Die immunregulatorischen Trigger-Rezeptoren auf myeloiden Zellen (TREM) beim Haushuhn

Einige auf myeloiden und lymphoiden Zellen bei Mensch und Maus identifizierte Rezeptorfamilien weisen sowohl aktivierende als auch inhibitorische Rezeptoren auf, die wichtige Funktionen bei der Regulation des Immunsystems haben. Die Triggering Receptors Expressed on Myeloid Cells (TREMs) stellen eine dieser immunregulatorischen Rezeptorfamilien dar und wurden beim Säuger bereits genauer untersucht. In der vorliegenden Doktorarbeit wurden die Mitglieder der TREMs beim Huhn eingehend charakterisiert. Der potentiell aktivierende TREM-A1 besaß eine extrazytoplasmatische Ig-Domäne und einen kurzen zytoplasmatischen Abschnitt, im transmembranen Bereich aber Lysin, als eine positiv geladene AS, die mit einem ITAM-haltigen Adaptormolekül assoziieren könnte. TREM-A1 hatte ein MR von etwa 25 kDa. Die mRNA für das membranständige TREM-A1 wurde vor allem in Makrophagen detektiert, aber auch in Gehirn, Knochenmark, Milz, Bursa und Thymus. Auf Proteinebene konnte die Expression durch einen neu hergestellten, spezifischen monoklonalen Antikörper auf Monozyten und Makrophagen, aber auch auf etwa 50% der B-Zellen und einer Subpopulation der T-Zellen nachgewiesen werden. Die mRNA der Ig-Domäne von TREM-A1 war in Thrombozyten viel höher exprimiert als die mRNA für den membranständigen Rezeptor, was einen Hinweis auf die Existenz einer löslichen Splice-Variante von TREM-A1 in diesen Zellen liefert. Mit Hilfe von Reportergenassays und löslichen Rezeptorkonstrukten konnte gezeigt werden, dass der Ligand von TREM-A1 auf stimulierten Milzleukozyten exprimiert wird, nicht jedoch auf stimulierten oder unstimulierten anderen Leukozyten. TREM-A1 wies in AS-Sequenz und Gewebeverteilung hohe Ähnlichkeit zu TREM-2 beim Säuger auf. Der inhibitorische TREM-B1 besaß zwei Ig-Domänen und einen langen zytoplasmatischen Bereich mit zwei ITIMs. TREM-B1 hatte ein MR von etwa 47 kDa und wurde mit Hilfe der qPCR vor allem auf Thrombozyten detektiert. Die ITIMs im zytoplasmatischen Anteil wurden nach Pervanadatbehandlung phosphoriliert und rekrutierten die Protein-Tyrosin-Phosphatasen SHP-1 und SHP-2. Der Ligand von TREM-B1 wurde auf mit PMA/CaIonophor stimulierten Milzleukozyten exprimiert, nicht jedoch auf mit ConA stimulierten Milzleukozyten oder auf stimulierten bzw. unstimulierten Leukozyten anderer Organe. TREM-B1 wies in AS-Sequenz und Gewebeverteilung hohe Ähnlichkeit zu TLT-1 beim Säuger auf. Vom inhibitorischen TREM-B2 existierten drei Varianten, die aber alle einen langen zytoplasmatischen Bereich mit zwei ITIMs besitzen. TREM-B2v1 besaß zwei extrazytoplasmatische Ig-Domänen, TREM-B2v2 nur eine, wobei die membran-proximale Ig-Domäne von TREM-B2v1 fehlte. TREM-B2v3 hatte die beiden Ig-Domänen von TREM-B2v1 doppelt. Die mRNA aller drei Varianten wurde vor allem in PBMC und Makrophagen exprimiert, etwas weniger hoch in Knochenmark, PBL, Milz und Caecaltonsillen.Some receptor families identified on myeloid and lymphoid cells in man und mouse have both inhibitory and activating receptors with important functions in the regulation of the immune system. The TREMs display one of these receptor families and were already analyzed in mammals. In the present thesis the members of the triggering receptors expressed on myeloid cells (TREMs) in chicken were closely characterized. The potential activating TREM-A1 had one extracytoplasmic Ig-domain and a short cytoplasmic tail, but a positively charged amino acid in the transmembrane region, which could associate to an ITAM-containing adaptor molecule. TREM-A1 had a relative molecular weight of about 25 kDa. The mRNA of the transmembrane TREM-A1 was mainly detected in macrophages, but also in brain, bone marrow, spleen, bursa and thymus. On protein-level the expression could be demonstrated on monocytes and macrophages by a newly produced specific monoclonal antibody, but also on about 50 % of B-cells and a subpopulation of T-cells. On thrombocytes the mRNA of the Ig-domain of TREM-A1 was expressed much higher than the mRNA of the transmembrane receptor, indicating the existence of a soluble splice variant of TREM-A1 in these cells. By reportergene assays and soluble receptor constructs could be shown, that the ligand of TREM-A1 was expressed by stimulated leukocytes from the spleen, but not by other stimulated and not stimulated leukocytes. TREM-A1 showed high similarities to TREM-2 in mammals in its amino acid sequence and gene expression pattern. The inhibitory TREM-B1 had two Ig-domains and a long cytoplasmic tail with two ITIMs. TREM-B1 had a relative molecular weight form about 47 kDa and was mainly detected in thrombocytes by qPCR. Upon pervanadate treatment the ITIMs in the cytoplasmic tail were phosphorylated and recruited the protein-tyrosine-phosphatases SHP-1 and SHP-2. The ligand of TREM-B1 was expressed on leukocytes from the spleen, stimulated with PMA/CaInonophore, but whether on leukocytes of the spleen stimulated with ConA nor on stimulated resp. not stimulated leukocytes form other organs. TREM-B1 showed high similarities to TLT-1 in mammals in its amino acid sequence and gene expression pattern. There were three variants of the inhibitory TREM-B2, all holding a long cytoplasmic tail with two ITIMs. TREM-B2v1 had two extracytoplasmic Ig-domains, TREM-B2v2 only one, which however failed the membrane-proximal Ig-domain from TREM-B2v1. TREM-B2v3 had both Ig-domains from TREM-B2v1 twice. The mRNA of all the variants was mainly expressed by PBMC and macrophages, less in bone marrow, PBL, spleen and caecal tonsils

