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1.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-35173051

RESUMEN

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.


Asunto(s)
Citrulina/farmacología , Mitocondrias/metabolismo , Sepsis/tratamiento farmacológico , Animales , Arginina/deficiencia , Arginina/metabolismo , Disponibilidad Biológica , Citrulina/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Tolerancia Inmunológica/inmunología , Terapia de Inmunosupresión/métodos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Sepsis/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología
2.
Respir Res ; 24(1): 185, 2023 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-37438806

RESUMEN

BACKGROUND: Bacterial pneumonia and related lung injury are among the most frequent causes of mortality in intensive care units, but also inflict serious and prolonged respiratory complications among survivors. Given that endoplasmic reticulum (ER) stress is a hallmark of sepsis-related alveolar epithelial cell (AEC) dysfunction, we tested if AMP-activated protein kinase (AMPK) affects recovery from ER stress and apoptosis of AECs during post-bacterial infection. METHODS: In a murine model of lung injury by P. aeruginosa non-lethal infection, therapeutic interventions included AMPK activator metformin or GSK-3ß inhibitor Tideglusib for 96 h. Recovery from AEC injury was evidenced by accumulation of soluble T-1α (AEC Type 1 marker) in BAL fluids along with fluorescence analysis of ER-stress (CHOP) and apoptosis (TUNEL) in lung sections. AMPK phosphorylation status and mediators of ER stress were determined via Immunoblot analysis from lung homogenates. Macrophage-dependent clearance of apoptotic cells was determined using flow cytometry assay. RESULTS: P. aeruginosa-induced lung injury resulted in accumulation of neutrophils and cellular debris in the alveolar space along with persistent (96 h) ER-stress and apoptosis of AECs. While lung infection triggered AMPK inactivation (de-phosphorylation of Thr172-AMPK), metformin and Tideglusib promptly restored the AMPK activation status. In post infected mice, AMPK activation reduced indices of lung injury, ER stress and related apoptosis of AECs, as early as 24 h post administration of AMPK activators. In addition, we demonstrate that the extent of apoptotic cell accumulation is also dependent on AMPK-mediated clearance of apoptotic cells by macrophages. CONCLUSIONS: Our study provides important insights into AMPK function in the preservation of AEC viability after bacterial infection, in particular due reduction of ER-stress and apoptosis, thereby promoting effective recovery from lung injury after pneumonia.


Asunto(s)
Células Epiteliales Alveolares , Lesión Pulmonar , Animales , Ratones , Proteínas Quinasas Activadas por AMP , Glucógeno Sintasa Quinasa 3 beta , Lesión Pulmonar/tratamiento farmacológico , Apoptosis
3.
Am J Respir Crit Care Med ; 204(6): 651-666, 2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34033525

RESUMEN

Rationale: Cigarette smoke (CS) inhalation triggers oxidative stress and inflammation, leading to accelerated lung aging, apoptosis, and emphysema, as well as systemic pathologies. Metformin is beneficial for protecting against aging-related diseases. Objectives: We sought to investigate whether metformin may ameliorate CS-induced pathologies of emphysematous chronic obstructive pulmonary disease (COPD). Methods: Mice were exposed chronically to CS and fed metformin-enriched chow for the second half of exposure. Lung, kidney, and muscle pathologies, lung proteostasis, endoplasmic reticulum (ER) stress, mitochondrial function, and mediators of metformin effects in vivo and/or in vitro were studied. We evaluated the association of metformin use with indices of emphysema progression over 5 years of follow-up among the COPDGene (Genetic Epidemiology of COPD) study participants. The association of metformin use with the percentage of emphysema and adjusted lung density was estimated by using a linear mixed model. Measurements and Main Results: Metformin protected against CS-induced pulmonary inflammation and airspace enlargement; small airway remodeling, glomerular shrinkage, oxidative stress, apoptosis, telomere damage, aging, dysmetabolism in vivo and in vitro; and ER stress. The AMPK (AMP-activated protein kinase) pathway was central to metformin's protective action. Within COPDGene, participants receiving metformin compared with those not receiving it had a slower progression of emphysema (-0.92%; 95% confidence interval [CI], -1.7% to -0.14%; P = 0.02) and a slower adjusted lung density decrease (2.2 g/L; 95% CI, 0.43 to 4.0 g/L; P = 0.01). Conclusions: Metformin protected against CS-induced lung, renal, and muscle injury; mitochondrial dysfunction; and unfolded protein responses and ER stress in mice. In humans, metformin use was associated with lesser emphysema progression over time. Our results provide a rationale for clinical trials testing the efficacy of metformin in limiting emphysema progression and its systemic consequences.


