Influence of Atrial Natriuretic Peptide on inflammatory pathways in the lung

The cardiovascular hormone ANP is known to exert anti-inflammatory properties in macrophages and endothelial cells. This work provides new insight into the inflammatory signalling pathways influenced by the ANP in the lung. For these purposes, the effects of ANP on both alveolar epithelial cells and a model of LPS-induced lung inflammation were characterized. In alveolar epithelial cells, ANP was shown to inhibit the activation of two major transcription factors, NF-kB and AP-1, in response to TNFa. Astonishingly, this did not result in a reduced expression of the adhesion molecule ICAM-1. ANP was also capable to diminish the activation of AP-1 and NF-kB in lung tissue in vivo using a mouse model of LPS-induced septic shock. The inhibition of NF-kB activation was caused by a delayed phosphorylation and subsequent degradation of IkBa. In addition, ANP treatment elevated total protein levels of IkBa. p38 MAPK and Akt are important mediators in LPS-induced signalling. We demonstrated an activation of these kinases in lung tissue in response to i.p. LPS challenge. ANP treatment was able to lessen this activation. Furthermore, exclusive ANP treatment resulted in an increased p38 MAPK activation, which might contribute to the observed impact on other pathways. ICAM-1 expression was not impaired in whole lung tissue. ANP strongly decreased TNFa serum levels dose-dependently, but had only a slight effect on TNFa tissue levels. Interestingly, TNFa mRNA expression was not significantly reduced. Taken together this work demonstrates that ANP is able to diminish several important inflammatory pathways which are involved in the development of acute respiratory distress syndrome in LPS-induced sepsis

Directed PCR-free engineering of highly repetitive DNA sequences

Background: Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products.Results: For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked byrestriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy wecloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays.Conclusion: Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highlyrepetitive DNA sequences of defined lengths by simple standard cloning procedures

Atrial Natriuretic Peptide, a Regulator of Nuclear Factor-κB Activation in Vivo

Natriuretic peptides (NPs) comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. Along this line, atrial NP (ANP) (international non-proprietary name: carperitide, HANP) is an approved drug for the treatment of acute heart failure. In recent years, evidence has been given that the NP system possesses a far broader biological spectrum than the regulation of blood pressure and volume homeostasis. In fact, a substantial amount of in vitro work indicates that ANP affects important inflammatory processes and signaling pathways. Quite surprisingly, however, no information exists on the in vivo antiinflammatory potential and signaling of ANP. We show here that pretreatment of lipopolysaccharide (Salmonella abortus equi, 2.5 mg/kg)-challenged mice with ANP (5μg/kg iv, 15 min) rapidly inhibits nuclear factor-κB activation via inhibition of phosphorylation and degradation of the IκB-α protein. ANP also reduces Akt activation upon lipopolysaccharide injection. In ANP-pretreated mice, the increase of TNF-α serum concentration is markedly prevented; most importantly, the survival of these animals improved. These findings demonstrate both in vitro and in vivo an antiinflammatory profile of ANP that deserves to be further investigated in a therapeutic perspective

Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1beta Release by Pulmonary Epithelial Cells

Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1beta. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1beta release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits alpha7, alpha9, and/or alpha10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae