RESUMEN

qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 (Il-6) and C-X-C motif ligand 1 (Cxcl-1) as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of Il-6 and Cxcl1 gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.

Asunto(s)

Lesión Pulmonar Aguda/genética , Algoritmos , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/normas , Regulación de la Expresión Génica , Marcadores Genéticos , Lesión Pulmonar Aguda/patología , Animales , Femenino , Ratones , Estándares de Referencia

RESUMEN

Retrograde lung vascular perfusion can appear in high-risk surgeries. The present report is the first to study long-term retrograde perfusion of isolated perfused mouse lungs (IPLs) and to use the tyrosine kinase ephB4 and its ligand ephrinB2 as potential markers for acute lung injury. Mouse lungs were subjected to anterograde or retrograde perfusion with normal-pressure ventilation (NV) or high-pressure ventilation (=overventilation, OV) for 4 hours. Outcome parameters were cytokine, ephrinB2 and ephB4 levels in perfusate samples and bronchoalveolar lavage (BAL), and the wet-to-dry ratio. Anterograde perfusion was feasible for 4 hours, while lungs receiving retrograde perfusion presented considerable collapse rates. Retrograde perfusion resulted in an increased wet-to-dry ratio when combined with high-pressure ventilation; other physiological parameters were not affected. Cytokine levels in BAL and perfusate, as well as levels of soluble ephB4 in BAL were increased in OV, while soluble ephrinB2 BAL levels were increased in retrograde perfusion. BAL levels of ephrinB2 and ephB4 were also determined in vivo, including mice ventilated for 7 hours with normal-volume ventilation (NVV) or high-volume ventilation (HVV) with increased levels of ephB4 in HVV BAL compared to NVV. Retrograde perfusion in IPL is limited as a routine method to investigate effects due to collapse for yet unclear reasons. If successful, retrograde perfusion has an influence on pulmonary oedema formation. In BAL, ephrinB2 seems to be up-regulated by flow reversal, while ephB4 is a marker for acute lung injury.

Asunto(s)

Lesión Pulmonar Aguda/diagnóstico , Citocinas/análisis , Edema/diagnóstico , Pulmón/cirugía , Perfusión/efectos adversos , Lesión Pulmonar Aguda/inmunología , Animales , Biomarcadores/análisis , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Edema/inmunología , Efrina-B2/análisis , Estudios de Factibilidad , Femenino , Humanos , Técnicas In Vitro/métodos , Pulmón/inmunología , Ratones , Perfusión/métodos , Receptor EphB4/análisis , Receptor EphB4/inmunología , Respiración Artificial/efectos adversos , Respiración Artificial/métodos , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

Neonates are extremely susceptible to bacterial infections, and evidences suggest that phagocytosis-induced cell death (PICD) is less frequently triggered in neonatal monocytes than in monocytes from adult donors. An insufficient termination of the inflammatory response, leading to a prolonged survival of neonatal monocytes with ongoing proinflammatory cytokine release, could be associated with the progression of various inflammatory diseases in neonates. Our previous data indicate that amphiregulin (AREG) is increasingly expressed on the cell surface of neonatal monocytes, resulting in remarkably higher soluble AREG levels after proteolytic shedding. In this study, we found that E. coli-infected neonatal monocytes show an increased phosphorylation of ERK, increased expression of Bcl-2 and Bcl-XL, and reduced levels of cleaved caspase-3 and caspase-9 compared to adult monocytes. In both cell types, additional stimulation with soluble AREG further increased ERK activation and expression of Bcl-2 and Bcl-XL and reduced levels of cleaved caspase-3 and caspase-9 in an EGFR-dependent manner. These data suggest that reduced PICD of neonatal monocytes could be due to reduced intrinsic apoptosis and that AREG can promote protection against PICD. This reduction of the intrinsic apoptosis pathway in neonatal monocytes could be relevant for severely prolonged inflammatory responses of neonates.

Asunto(s)

Anfirregulina/farmacología , Fagocitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína C-Reactiva/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Muerte Celular/efectos de los fármacos , Citocromos c/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Citometría de Flujo , Humanos , Interleucina-6/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Fagocitosis/fisiología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética

RESUMEN

Neonates are highly susceptible to microbial infections which is partially attributable to fundamental phenotypic and functional differences between effector cells of the adult and neonatal immune system. The resolution of the inflammation is essential to return to tissue homeostasis, but given that various neonatal diseases, such as periventricular leukomalacia, necrotizing enterocolitis, or bronchopulmonary dysplasia, are characterized by sustained inflammation, newborns seem predisposed to a dysregulation of the inflammatory response. Targeted apoptosis of effector cells is generally known to control the length and extent of the inflammation, and previous studies have demonstrated that phagocytosis-induced cell death (PICD), a special type of apoptosis in phagocytic immune cells, is less frequently triggered in neonatal monocytes than in adult monocytes. We concluded that a rescue of monocyte PICD could be a potential therapeutic approach to target sustained inflammation in neonates. The EGFR ligand amphiregulin (AREG) is shed in response to bacterial infection and was shown to mediate cellular apoptosis resistance. We hypothesized that AREG might contribute to the reduced PICD of neonatal monocytes by affecting apoptosis signaling. In this study, we have examined a cascade of signaling events involved in extrinsic apoptosis by using a well-established in vitro E. coli infection model in monocytes from human peripheral blood (PBMO) and cord blood (CBMO). We found that CBMO shows remarkably higher pro-AREG surface expression as well as soluble AREG levels in response to infection as compared to PBMO. AREG increases intracellular MMP-2 and MMP-9 levels and induces cleavage of membrane-bound FasL through engagement with the EGF receptor. Our results demonstrate that loss of AREG rescues PICD in CBMO to the level comparable to adult monocytes. These findings identify AREG as a potential target for the prevention of prolonged inflammation in neonates.

Asunto(s)

Anfirregulina/metabolismo , Muerte Celular/fisiología , Monocitos/citología , Monocitos/metabolismo , Fagocitosis/fisiología , Anfirregulina/genética , Apoptosis/genética , Apoptosis/fisiología , Muerte Celular/genética , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Escherichia coli/patogenicidad , Citometría de Flujo , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Fagocitosis/genética

Asunto(s)

Respiración Artificial , Síndrome de Dificultad Respiratoria , Humanos

RESUMEN

Mechanical ventilation (MV) elicits complex and clinically relevant cellular responses in the lungs. The current study was designed to define the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a major regulator of the cellular antioxidant defense system, in the pulmonary response to MV. Nrf2 activity was quantified in ventilated isolated perfused mouse lungs (IPL). Regulation of amphiregulin (AREG) was investigated in BEAS-2B cells with inactivated Nrf2 or Keap1, the inhibitor of Nrf2, using a luciferase vector with AREG promoter. AREG-dependent Nrf2 activity was examined in BEAS-2B cells, murine precision-cut lung slices (PCLS), and IPL. Finally, Nrf2 knockout and wild-type mice were ventilated to investigate the interplay between Nrf2 and AREG during MV in vivo. Lung functions and inflammatory parameters were measured. Nrf2 was activated in a ventilation-dependent manner. The knockdown of Nrf2 and Keap1 via short hairpin RNA in BEAS-2B cells and an EMSA with lung tissue revealed that AREG is regulated by Nrf2. Conversely, AREG application induced a significant Nrf2 activation in BEAS-2B cells, PCLS, and IPL. The signal transduction of ventilation-induced Nrf2 activation was shown to be p38 MAP kinase-dependent. In vivo ventilation experiments indicated that AREG is regulated by Nrf2 during MV. We conclude that Areg expression is regulated by Nrf2. During high-pressure ventilation, Nrf2 becomes activated and induces AREG, leading to a positive feedback loop between Nrf2 and AREG, which involves the p38 MAPK and results in the expression of cytoprotective genes.

Asunto(s)

Bronquios/fisiología , Familia de Proteínas EGF/metabolismo , Factor 2 Relacionado con NF-E2/genética , Respiración Artificial , Anfirregulina , Animales , Elementos de Respuesta Antioxidante/fisiología , Bronquios/citología , Células Cultivadas , Retroalimentación Fisiológica/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Técnicas de Cultivo de Órganos , Regiones Promotoras Genéticas/fisiología , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