Asunto(s)
Metformina/uso terapéutico , Sustancias Protectoras/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfisema Pulmonar/prevención & control , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/metabolismo , Fumar Cigarrillos/efectos adversos , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfisema Pulmonar/etiología , Enfisema Pulmonar/metabolismo , Resultado del Tratamiento
4.
Lab Invest ; 101(11): 1467-1474, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34504306

RESUMEN

The mortality rates among patients who initially survive sepsis are, in part, associated with a high risk of secondary lung infections and respiratory failure. Given that phagolysosomes are important for intracellular killing of pathogenic microbes, we investigated how severe lung infections associated with post-sepsis immunosuppression affect phagolysosome biogenesis. In mice with P. aeruginosa-induced pneumonia, we found a depletion of both phagosomes and lysosomes, as evidenced by decreased amounts of microtubule associated protein light chain 3-II (LC3-II) and lysosomal-associated membrane protein (LAMP1). We also found a loss of transcription factor E3 (TFE3) and transcription factor EB (TFEB), which are important activators for transcription of genes encoding autophagy and lysosomal proteins. These events were associated with increased expression of ZKSCAN3, a repressor for transcription of genes encoding autophagy and lysosomal proteins. Zkscan3-/- mice had increased expression of genes involved in the autophagy-lysosomal pathway along with enhanced killing of P. aeruginosa in the lungs, as compared to wild-type mice. These findings highlight the involvement of ZKSCAN3 in response to severe lung infection, including susceptibility to secondary bacterial infections due to immunosuppression.


Asunto(s)
Fagosomas/fisiología , Neumonía Bacteriana/complicaciones , Infecciones por Pseudomonas/complicaciones , Sepsis/inmunología , Factores de Transcripción/deficiencia , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Tolerancia Inmunológica , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Neumonía Bacteriana/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Sepsis/microbiología
5.
Am J Transplant ; 21(9): 2964-2977, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33724664

RESUMEN

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.


Asunto(s)
Inhibidores de la Calcineurina , Proteína HMGB1 , Animales , Inhibidores de la Calcineurina/efectos adversos , Ciclosporina/efectos adversos , Metabolismo Energético , Inmunosupresores/efectos adversos , Ratones , Tacrolimus/toxicidad
8.
Eur Respir J ; 52(2)2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29946009

RESUMEN

Exaggerated release of neutrophil extracellular traps (NETs) along with decreased NET clearance and inability to remove apoptotic cells (efferocytosis) may contribute to sustained inflammation in acute respiratory distress syndrome (ARDS). Recent studies in experimental models of ARDS have revealed the crosstalk between AMP-activated protein kinase (AMPK) and high-mobility group box 1 (HMGB1), which may contribute to effectiveness of efferocytosis, thereby reducing inflammation and ARDS severity.We investigated neutrophil and NET clearance by macrophages from control and ARDS patients and examined how bronchoalveolar lavage (BAL) fluid from control and ARDS patients could affect NET formation and efferocytosis. Metformin (an AMPK activator) and neutralising antibody against HMGB1 were applied to improve efferocytosis and NET clearance.Neutrophils from ARDS patients showed significantly reduced apoptosis. Conversely, NET formation was significantly enhanced in ARDS patients. Exposure of neutrophils to ARDS BAL fluid promoted NET production, while control BAL fluid had no effect. Macrophage engulfment of NETs and apoptotic neutrophils was diminished in ARDS patients. Notably, activation of AMPK in macrophages or neutralisation of HMGB1 in BAL fluid improved efferocytosis and NET clearance.In conclusion, restoration of AMPK activity with metformin or specific neutralisation of HMGB1 in BAL fluid represent promising therapeutic strategies to decrease sustained lung inflammation during ARDS.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Trampas Extracelulares/metabolismo , Proteína HMGB1/metabolismo , Macrófagos/citología , Síndrome de Dificultad Respiratoria/metabolismo , Anciano , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Femenino , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Fagocitosis , Neumonía/metabolismo , Síndrome de Dificultad Respiratoria/fisiopatología
9.
Cell Mol Life Sci ; 74(21): 3913-3925, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28803347

