macrophage inflammatory protein (MIP)-1α gene expression and protein secretion were higher in whole-lung digest from uninjected gp91phox-/y mice compared to the WT mice. Gene expression of MIP-1α, MCP-1, and MIP-2 was upregulated in alveolar macrophages obtained from gp91phox-/y mice at baseline compared with WT mice. Further, ex vivo analysis of alveolar macrophages, but not bone marrow-derived macrophages or peritoneal macrophages, demonstrated higher gene expression of MIP-1α and MIP-2. Moreover, isolated lung polymorphonuclear neutrophils migrate to BALf obtained from gp91phox-/y mice, further providing evidence of a cell-specific anti-inflammatory role for Nox2 in alveolar macrophages. We speculate that Nox2 represses the development of inflammatory lung injury by modulating chemokine expression by the alveolar macrophage.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Macrófagos Alveolares/metabolismo , NADPH Oxidase 2/fisiologia , Neutrófilos/patologia , Lesão Pulmonar Aguda/patologia , Animais , Movimento Celular , Quimiocinas/metabolismo , Inflamação/prevenção & controle , Pulmão/enzimologia , Macrófagos Alveolares/enzimologia , Camundongos , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , Espécies Reativas de Oxigênio , Síndrome de Resposta Inflamatória Sistêmica/patologia
13.
Pediatr Crit Care Med ; 18(12): 1145-1152, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29068910

RESUMO

OBJECTIVES: Infants with congenital heart disease frequently require cardiopulmonary bypass, which causes systemic inflammation. The goal of this study was to determine if neutrophil phenotype and activation status predicts the development of inflammatory complications following cardiopulmonary bypass. DESIGN: Prospective cohort study. SETTING: Tertiary care PICU with postoperative cardiac care. PATIENTS: Thirty-seven patients 5 days to 10 months old with congenital heart disease requiring cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Laboratory and clinical data collected included length of mechanical ventilation, acute kidney injury, and fluid overload. Neutrophils were isolated from whole blood at three time points surrounding cardiopulmonary bypass. Functional analyses included measurement of cell surface protein expression and nicotinamide adenine dinucleotide phosphate oxidase activity. Of all patients studied, 40.5% displayed priming of nicotinamide adenine dinucleotide phosphate oxidase activity in response to N-formyl-Met-Leu-Phe stimulation 24 hours post cardiopulmonary bypass as compared to pre bypass. Neonates who received steroids prior to bypass demonstrated enhanced priming of nicotinamide adenine dinucleotide phosphate oxidase activity at 48 hours. Patients who displayed priming post cardiopulmonary bypass were 8.8 times more likely to develop severe acute kidney injury as compared to nonprimers. Up-regulation of neutrophil surface CD11b levels pre- to postbypass occurred in 51.4% of patients, but this measure of neutrophil priming was not associated with acute kidney injury. Subsequent analyses of the basal neutrophil phenotype revealed that those with higher basal CD11b expression were significantly less likely to develop acute kidney injury. CONCLUSIONS: Neutrophil priming occurs in a subset of infants undergoing cardiopulmonary bypass. Acute kidney injury was more frequent in those patients who displayed priming of nicotinamide adenine dinucleotide phosphate oxidase activity after cardiopulmonary bypass. This pilot study suggests that neutrophil phenotypic signature could be used to predict inflammatory organ dysfunction.


Assuntos
Injúria Renal Aguda/etiologia , Ponte Cardiopulmonar/efeitos adversos , Cardiopatias Congênitas/cirurgia , Inflamação/etiologia , Neutrófilos/metabolismo , Fenótipo , Complicações Pós-Operatórias/etiologia , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/metabolismo , Biomarcadores/metabolismo , Feminino , Cardiopatias Congênitas/imunologia , Humanos , Lactente , Recém-Nascido , Inflamação/diagnóstico , Inflamação/metabolismo , Unidades de Terapia Intensiva Pediátrica , Modelos Logísticos , Masculino , Ativação de Neutrófilo , Neutrófilos/imunologia , Projetos Piloto , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/metabolismo , Estudos Prospectivos
14.
Innate Immun ; 22(8): 635-646, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27655046

RESUMO

Neutrophil (polymorphonuclear leukocyte) activation with release of granule contents plays an important role in the pathogenesis of acute lung injury, prompting clinical trials of inhibitors of neutrophil elastase. Despite mounting evidence for neutrophil-mediated host tissue damage in a variety of disease processes, mechanisms regulating azurophilic granule exocytosis at the plasma membrane, and thus release of elastase and other proteases, are poorly characterized. We hypothesized that azurophilic granule exocytosis would be enhanced under priming conditions similar to those seen during acute inflammatory events and during chronic inflammatory disease, and selected the cytokine TNF-α to model this in vitro. Neutrophils stimulated with TNF-α alone elicited intracellular reactive oxygen species (ROS) generation and mobilization of secretory vesicles, specific, and gelatinase granules. p38 and ERK1/2 MAPK were involved in these components of priming. TNF-α priming alone did not mobilize azurophilic granules to the cell surface, but did markedly increase elastase release into the extracellular space in response to secondary stimulation with N-formyl-Met-Leu-Phe (fMLF). Priming of fMLF-stimulated elastase release was further augmented in the absence of NADPH oxidase-derived ROS. Our findings provide a mechanism for host tissue damage during neutrophil-mediated inflammation and suggest a novel anti-inflammatory role for the NADPH oxidase.


Assuntos
Lesão Pulmonar Aguda/imunologia , Elastase de Leucócito/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Vesículas Secretórias/metabolismo , Corantes Azur/química , Degranulação Celular , Células Cultivadas , Exocitose , Gelatinases/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Ativação de Neutrófilo , Espécies Reativas de Oxigênio/metabolismo , Vesículas Secretórias/química , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Immunol ; 196(3): 1376-86, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26729809

RESUMO

Polymorphonuclear leukocytes (PMN) achieve an intermediate or primed state of activation following stimulation with certain agonists. Primed PMN have enhanced responsiveness to subsequent stimuli, which can be beneficial in eliminating microbes but may cause host tissue damage in certain disease contexts, including sepsis. As PMN priming by TLR4 agonists is well described, we hypothesized that ligation of TLR2/1 or TLR2/6 would prime PMN. Surprisingly, PMN from only a subset of donors were primed in response to the TLR2/1 agonist, Pam3CSK4, although PMN from all donors were primed by the TLR2/6 agonist, FSL-1. Priming responses included generation of intracellular and extracellular reactive oxygen species, MAPK phosphorylation, integrin activation, secondary granule exocytosis, and cytokine secretion. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was enhanced by a common single-nucleotide polymorphism (SNP) in TLR1 (rs5743618). Notably, PMN from donors with the SNP had higher surface levels of TLR1 and were demonstrated to have enhanced association of TLR1 with the endoplasmic reticulum chaperone gp96. We analyzed TLR1 genotypes in a pediatric sepsis database and found that patients with sepsis or septic shock who had a positive blood culture and were homozygous for the SNP associated with neutrophil priming had prolonged pediatric intensive care unit length of stay. We conclude that this TLR1 SNP leads to excessive PMN priming in response to cell stimulation. Based on our finding that septic children with this SNP had longer pediatric intensive care unit stays, we speculate that this SNP results in hyperinflammation in diseases such as sepsis.


Assuntos
Ativação de Neutrófilo/genética , Receptor 1 Toll-Like/genética , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genótipo , Humanos , Immunoblotting , Unidades de Terapia Intensiva , Tempo de Internação , Neutrófilos/imunologia , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse , Receptor 1 Toll-Like/imunologia
16.
Am J Physiol Lung Cell Mol Physiol ; 307(1): L71-82, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24793165

RESUMO

Systemic inflammatory response syndrome (SIRS) is a common clinical condition in patients in intensive care units that can lead to complications, including multiple organ dysfunction syndrome (MODS). MODS carries a high mortality rate, and it is unclear why some patients resolve SIRS, whereas others develop MODS. Although oxidant stress has been implicated in the development of MODS, several recent studies have demonstrated a requirement for NADPH oxidase 2 (NOX2)-derived oxidants in limiting inflammation. We recently demonstrated that NOX2 protects against lung injury and mortality in a murine model of SIRS. In the present study, we investigated the role of NOX2-derived oxidants in the progression from SIRS to MODS. Using a murine model of sterile systemic inflammation, we observed significantly greater illness and subacute mortality in gp91(phox-/y) (NOX2-deficient) mice compared with wild-type mice. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histological examination showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation.


Assuntos
Lesão Pulmonar/genética , Glicoproteínas de Membrana/genética , Insuficiência de Múltiplos Órgãos/genética , NADPH Oxidases/genética , Síndrome de Resposta Inflamatória Sistêmica/patologia , Animais , Células da Medula Óssea/imunologia , Líquido da Lavagem Broncoalveolar/química , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Pulmão/patologia , Lesão Pulmonar/mortalidade , Lesão Pulmonar/prevenção & controle , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/mortalidade , Insuficiência de Múltiplos Órgãos/prevenção & controle , NADPH Oxidase 2 , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Síndrome de Resposta Inflamatória Sistêmica/genética , Síndrome de Resposta Inflamatória Sistêmica/mortalidade , Zimosan
17.
J Biol Chem ; 287(15): 12395-404, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22235113

RESUMO

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91(phox), p40(phox), p67(phox), and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.


Assuntos
Endocitose , Endossomos/metabolismo , Lipopolissacarídeos/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Fosfatase Alcalina/metabolismo , Citocromos c/química , Endossomos/enzimologia , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Microscopia Confocal , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/enzimologia , Neutrófilos/fisiologia , Oxirredução , Consumo de Oxigênio , Espécies Reativas de Oxigênio/química , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismo
18.
J Innate Immun ; 3(3): 298-314, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21088376

RESUMO

Activation of polymorphonuclear leukocytes (PMN) can be modulated to intermediate 'primed' states characterized by enhanced responsiveness to subsequent stimuli. We studied priming in response to TNF-α in human PMN and PLB-985 cells, a myeloid cell line differentiated to a neutrophilic phenotype (PLB-D). PMN generated reactive oxygen species (ROS) in response to TNF-α alone, and NADPH oxidase activity increased in response to stimulation with formyl-Met-Leu-Phe after priming. PLB-D cells also demonstrated priming of NADPH oxidase activity. Similar to priming by endotoxin, priming of the respiratory burst by TNF-α was predominantly oxygen dependent, with marked attenuation of ROS generation if primed anaerobically. Both PMN and PLB-D cells displayed significant increases in cell surface CD11b and gp91(phox) expression after TNF-α priming and PMN displayed activation of MAPK. In response to TNF-α priming, neither mobilization of intracellular proteins nor activation of MAPK pathways was NADPH oxidase dependent. Priming of PMN and PLB-D cells by low TNF-α concentrations enhanced chemotaxis. These data demonstrate that pathophysiological concentrations of TNF-α elicit NADPH oxidase-derived ROS and prime cells for enhanced surface protein expression, activation of p38 and ERK1/2 MAPK pathways, and increased chemotaxis. Furthermore, PLB-D cells undergo TNF-α priming and provide a genetically modifiable model to study priming mechanisms.


Assuntos
NADP/metabolismo , Neutrófilos/metabolismo , Oxigênio/metabolismo , Sepse/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular/imunologia , Ativação Enzimática/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NADP/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/patologia , Oxigênio/imunologia , Sepse/prevenção & controle , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Am J Physiol Lung Cell Mol Physiol ; 296(6): L1076-84, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19346432

RESUMO

Francisella tularensis, the causative agent of tularemia, is a highly virulent organism, especially when exposure occurs by inhalation. Recent data suggest that Francisella interacts directly with alveolar epithelial cells. Although F. tularensis causes septicemia and can live extracellularly in a murine infection model, there is little information about the role of the vascular endothelium in the host response. We hypothesized that F. tularensis would interact with pulmonary endothelial cells as a prerequisite to the clinically observed recruitment of neutrophils to the lung. Using an in vitro Transwell model system, we studied interactions between F. tularensis live vaccine strain (Ft LVS) and a pulmonary microvascular endothelial cell (PMVEC) monolayer. Organisms invaded the endothelium and were visualized within individual endothelial cells by confocal microscopy. Although these bacteria-endothelial cell interactions did not elicit production of the proinflammatory chemokines, polymorphonuclear leukocytes (PMN) were stimulated to transmigrate across the endothelium in response to Ft LVS. Moreover, transendothelial migration altered the phenotype of recruited PMN; i.e., the capacity of these PMN to activate NADPH oxidase and release elastase in response to subsequent stimulation was reduced compared with PMN that traversed PMVEC in response to Streptococcus pneumoniae. The blunting of PMN responsiveness required PMN transendothelial migration but did not require PMN uptake of Ft LVS, was not dependent on the presence of serum-derived factors, and was not reproduced by Ft LVS-conditioned medium. We speculate that the capacity of Ft LVS-stimulated PMVEC to support transendothelial migration of PMN without triggering release of IL-8 and monocyte chemotactic protein-1 and to suppress the responsiveness of transmigrated PMN to subsequent stimulation could contribute to the dramatic virulence during inhalational challenge with Francisella.


Assuntos
Células Endoteliais/microbiologia , Francisella tularensis/imunologia , Pulmão/microbiologia , Neutrófilos/microbiologia , Tularemia/imunologia , Movimento Celular/imunologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Francisella tularensis/patogenicidade , Humanos , Imunofenotipagem , Elastase de Leucócito/metabolismo , Pulmão/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Fagocitose/imunologia , Tularemia/patologia , Virulência
20.
J Biol Chem ; 282(47): 33958-67, 2007 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-17908687

RESUMO

Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.


Assuntos
Canais de Cloreto/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Vesículas Secretórias/enzimologia , Anaerobiose , Animais , Linhagem Celular , Canais de Cloreto/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Camundongos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neisseria meningitidis/química , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/genética , Neutrófilos/citologia , Oxirredução/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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