In acute pulmonary inflammation, danger is first recognized by epithelial cells lining the alveolar lumen and relayed to vascular responses, including leukocyte recruitment and increased endothelial permeability. We supposed that this inflammatory relay critically depends on the immunological function of lung interstitial cells such as smooth muscle cells (SMC). Mice with smooth muscle protein-22α promotor-driven deficiency of the disintegrin and metalloproteinase (ADAM) 17 (SM22-Adam17(-/-)) were investigated in models of acute pulmonary inflammation (LPS, cytokine, and acid instillation). Underlying signaling mechanisms were identified in cultured tracheal SMC and verified by in vivo reconstitution experiments. SM22-Adam17(-/-) mice showed considerably decreased cytokine production and vascular responses in LPS- or acid-induced pulmonary inflammation. In vitro, ADAM17 deficiency abrogated cytokine release of primary SMC stimulated with LPS or supernatant of acid-exposed epithelial cells. This was explained by a loss of ADAM17-mediated growth factor shedding. LPS responses required ErbB1/epidermal growth factor receptor transactivation by TGFα, whereas acid responses required ErbB4 transactivation by neuregulins. Finally, LPS-induced pulmonary inflammation in SM22-Adam17(-/-) mice was restored by exogenous TGFα application, confirming the involvement of transactivation pathways in vivo. This highlights a new decisive immunological role of lung interstitial cells such as SMC in promoting acute pulmonary inflammation by ADAM17-dependent transactivation.

Asunto(s)

Proteínas ADAM/metabolismo , Receptores ErbB/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Oncogénicas v-erbB/metabolismo , Neumonía/metabolismo , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Receptores ErbB/genética , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Oncogénicas v-erbB/genética , Neumonía/genética , Regiones Promotoras Genéticas/genética , Receptor ErbB-4 , Activación Transcripcional/genética , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo

RESUMEN

INTRODUCTION: Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with rampantly proliferating synoviocytes and joint destruction due to oxidative stress. Recently, we described nuclear factor erythroid 2-related factor 2 (Nrf2) as a major requirement for limiting cartilage destruction. NF-κB and AP-1 are the main transcription factors triggering the inflammatory progression in RA. We used sulforaphane, an isothiocyanate, which is both an Nrf2 inducer and a NF-κB and AP-1 inhibitor. METHODS: Cultured synoviocytes were stimulated with sulforaphane (SFN) with or without TNF-α pre-treatment. NF-κB, AP-1, and Nrf2 activation was investigated via dual luciferase reporter gene assays. Matrix metalloproteinases (MMPs) were measured via zymography and luminex technique. Cytokine levels were detected using ELISA. Cell viability, apoptosis and caspase activity were studied. Cell proliferation was analysed by real-time cell analysis. RESULTS: SFN treatment decreased inflammation and proliferation dose-dependently in TNF-α-stimulated synoviocytes. SFN did not reduce MMP-3 and MMP-9 activity or expression significantly. Interestingly, we demonstrated that SFN has opposing effects on naïve and TNF-α-stimulated synoviocytes. In naïve cells, SFN activated the cytoprotective transcription factor Nrf2. In marked contrast to this, SFN induced apoptosis in TNF-α-pre-stimulated synoviocytes. CONCLUSIONS: We were able to show that SFN treatment acts contrary on naïve and inflammatory synoviocytes. SFN induces the cytoprotective transcription factor Nrf2 in naïve synoviocytes, whereas it induces apoptosis in inflamed synoviocytes. These findings indicate that the use of sulforaphane might be considered as an adjunctive therapeutic strategy to combat inflammation, pannus formation, and cartilage destruction in RA.

Asunto(s)

Isotiocianatos/farmacología , Factor 2 Relacionado con NF-E2/agonistas , FN-kappa B/antagonistas & inhibidores , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Sulfóxidos , Membrana Sinovial/citología , Factor de Transcripción AP-1/metabolismo

RESUMEN

Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are life-threatening diseases that are characterized by acute onset, pulmonary inflammation, oedema due to increased vascular permeability and severe hypoxemia. Clinically, ARDS can be divided into ARDS due to direct causes such as pneumonia, aspiration or injurious ventilation, and due to extrapulmonary indirect causes such as sepsis, severe burns or pancreatitis. In order to identify potential therapeutic targets, we asked here whether common molecular mechanisms can be identified that are relevant in different models of the direct form of ALI/ARDS. To this end, we reviewed three widely used models: (a) one based on a biological insult, i.e. instillation of bacterial endotoxins; (b) one based on a chemical insult, i.e. instillation of acid; and (c) one based on a mechanical insult, i.e. injurious ventilation. Studies were included only if the mediator or mechanism of interest was studied in at least two of the three animal models listed above. As endpoints, we selected neutrophil sequestration, permeability, hypoxemia (physiological dysfunction) and survival. Our analysis showed that most studies have focused on mechanisms of pulmonary neutrophil sequestration and models with moderate forms of oedema. The underlying mechanisms that involve canonical inflammatory pathways such as MAP kinases, CXCR2 chemokines, PAF, leukotrienes, adhesions molecules (CD18, ICAM-1) and elastase have been defined relatively well. Further mechanisms including TNF, DARC, HMGB1, PARP, GADD45 and collagenase are under investigation. Such mechanisms that are shared between the three ALI models may represent viable therapeutic targets. However, only few studies have linked these pathways to hypoxemia, the most important clinical aspect of ALI/ARDS. Since moderate oedema does not necessarily lead to hypoxemia, we suggest that the clinical relevance of experimental studies can be further improved by putting greater emphasis on gas exchange.

