RESUMO
We previously reported that maternal alcohol use increased the risk of sepsis in premature and term newborns. In the neonatal mouse, fetal ethanol (ETOH) exposure depleted the antioxidant glutathione (GSH), which promoted alveolar macrophage (AM) immunosuppression and respiratory syncytial virus (RSV) infections. In this study, we explored if oral liposomal GSH (LGSH) would attenuate oxidant stress and RSV infections in the ETOH-exposed mouse pups. C57BL/6 female mice were pair-fed a liquid diet with 25% of calories from ethanol or maltose-dextrin. Postnatal day 10 pups were randomized to intranasal saline, LGSH, and RSV. After 48 h, we assessed oxidant stress, AM immunosuppression, pulmonary RSV burden, and acute lung injury. Fetal ETOH exposure increased oxidant stress threefold, lung RSV burden twofold and acute lung injury threefold. AMs were immunosuppressed with decreased RSV clearance. However, LGSH treatments of the ETOH group normalized oxidant stress, AM immune phenotype, the RSV burden, and acute lung injury. These studies suggest that the oxidant stress caused by fetal ETOH exposure impaired AM clearance of infectious agents, thereby increasing the viral infection and acute lung injury. LGSH treatments reversed the oxidative stress and restored AM immune functions, which decreased the RSV infection and subsequent acute lung injury.
RESUMO
Cardiovascular disease is the leading cause of death in chronic kidney disease (CKD). Arginine, the endogenous precursor for nitric oxide synthesis, is produced in the kidneys. Arginine bioavailability contributes to endothelial and myocardial dysfunction in CKD. Plasma from 129X1/SvJ mice with and without CKD (5/6th nephrectomy), and banked plasma from children with and without CKD were analyzed for amino acids involved in arginine metabolism, ADMA, and arginase activity. Echocardiographic measures of myocardial function were compared with plasma analytes. In a separate experiment, a non-specific arginase inhibitor was administered to mice with and without CKD. Plasma citrulline and glutamine concentrations correlated with multiple measures of myocardial dysfunction. Plasma arginase activity was significantly increased in CKD mice at 16 weeks vs. 8 weeks (p = 0.002) and ventricular strain improved after arginase inhibition in mice with CKD (p = 0.03). In children on dialysis, arginase activity was significantly increased vs. healthy controls (p = 0.04). Increasing ADMA correlated with increasing RWT in children with CKD (r = 0.54; p = 0.003). In a mouse model, and children, with CKD, arginine dysregulation correlates with myocardial dysfunction.
Assuntos
Arginina , Insuficiência Renal Crônica , Animais , Camundongos , Arginase , Diálise Renal , Modelos Animais de Doenças , CitrulinaRESUMO
The lung microenvironment plays a crucial role in maintaining lung homeostasis as well as the initiation and resolution of both acute and chronic lung injury. Acute chest syndrome (ACS) is a complication of sickle cell disease (SCD) like acute lung injury. Both the endothelial cells and peripheral blood mononuclear cells are known to secrete proinflammatory cytokines elevated during ACS episodes. However, in SCD, the lung microenvironment that may favor excessive production of proinflammatory cytokines and the contribution of other lung resident cells, such as alveolar macrophages and alveolar type 2 epithelial (AT-2) cells, to ACS pathogenesis is not completely understood. Here, we sought to understand the pulmonary microenvironment and the proinflammatory profile of lung alveolar macrophages (LAMs) and AT-2 cells at steady state in Townes sickle cell (SS) mice compared to control mice (AA). In addition, we examined lung function and micromechanics molecules essential for pulmonary epithelial barrier function in these mice. Our results showed that bronchoalveolar lavage (BAL) fluid in SS mice had elevated protein levels of pro-inflammatory cytokines interleukin (IL)-1ß and IL-12 (p ⩽ 0.05) compared to AA controls. We showed for the first time, significantly increased protein levels of inflammatory mediators (Human antigen R (HuR), Toll-like receptor 4 (TLR4), MyD88, and PU.1) in AT-2 cells (1.4 to 2.2-fold) and LAM (17-21%) isolated from SS mice compared to AA control mice at steady state. There were also low levels of anti-inflammatory transcription factors (Nrf2 and PPARy) in SS mice compared to AA controls (p ⩽ 0.05). Finally, we found impaired lung function and a dysregulated composition of surfactant proteins (B and C). Our results demonstrate that SS mice at steady state had a compromised lung microenvironment with elevated expression of proinflammatory cytokines by AT-2 cells and LAM, as well as dysregulated expression of surfactant proteins necessary for maintaining the alveolar barrier integrity and lung function.
