IFN-γ and TNF-α synergize to inhibit CTGF expression in human lung endothelial cells.

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-β, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma

Detrimental effects of an inhaled phosphodiesterase-4 inhibitor on lung inflammation in ventilated preterm lambs exposed to chorioamnionitis are dose dependent

Background: Treatment of bronchopulmonary dysplasia in preterm infants is challenging due to its multifactorial origin. In rodent models of neonatal lung injury, selective inhibition of phosphodiesterase 4 (PDE4) has been shown to exert anti-inflammatory properties in the lung. We hypothesized that GSK256066, a highly selective, inhalable PDE4 inhibitor, would have beneficial effects on lung injury and inflammation in a triple hit lamb model of Ureaplasma parvum (UP)-induced chorioamnionitis, prematurity, and mechanical ventilation. Methods: Twenty-one preterm lambs were surgically delivered preterm at 129 days after 7 days intrauterine exposure to UP. Sixteen animals were subsequently ventilated for 24 hours and received endotracheal surfactant and intravenous caffeine citrate. Ten animals were randomized to receive twice a high (10 μg/kg) or low dose (1 μg/kg) of nebulized PDE4 inhibitor. Results: Nebulization of high, but not low, doses of PDE4 inhibitor led to a significant decrease in pulmonary PDE activity, and was associated with lung injury and vasculitis, influx of neutrophils, and increased proinflammatory cytokine messenger RNA levels. Conclusion: Contrary to our hypothesis, we found in our model a dose-dependent proinflammatory effect of an inhaled highly selective PDE4 inhibitor in the lung. Our findings indicate the narrow therapeutic range of inhaled PDE4 inhibitors in the preterm population

APOBEC3G-mediated Inhibition of Measles Virus Replication

Das Masernvirus (MV) ist ein negativ-strängiges RNA-Virus aus der Familie der Paramyxovi-ridae und zählt immer noch zu den häufigsten Todesursachen bei Kindern in Entwicklungs-ländern. Nach dem Eintritt in den Körper führt eine Infektion von CD150-exprimierenden B- und T-Lymphozyten sowie dendritischen Zellen (DCs) und Monozyten bzw. Makrophagen zu einer systemischen Infektion. Viele Mitglieder der Familie der APOBEC-Proteine („apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like proteins“), u. a. auch APOBEC3G, werden als Teil der angeborenen Immunantwort in diesen Zellen und infizierten Geweben exprimiert. In früheren Studien wurde bereits gezeigt, dass diese Proteine durch ihre RNA-bindenden Eigenschaften und ihre Fähigkeit zur Desaminierung von Cytosin zu Uracil zu einer Inhibition von verschiedenen Retroelementen und Retroviren, wie beispielsweise den „long interspersed nuclear elements 1“ und dem humanen Im-mundefizienz-Virus (HIV) führen. Das Ziel dieser Arbeit war es, mögliche antivirale Mechanismen von humanem APOBEC3G gegen das MV als Vertreter der negativ-strängigen RNA-Viren zu identifizieren. Hierzu wurden rekombinante MV-Wildtyp und -Impfstämme, sowie APOBEC3G-überexprimierende Vero-Zelllinien verwendet. Es zeigte sich, dass die Replikation des verwendeten rekombinanten Masern Wildtyp- und Impfvirus durch humanes APOBEC3G inhibiert wird. Diese Inhibition äußerte sich in einer reduzierten Synzytienbildung, einer mindestens 50 %igen Reduktion viral-exprimierter Proteine, sowie in einer 90-99 %igen Reduktion der auf APOBEC3G-exprimierenden Zellen entstandenen viralen Titer. Durch Sequenzanalysen konnte festgestellt werde, dass es zu einem Anstieg von 0,2 auf 0,95 unspezifischer Mutationen pro 1.000 Basenpaaren in MV-Transkripten kam, deren Muster mit dem in Kontrollzellen vergleichbar war, wohingegen typische C zu U(T) bzw. G zu A Hypermutationen nicht auftraten. Eine Kolokalisation von humanem APOBEC3G mit MV-spezifischen Proteinen konnte ebenfalls nicht eindeutig beobachtet werden. Es zeigte sich allerdings, dass APOBEC3G an virale RNA binden konnte. Außerdem wurde APOBEC3G in aufgereinigten viralen Partikeln um etwa den Faktor 4 angereichert. Versuche mit einem MV-Minireplikon-System ergaben, dass APOBEC3G eine Inhibition der viralen RNA-abhängigen RNA-Polymerase bewirkt, vermutlich aufgrund der Fähigkeit des Proteins an virale RNA zu binden. Immunfluoreszenzfärbungen mit mutierten APOBEC3G-Proteinen haben auch in die-ser Arbeit erneut belegt, dass der RNA-bindenden Desaminase-Domäne bei der zellulären Lokalisation des Proteins eine besondere Rolle zukommt, da einige Mutationen innerhalb dieses Bereiches zu einem hohen Verlust der Proteinexpression sowie zu einer Ansammlung der mutierten Proteine am rauen endoplasmatischen Retikulum führen. Die in dieser Arbeit gezeigte Inhibition der Replikation von MV durch humanes APO-BEC3G lässt eine generelle antivirale Aktivität von Mitgliedern der APOBEC-Familie gegen negativ-strängige RNA-Viren vermuten, welche auf der Fähigkeit des Proteins beruht RNA zu binden. Weitere Untersuchungen bezüglich der Inhibition anderer Vertreter der Mononegavirales durch verschiedene APOBEC-Proteine könnten Aufschluss über die beteiligten Mechanismen geben.Measles virus (MV) is a negative-strand RNA virus which belongs to the family Para-myxoviridae and remains one of the leading causes of morbidity and mortality in the develop-ing world. Following entry of MV into the body, infection of CD150-positive B- and T-cells as well as dendritic cells and monocytes/macrophages leads to a systemic infection. Members of the APOBEC-protein family (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like proteins), such as APOBEC3G, are expressed in these cells and infected tissues and form an important component of the innate immune response to viral infections. A number of studies have previously demonstrated that these proteins are able to inhibit various retroelements and retroviruses, such as long interspersed nuclear elements 1 and human immunodeficiency virus (HIV). This inhibition is based on their ability to bind RNA and to deaminate cytosine to uracil. The goal of this thesis was to identify possible antiviral mechanisms of human APOBEC3G against MV as an example for negative-strand RNA viruses. This was achieved through the use of recombinant wild-type and vaccine strains of MV and Vero cell lines overexpressing APOBEC3G. We could show that the replication of recombinant wild-type and vaccine strains of MV was inhibited by APOBEC3G, resulting in a 50 % reduction in the expression of viral proteins and syncytia formation, and a 90 to 99 % reduction in viral titers produced following infection of APOBEC3G expressing cells. Sequence analysis revealed that random mutations in viral transcripts increased from 0.2 to 0.95 mutations per 1000 bp and that the pattern of mutations was similar to that in control cells. Although typical C to U(T) or G to A hypermutations and co-localization of APOBEC3G and MV-specific proteins were not observed, APOBEC3G was able to bind viral RNA and was found to be enriched approximately 4-fold in viral particles. Further studies with a MV-specific minireplicon-system revealed that APOBEC3G led to an impairment of the viral RNA-dependent RNA-polymerase, probably due to the ability of the protein to bind RNA. Immunofluorescence immunostaining of mutated APOBEC3G-proteins showed that the RNA binding deaminase domain plays an important role in determining the cellular localization of the protein. Amino-acid changes in this domain led to a considerable loss of protein expression and to accumulation of the mutated proteins at the rough endoplasmic reticulum. The inhibition of MV replication described here suggests a general antiviral activity of members of the APOBEC family against negative-strand RNA viruses, based on the ability of these proteins to bind RNA. Further examinations including other members of the Mononegavirales may provide novel insights into the underlying mechanisms leading to inhibition of viral replication

Caffeine and Rolipram Affect Smad Signalling and TGFβ1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells

Caffeine administration is an important part of the therapeutic treatment of bronchopulmonary dysplasia (BPD) in preterm infants. However, caffeine mediated effects on airway remodelling are still undefined. The TGF-β/Smad signalling pathway is one of the key pathways involved in airway remodelling. Connective tissue growth factor (CTGF), a downstream mediator of TGF-β, and transgelin, a binding and stabilising protein of the cytoskeleton, are both regulated by TGF-b1 and play an important role in airway remodelling. Both have also been implicated in the pathogenesis of BPD. The aim of the present study was to clarify whether caffeine, an unspecific phosphodiesterase (PDE) inhibitor, and rolipram, a prototypical PDE-4 selective inhibitor, were both able to affect TGF-β1-induced Smad signalling and CTGF/transgelin expression in lung epithelial cells. Furthermore, the effect of transgelin knock-down on Smad signalling was studied. The pharmacological effect of caffeine and rolipram on Smad signalling was investigated by means of a luciferase assay via transfection of a TGFβ1- inducible reporter plasmid in A549 cells. The regulation of CTGF and transgelin expression by caffeine and rolipram were studied by promoter analysis, real-time PCR and Western blot. Endogenous transgelin expression was down-regulated by lentiviral transduction mediating transgelin-specific shRNA expression. The addition of caffeine and rolipram inhibited TGFβ1 induced reporter gene activity in a concentration-related manner. They also antagonized the TGF-b1 induced upregulation of CTGF and transgelin on the promoter-, the mRNA-, and the protein-level. Functional analysis showed that transgelin silencing reduced TGF-β1 induced Smad-signalling and CTGF induction in lung epithelial cells. The present study highlights possible new molecular mechanisms of caffeine and rolipram including an inhibition of Smad signalling and of TGF-β1 regulated genes involved in airway remodelling. An understanding of these mechanisms might help to explain the protective effects of caffeine in prevention of BPD and suggests rolipram to be a potent replacement for caffeine

Synergistic Effect of Caffeine and Glucocorticoids on Expression of Surfactant Protein B (SP-B) mRNA

Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 mM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8611.1-fold and 5.261.4-fold increase, respectively. Synergistic induction was achieved after coadministration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206659.7-fold increase, p,0.0001) or cAMP (1 mM) (2136111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9639.0), prednisolone (154666.8), and betamethasone (12366.4). Rolipram also induced SP-B mRNA (64.9621.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3a mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.869.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeinedependent phosphodiesterase inhibition and by upregulation of both ErbB4 and the GR. These results suggested that caffeine is able to induce the expression of SP-transcription factors and affects the signaling pathways of glucocorticoids, amplifying their effects. Co-administration of caffeine and corticosteroids may therefore be of benefit in surfactant homeostasis

KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport

Recently, we found that the cytidine deaminase APOBEC3G (A3G) inhibits measles (MV) replication. Using a microarray, we identified differential regulation of several host genes upon ectopic expression of A3G. One of the up-regulated genes, the endoplasmic reticulum (ER) protein retention receptor KDELR2, reduced MV replication ~5 fold when it was over-expressed individually in Vero and CEM-SS T cells. Silencing of KDELR2 in A3G-expressing Vero cells abrogated the antiviral activity induced by A3G, confirming its role as an A3G-regulated antiviral host factor. Recognition of the KDEL (Lys-Asp-Glu-Leu) motif by KDEL receptors initiates the retrograde transport of soluble proteins that have escaped the ER and play an important role in ER quality control. Although KDELR2 over-expression reduced MV titers in cell cultures, we observed no interaction between KDELR2 and the MV hemagglutinin (H) protein. Instead, KDELR2 retained chaperones in the ER, which are required for the correct folding and transport of the MV envelope glycoproteins H and fusion protein (F) to the cell surface. Our data indicate that KDELR2 competes with MV envelope proteins for binding to calnexin and GRP78/Bip, and that this interaction limits the availability of the chaperones for MV proteins, causing the reduction of virus spread and titers

Expression of surfactant protein B is dependent on cell density in H441 lung epithelial cells