Draft genome sequences of two Bulgarian Bacillus anthracis strains

Bacillus anthracis strains previously isolated from Bulgaria form a unique subcluster within the A1.a cluster that is typical for isolates from southeastern Europe. Here, we report the draft genome sequences of two Bulgarian B. anthracis strains belonging to the A branch (A.Br.) 008/009 canonical single nucleotide polymorphism (SNP) group of the major A branch

Mycobacterium microti Infections in Free-Ranging Red Deer (Cervus elaphus)

Infections with Mycobacterium microti, a member of the M. tuberculosis complex, have been increasingly reported in humans and in domestic and free-ranging wild animals. At postmortem examination, infected animals may display histopathologic lesions indistinguishable from those caused by M. bovis or M. caprae, potentially leading to misidentification of bovine tuberculosis. We report 3 cases of M. microti infections in free-ranging red deer (Cervus elaphus) from western Austria and southern Germany. One diseased animal displayed severe pyogranulomatous pleuropneumonia and multifocal granulomas on the surface of the pericardium. Two other animals showed alterations of the lungs and associated lymph nodes compatible with parasitic infestation. Results of the phylogenetic analysis including multiple animal strains from the study area showed independent infection events, but no host-adapted genotype. Personnel involved in bovine tuberculosis–monitoring programs should be aware of the fastidious nature of M. microti, its pathogenicity in wildlife, and zoonotic potential

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50-70%

Whole-Genome Investigation of <i>Salmonella</i> Dublin Considering Mountain Pastures as Reservoirs in Southern Bavaria, Germany

Worldwide, Salmonella Dublin (S. Dublin) is responsible for clinical disease in cattle and also in humans. In Southern Bavaria, Germany, the serovar was identified as a causative agent for 54 animal disease outbreaks in herds between 2017 and 2021. Most of these emerged from cattle herds (n = 50). Two occurred in pig farms and two in bovine herds other than cattle. Genomic analysis of 88 S. Dublin strains isolated during these animal disease outbreaks revealed 7 clusters with 3 different MLST-based sequence types and 16 subordinate cgMLST-based complex types. Antimicrobial susceptibility investigation revealed one resistant and three intermediate strains. Furthermore, only a few genes coding for bacterial virulence were found among the isolates. Genome analysis enables pathogen identification and antimicrobial susceptibility, serotyping, phylogeny, and follow-up traceback analysis. Mountain pastures turned out to be the most likely locations for transmission between cattle of different herd origins, as indicated by epidemiological data and genomic traceback analyses. In this context, S. Dublin shedding was also detected in asymptomatic herding dogs. Due to the high prevalence of S. Dublin in Upper Bavaria over the years, we suggest referring to this administrative region as “endemic”. Consequently, cattle should be screened for salmonellosis before and after mountain pasturing

Draft genome sequence of Bacillus anthracis BF-1, Isolated from Bavarian cattle

Antwerpen M, Proença DN, Rückert C, et al. Draft genome sequence of Bacillus anthracis BF-1, Isolated from Bavarian cattle. Journal of bacteriology. 2012;194(22):6360-6361.Bacillus anthracis BF-1 was isolated from a cow in Bavaria (Germany) that had succumbed to anthrax. Here, we report the draft genome sequence of this strain, which belongs to the European B2 subclade of B. anthracis. The closest phylogenetic neighbor of strain BF-1 is a strain isolated from cattle in France

TREM-B1 is predominantly expressed on PBMC.

<p>Leukocytes obtained from bursa, thymus, caecal tonsils, spleen, bone marrow and blood (PBMC) were incubated with the 7E8 mab and analyzed by flow cytometry. The markers were set according to an isotype-matched negative control and the percentage of positive cells is indicated as a number. One of three representative experiments is shown.</p

TREM-B1 recruits SHP-2 upon phosphorylation.

<p>TREM-B1-FLAG transfected 2D8 cells were treated with pervanadate for the times indicated, lysed and immunoprecipitated using an anti-FLAG mab. Indicated antibodies were used for detection of the proteins.</p

TREM-B1 is expressed on thrombocytes.

<p>(A) Viable PBMC were discriminated by 7-AAD staining and gated on lymphocytes/thrombocytes (R1), monocytes/NK cells (R2) or heterophils (R3) in FSC/SSC. Cells were double immunofluorescence stained with either the 1E9 (upper panel) or the 7E8 (lower panel) mab in combination with several cell surface markers as indicated. Numbers indicate the percentage of cells in the respective quadrants. Isotype-matched controls were included in each experiment. One of ten representative experiments is shown. (B) Macrophages were single stained with the TREM-B1 specific mab 7E9 and the macrophage/monocyte specific marker KUL01 (filled histograms). Isotype-matched controls are shown as open histograms and percentages of positive cells are indicated as numbers.</p

Oligonucleotides used for real-time RT-PCR.

<p>Oligonucleotides used for real-time RT-PCR.</p