Assuntos
Anemia Falciforme , Macrófagos Alveolares , Camundongos , Humanos , Animais , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Células Endoteliais/metabolismo , Leucócitos Mononucleares/metabolismo , Pulmão/patologia , Citocinas/metabolismo , Anemia Falciforme/patologia , Tensoativos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Alcohol use disorders (AUD) cause alveolar macrophage (AM) immune dysfunction and increase risk of lung infections. Excessive alcohol use causes AM oxidative stress, which impairs AM phagocytosis and pathogen clearance from the alveolar space. Alcohol induces expression of NADPH oxidases (Noxes), primary sources of oxidative stress in AM. In contrast, alcohol decreases AM peroxisome proliferator-activated receptor gamma (PPARγ), a critical regulator of AM immune function. To explore the underlying molecular mechanisms for these effects of alcohol, we hypothesized that ethanol promotes CCAAT/enhancer-binding protein beta (C/EBPß)-mediated suppression of Nox-related microRNAs (miRs), in turn enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. We also hypothesized that PPARγ activation with pioglitazone (PIO) would reverse alcohol-induced C/EBPß expression and attenuate AM oxidative stress and phagocytic dysfunction. Cells from the mouse AM cell line (MH-S) were exposed to ethanol in vitro or primary AM were isolated from mice fed ethanol in vivo. Ethanol enhanced C/EBPß expression, decreased Nox 1-related miR-1264 and Nox 2-related miR-107 levels, and increased Nox1, Nox2, and Nox 4 expression in MH-S cells in vitro and mouse AM in vivo. These alcohol-induced AM derangements were abrogated by loss of C/EBPß, overexpression of miRs-1264 or -107, or PIO treatment. These findings identify C/EBPß and Nox-related miRs as novel therapeutic targets for PPARγ ligands, which could provide a translatable strategy to mitigate susceptibility to lung infections in people with a history of AUD. These studies further clarify the molecular underpinnings for a previous clinical trial using short-term PIO treatment to improve AM immunity in AUD individuals.
Assuntos
Etanol , Macrófagos Alveolares , MicroRNAs , Processamento Pós-Transcricional do RNA , Animais , Camundongos , Alcoolismo/tratamento farmacológico , Alcoolismo/genética , Etanol/efeitos adversos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , PPAR gama/genética , PPAR gama/metabolismoRESUMO
Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5-7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5-7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4-5), increased necrosis score (P < 0.05, n = 4-5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4-5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.
Assuntos
Anemia Falciforme , Água Potável , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Animais , Arginina/metabolismo , Arginina/farmacologia , Disponibilidade Biológica , Água Potável/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia , Camundongos , Músculo Esquelético/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Necrose/metabolismo , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Fluxo Sanguíneo RegionalRESUMO
Secretory phospholipase 2 (sPLA2) acts as a mediator between proximal and distal events of the inflammatory cascade. Its role in SARS-CoV-2 infection is unknown, but could contribute to COVID-19 inflammasome activation and cellular damage. We present the first report of plasma sPLA2 levels in adults and children with COVID-19 compared with controls. Currently asymptomatic adults with a history of recent COVID-19 infection (≥4 weeks before) identified by SARS-CoV-2 IgG antibodies had sPLA2 levels similar to those who were seronegative (9 ± 6 vs.17 ± 28 ng/mL, P = 0.26). In contrast, children hospitalized with severe COVID-19 had significantly elevated sPLA2 compared with those with mild or asymptomatic SARS-CoV-2 infection (269 ± 137 vs. 2 ± 3 ng/mL, P = 0.01). Among children hospitalized with multisystem inflammatory syndrome in children (MIS-C), all had severe disease requiring pediatric intensive care unit (PICU) admission. sPLA2 levels were significantly higher in those with acute illness <10 days versus convalescent disease ≥10 days (540 ± 510 vs. 2 ± 1, P = 0.04). Thus, sPLA2 levels correlated with COVID-19 severity and acute MIS-C in children, implicating a role in inflammasome activation and disease pathogenesis. sPLA2 may be a useful biomarker to stratify risk and guide patient management for children with acute COVID-19 and MIS-C. Therapeutic compounds targeting sPLA2 and inflammasome activation warrant consideration.
Assuntos
COVID-19/sangue , Fosfolipases A2 Secretórias/sangue , Síndrome de Resposta Inflamatória Sistêmica/sangue , Adolescente , Adulto , Fatores Etários , Biomarcadores/sangue , COVID-19/complicações , COVID-19/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Síndrome de Resposta Inflamatória Sistêmica/patologia , Síndrome de Resposta Inflamatória Sistêmica/virologiaRESUMO
Alcohol use disorders (AUD) increase susceptibility to respiratory infections by 2- to 4-fold in part because of impaired alveolar macrophage (AM) immune function. Alcohol causes AM oxidative stress, diminishing AM phagocytic capacity and clearance of microbes from the alveolar space. Alcohol increases AM NADPH oxidases (Noxes), primary sources of AM oxidative stress, and reduces peroxisome proliferator-activated receptor γ (PPARγ) expression, a critical regulator of AM immune function. To investigate the underlying mechanisms of these alcohol-induced AM derangements, we hypothesized that alcohol stimulates CCAAT/enhancer-binding protein ß (C/EBPß) to suppress Nox-related microRNAs (miRs), thereby enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. Furthermore, we postulated that pharmacologic PPARγ activation with pioglitazone would inhibit C/EBPß and attenuate alcohol-induced AM dysfunction. AM isolated from human AUD subjects or otherwise healthy control subjects were examined. Compared with control AM, alcohol activated AM C/EBPß, decreased Nox1-related miR-1264 and Nox2-related miR-107, and increased Nox1, Nox2, and Nox4 expression and activity. These alcohol-induced AM derangements were abrogated by inhibition of C/EBPß, overexpression of miR-1264 or miR-107, or pioglitazone treatment. These findings define novel molecular mechanisms of alcohol-induced AM dysfunction mediated by C/EBPß and Nox-related miRs that are amenable to therapeutic targeting with PPARγ ligands. These results demonstrate that PPARγ ligands provide a novel and rapidly translatable strategy to mitigate susceptibility to respiratory infections and related morbidity in individuals with AUD.
Assuntos
Alcoolismo/tratamento farmacológico , Alcoolismo/metabolismo , Etanol/efeitos adversos , Macrófagos Alveolares/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Pioglitazona/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Fagócitos/metabolismoRESUMO
Low plasma arginine bioavailability has been implicated in endothelial dysfunction and immune dysregulation. The role of arginine in COVID-19 is unknown, but could contribute to cellular damage if low. Our objective was to determine arginine bioavailability in adults and children with COVID-19 vs. healthy controls. We hypothesized that arginine bioavailability would be low in patients with COVID-19 and multisystem inflammatory syndrome in children (MIS-C). We conducted a prospective observational study of three patient cohorts; arginine bioavailability was determined in asymptomatic healthy controls, adults hospitalized with COVID-19, and hospitalized children/adolescents <21 y old with COVID-19, MIS-C, or asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection identified on admission screen. Mean patient plasma amino acids were compared to controls using the Student's t test. Arginine-to-ornithine ratio, a biomarker of arginase activity, and global arginine bioavailability ratio (GABR, arginine/[ornithine+citrulline]) were assessed in all three groups. A total of 80 patients were included (28 controls, 32 adults with COVID-19, and 20 pediatric patients with COVID-19/MIS-C). Mean plasma arginine and arginine bioavailability ratios were lower among adult and pediatric patients with COVID-19/MIS-C compared to controls. There was no difference between arginine bioavailability in children with COVID-19 vs. MIS-C. Adults and children with COVID-19 and MIS-C in our cohort had low arginine bioavailability compared to healthy adult controls. This may contribute to immune dysregulation and endothelial dysfunction in COVID-19. Low arginine-to-ornithine ratio in patients with COVID-19 or MIS-C suggests an elevation of arginase activity. Further study is merited to explore the role of arginine dysregulation in COVID-19.
Assuntos
Aminoácidos/sangue , COVID-19/sangue , Hospitalização , SARS-CoV-2/metabolismo , Adulto , COVID-19/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Excessive alcohol users have increased risk of developing respiratory infections in part due to oxidative stress-induced alveolar macrophage (AM) phagocytic dysfunction. Chronic ethanol exposure increases cellular oxidative stress in AMs via upregulation of NADPH oxidase (Nox) 4, and treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, rosiglitazone, decreases ethanol-induced Nox4. However, the mechanism by which ethanol induces Nox4 expression and the PPARγ ligand reverses this defect has not been elucidated. Since microRNA (miR)-92a has been predicted to target Nox4 for destabilization, we hypothesized that ethanol exposure decreases miR-92a expression and leads to Nox4 upregulation. Previous studies have implicated mitochondrial-derived oxidative stress in AM dysfunction. We further hypothesized that ethanol increases mitochondrial-derived AM oxidative stress and dysfunction via miR-92a, and that treatment with the PPARγ ligand, pioglitazone, could reverse these derangements. To test these hypotheses, a mouse AM cell line, MH-S cells, was exposed to ethanol in vitro, and primary AMs were isolated from a mouse model of chronic ethanol consumption to measure Nox4, mitochondrial target mRNA (qRT-PCR) and protein levels (confocal microscopy), mitochondria-derived reactive oxygen species (confocal immunofluorescence), mitochondrial fission (electron microscopy), and mitochondrial bioenergetics (extracellular flux analyzer). Ethanol exposure increased Nox4, enhanced mitochondria-derived oxidative stress, augmented mitochondrial fission, and impaired mitochondrial bioenergetics. Transfection with a miR-92a mimic in vitro or pioglitazone treatment in vivo diminished Nox4 levels, resulting in improvements in these ethanol-mediated derangements. These findings demonstrate that pioglitazone may provide a novel therapeutic approach to mitigate ethanol-induced AM mitochondrial derangements.
Assuntos
Etanol , Macrófagos Alveolares/patologia , NADPH Oxidase 4/metabolismo , Animais , Linhagem Celular , Etanol/toxicidade , Macrófagos Alveolares/metabolismo , Camundongos , NADPH Oxidase 4/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Altered mitochondrial function occurs in sickle cell disease (SCD), due in part to low nitric oxide (NO) bioavailability. Arginine, the substrate for NO production, becomes acutely deficient in SCD patients with vaso-occlusive pain episodes (VOE). To determine if arginine improves mitochondrial function, 12 children with SCD-VOE (13.6 ± 3 years; 67% male; 75% hemoglobin-SS) were randomized to 1 of 3 arginine doses: (1) 100 mg/kg IV 3 times/day (TID); (2) loading dose (200 mg/kg) then 100 mg/kg TID; or (3) loading dose (200 mg/kg) followed by continuous infusion (300 mg/kg per day) until discharge. Platelet-rich plasma mitochondrial activity, protein expression, and protein-carbonyls were measured from emergency department (ED) presentation vs discharge. All VOE subjects at ED presentation had significantly decreased complex-V activity compared to a steady-state cohort. Notably, complex-V activity was increased at discharge in subjects from all 3 arginine-dosing schemes; greatest increase occurred with a loading dose (P < .001). Although complex-IV and citrate synthase activities were similar in VOE platelets vs steady state, enzyme activities were significantly increased in VOE subjects after arginine-loading dose treatment. Arginine also decreased protein-carbonyl levels across all treatment doses (P < .01), suggesting a decrease in oxidative stress. Arginine therapy increases mitochondrial activity and reduces oxidative stress in children with SCD/VOE. This trial was registered at www.clinicaltrials.gov as #NCT02536170.
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Anemia Falciforme/tratamento farmacológico , Arginina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Adolescente , Analgésicos Opioides/uso terapêutico , Anemia Falciforme/complicações , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Arginina/administração & dosagem , Criança , Feminino , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/etiologia , Estudos ProspectivosRESUMO
Alcohol use prior to and during pregnancy remains a significant societal problem and can lead to developmental fetal abnormalities including compromised myocardia function and increased risk for heart disease later in life. Alcohol-induced cardiac toxicity has traditionally been studied in animal-based models. These models have limitations due to physiological differences from human cardiomyocytes (CMs) and are also not suitable for high-throughput screening. We hypothesized that human-induced pluripotent stem cell-derived CMs (hiPSC-CMs) could serve as a useful tool to study alcohol-induced cardiac defects and/or toxicity. In this study, hiPSC-CMs were treated with ethanol at doses corresponding to the clinically relevant levels of alcohol intoxication. hiPSC-CMs exposed to ethanol showed a dose-dependent increase in cellular damage and decrease in cell viability, corresponding to increased production of reactive oxygen species. Furthermore, ethanol exposure also generated dose-dependent increased irregular Ca2+ transients and contractility in hiPSC-CMs. RNA-seq analysis showed significant alteration in genes belonging to the potassium voltage-gated channel family or solute carrier family, partially explaining the irregular Ca2+ transients and contractility in ethanol-treated hiPSC-CMs. RNA-seq also showed significant upregulation in the expression of genes associated with collagen and extracellular matrix modeling, and downregulation of genes involved in cardiovascular system development and actin filament-based process. These results suggest that hiPSC-CMs can be a novel and physiologically relevant system for the study of alcohol-induced cardiac toxicity.
Assuntos
Etanol/toxicidade , Cardiopatias/induzido quimicamente , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cardiotoxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Medição de RiscoRESUMO
Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca2+ transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.
Assuntos
Benzamidas/farmacologia , Sinergismo Farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/fisiologia , PPAR alfa/agonistas , Anti-Inflamatórios/farmacologia , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Dexametasona/farmacologia , Metabolismo Energético , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Metabolismo dos Lipídeos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Transcriptoma , Tri-Iodotironina/farmacologiaRESUMO
BACKGROUND: We previously reported that maternal alcohol use significantly increases the risk of sepsis in premature and term newborns. In the mouse, fetal ethanol exposure results in an immunosuppressed phenotype for the alveolar macrophage (AM) and decreases bacterial phagocytosis. In pregnant mice, ethanol decreased AM zinc homeostasis, which contributed to immunosuppression and impaired AM phagocytosis. In this study, we explored whether ethanol-induced zinc insufficiency extended to the pup AMs and contributed to immunosuppression and exacerbated viral lung infections. METHODS: C57BL/6 female mice were fed a liquid diet with 25% ethanol-derived calories or pair-fed a control diet with 25% of calories as maltose-dextrin. Some pup AMs were treated in vitro with zinc acetate before measuring zinc pools or transporter expression and bacteria phagocytosis. Some dams were fed additional zinc supplements in the ethanol or control diets, and then we assessed pup AM zinc pools, zinc transporters, and the immunosuppressant TGFß1. On postnatal day 10, some pups were given intranasal saline or respiratory syncytial virus (RSV), and then AM RSV phagocytosis and the RSV burden in the airway lining fluid were assessed. RESULTS: Fetal ethanol exposure decreased pup AM zinc pools, zinc transporter expression, and bacterial clearance, but in vitro zinc treatments reversed these alterations. In addition, the expected ethanol-induced increase in TGFß1 and immunosuppression were associated with decreased RSV phagocytosis and exacerbated RSV infections. However, additional maternal zinc supplements blocked the ethanol-induced perturbations in the pup AM zinc homeostasis and TGFß1 immunosuppression, thereby improving RSV phagocytosis and attenuating the RSV burden in the lung. CONCLUSION: These studies suggest that, despite normal maternal dietary zinc intake, in utero alcohol exposure results in zinc insufficiency, which contributes to compromised neonatal AM immune functions, thereby increasing the risk of bacterial and viral infections.
