Robert Geffers

TRANSFAC®: transcriptional regulation, from patterns to profiles

The TRANSFAC database on eukaryotic transcriptional regulation, comprising data on transcription factors, their target genes and regulatory binding sites, has been extended and further developed, both in number of entries and in the scope and structure of the collected data. Structured fields for expression patterns have been introduced for transcription factors from human and mouse, using the CYTOMER database on anatomical structures and developmental stages. The functionality of Match, a tool for matrix-based search of transcription factor binding sites, has been enhanced. For instance, the program now comes along with a number of tissue-(or state-)specific profiles and new profiles can be created and modified with Match Profiler. The GENE table was extended and gained in importance, containing amongst others links to LocusLink, RefSeq and OMIM now. Further, (direct) links between factor and target gene on one hand and between gene and encoded factor on the other hand were introduced. The TRANSFAC public release is available at http://www.gene-regulation.com. For yeast an additional release including the latest data was made available separately as TRANSFAC Saccharomyces Module (TSM) at http://transfac.gbf.de. For CYTOMER free download versions are available at http://www.biobase.de:8080/index.html.

The TRANSFAC system on gene expression regulation

The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

Frontline: Neuropilin-1: a surface marker of regulatory T cells

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up‐regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin‐1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down‐regulated in naive CD4+CD25– T cells after TCR stimulation. Furthermore, CD4+Nrp1high T cells express high levels of Foxp3 and suppress CD4+CD25– T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non‐regulatory T cells.

Gene expression profiling of human decidual macrophages: evidence for immunosuppressive phenotype.

Background Although uterine macrophages are thought to play an important regulatory role at the maternal-fetal interface, their global gene expression profile is not known. Methodology/Principal Findings Using micro-array comprising approximately 14,000 genes, the gene expression pattern of human first trimester decidual CD14+ monocytes/macrophages was characterized and compared with the expression profile of the corresponding cells in blood. Some of the key findings were confirmed by real time PCR or by secreted protein. A unique gene expression pattern intrinsic of first trimester decidual CD14+ cells was demonstrated. A large number of regulated genes were functionally related to immunomodulation and tissue remodelling, corroborating polarization patterns of differentiated macrophages mainly of the alternatively activated M2 phenotype. These include known M2 markers such as CCL-18, CD209, insulin-like growth factor (IGF)-1, mannose receptor c type (MRC)-1 and fibronectin-1. Further, the selective up-regulation of triggering receptor expressed on myeloid cells (TREM)-2, alpha-2-macroglobulin (A2M) and prostaglandin D2 synthase (PGDS) provides new insights into the regulatory function of decidual macrophages in pregnancy that may have implications in pregnancy complications. Conclusions/Significance The molecular characterization of decidual macrophages presents a unique transcriptional profile replete with important components for fetal immunoprotection and provides several clues for further studies of these cells.

Quorum-Sensing Antagonistic Activities of Azithromycin in Pseudomonas aeruginosa PAO1: a Global Approach

ABSTRACT The administration of macrolides such as azithromycin for chronic pulmonary infection of cystic fibrosis patients has been reported to be of benefit. Although the mechanisms of action remain obscure, anti-inflammatory effects as well as interference of the macrolide with Pseudomonas aeruginosa virulence factor production have been suggested to contribute to an improved clinical outcome. In this study we used a systematic approach and analyzed the impact of azithromycin on the global transcriptional pattern and the protein expression profile of P. aeruginosa PAO1 cultures versus those in untreated controls. The most remarkable result of this study is the finding that azithromycin exhibited extensive quorum-sensing antagonistic activities. In accordance with the inhibition of the quorum-sensing systems, virulence factor production was diminished and the oxidative stress response was impaired, whereas the type III secretion system was strongly induced. Moreover, P. aeruginosa motility was reduced, which probably accounts for the previously observed impaired biofilm formation capabilities of azithromycin-treated cultures. The interference of azithromycin with quorum-sensing-dependent virulence factor production, biofilm formation, and oxidative stress resistance in P. aeruginosa holds great promise for macrolide therapy in cystic fibrosis. Clearly quorum-sensing antagonist macrolides should be paid more attention in the management of chronic P. aeruginosa infections, and as quorum-sensing antagonists, macrolides might gain vital importance for more general application against chronic infections.

