Charakterisierung von TNF-α beim Huhn

Bereits Ende des 19. Jahrhunderts wurde die Wirkung des TNF-α als erstes Zytokin beschrieben und ist bis heute eines der am besten untersuchten Signalmoleküle im Immunsystem. Inzwischen konnte eine große Familie strukturell ähnlicher Moleküle identifiziert werden, die in ihrer Gesamtheit die TNF-Superfamilie bilden. Trotz seiner wichtigen Funktion in der Regulation des Immunsystems konnte ein homologes Protein beim Huhn bis heute nicht identifiziert werden, obwohl Orthologe bei zahlreichen Säugern, aber auch bei Amphibien und Fischen charakterisiert wurden. Die Verfügbarkeit großer genomischer Datensätze einer Vielzahl von Vogelspezies hat in den letzten Jahren die Identifizierung zahlreicher sogenannter „missing genes“ in Vogelgenomen ermöglicht, darunter wichtiger Zytokine wie Erythropoetin oder Leptin. Unter Verwendung dieser Datensätze und vergleichbarer Methoden ist es in der vorliegenden Arbeit gelungen, die vollständige Sequenz von TNF-α beim Huhn (chTNF-α) und weiteren Vogelarten zu identifizieren und chTNF-α im Weiteren zu charakterisieren. Die Sequenzen des TNF-α des Huhns und fünf weiterer Vogelspezies weisen eine konservierte extrazelluläre TNF-Homologie Domäne auf, welche eine ca. 45 %ige Übereinstimmung mit der Aminosäuresequenz des Zytokins der Säuger zeigt. Neben der Transmembran-Domäne besitzen die aviären TNF-α Orthologe einen verlängerten intrazellulären Abschnitt. Zusätzlich wurden die bereits beim Huhn bekannten Sequenzen der TNF-Rezeptoren (TNFR1, TNFR2) in Bezug auf ihre phylogenetische Verwandtschaft mit Sequenzen anderer Vertebraten untersucht. Um erste Informationen über die Regulation von TNF-α beim Huhn zu erhalten, wurden Expressionsanalysen von chTNF-α mittels qRT-PCR durchgeführt. Hierzu wurden primäre Makrophagen isoliert und mit Lipopolysaccharid (LPS) für unterschiedliche Zeiträume stimuliert. Wie auch für andere Vertebraten beschrieben, wird TNF-α beim Huhn sehr schnell induziert. Eine signifikante Hochregulation der spezifischen mRNA wurde bereits nach zwei Stunden beobachtet. Nahezu identische Kinetiken wurden für die Induktion in Makrophagen beobachtet, die aus Monozyten, der Milz oder aus Knochenmarksvorläuferzellen gewonnen wurden. Die Analyse der chTNF-α Expression in Lymphozyten zeigte eine signifikante Induktion in isolierten CD4+ T-Helferzellen, nicht aber in CD8+ zytotoxischen T-Zellen. Auch die intravenöse Applikation von LPS führte in vivo nach drei Stunden zu einer signifikanten Induktion der TNF-α mRNA in der Milz, nicht aber in der Leber. Um die biologische Aktivität des Zytokins zu analysieren, wurde die vollständige chTNF-α-Sequenz sowohl in einem eukaryotischen (HEK293) als auch einem prokaryotischen Expressionssystem exprimiert. Die biologische Aktivität der rekombinanten Proteine wurde mit Hilfe einer NF-κB-Reporter-Zelllinie (CEC-NF-κB) quantifiziert. ChTNF-α aus beiden Expressionssystemen bewirkte eine starke Induktion des Reporters. Auch ein Konstrukt, welches lediglich die extrazelluläre Domäne des chTNF-α beinhaltete, führte zu einer hoch signifikanten Reporteraktivierung. Zusammenfassend konnte somit die Existenz eines Orthologs von TNF-α beim Huhn und dessen biologische Aktivität nachgewiesen werden. Damit wurde die Grundlage für weitere umfassende Untersuchungen zur funktionellen Relevanz von TNF-α bei inflammatorischen Prozessen beim Huhn und die Nutzung dieses Zytokins als Biomarker gelegt.TNF-α was the first cytokine to be described and early reports date back to the end of the 19th century. To date it is one of the most intensively investigated signaling molecules of the immune system. Over the last 40 years a large family of structurally similar molecules has been identified, providing the entirety of the TNF-superfamily. Despite its important function in the regulation of the immune system, a homologues protein has not yet been identified in the chicken, although orthologues have been characterized in numerous mammals and also in amphibians and fish. The availability of large genomic databases in a variety of avian species has enabled the identification of numerous so-called “missing genes” in the avian genomes. In recent years this has included important cytokines like erythropoietin and leptin. Using these datasets and comparable methods, this study succeeded in indentifying the complete sequence of TNF-α in the chicken and other bird species and characterizing chTNF-α. The sequences of TNF-α of the chicken and five other bird species show a conserved extracellular TNF-homology domain with 45 % amino acid similarity compared to the mammalian cytokines. In addition to the transmembrane domain the avian TNF-α orthologues contain an extended intracellular part. Furthermore, sequences of the TNF receptors (TNFR1, TNFR2) in the chicken already described by others were analysed for their phylogenetic relationship to sequences of other vertebrates. To obtain first information about the regulation of chTNF-α, expression analyses were performed using qRT-PCR. Primary macrophages were isolated and stimulated with lipopolysaccharide (LPS) for different periods of time. As described for other vertebrates, TNF-α is induced rapidly in chicken cells. A significant upregulation of the specific mRNA was already observed after 2 hours. Almost identical kinetics were observed for the induction in macrophages, which were obtained from monocytes, spleen and bone marrow precursor cells. The analysis of the chTNF-α expression in lymphocytes showed a significant induction in isolated CD4+ T-helper cells, but not in CD8+ cytotoxic T-cells. After three hours the intravenous application of LPS also resulted in a significant induction of chTNF-α mRNA in the spleen but not in the liver. To analyse the biological activity of the cytokine, the entire sequence of chTNF-α was expressed both in eukaryotic (HEK293) and prokaryotic expression systems. The biological activity of the recombinant protein was quantified using a NF-κB-reporter-cell line (CEC-NF-κB). chTNF-α derived from both expression systems caused a strong induction of the reporter. A construct, representing the extracellular domain of chTNF-α, also induced a significantly high activation of the reporter. In summary, the existence of an orthologue of TNF-α in the chicken and its biological activity could be demonstrated. This study lays the foundation for further comprehensive investigations of the functional relevance of TNF-α during inflammatory processes in chicken and the use of this cytokine as a biomarker