Assuntos
Transtornos do Espectro Alcoólico Fetal/etiologia , Macrófagos Alveolares/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/etiologia , Zinco/deficiência , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Feminino , Transtornos do Espectro Alcoólico Fetal/imunologia , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Tolerância Imunológica , Macrófagos Alveolares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/fisiopatologiaRESUMO
BACKGROUND: Airway neutrophils are abundant in some children with severe asthma, but their functions are poorly understood. OBJECTIVE: To characterize that the inflammatory airway environment of children with neutrophil-predominant severe asthma promotes neutrophil survival and disrupts neutrophil-associated innate immune defenses. METHODS: Sixty-seven children with severe asthma refractory to high-dose inhaled corticosteroid treatment undergoing bronchoscopy with bronchoalveolar lavage (BAL) for clinical indications were stratified into neutrophil "high" versus "low" groups on the basis of BAL differential counts. Neutrophil activation markers, functional assays, and phenotyping studies were performed, as well as airway macrophage functional assays. Results were compared with those from children with moderate asthma treated with inhaled corticosteroids. RESULTS: Children with neutrophil-predominant severe asthma had increased markers of neutrophil activation/degranulation and a greater magnitude of airway proinflammatory cytokine and chemokine release. Primary neutrophils exposed to BAL of these children exhibited greater phagocytic capability and greater neutrophil extracellular trap formation, but a more impaired respiratory burst. Despite greater abundance of airway TGF-ß1, the neutrophils were not more apoptotic. Instead, neutrophils had a highly proinflammatory phenotype associated with a number of surface markers that regulate neutrophil activation, recruitment/migration, and granule release. Airway macrophages from children with neutrophil-predominant severe asthma were also more proinflammatory with impaired phagocytosis and increased apoptosis. CONCLUSIONS: Children with neutrophil-predominant severe asthma have proinflammatory neutrophils with enhanced survival. Airway macrophages are also proinflammatory and dysfunctional and may contribute to global innate immune impairment. Therapies that target neutrophils and related inflammation may be warranted in this subset of children.
Assuntos
Asma/imunologia , Neutrófilos/imunologia , Adolescente , Corticosteroides/uso terapêutico , Adulto , Asma/tratamento farmacológico , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Criança , Citocinas/imunologia , Resistência a Medicamentos , Feminino , Células HL-60 , Humanos , Macrófagos/imunologia , Masculino , Fagocitose , Fenótipo , Adulto JovemRESUMO
TGF-ß1 is a pleiotropic cytokine with an established role in fibrosis; however, the immunosuppressive effects of TGF-ß1 are less characterized. Elevated levels of TGF-ß1 are found in patients with acute and chronic lung diseases, and the underlying disease processes are exacerbated by respiratory viral infections. The alveolar macrophage is the first line of cellular defense against respiratory viral infections, and its response to infections is dependent on environmental cues. Using the mouse alveolar macrophage line, MH-S, and human CD14+ monocyte-derived macrophages, we examined the effects of TGF-ß1 on the type I IFN antiviral response, macrophage polarization, and mitochondrial bioenergetics following a challenge with human respiratory syncytial virus (RSV). Our results showed that TGF-ß1 treatment of macrophages decreased the antiviral and proinflammatory response, and suppressed basal, maximal, spare mitochondrial respiration, and mitochondrial ATP production. Challenge with RSV following TGF-ß1 treatment further exacerbated mitochondrial dysfunction. The TGF-ß1 and TGF-ß1+RSV-treated macrophages had a higher frequency of apoptosis and diminished phagocytic capacity, potentially through mitochondrial stress. Disruption of TGF-ß1 signaling or rescue of mitochondrial respiration may be novel therapeutically targetable pathways to improve macrophage function and prevent secondary bacterial infections that complicate viral respiratory infections.
Assuntos
Interferon Tipo I/metabolismo , Macrófagos Alveolares/metabolismo , Mitocôndrias/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/patogenicidade , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Premature neonates frequently require oxygen supplementation as a therapeutic intervention that, while necessary, also exposes the lung to significant oxidant stress. We hypothesized that hyperoxia has a deleterious effect on alveolar epithelial barrier function rendering the neonatal lung susceptible to injury and/or bronchopulmonary dysplasia (BPD). MATERIALS AND METHODS: We examined the effects of exposure to 85% oxygen on neonatal rat alveolar barrier function in vitro and in vivo. Whole lung was measured using wet-to-dry weight ratios and bronchoalveolar lavage protein content and cultured primary neonatal alveolar epithelial cells (AECs) were measured using transepithelial electrical resistance (TEER) and paracellular flux measurements. Expression of claudin-family tight junction proteins, E-cadherin and the Snail transcription factor SNAI1 were measured by Q-PCR, immunoblot and confocal immunofluorescence microscopy. RESULTS: Cultured neonatal AECs exposed to 85% oxygen showed impaired barrier function. This oxygen-induced increase in paracellular leak was associated with altered claudin expression, where claudin-3 and -18 were downregulated at both the mRNA and protein level. Claudin-4 and -5 mRNA were also decreased, although protein expression of these claudins was largely maintained. Lung alveolarization and barrier function in vivo were impaired in response to hyperoxia. Oxygen exposure also significantly decreased E-cadherin expression and induced expression of the SNAI1 transcription factor in vivo and in vitro. CONCLUSIONS: These data support a model in which hyperoxia has a direct impact on alveolar tight and adherens junctions to impair barrier function. Strategies to antagonize the effects of high oxygen on alveolar junctions may potentially reverse this deleterious effect.
Assuntos
Células Epiteliais Alveolares/metabolismo , Caderinas/metabolismo , Claudinas/metabolismo , Hiperóxia/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/metabolismo , Caderinas/genética , Células Cultivadas , Claudinas/genética , Ratos Sprague-Dawley , Fatores de Transcrição da Família Snail/genética , Junções Íntimas/metabolismoRESUMO
BACKGROUND/OBJECTIVES: Disruptions in redox balance lead to oxidative stress, a promoter of morbidity in critical illness. This study aimed to: (1) characterize the plasma and alveolar thiol/disulfide redox pools, (2) examine their associations with alveolar macrophage phagocytosis, and (3) determine the effect of high dose vitamin D3 on plasma thiol/disulfide redox. SUBJECTS/METHODS: Subjects were 30 critically ill, ventilated adults in a double-blind randomized trial of high-dose (250 000 or 500 000 IU) vitamin D3 or placebo. Baseline bronchoalveolar lavage fluid (BALF) samples were analyzed for determination of alveolar phagocytosis index (PI) and for concentrations of glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), cystine (CySS), and their respective redox potentials (EhGSSG and EhCySS). Plasma redox outcomes were assessed at baseline and days 7 and 14. RESULTS: Baseline plasma Cys was inversely associated with alveolar PI (ρ = -0.69, P = 0.003), and EhCySS was positively associated with PI (ρ = 0.61, P = 0.01). Over time, among all subjects there was an increase in plasma GSH levels and a decrease in EhGSSG (P < 0.01 for both), with no difference by treatment group. Vitamin D3 decreased oxidized plasma GSSG to a more normal state (P for group x time = 0.009). CONCLUSIONS: Oxidative stress indicators were positively associated with alveolar macrophage phagocytic function in acutely ill ventilated adults. High-dose vitamin D3 decreased plasma GSSG concentrations, which suggests that vitamin D can possibly improve the oxidative stress environment.
Assuntos
Colecalciferol/uso terapêutico , Estado Terminal/terapia , Macrófagos Alveolares/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Respiração Artificial , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Cisteína/sangue , Cistina/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Glutationa/sangue , Dissulfeto de Glutationa/sangue , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: High-dose vitamin D3 increases plasma total 25-hydroxyvitamin D [25(OH)D] in critically ill, ventilated patients; however, to our knowledge, the effect on plasma levels of free (nonprotein-bound) 25(OH)D has not been investigated in critical illness. Moreover, the relationship of free 25(OH)D and the regulation of endogenous antimicrobial peptides (AMPs) remains unknown. The aims of this study were to determine in critically ill adults with respiratory failure the effect of previous high-dose regimens of vitamin D3 on free 25(OH)D concentrations, the relationship of free 25(OH)D with circulating cathelicidin (LL-37) and human beta-defensin-2 (hBD-2), and the associations between plasma levels of free 25(OH)D and these AMPs to alveolar macrophage phagocytosis function. METHODS: In a double blind, randomized controlled trial, critically ill ventilator-dependent adults (N = 30) received enteral vitamin D3 (250,000 or 500,000 IU total over 5 d) or placebo. Plasma was obtained serially for concentrations of free 25(OH)D, LL-37, hBD-2, and expression of peripheral blood mononuclear cell human cationic antimicrobial protein (hCAP18) mRNA. Total 25(OH)D and LL-37 concentrations and alveolar macrophage phagocytosis were determined in bronchoalveolar lavage fluid. RESULTS: Plasma concentrations of free 25(OH)D over time were correlated with total 25(OH)D levels (r= 0.82; P < 0.001). The increase in free 25(OH)D was greater with the 500 000 IU vitamin D3 dose than with the lower dose. The percent change in mRNA expression of hCAP18 was positively associated with percent change in free 25(OH)D at days 7 and 14 (ρ = 0.48; P = 0.04 and ρ = 0.59; P = 0.03, respectively). Additionally, plasma LL-37 levels correlated with the percentage of alveolar macrophages exhibiting phagocytosis (ρ = 0.51; P = 0.04). CONCLUSIONS: The present study found a dose-related increase in plasma free-25(OH)D levels, which was associated with increasing circulating mRNA expression of hCAP18 over time. There were no correlations between changes in total and free 25(OH)D against plasma LL-37 and hBD-2 concentrations. Larger studies appear warranted to determine the impact of high-dose vitamin D3 administration on endogenous AMPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Colecalciferol/farmacologia , Cuidados Críticos/métodos , Respiração Artificial , Vitamina D/análogos & derivados , Idoso , Colecalciferol/sangue , Estado Terminal , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vitamina D/sangue , Vitaminas/farmacologiaRESUMO
Our understanding of the intrinsic effects of cystic fibrosis (CF) transmembrane conductance regulator (cftr) deletion on resident neonatal alveolar macrophage (AM) remains limited. We previously demonstrated that diminished glutathione (GSH) or excessive AM transforming growth factor beta one (TGFß1) contributes to AM dysfunction in a variety of disease states. In this study, using a gut-corrected cftr neonatal knockout (KO) mouse model and a siRNA-manipulated macrophage-like cell line (THP-1 cell), we hypothesized (1) that cftr mutation alone increases neonatal AM oxidant stress and cellular TGFß1 signaling via altered GSH, thereby impairing cellular function, and (2) that exogenous GSH attenuates AM alterations and dysfunction in the KO AM In neonatal KO mice, the baseline bronchoalveolar lavage fluid demonstrated a near doubling in mixed disulfides (P ≤ 0.05) and oxidized GSSG (P ≤ 0.05) without concurrent inflammation compared to WT littermates. KO AM demonstrated diminished AM thiols (P ≤ 0.05), increased AM mitochondrial ROS (P ≤ 0.05), increased AM TGFß1 (P ≤ 0.05) with increased TGFß1 signaling (P ≤ 0.05), and impaired phagocytosis (P ≤ 0.05). KO AM mitochondrial ROS was modulated by exogenous GSH (P ≤ 0.05). Conversely, TGFß1 was reduced (P ≤ 0.05) and impaired phagocytosis was rescued (P ≤ 0.05) by exogenous GSH in the KO AM These results suggest that an altered neonatal AM phenotype may contribute to the initiation of lung inflammation/infection in the CF lung. Modulation of the AM in the neonatal CF lung may potentially alter progression of disease.