RESUMEN
The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta (PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not. In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice coincides with upregulation of PIK3CG expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals instructing commitment to the ASC fate.
Asunto(s)
Formación de Anticuerpos , Linfocitos B , Diferenciación Celular , Fosfatidilinositol 3-Quinasa Clase Ib , Animales , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/inmunología , Ratones , Diferenciación Celular/inmunología , Humanos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Formación de Anticuerpos/inmunología , Ratones Noqueados , Células Productoras de Anticuerpos/inmunología , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Células B de Memoria/inmunología , Células B de Memoria/metabolismoRESUMEN
Pulmonary hypertension (PH) is a debilitating condition characterized by increased pulmonary arterial pressures and remodeling of pulmonary arteries, leading to right heart failure. Women have a higher prevalence of PH, whereas men have more severe disease and poorer outcomes. Animal models also show female-predominant disease. Despite the known sex differences in PH, little is known about how pathogenesis differs between the sexes. There is growing evidence of mitochondrial dysfunction, as well as altered mitophagy in PH. We hypothesized that sexual dimorphism contributes to mitochondrial dysfunction and altered mitophagy in PH. Using mouse lung endothelial cells, we exposed both wild-type and Parkin-/- cells to hypoxia and measured the effects on mitochondrial function and mitophagy-associated proteins. Our results show that females have more Parkin expression at baseline as well as increased mitochondrial respiratory capacity when exposed to oxidative stress. Inhibition of Parkin increased metabolic activity but reduced cell proliferation but to different degrees depending on sex, with results differing by sex. Our findings demonstrate sexual dimorphism in mitophagy-associated proteins and in mitochondrial respiration, which may help shed light on how the pathogenesis of PH may differ between the sexes.
Asunto(s)
Hipertensión Pulmonar , Mitofagia , Animales , Células Endoteliales/metabolismo , Femenino , Humanos , Hipertensión Pulmonar/metabolismo , Masculino , Ratones , Mitofagia/fisiología , Caracteres Sexuales , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
We previously reported that the canonical innate immune receptor toll-like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic-specific, lung- and cell-targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4-/- mice. Lung Ec-silencing of TLR4 in wild-type mice induced emphysema, highlighting the specific and distinct role of Ec-expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16INK4a , a senescence-associated gene. Lung Ec-p16INK4a -silencing prevented TLR4-/- induced emphysema, revealing a new functional role for p16INK4a in lungs. TLR4 suppressed endogenous p16INK4a expression via HDAC2-mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence-related gene expression.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Células Endoteliales/metabolismo , Epigénesis Genética/genética , Pulmón/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptor Toll-Like 4/deficienciaRESUMEN
AIMS: Oxidant-induced endothelial injury plays a critical role in the pathogenesis of acute lung injury (ALI) and subsequent respiratory failure. Our previous studies revealed an endogenous antioxidant and protective pathway in lung endothelium mediated by heat shock protein 70 (Hsp70)-toll-like receptor 4 (TLR4) signaling. However, the downstream effector mechanisms remained unclear. Stanniocalcin 1 (STC1) has been reported to mediate antioxidant responses in tissues such as the lungs. However, regulators of STC1 expression as well as its physiological function in the lungs were unknown. We sought to elucidate the relationship between TLR4 and STC1 in hyperoxia-induced lung injury in vitro and in vivo and to define the functional role of STC1 expression in lung endothelium. RESULTS: We identified significantly decreased STC1 expression in TLR4 knockout mouse lungs and primary lung endothelium isolated from TLR4 knockout mice. Overexpression of STC1 was associated with endothelial cytoprotection, whereas decreased or insufficient expression was associated with increased oxidant-induced injury and death. An Hsp70-TLR4-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signal mediates STC1 induction in the lungs and endothelial cells. We also demonstrated a previously unrecognized role for mitochondrial-associated STC1, via TLR4, in maintaining normal glycolysis, mitochondrial bioenergetics, and mitochondrial calcium levels. INNOVATION: To date, a physiological role for STC1 in oxidant-induced ALI has not been identified. In addition, our studies show that STC1 is regulated by TLR4 and exerts lung and endothelial protection in response to sterile oxidant-induced lung injury. CONCLUSIONS: Our studies reveal a novel TLR4-STC1-mediated mitochondrial pathway that has homeostatic as well as oxidant-induced cytoprotective functions in lung endothelium.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Células Endoteliales/metabolismo , Glicoproteínas/genética , Mitocondrias/metabolismo , Oxidantes/metabolismo , Receptor Toll-Like 4/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Antioxidantes/metabolismo , Calcio/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Glucólisis , Glicoproteínas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
GWAS have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single-cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers and loss of distal alveoli and airspace enlargement of over 20% compared with controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single-cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts was conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets and that disruption of fibroblast identity can alter the alveolar stem cell niche, leading to emphysematous changes in the murine lung.
