• Common and Unique Gene Expression Signatures of Human Macrophages in Response to Four Strains of Mycobacterium avium That Differ in Their Growth and Persistence Characteristics

      Blumenthal, Antje; Lauber, Jörg; Hoffmann, Reinhard; Ernst, Martin; Keller, Christine; Buer, Jan; Ehlers, Stefan; Reiling, Norbert (American Society for Microbiology, 2005-06)
    • The host response to the probiotic Escherichia coli strain Nissle 1917: specific up-regulation of the proinflammatory chemokine MCP-1.

      Ukena, Sya N; Westendorf, Astrid M; Hansen, Wiebke; Rohde, Manfred; Geffers, Robert; Coldewey, Sina; Suerbaum, Sebastian; Buer, Jan; Gunzer, Florian; German Research Centre for Biotechnology, Mucosal Immunity Group, Mascheroder Weg 1, 38124 Braunschweig, Germany. suk@gbf.de (2005)
      BACKGROUND: The use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells. METHODS: Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins. RESULTS: Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2alpha) and macrophage inflammatory protein-2 beta (MIP-2beta) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2alpha mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue. CONCLUSION: Thus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells.
    • Identification of novel regulators in T-cell differentiation of aplastic anemia patients.

      Franzke, Anke; Geffers, Robert; Hunger, J Katrin; Pförtner, Susanne; Piao, Wenji; Ivanyi, Philipp; Grosse, Jens; Probst-Kepper, Michael; Ganser, Arnold; Buer, Jan (2006)
      BACKGROUND: Aplastic anemia (AA) is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. The resulting hypocellularity limits a detailed analysis of the cellular immune response. To overcome this technical problem we performed a microarray analysis of CD3+ T-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers. Two AA patients were additionally analyzed after achieving a partial remission following immunosuppression. The regulation of selected candidate genes was confirmed by real-time RT-PCR. RESULTS: Among more than 22,200 transcripts, 583 genes were differentially expressed in the bone marrow of AA patients compared to healthy controls. Dysregulated genes are involved in T-cell mediated cytotoxicity, immune response of Th1 differentiated T-cells, and major regulators of immune function. In hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response. CONCLUSION: Our study suggests a pivotal role of Th1/Tc1 differentiated T-cells in immune-mediated marrow destruction of AA patients. Most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes.
    • Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity.

      Ukena, Sya N; Singh, Anurag; Dringenberg, Ulrike; Engelhardt, Regina; Seidler, Ursula; Hansen, Wiebke; Bleich, André; Bruder, Dunja; Franzke, Anke; Rogler, Gerhard; et al. (2007)
      BACKGROUND: Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs). METHODOLOGY AND PRINCIPAL FINDINGS: To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1. CONCLUSION AND SIGNIFICANCE: This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances.
    • Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota.

      Stecher, Bärbel; Robbiani, Riccardo; Walker, Alan W; Westendorf, Astrid M; Barthel, Manja; Kremer, Marcus; Chaffron, Samuel; Macpherson, Andrew J; Buer, Jan; Parkhill, Julian; et al. (2007-10)
      Most mucosal surfaces of the mammalian body are colonized by microbial communities ("microbiota"). A high density of commensal microbiota inhabits the intestine and shields from infection ("colonization resistance"). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10(-/-), VILLIN-HA(CL4-CD8)) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.
    • Signatures of human regulatory T cells: an encounter with old friends and new players.

      Pfoertner, Susanne; Jeron, Andreas; Probst-Kepper, Michael; Guzman, Carlos A; Hansen, Wiebke; Westendorf, Astrid M; Toepfer, Tanja; Schrader, Andres J; Franzke, Anke; Buer, Jan; et al. (2006)
      BACKGROUND: Naturally occurring CD4+ CD25+ regulatory T cells (TReg) are involved in the control of autoimmune diseases, transplantation tolerance, and anti-tumor immunity. Thus far, genomic studies on TReg cells were restricted to murine systems, and requirements for their development, maintenance, and mode of action in humans are poorly defined. RESULTS: To improve characterization of human TReg cells, we compiled a unique microarray consisting of 350 TReg cell associated genes (Human TReg Chip) based on whole genome transcription data from human and mouse TReg cells. TReg cell specific gene signatures were created from 11 individual healthy donors. Statistical analysis identified 62 genes differentially expressed in TReg cells, emphasizing some cross-species differences between mice and humans. Among them, several 'old friends' (including FOXP3, CTLA4, and CCR7) that are known to be involved in TReg cell function were recovered. Strikingly, the vast majority of genes identified had not previously been associated with human TReg cells (including LGALS3, TIAF1, and TRAF1). Most of these 'new players' however, have been described in the pathogenesis of autoimmunity. Real-time RT-PCR of selected genes validated our microarray results. Pathway analysis was applied to extract signaling modules underlying human TReg cell function. CONCLUSION: The comprehensive set of genes reported here provides a defined starting point to unravel the unique characteristics of human TReg cells. The Human TReg Chip constructed and validated here is available to the scientific community and is a useful tool with which to study the molecular mechanisms that orchestrate TReg cells under physiologic and diseased conditions.
    • Skp2-dependent degradation of p27kip1 is essential for cell cycle progression

      Kossatz, Uta; Dietrich, Nils; Zender, Lars; Buer, Jan; Manns, Michael P; Malek, Nisar P. (Cold Spring Harbor Laboratory Press, 2004-11-01)
    • Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer.

      Timmerbeul, Inke; Garrett-Engele, Carrie M; Kossatz, Uta; Chen, Xueyan; Firpo, Eduardo; Grünwald, Viktor; Kamino, Kenji; Wilkens, Ludwig; Lehmann, Ulrich; Buer, Jan; et al. (2006-09-19)
      Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.