Background: Expression of surfactant protein (SP)-B, which assures the structural stability of the pulmonary surfactant film, is influenced by various stimuli, including glucocorticoids; however, the role that cell-cell contact plays in SP-B transcription remains unknown. The aim of the current study was to investigate the impact of cell-cell contact on SP-B mRNA and mature SP-B expression in the lung epithelial cell line H441. Methods: Different quantities of H441 cells per growth area were either left untreated or incubated with dexamethasone. The expression of SP-B, SP-B transcription factors, and tight junction proteins were determined by qPCR and immunoblotting. The influence of cell density on SP-B mRNA stability was investigated using the transcription inhibitor actinomycin D. Results: SP-B mRNA and mature SP-B expression levels were significantly elevated in untreated and dexamethasone-treated H441 cells with increasing cell density. High cell density as a sole stimulus was found to barely have an impact on SP-B transcription factor and tight junction mRNA levels, while its stimulatory ability on SP-B mRNA expression could be mimicked using SP-B-negative cells. SP-B mRNA stability was significantly increased in high-density cells, but not by dexamethasone alone. Conclusion: SP-B expression in H441 cells is dependent on cell-cell contact, which increases mRNA stability and thereby potentiates the glucocorticoid-mediated induction of transcription. Loss of cell integrity might contribute to reduced SP-B secretion in damaged lung cells via downregulation of SP-B transcription. Cell density-mediated effects should thus receive greater attention in future cell culture-based research

Caffeine and rolipram affect Smad signalling and TGF-β1 stimulated CTGF and transgelin expression in lung epithelial cells.

Caffeine administration is an important part of the therapeutic treatment of bronchopulmonary dysplasia (BPD) in preterm infants. However, caffeine mediated effects on airway remodelling are still undefined. The TGF-β/Smad signalling pathway is one of the key pathways involved in airway remodelling. Connective tissue growth factor (CTGF), a downstream mediator of TGF-β, and transgelin, a binding and stabilising protein of the cytoskeleton, are both regulated by TGF-β1 and play an important role in airway remodelling. Both have also been implicated in the pathogenesis of BPD. The aim of the present study was to clarify whether caffeine, an unspecific phosphodiesterase (PDE) inhibitor, and rolipram, a prototypical PDE-4 selective inhibitor, were both able to affect TGF-β1-induced Smad signalling and CTGF/transgelin expression in lung epithelial cells. Furthermore, the effect of transgelin knock-down on Smad signalling was studied. The pharmacological effect of caffeine and rolipram on Smad signalling was investigated by means of a luciferase assay via transfection of a TGF-β1-inducible reporter plasmid in A549 cells. The regulation of CTGF and transgelin expression by caffeine and rolipram were studied by promoter analysis, real-time PCR and Western blot. Endogenous transgelin expression was down-regulated by lentiviral transduction mediating transgelin-specific shRNA expression. The addition of caffeine and rolipram inhibited TGF-β1 induced reporter gene activity in a concentration-related manner. They also antagonized the TGF-β1 induced up-regulation of CTGF and transgelin on the promoter-, the mRNA-, and the protein-level. Functional analysis showed that transgelin silencing reduced TGF-β1 induced Smad-signalling and CTGF induction in lung epithelial cells. The present study highlights possible new molecular mechanisms of caffeine and rolipram including an inhibition of Smad signalling and of TGF-β1 regulated genes involved in airway remodelling. An understanding of these mechanisms might help to explain the protective effects of caffeine in prevention of BPD and suggests rolipram to be a potent replacement for caffeine

Impact of cell density on mature SP-B expression in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated or incubated with 1 μM dexamethasone. After 48 h immunoblotting against SP-B and β-actin was performed. SP-B levels were normalized to β-actin and fold differences compared to 2.5 × 10⁴ dexamethasone-treated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. ** <i>p</i> < 0.01 compared to 5 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

Immunoblots against claudin-5 and claudin-8 from H441 cells at different densities.

<p>H441 cells were seeded at low (1 × 10⁴ cells per cm²) and high (7.5 × 10⁴ cells per cm²) quantities and either left untreated or incubated with 1 μM dexamethasone for 48 h. Immunoblots against claudin-5 (<b>A</b>) and claudin-8 (<b>B</b>) were performed, protein levels were normalized to total blotted protein, and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. * <i>p</i> < 0.05 compared to 1 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p