Asunto(s)

Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Humanos , Transducción de Señal

RESUMEN

Several studies report immunomodulatory effects of endogenous IL-10 after trauma. The present study investigates the effect of inhalative IL-10 administration on systemic and pulmonary inflammation in hemorrhagic shock. Male C57/BL6 mice (8 animals per group) were subjected to pressure-controlled hemorrhagic shock for 1.5 hrs followed by resuscitation and inhalative administration of either 50 µL PBS (Shock group) or 50 µg/kg recombinant mouse IL-10 dissolved in 50 µL PBS (Shock + IL-10 group). Animals were sacrificed after 4.5 hrs of recovery and serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with ELISA kits. Acute pulmonary inflammation was assessed by pulmonary myeloperoxidase (MPO) activity and pulmonary H&E histopathology. Inhalative IL-10 administration decreased pulmonary inflammation without altering the systemic concentrations of IL-6, IL-10, and KC. Serum MCP-1 levels were significantly reduced following inhalative IL-10 administration. These findings suggest that inhalative IL-10 administration may modulate the pulmonary microenvironment without major alterations of the systemic inflammatory response, thus minimizing the potential susceptibility to infection and sepsis.

Asunto(s)

Interleucina-10/administración & dosificación , Neumonía/etiología , Neumonía/prevención & control , Choque Hemorrágico/complicaciones , Administración por Inhalación , Animales , Citocinas/sangre , Interleucina-10/sangre , Interleucina-10/inmunología , Interleucina-10/uso terapéutico , Interleucina-6/sangre , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía/sangre , Neumonía/inmunología , Choque Hemorrágico/sangre , Choque Hemorrágico/inmunología

RESUMEN

INTRODUCTION: Mechanical ventilation (MV) of mice is increasingly required in experimental studies, but the conditions that allow stable ventilation of mice over several hours have not yet been fully defined. In addition, most previous studies documented vital parameters and lung mechanics only incompletely. The aim of the present study was to establish experimental conditions that keep these parameters within their physiological range over a period of 6 h. For this purpose, we also examined the effects of frequent short recruitment manoeuvres (RM) in healthy mice. METHODS: Mice were ventilated at low tidal volume V(T) = 8 mL/kg or high tidal volume V(T) = 16 mL/kg and a positive end-expiratory pressure (PEEP) of 2 or 6 cm H(2)O. RM were performed every 5 min, 60 min or not at all. Lung mechanics were followed by the forced oscillation technique. Blood pressure (BP), electrocardiogram (ECG), heart frequency (HF), oxygen saturation and body temperature were monitored. Blood gases, neutrophil-recruitment, microvascular permeability and pro-inflammatory cytokines in bronchoalveolar lavage (BAL) and blood serum as well as histopathology of the lung were examined. RESULTS: MV with repetitive RM every 5 min resulted in stable respiratory mechanics. Ventilation without RM worsened lung mechanics due to alveolar collapse, leading to impaired gas exchange. HF and BP were affected by anaesthesia, but not by ventilation. Microvascular permeability was highest in atelectatic lungs, whereas neutrophil-recruitment and structural changes were strongest in lungs ventilated with high tidal volume. The cytokines IL-6 and KC, but neither TNF nor IP-10, were elevated in the BAL and serum of all ventilated mice and were reduced by recurrent RM. Lung mechanics, oxygenation and pulmonary inflammation were improved by increased PEEP. CONCLUSIONS: Recurrent RM maintain lung mechanics in their physiological range during low tidal volume ventilation of healthy mice by preventing atelectasis and reduce the development of pulmonary inflammation.

Asunto(s)

Lesión Pulmonar/etiología , Lesión Pulmonar/metabolismo , Pulmón/metabolismo , Pulmón/patología , Respiración Artificial/efectos adversos , Animales , Femenino , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Respiración con Presión Positiva