RESUMEN

The skin being a protective barrier between external and internal (body) environments has the sensory and adaptive capacity to maintain local and global body homeostasis in response to noxious factors. An important part of the skin response to stress is its ability for melatonin synthesis and subsequent metabolism through the indolic and kynuric pathways. Indeed, melatonin and its metabolites have emerged as indispensable for physiological skin functions and for effective protection of a cutaneous homeostasis from hostile environmental factors. Moreover, they attenuate the pathological processes including carcinogenesis and other hyperproliferative/inflammatory conditions. Interestingly, mitochondria appear to be a central hub of melatonin metabolism in the skin cells. Furthermore, substantial evidence has accumulated on the protective role of the melatonin against ultraviolet radiation and the attendant mitochondrial dysfunction. Melatonin and its metabolites appear to have a modulatory impact on mitochondrion redox and bioenergetic homeostasis, as well as the anti-apoptotic effects. Of note, some metabolites exhibit even greater impact than melatonin alone. Herein, we emphasize that melatonin-mitochondria axis would control integumental functions designed to protect local and perhaps global homeostasis. Given the phylogenetic origin and primordial actions of melatonin, we propose that the melatonin-related mitochondrial functions represent an evolutionary conserved mechanism involved in cellular adaptive response to skin injury and repair.


Asunto(s)
Antioxidantes/farmacología , Melatonina/farmacología , Mitocondrias/metabolismo , Piel/metabolismo , Animales , Humanos , Mitocondrias/efectos de los fármacos , Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel
10.
FASEB J ; 30(6): 2135-50, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26884454

RESUMEN

Matricellular proteins mediate pleiotropic effects during tissue injury and repair. CCN1 is a matricellular protein that has been implicated in angiogenesis, inflammation, and wound repair. In this study, we identified CCN1 as a gene that is differentially up-regulated in alveolar mesenchymal cells of human subjects with rapidly progressive idiopathic pulmonary fibrosis (IPF). Elevated levels of CCN1 mRNA were confirmed in lung tissues of IPF subjects undergoing lung transplantation, and CCN1 protein was predominantly localized to fibroblastic foci. CCN1 expression in ex vivo IPF lung fibroblasts correlated with gene expression of the extracellular matrix proteins, collagen (Col)1a1, Col1a2, and fibronectin as well as the myofibroblast marker, α-smooth muscle actin. RNA interference (RNAi)-mediated knockdown of CCN1 down-regulated the constitutive expression of these profibrotic genes in IPF fibroblasts. TGF-ß1, a known mediator of tissue fibrogenesis, induces gene and protein expression of CCN1 via a mothers against decapentaplegic homolog 3 (SMAD3)-dependent mechanism. Importantly, endogenous CCN1 potentiates TGF-ß1-induced SMAD3 activation and induction of profibrotic genes, supporting a positive feedback loop leading to myofibroblast activation. In vivo RNAi-mediated silencing of CCN1 attenuates fibrogenic responses to bleomycin-induced lung injury. These studies support previously unrecognized, cooperative interaction between the CCN1 matricellular protein and canonical TGF-ß1/SMAD3 signaling that promotes lung fibrosis.-Kurundkar, A. R., Kurundkar, D., Rangarajan, S., Locy, M. L., Zhou, Y., Liu, R.-M., Zmijewski, J., Thannickal, V. J. The matricellular protein CCN1 enhances TGF-ß1/SMAD3-dependent profibrotic signaling in fibroblasts and contributes to fibrogenic responses to lung injury.


Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Lesión Pulmonar/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células Cultivadas , Proteína 61 Rica en Cisteína/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Proteína smad3/genética , Factor de Crecimiento Transformador beta1/genética , Regulación hacia Arriba
11.
Am J Respir Cell Mol Biol ; 54(1): 51-9, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26072676

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a disease with relentless course and limited therapeutic options. Nintedanib (BIBF-1120) is a multiple tyrosine kinase inhibitor recently approved by the U.S. Food and Drug Administration for the treatment of IPF. The precise antifibrotic mechanism(s) of action of nintedanib, however, is not known. Therefore, we studied the effects of nintedanib on fibroblasts isolated from the lungs of patients with IPF. Protein and gene expression of profibrotic markers were assessed by Western immunoblotting and real-time PCR. Autophagy markers and signaling events were monitored by biochemical assays, Western immunoblotting, microscopy, and immunofluorescence staining. Silencing of autophagy effector proteins was achieved with small interfering RNAs. Nintedanib down-regulated protein and mRNA expression of extracellular matrix (ECM) proteins, fibronectin, and collagen 1a1 while inhibiting transforming growth factor (TGF)-ß1-induced myofibroblast differentiation. Nintedanib also induced beclin-1-dependent, ATG7-independent autophagy. Nintedanib's ECM-suppressive actions were not mediated by canonical autophagy. Nintedanib inhibited early events in TGF-ß signaling, specifically tyrosine phosphorylation of the type II TGF-ß receptor, activation of SMAD3, and p38 mitogen-activated protein kinase. Nintedanib down-regulates ECM production and induces noncanonical autophagy in IPF fibroblasts while inhibiting TGF-ß signaling. These mechanisms appear to be uncoupled and function independently to mediate its putative antifibrotic effects.


Asunto(s)
Fibrosis Pulmonar Idiopática/prevención & control , Indoles/farmacología , Pulmón/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Relación Dosis-Respuesta a Droga , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Pulmón/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína smad3/metabolismo , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta1/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
12.
J Biol Chem ; 290(42): 25427-38, 2015 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-26318453

RESUMEN

Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor ß1 (TGF-ß1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-ß1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-ß1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation.


Asunto(s)
Diferenciación Celular , Metabolismo Energético , Miofibroblastos/metabolismo , Línea Celular , Transporte de Electrón , Glucólisis , Humanos , Pulmón/citología , Pulmón/metabolismo , Mitocondrias/metabolismo , Miofibroblastos/citología , Consumo de Oxígeno , Factor de Crecimiento Transformador beta1/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
13.
Am J Physiol Lung Cell Mol Physiol ; 310(1): L71-85, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26545901

RESUMEN

Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherin's role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherin's interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherin's intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease.


Asunto(s)
Adenilato Quinasa/metabolismo , Cadherinas/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Pulmón/irrigación sanguínea , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animales , Células Cultivadas , Activación Enzimática/inmunología , Pulmón/metabolismo , Masculino , Ratas Sprague-Dawley , Ribonucleótidos/metabolismo
14.
Mol Med ; 21(1): 937-950, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26650187

RESUMEN

Alterations in metabolic and bioenergetic homeostasis contribute to sepsis-mediated organ injury. However, how AMP-activated protein kinase (AMPK), a major sensor and regulator of energy expenditure and production, affects development of organ injury and loss of innate capacity during polymicrobial sepsis remains unclear. In the present experiments, we found that cross-talk between the AMPK and GSK3ß signaling pathways controls chemotaxis and the ability of neutrophils and macrophages to kill bacteria ex vivo. In mice with polymicrobial abdominal sepsis or more severe sepsis induced by the combination of hemorrhage and intraabdominal infection, administration of the AMPK activator metformin or the GSK3ß inhibitor SB216763 reduced the severity of acute lung injury (ALI). Improved survival in metformin-treated septic mice was correlated with preservation of mitochondrial complex V (ATP synthase) function and increased amounts of ETC complex III and IV. Although immunosuppression is a consequence of sepsis, metformin effectively increased innate immune capacity to eradicate P. aeruginosa in the lungs of septic mice. We also found that AMPK activation diminished accumulation of the immunosuppressive transcriptional factor HIF-1α as well as the development of endotoxin tolerance in LPS-treated macrophages. Furthermore, AMPK-dependent preservation of mitochondrial membrane potential also prevented LPS-mediated dysfunction of neutrophil chemotaxis. These results indicate that AMPK activation reduces the severity of polymicrobial sepsis-induced lung injury and prevents the development of sepsis-associated immunosuppression.