Asunto(s)
Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Alveolos Pulmonares/metabolismo , Enfisema Pulmonar/metabolismo , Transducción de Señal , Animales , Fibroblastos/patología , Proteínas Hedgehog/genética , Humanos , Ratones , Ratones Noqueados , Alveolos Pulmonares/patología , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologíaRESUMEN
Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.
Asunto(s)
Asma/inmunología , Plaquetas/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Células Th2/inmunología , Proteínas Wnt/antagonistas & inhibidores , Animales , Antígenos Dermatofagoides/inmunología , Antígenos de Protozoos/inmunología , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Pyroglyphidae , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
AIMS: Oxidants play a critical role in the pathogenesis of acute lung injury (ALI). Nox3 is a novel member of the NADPH oxidase (Nox) family of oxidant-generating enzymes, which our laboratory had previously identified to be induced in the lungs of TLR4(-/-) mice. However, the physiologic role of Nox3 induction in lungs and its precise relationship to TLR4 are unknown. Furthermore, the cell compartment involved and the signaling mechanisms of Nox3 induction are unknown. RESULTS: We identified that Nox3 is regulated by heat shock protein 70 (Hsp70) signaling via a TLR4-Trif-signal transducer and activator of transcription 3 (Stat3) pathway and that Nox3 induction leads to increased oxidant injury and death in mice and lung endothelial cells. We generated Nox3(-/-)/TLR4(-/-) double knockout mice, endothelial-targeting lentiviral silencing constructs, and endothelial-targeted Stat3(-/-) mice to specifically demonstrate that Nox3 induction is responsible for the pro-oxidant, proapoptotic phenotype of TLR4(-/-) mice. We also show that an endothelial Hsp70-TLR4-Trif-Stat3 axis is required to suppress deleterious Nox3 induction. INNOVATION: To date, a physiologic role for Nox3 in oxidant-induced ALI has not been identified. In addition, we generated unique double knockout mice and endothelial-targeted lentiviral silencing constructs to specifically demonstrate the role of a TLR4 signaling pathway in regulating pro-oxidant generation. CONCLUSIONS: We identified an endothelial TLR4-Trif antioxidant pathway that leads to the inhibition of a novel NADPH oxidase, Nox3, in lungs and lung endothelial cells. We also identified the role of a TLR4 ligand, Hsp70, in suppressing Nox3 in basal and pro-oxidant conditions. These studies identify potentially new therapeutic targets in oxidant-induced ALI. Antioxid. Redox Signal. 24, 991-1012.
Asunto(s)
Lesión Pulmonar Aguda/enzimología , Proteínas HSP70 de Choque Térmico/metabolismo , NADPH Oxidasas/metabolismo , Receptor Toll-Like 4/metabolismo , Células Epiteliales Alveolares/enzimología , Animales , Apoptosis , Secuencia de Bases , Sitios de Unión , Represión Enzimática , Expresión Génica , Hiperoxia/enzimología , Pulmón/enzimología , Pulmón/patología , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasas/genética , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Receptor Toll-Like 4/genéticaRESUMEN
TLR4 deficiency causes hypersusceptibility to oxidant-induced injury. We investigated the role of TLR4 in lung protection, using used bone marrow chimeras; cell-specific transgenic modeling; and lentiviral delivery in vivo to knock down or express TLR4 in various lung compartments; and lung-specific VEGF transgenic mice to investigate the effect of TLR4 on VEGF-mediated protection. C57/BL6 mice were exposed to 100% oxygen in an enclosed chamber and assessed for survival and lung injury. Primary endothelial cells were stimulated with recombinant VEGF and exposed to hyperoxia or hydrogen peroxide. Endothelium-specific expression of human TLR4 (as opposed to its expression in epithelium or immune cells) increased the survival of TLR4-deficent mice in hyperoxia by 24 h and decreased LDH release and lung cell apoptosis after 72 h of exposure by 30%. TLR4 expression was necessary and sufficient for the protective effect of VEGF in the lungs and in primary endothelial cells in culture. TLR4 knockdown inhibited VEGF signaling through VEGF receptor 2 (VEGFR2), Akt, and ERK pathways in lungs and primary endothelial cells and decreased the availability of VEGFR2 at the cell surface. These findings demonstrate a novel mechanism through which TLR4, an innate pattern receptor, interacts with an endothelial survival pathway.
Asunto(s)
Células Endoteliales/metabolismo , Hiperoxia/metabolismo , Lesión Pulmonar/metabolismo , Pulmón/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Pulmón/efectos de los fármacos , Lesión Pulmonar/inducido químicamente , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos/metabolismo , Oxidantes/efectos adversos , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Exposure to hyperoxia results in acute lung injury. A pathogenic consequence of hyperoxia is endothelial injury. Macrophage migration inhibitory factor (MIF) has a cytoprotective effect on lung endothelial cells; however, the mechanism is uncertain. We postulate that the MIF receptor CD74 mediates this protective effect. Using adult wild-type (WT), MIF-deficient (Mif(-/-)), CD74-deficient (Cd74(-/-)) mice and MIF receptor inhibitor treated mice, we report that MIF deficiency or inhibition of MIF receptor binding results in increased sensitivity to hyperoxia. Mif(-/-) and Cd74(-/-) mice demonstrated decreased median survival following hyperoxia compared to WT mice. Mif(-/-) mice demonstrated an increase in bronchoalveolar protein (48%) and lactate dehydrogenase (LDH) (68%) following 72 hours of hyperoxia. Similarly, treatment with MIF receptor antagonist resulted in a 59% and 91% increase in bronchoalveolar lavage protein and LDH, respectively. Inhibition of CD74 in primary murine lung endothelial cells (MLECs) abrogated the protective effect of MIF, including decreased hyperoxia-mediated AKT phosphorylation and a 20% reduction in the antiapoptotic effect of exogenous MIF. Treatment with MIF decreased hyperoxia-mediated H2AX phosphorylation in a CD74-dependent manner. These data suggest that therapeutic manipulation of the MIF-CD74 axis in lung endothelial cells may be a novel approach to protect against acute oxidative stress.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Endotelio Vascular/metabolismo , Hiperoxia/complicaciones , Oxidorreductasas Intramoleculares/fisiología , Factores Inhibidores de la Migración de Macrófagos/fisiología , Receptores Inmunológicos/fisiología , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Animales , Apoptosis , Western Blotting , Proliferación Celular , Células Cultivadas , Endotelio Vascular/citología , Femenino , Técnica del Anticuerpo Fluorescente , Hiperoxia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets.
Asunto(s)
Catepsina E/metabolismo , Dinámicas Mitocondriales , Enfisema Pulmonar/metabolismo , Animales , Apoptosis , Lavado Broncoalveolar , Catepsina E/genética , Humanos , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/farmacología , Enfisema Pulmonar/fisiopatología , Quinazolinonas/farmacología , Humo/efectos adversos , Contaminación por Humo de Tabaco/efectos adversosRESUMEN
High levels of inspired oxygen, hyperoxia, are frequently used in patients with acute respiratory failure. Hyperoxia can exacerbate acute respiratory failure, which has high mortality and no specific therapies. We identified novel roles for PTEN-induced putative kinase 1 (PINK1), a mitochondrial protein, and the cytosolic innate immune protein NLRP3 in the lung and endothelium. We generated double knockouts (PINK1(-/-)/NLRP3(-/-)), as well as cell-targeted PINK1 silencing and lung-targeted overexpression constructs, to specifically show that PINK1 mediates cytoprotection in wild-type and NLRP3(-/-) mice. The ability to resist hyperoxia is proportional to PINK1 expression. PINK1(-/-) mice were the most susceptible; wild-type mice, which induced PINK1 after hyperoxia, had intermediate susceptibility; and NLRP3(-/-) mice, which had high basal and hyperoxia-induced PINK1, were the least susceptible. Genetic deletion of PINK1 or PINK1 silencing in the lung endothelium increased susceptibility to hyperoxia via alterations in autophagy/mitophagy, proteasome activation, apoptosis, and oxidant generation.
Asunto(s)
Proteínas Portadoras/inmunología , Endotelio/inmunología , Hiperoxia/inmunología , Pulmón/inmunología , Oxidantes/efectos adversos , Proteínas Quinasas/inmunología , Animales , Proteínas Portadoras/genética , Endotelio/patología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/genética , Inducción Enzimática/inmunología , Hiperoxia/genética , Hiperoxia/patología , Hiperoxia/prevención & control , Pulmón/patología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Quinasas/genéticaRESUMEN
The pathogenesis of chronic obstructive pulmonary disease (COPD) remains poorly understood. Cellular senescence and apoptosis contribute to the development of COPD; however, crucial regulators of these underlying mechanisms remain unknown. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that antagonizes both apoptosis and premature senescence and may be important in the pathogenesis of COPD. This study examines the role of MIF in the pathogenesis of COPD. Mice deficient in MIF (Mif(-/-)) or the MIF receptor CD74 (Cd74(-/-)) and wild-type (WT) controls were aged for 6 mo. Both Mif(-/-) and Cd74(-/-) mice developed spontaneous emphysema by 6 mo of age compared with WT mice as measured by lung volume and chord length. This was associated with activation of the senescent pathway markers p53/21 and p16. Following exposure to cigarette smoke, Mif(-/-) mice were more susceptible to the development of COPD and apoptosis compared with WT mice. MIF plasma concentrations were measured in a cohort of 224 human participants. Within a subgroup of older current and former smokers (n = 72), MIF concentrations were significantly lower in those with COPD [8.8, 95%CI (6.7-11.0)] compared with those who did not exhibit COPD [12.7 ng/ml, 95%CI (10.6-14.8)]. Our results suggest that both MIF and the MIF receptor CD74 are required for maintenance of normal alveolar structure in mice and that decreases in MIF are associated with COPD in human subjects.
Asunto(s)
Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Receptores Inmunológicos/sangre , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/efectos de los fármacos , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Enfisema/genética , Femenino , Humanos , Oxidorreductasas Intramoleculares/deficiencia , Pulmón/metabolismo , Factores Inhibidores de la Migración de Macrófagos/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Humo/efectos adversos , Fumar/efectos adversos , Nicotiana/efectos adversos , Proteína p53 Supresora de Tumor/metabolismo , Adulto JovenRESUMEN
Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can exacerbate respiratory failure. Our previous study showed that TLR4 confers protection against hyperoxia-induced lung injury and mortality. Hsp70 has potent cytoprotective properties and has been described as a TLR4 ligand in cell lines. We sought to elucidate the relationship between TLR4 and Hsp70 in hyperoxia-induced lung injury in vitro and in vivo and to define the signaling mechanisms involved. Wild-type, TLR4(-/-), and Trif(-/-) (a TLR4 adapter protein) murine lung endothelial cells (MLECs) were exposed to hyperoxia. We found markedly elevated levels of intracellular and secreted Hsp70 from wild-type mice lungs and MLECs after hyperoxia. We confirmed that Hsp70 and TLR4 coimmunoprecipitate in lung tissue and MLECs. Hsp70-mediated NF-κB activation appears to depend upon TLR4. In the absence of TLR4, Hsp70 loses its protective effects in endothelial cells. Furthermore, these protective properties of Hsp70 are TLR4 adapter Trif dependent and MyD88 independent. Hsp70-deficient mice have increased mortality during hyperoxia, and lung-targeted adenoviral delivery of Hsp70 effectively rescues both Hsp70-deficient and wild-type mice. To our knowledge, our studies are the first to define an Hsp70-TLR4-Trif cytoprotective axis in the lung and endothelial cells. This pathway is a potential therapeutic target against a range of oxidant-induced lung injuries.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Hiperoxia/metabolismo , Lesión Pulmonar/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Oxígeno/administración & dosificación , ARN Mensajero/biosíntesis , Transducción de Señal , Receptor Toll-Like 4/genéticaRESUMEN
Sepsis is a leading cause of intensive care unit admissions, with high mortality and morbidity. Although outcomes have improved with better supportive care, specific therapies are limited. Endothelial activation and oxidant injury are key events in the pathogenesis of sepsis-induced lung injury. The signaling pathways leading to these events remain poorly defined. We sought to determine the role of MAPK kinase 3 (MKK3), a kinase of the p38 group, in the pathogenesis of sepsis. We used a murine i.p. LPS model of systemic inflammation to mimic sepsis. Lung injury parameters were assessed in lung tissue and bronchoalveolar lavage specimens. Primary lung endothelial cells were cultured and assessed for mediators of inflammation and injury, such as ICAM-1, AP-1, NF-κB, and mitochondrial reactive oxygen species. Our studies demonstrate that MKK3 deficiency confers virtually complete protection against organ injury after i.p. LPS. Specifically, MKK3(-/-) mice were protected against acute lung injury, as assessed by reduced inflammation, mitochondrial reactive oxygen species generation, endothelial injury, and ICAM-1 expression after LPS administration. Our results show that endothelial MKK3 is required for inflammatory cell recruitment to the lungs, mitochondrial oxidant-mediated AP-1, NF-κB activation, and ICAM-1 expression during LPS challenge. Collectively, these studies identify a novel role for MKK3 in lethal LPS responses and provide new therapeutic targets against sepsis and acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda/enzimología , Células Endoteliales/enzimología , Endotoxemia/enzimología , MAP Quinasa Quinasa 3/fisiología , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/prevención & control , Animales , Apoptosis , Líquido del Lavado Bronquioalveolar , Quimiotaxis de Leucocito/fisiología , Modelos Animales de Enfermedad , Células Endoteliales/patología , Endotoxemia/patología , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Lipopolisacáridos/toxicidad , Pulmón/patología , MAP Quinasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa 3/deficiencia , MAP Quinasa Quinasa 3/genética , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Activación Neutrófila , Peritonitis/inducido químicamente , Peritonitis/enzimología , ARN Interferente Pequeño/farmacología , Quimera por Radiación , Especies Reactivas de Oxígeno/metabolismo , Sepsis/enzimología , Factor de Transcripción AP-1/metabolismoRESUMEN
OBJECTIVE: Increased circulating cytokine levels are a prominent feature of aging that may contribute to atherosclerosis. However, the role vascular cells play in chronic inflammation induced by aging is not clear. Here, we examined the role of aging on inflammatory responses of vascular cells. METHODS AND RESULTS: In an ex vivo culture system, we examined the inflammatory response of aortas from young (2-4 months) and aged (16-18 months) mice under nonstimulatory conditions. We found that basal levels of interleukin-6 were increased in aged aortas. Aged aortic vascular smooth muscle cells (VSMC) exhibited a higher basal secretion of interleukin-6 than young VSMC. Gene and protein expression analysis revealed that aged VSMC exhibited upregulation of chemokines (eg, CCL2), adhesion molecules (eg, intracellular adhesion molecule 1), and innate immune receptors (eg, Toll-like receptor [TLR] 4), which all contribute to atherosclerosis. Using VSMC from aged TL4(-/-) and Myd88(-/-) mice, we demonstrate that signaling via TLR4 and its signal adaptor, MyD88, are in part responsible for the age-elevated basal interleukin-6 response. CONCLUSIONS: Aging induces a proinflammatory phenotype in VSMC due in part to increased signaling of TLR4 and MyD88. Our results provide a potential explanation as to why aging leads to chronic inflammation and enhanced atherosclerosis.
Asunto(s)
Envejecimiento/inmunología , Quimiocina CCL2/biosíntesis , Interleucina-6/biosíntesis , Miocitos del Músculo Liso/inmunología , Envejecimiento/genética , Animales , Aorta Torácica/inmunología , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/inmunología , Células Cultivadas , Quimiocina CCL2/genética , Femenino , Interleucina-6/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Transducción de Señal , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Regulación hacia ArribaRESUMEN
Prolonged exposure to hyperoxia results in hyperoxic acute lung injury (HALI). Vascular endothelial growth factor (VEGF) has been shown to have cytoprotective effects and prolong survival in an in vivo model of HALI. Heme oxygenase-1 (HO-1) has protective effects in hyperoxia; therefore, we hypothesized that induction of HO-1 would be an important contributor to VEGF-induced cytoprotection. Using inducible, lung-specific VEGF overexpressing transgenic mice, we demonstrated that VEGF is a potent inducer of HO-1 mRNA and protein in mouse lung. To evaluate the effect of inhibition of HO-1 on injury, VEGF transgenic mice were treated with HO-1 short interfering RNA (HO-1 siRNA) and exposed to hyperoxia. Total lung lavage protein concentration, TUNEL staining, lipid peroxidation, and wet-to-dry ratio were all increased, consistent with increased injury. These findings were corroborated by survival studies in which inhibition of HO-1 function using tin-protoporphryin or silencing of HO-1 with lentiviral HO-1 short hairpin RNA (ShRNA) significantly decreased survival in hyperoxia. We conclude from these data that VEGF-induced HO-1 is an important contributor to cytoprotection in this in vivo model of acute lung injury and that alterations in VEGF function in the lung are likely to be important determinants of the outcome of acute lung injury.
Asunto(s)
Citoprotección , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hiperoxia/prevención & control , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Secuencia de Bases , Líquido del Lavado Bronquioalveolar , Cartilla de ADN , Modelos Animales de Enfermedad , Inducción Enzimática , Hemo Oxigenasa (Desciclizante)/genética , Humanos , Hiperoxia/enzimología , Etiquetado Corte-Fin in Situ , Peroxidación de Lípido , Pulmón/enzimología , Ratones , Ratones Transgénicos , ARN Interferente Pequeño , Síndrome de Dificultad Respiratoria/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
TLRs have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unknown. Given their direct interface with the external environment, TLRs in the lungs are prime candidates to respond to air constituents, namely particulates and oxygen. The mechanism whereby the lung maintains structural integrity in the face of constant ambient exposures is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, usually in the later decades of life and after years of insult, most commonly cigarette smoke. Here we show Tlr4(-/-) mice exhibited emphysema as they aged. Adoptive transfer experiments revealed that TLR4 expression in lung structural cells was required for maintaining normal lung architecture. TLR4 deficiency led to the upregulation of what we believe to be a novel NADPH oxidase (Nox), Nox3, in lungs and endothelial cells, resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4(-/- )mice or endothelial cells with chemical NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Our data identify a role for TLR4 in maintaining constitutive lung integrity by modulating oxidant generation and provide insights into the development of emphysema.
Asunto(s)
Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/metabolismo , Animales , Antioxidantes/metabolismo , Células Cultivadas , Elastina/metabolismo , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , NADPH Oxidasas/metabolismo , Oxidantes/metabolismo , Elastasa Pancreática/metabolismo , Enfisema Pulmonar/genética , Receptor Toll-Like 4/genéticaRESUMEN
Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. Unfortunately, the oxidant stress generated by prolonged hyperoxia can lead to respiratory failure, multiorgan failure, and death. Although the endothelial cell is known to be a target for hyperoxia-induced injury, its precise role is unclear. Heme oxygenase-1 (HO-1) and "signal transducer and activator of transcription 3" (STAT3) have been found to confer protection against endothelial cell injury. We sought to elucidate the specific roles of HO-1 and STAT3 in hyperoxic lung and endothelial cell injury. Mice or murine lung endothelial cells (MLEC) administered HO-1 siRNA exhibited marked injury and death compared with nonspecific siRNA. Overexpression of either HO-1 or STAT3 confers protection. However, HO-1 and its reaction product carbon monoxide (CO) lose their protective effects in the presence of STAT3 siRNA in MLEC or in endothelial-specific, STAT3-deficient mice. STAT3 overexpression is able to partially rescue HO-1-deficient MLEC from hyperoxia-induced cell death. Our results demonstrate 1) the importance of the endothelium in lethal hyperoxic injury, 2) HO-1 and CO require endothelial STAT3 for their protective effects, and 3) STAT3 confers endothelial cell protection via both HO-1-dependent and independent mechanisms.