Interferon-α–Induced TRAIL on Natural Killer Cells Is Associated With Control of Hepatitis C Virus Infection

BACKGROUND & AIMS Pegylated interferon-alpha (PEG-IFNalpha), in combination with ribavirin, controls hepatitis C virus (HCV) infection in approximately 50% of patients by mechanisms that are not completely understood. Beside a direct antiviral effect, different immunomodulatory effects have been discussed. Natural killer (NK) cells might be associated with control of HCV infection. We examined the effects of IFNalpha on human NK cells and its relevance to HCV infection. METHODS We performed gene expression profiling studies of NK cells following stimulation of peripheral blood mononuclear cells with IFNalpha. We evaluated IFNalpha-induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression using flow cytometry analyses of NK cells isolated from patients with acute or chronic hepatitis C that had received PEG-IFNalpha therapy. RESULTS TRAIL was among the most up-regulated genes after IFNalpha stimulation of NK cells from healthy controls. After in vitro stimulation with IFNalpha, CD56(dim) NK cells from patients who had responded to PEG-IFNalpha therapy expressed higher levels of TRAIL than cells from patients with chronic hepatitis C. TRAIL expression, ex vivo, was inversely correlated with HCV-RNA levels during the early phase of PEG-IFNalpha therapy. In patients with acute hepatitis C, TRAIL expression on CD56(bright) NK cells increased significantly compared with cells from controls. In in vitro studies, IFNalpha-stimulated NK cells eliminated HCV-replicating hepatoma cells by a TRAIL-mediated mechanism. CONCLUSIONS IFNalpha-induced expression of TRAIL on NK cells is associated with control of HCV infection; these observations might account for the second-phase decline in HCV-RNA levels during PEG-IFNalpha therapy.

Macrophages at the Fetal–Maternal Interface Express Markers of Alternative Activation and Are Induced by M-CSF and IL-10

During pregnancy, the maternal immune system is challenged by the presence of the fetus, which must be tolerated despite being semiallogeneic. Uterine mucosal (or decidual) macrophages (Mϕ), one of the major leukocyte populations at the fetal–maternal interface, have been implicated in fetal tolerance, but information regarding their regulation is scarce. In this study, we investigated the role of several factors potentially involved in the differentiation and polarization of decidual Mϕ with an in vitro Mϕ differentiation model. By using flow cytometry, we showed that M-CSF and IL-10 were potent inducers of M2 (immunoregulatory) Mϕ markers expressed on human decidual Mϕ (CD14, CD163, CD206, CD209). In contrast, proinflammatory stimuli, and unexpectedly also the Th2-associated IL-4 and IL-13, induced different patterns of expression, indicating that a Th2-dominated environment is not required for decidual Mϕ polarization. M-CSF/IL-10–stimulated and decidual Mϕ also showed similar cytokine secretion patterns, with production of IL-10 as well as IL-6, TNF, and CCL4. Conversely, the proinflammatory, LPS/IFN-γ–stimulated Mϕ produced significantly higher levels of TNF and no IL-10. We also used a gene array with 420 Mϕ-related genes, of which 100 were previously reported to be regulated in a global gene expression profiling of decidual Mϕ, confirming that M-CSF/IL-10–induced Mϕ are closely related to decidual Mϕ. Taken together, our results consistently point to a central role for M-CSF and in particular IL-10 in the shaping of decidual Mϕ with regulatory properties. These cytokines may therefore play an important role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3′ untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Host Genetic Background Strongly Influences the Response to Influenza A Virus Infections

The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD50 to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses.

The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing.

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.