15.
J Immunol ; 192(10): 4795-803, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24719460

RESUMEN

Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies also suggested that resistin has proinflammatory properties. We examined whether the human-specific variant of resistin affects neutrophil activation and the severity of LPS-induced acute lung injury. Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using humanized resistin mice that exclusively express human resistin (hRTN(+/-)(/-)) but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn(+/-/-) neutrophils compared with control Rtn(-/-/-) neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase, a major sensor and regulator of cellular bioenergetics that also is implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin enhanced neutrophil extracellular trap formation. In LPS-induced acute lung injury, humanized resistin mice demonstrated enhanced production of proinflammatory cytokines, more severe pulmonary edema, increased neutrophil extracellular trap formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4-induced inflammatory responses, and it may be a target for future therapies aimed at reducing the severity of acute lung injury and other inflammatory situations in which neutrophils play a major role.


Asunto(s)
Lesión Pulmonar Aguda/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Resistina/inmunología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/terapia , Animales , Quimiocina CXCL2/genética , Quimiocina CXCL2/inmunología , Modelos Animales de Enfermedad , Femenino , Proteína HMGB1/genética , Proteína HMGB1/inmunología , Histonas/genética , Histonas/inmunología , Humanos , Lipopolisacáridos/toxicidad , Pulmón/inmunología , Pulmón/patología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Noqueados , Neutrófilos/patología , Resistina/genética , Índice de Severidad de la Enfermedad , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología
16.
J Allergy Clin Immunol ; 135(2): 413-424.e15, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25420684

RESUMEN

BACKGROUND: Subsets of myeloid-derived regulatory cells (MDRCs), which are phenotypically similar to the myeloid-derived suppressor cells found in patients with cancer, have recently been appreciated as critical regulators of airway inflammation in mouse models of asthma. OBJECTIVE: We test the hypothesis that subsets of airway MDRCs contribute differentially to the inflammatory milieu in human asthma and chronic obstructive pulmonary disease (COPD). METHODS: We used bronchoalveolar lavage to identify and characterize human airway MDRCs from 10 healthy subjects, 9 patients with mild asthma, and 8 patients with COPD, none of whom were treated with inhaled or systemic corticosteroids. We defined subsets of airway MDRCs using flow cytometry, the molecular mediators they produce, and their abilities to regulate proliferation of polyclonally activated autologous T lymphocytes. RESULTS: We found substantial differences in the functional potential of MDRC subsets in healthy subjects, patients with asthma, and patients with COPD, with these differences regulated by the nitrosative and oxidative free radicals and cytokines they produced. Nitric oxide-producing MDRCs suppressed and superoxide-producing MDRCs enhanced proliferation of polyclonally activated autologous CD4 T cells. HLA-DR(+)CD11b(+)CD11c(+)CD163(-) superoxide-producing MDRCs, which stimulated proliferation of autologous T cells, comprised a high fraction of MDRCs in the airways of patients with mild asthma or COPD but not those of healthy control subjects. CD11b(+)CD14(+)CD16(-)HLA-DR(-) nitric oxide-producing MDRCs, which suppressed T-cell proliferation, were present in high numbers in airways of patients with mild asthma but not patients with COPD or healthy control subjects. CONCLUSION: Subsets of airway MDRCs conclusively discriminate patients with mild asthma, patients with COPD, and healthy subjects from each other. The distinctive activities of these MDRCs in patients with asthma or COPD might provide novel targets for new therapeutics for these common disorders. [Corrected]


Asunto(s)
Asma/diagnóstico , Asma/inmunología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Fenotipo , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Adulto , Antígenos de Superficie/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Estudios de Casos y Controles , Comunicación Celular , Diagnóstico Diferencial , Femenino , Volumen Espiratorio Forzado , Radicales Libres/metabolismo , Humanos , Inmunomodulación , Inmunofenotipificación , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Factores de Riesgo , Linfocitos T/inmunología
17.
J Neurosci ; 34(44): 14606-23, 2014 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-25355214

RESUMEN

The cell adhesion molecule close homolog of L1 (CHL1) plays important functional roles in the developing and adult nervous system. In search of the binding partners that mediate the diverse and sometimes opposing functions of CHL1, the extracellular matrix-associated proteins vitronectin and plasminogen activator inhibitor-2 (PAI-2) were identified as novel CHL1 interaction partners and tested for involvement in CHL1-dependent functions during mouse cerebellar development. CHL1-induced cerebellar neurite outgrowth and cell migration at postnatal days 6-8 were inhibited by a CHL1-derived peptide comprising the integrin binding RGD motif, and by antibodies against vitronectin or several integrins, indicating a vitronectin-dependent integrin-mediated pathway. A PAI-2-derived peptide, or antibodies against PAI-2, urokinase type plasminogen activator (uPA), uPA receptor, and several integrins reduced cell migration. CHL1 colocalized with vitronectin, PAI-2, and several integrins in cerebellar granule cells, suggesting an association among these proteins. Interestingly, at the slightly earlier age of 4-5 d, cerebellar neurons did not depend on CHL1 for neuritogenesis and cell migration. However, differentiation of progenitor cells into neurons at this stage was dependent on homophilic CHL1-CHL1 interactions. These observations indicate that homophilic CHL1 trans-interactions regulate differentiation of neuronal progenitor cells at early postnatal stages, while heterophilic trans-interactions of CHL1 with vitronectin, integrins, and the plasminogen activator system regulate neuritogenesis and neuronal cell migration at a later postnatal stage of cerebellar morphogenesis. Thus, within very narrow time windows in postnatal cerebellar development, distinct types of molecular interactions mediated by CHL1 underlie the diverse functions of this protein.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Integrinas/metabolismo , Neuritas/metabolismo , Inhibidor 2 de Activador Plasminogénico/metabolismo , Vitronectina/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Ratones , Ratones Noqueados , Neuritas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo
18.
Chem Res Toxicol ; 28(2): 175-81, 2015 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-25590513

RESUMEN

1-Hydroxyphenazine (1-HP) is a virulence factor produced by Pseudomonas aeruginosa. In this study,supercoiled plasmid DNA was employed as an analytical tool for the detection of ROS generation mediated by 1-HP. These assays provided evidence that 1-HP, in conjunction with NADPH alone or NADPH and the enzyme NADPH:cytochrome P450 reductase, mediated the production of superoxide radical under physiological conditions. Experiments with murine macrophage RAW264.7 cells and profluorescent ROS probes dichlorodihydrofluorescein or dihydroethidine provided preliminary evidence that 1-HP mediates the generation of intracellular oxidants. Generation of reactive oxygen species may contribute to the virulence properties of 1-HP in P. aeruginosa infections.


Asunto(s)
Fenazinas/química , Fenazinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Virulencia/metabolismo , Animales , Células Cultivadas , Ratones , Estructura Molecular , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Pseudomonas aeruginosa/química , Especies Reactivas de Oxígeno/química , Factores de Virulencia/química
19.
J Immunol ; 190(5): 2273-81, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23345331

RESUMEN

Effective removal of apoptotic cells, particularly apoptotic neutrophils, is essential for the successful resolution of acute inflammatory conditions. In these experiments, we found that whereas interaction between vitronectin and integrins diminished the ability of macrophages to ingest apoptotic cells, interaction between vitronectin with urokinase-type plasminogen activator receptor (uPAR) on the surface of apoptotic cells also had equally important inhibitory effects on efferocytosis. Preincubation of vitronectin with plasminogen activator inhibitor-1 eliminated its ability to inhibit phagocytosis of apoptotic cells. Similarly, incubation of apoptotic cells with soluble uPAR or Abs to uPAR significantly diminished efferocytosis. In the setting of LPS-induced ALI, enhanced efferocytosis and decreased numbers of neutrophils were found in bronchoalveolar lavage obtained from vitronectin-deficient (vtn(-/-)) mice compared with wild type (vtn(+/+)) mice. Furthermore, there was increased clearance of apoptotic vtn(-/-) as compared with vtn(+/+) neutrophils after introduction into the lungs of vtn(-/-) mice. Incubation of apoptotic vtn(-/-) neutrophils with purified vitronectin before intratracheal instillation decreased efferocytosis in vivo. These findings demonstrate that the inhibitory effects of vitronectin on efferocytosis involve interactions with both the engulfing phagocyte and the apoptotic target cell.


Asunto(s)
Lesión Pulmonar Aguda/inmunología , Apoptosis/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Timocitos/efectos de los fármacos , Vitronectina/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Anticuerpos/farmacología , Apoptosis/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Recuento de Células , Técnicas de Cocultivo , Femenino , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/patología , Fagocitosis/inmunología , Inhibidor 1 de Activador Plasminogénico/farmacología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/inmunología , Timocitos/inmunología , Timocitos/patología , Vitronectina/deficiencia , Vitronectina/genética
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