RESUMEN
Acute lung injury during bacterial infection is associated with neutrophilic inflammation, epithelial cell apoptosis, and disruption of the alveolar-capillary barrier. TLR4 is required for lung injury in animals exposed to bacterial LPS and initiates proinflammatory responses in part via the transcription factor NF-κB. Ligation of TLR4 also initiates a proapoptotic response by activating IFN-ß and STAT1-dependent genes. We recently demonstrated that mammalian target of rapamycin (mTOR), a key controller of cell growth and survival, can physically interact with STAT1 and suppress the induction of STAT1-dependent apoptosis genes. We therefore hypothesized that the mTOR inhibitor rapamycin would increase LPS-induced apoptosis and lung injury in vivo. Rapamycin increased lung injury and cellular apoptosis in C57BL/6J mice exposed to intratracheal LPS for 24 h. Rapamycin also augmented STAT1 activation, and the induction of STAT1-dependent genes that mediate cellular apoptosis (i.e., Fas, caspase-3). LPS-induced lung injury was attenuated in STAT1 knockout mice. In addition, LPS and IFN-ß-induced apoptosis was absent in cultured cells lacking STAT1, and, unlike in wild-type cells, a permissive effect of rapamycin was not observed. In contrast to its effect on STAT1, rapamycin inhibited NF-κB activation in vivo and reduced selected markers of inflammation (i.e., neutrophils in the bronchoalveolar lavage fluid, TNF-α). Therefore, although it inhibits NF-κB and neutrophilic inflammation, rapamycin augments LPS-induced lung injury and apoptosis in a mechanism that involves STAT1 and the induction of STAT1-dependent apoptosis genes.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Apoptosis/efectos de los fármacos , Lipopolisacáridos/toxicidad , Serina-Treonina Quinasas TOR/inmunología , Receptor Toll-Like 4/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Antibacterianos/farmacología , Apoptosis/genética , Apoptosis/inmunología , Lavado Broncoalveolar , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-kappaB) to its 5'-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-gamma). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN-gamma-stimulated hiNOS promoter activity. In response to LPS/IFN-gamma, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-gamma- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products.
Asunto(s)
Factores de Transcripción Forkhead/fisiología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , Fosfatidilinositol 3-Quinasas/fisiología , Regiones Promotoras Genéticas , Proteínas Represoras/fisiología , Androstadienos/farmacología , Sitios de Unión/genética , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Humanos , Mediadores de Inflamación/fisiología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/genética , Transactivadores/fisiología , Factor de Transcripción AP-1/fisiología , WortmaninaRESUMEN
Only 20% of smokers develop chronic obstructive pulmonary disease. An important determinant of susceptibility is genomic variation. We undertook this study to define strains of mice with different susceptibilities for the development of smoking-induced emphysema because they could help identify genetic factors of susceptibility. NZWLac/J, C57BL6/J, A/J, SJ/L, and AKR/J strains were exposed to cigarette smoke for 6 months. Elastance (Htis), the extent of emphysema (mean linear intercept [Lm]), and the inflammatory cell and cytokine response were measured. NZWLac/J had no change in Lm or Htis (resistant). C57BL6/J, A/J, and SJ/L increased Lm, but not Htis (mildly susceptible). AKR/J increased Lm and Htis (super-susceptible). Only AKR/J had significant inflammation comprising macrophages, neutrophils, and T cells. The AKR/J showed an upregulation of Th1 cytokines whereas in the C57BL/6/J and NZWlac/J, cytokines did not change or were downregulated. We conclude that Lm, elastance, and inflammation are features that are needed to phenotype emphysema in mice. The inflammatory cell and cytokine profile may be an important determinant of the phenotype in response to cigarette smoke exposure. The identification of resistant and susceptible strains for the development of emphysema could be useful for genomic studies of emphysema susceptibility in mice and eventually in humans.
Asunto(s)
Quimiocinas/análisis , Pulmón/patología , Ratones Endogámicos/clasificación , Enfisema Pulmonar/etiología , Contaminación por Humo de Tabaco/efectos adversos , Animales , Biopsia con Aguja , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Mediadores de Inflamación/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Probabilidad , Enfisema Pulmonar/fisiopatología , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Especificidad de la EspecieRESUMEN
Cigarette smoking in humans is associated with various patterns of emphysema and functional consequences. We tested the hypothesis that variations in alpha1-antitrypsin expression modulate the pattern of emphysema and functional consequences in cigarette smoke-exposed mice. We compared the effects of up to 6 months of cigarette smoke exposure in C57BL/6J (C57) mice and in low-alpha1-antitrypsin, C57BL/6J pa+/pa+ (pallid) mice. At the end of the experiment, we determined lung mechanical properties, the extent (mean linear intercept) and type of emphysema, and the cellular inflammatory response. After 4 months of cigarette smoking, pallid smoking mice, but not C57 smoking mice, had a significant increase in mean linear intercept. After 6 months of smoke exposure, C57 smoking mice and pallid smoking mice had similar degrees of emphysema. The pattern of emphysema in pallid smoking mice was more diffuse than in C57 smoking mice, affecting all airspaces. Pallid mice, but not C57 mice, developed a T cell inflammation in the alveolar wall after 6 months of smoking (p < 0.01). Although lung compliance was not changed in C57 smoking mice after smoke exposure, it increased significantly in pallid smoking mice over the 6 months of exposure (p < 0.0082). In summary, cigarette smoking induces emphysema in C57 and pallid mice, but the emphysema, inflammatory infiltrate, and resulting physiologic abnormalities were substantially different in the two strains, with the C57 and pallid mice exhibiting features similar to centrilobular and panlobular emphysema, respectively.