Characterization of Chicken Tumor Necrosis Factor-alpha, a Long Missed Cytokine in Birds

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-alpha (chTNF-alpha) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-alpha were identified in the genomes of 12 additional avian species including Palaeognathae and Neognathae, and the synteny of the closely adjacent loci with mammalian TNF-alpha orthologs was demonstrated in the crow (Corvus cornix) genome. In addition to chTNF-alpha, we obtained full sequences for homologs of TNF-alpha receptors 1 and 2 (TNFR1, TNFR2). chTNF-a mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-alpha expression in CD4(+) but not in CD8(+) cells. To gain insights into its biological activity, we generated recombinant chTNF-alpha in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NF kappa B-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-alpha/TNF-alpha receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems

A hypermorphic epithelial β-catenin mutation facilitates intestinal tumorigenesis in mice in response to compounding WNT-pathway mutations

Activation of the Wnt/β-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant β-catenin (Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-β-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33ΔN-Bcat mice showed an increase in the constitutive Wnt/β-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33ΔN-Bcat mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7  and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33ΔN-Bcat mice, but those Wnt target genes associated with excessive proliferation (i.e. Cdnn1, myc) were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33ΔN-Bcat mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-β-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/β-catenin pathway to facilitate and promote tumorigenesis

Genomic basis of a social polymorphism in a halictid bee

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Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe