Claus Vogl

Nonconventional Initiation Complex Assembly by STAT and NF-κB Transcription Factors Regulates Nitric Oxide Synthase Expression

Summary Transcriptional regulation of the Nos2 gene encoding inducible nitric oxide synthase (iNOS) requires type I interferon (IFN-I) signaling and additional signals emanating from pattern recognition receptors. Here we showed sequential and cooperative contributions of the transcription factors ISGF3 (a complex containing STAT1, STAT2, and IRF9 subunits) and NF-κB to the transcriptional induction of the Nos2 gene in macrophages infected with the intracellular bacterial pathogen Listeria monocytogenes. NF-κB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 kinase for serine 5 phosphorylation of the RNA polymerase II (pol II) carboxyterminal domain (CTD). Subsequent to TFIIH deposition by NF-κB, ISGF3 attracted the pol II enzyme and phosphorylation at CTD S5 occurred. Thus, STATs and NF-κB cooperate through pol II promoter recruitment and the phosphorylation of its CTD, respectively, as a prerequisite for productive elongation of iNOS mRNA.

Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes.

Production of type I interferons (IFN-I, mainly IFNα and IFNβ) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized “interferon-producing cell” (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-β, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro–differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.

Tristetraprolin Is Required for Full Anti-Inflammatory Response of Murine Macrophages to IL-10

IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-α, a potent proinflammatory mediator that is the target for multiple anti-TNF-α clinical strategies in Crohn’s disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-α and IL-1α in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3′ untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.

Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation

For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA‐destabilizing function of the AU‐rich element‐binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP‐dependent decay is initially limited to few mRNAs. With time, the TTP‐dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation‐induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS‐treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re‐installment but not maintenance of immune homeostasis. These findings reveal a TTP‐ and p38 MAPK‐dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.

Insulin-like vs. non-insulin-like stimulation of glucose metabolism by vanadium, tungsten, and selenium compounds in rat muscle

The direct impact of vanadate, tungstate, selenate, and selenite on glucose metabolism of isolated rat soleus muscle was investigated. All compounds stimulated glucose transport, but only vanadate exerted an insulin-like effect on glycogen synthesis (mumol glucose into glycogen*g-1*h-1: control 1.43 +/- 0.11 vs. 1 mmol/l vanadate, 2.08 +/- 0.11, p < 0.0001), which was more distinct in the presence of 1 mmol/l H2O2 (control, 1.44 +/- 0.13 vs. 1 mmol/l vanadate, 3.49 +/- 0.12, p < 0.001). Glucose handling of muscles exposed to tungstate, selenate, or selenite resembled that of hypoxic muscle, i.e. the induced rise in glucose uptake was inhibited by dantrolene and associated with high rates of glycolysis and rapid glycogen depletion (glycogen content after incubation, mumol glucosyl units/g: control, 16.2 +/- 0.7 vs. hypoxia, 2.7 +/- 0.5, p < 0.0001; control, 17.0 +/- 0.5 vs. 100 mmol/l tungstate, 5.5 +/- 0.4, p < 0.001; control, 16.2 +/- 0.7 vs. 100 mmol/l selenate, 1.5 +/- 0.3, and vs. 300 mumol/l selenite, 1.7 +/- 0.3, p < 0.0001 each). The results suggest that vanadate (and more pronounced its peroxides) exerts true insulin-like action on isolated muscle glucose metabolism, whereas tungsten and selenium salts trigger glucose transport in association with a catabolic response, which may represent an unspecific response to toxic/osmotic stress.

Identification of Genetic Variation on the Horse Y Chromosome and the Tracing of Male Founder Lineages in Modern Breeds

The paternally inherited Y chromosome displays the population genetic history of males. While modern domestic horses (Equus caballus) exhibit abundant diversity within maternally inherited mitochondrial DNA, no significant Y-chromosomal sequence diversity has been detected. We used high throughput sequencing technology to identify the first polymorphic Y-chromosomal markers useful for tracing paternal lines. The nucleotide variability of the modern horse Y chromosome is extremely low, resulting in six haplotypes (HT), all clearly distinct from the Przewalski horse (E. przewalskii). The most widespread HT1 is ancestral and the other five haplotypes apparently arose on the background of HT1 by mutation or gene conversion after domestication. Two haplotypes (HT2 and HT3) are widely distributed at high frequencies among modern European horse breeds. Using pedigree information, we trace the distribution of Y-haplotype diversity to particular founders. The mutation leading to HT3 occurred in the germline of the famous English Thoroughbred stallion “Eclipse” or his son or grandson and its prevalence demonstrates the influence of this popular paternal line on modern sport horse breeds. The pervasive introgression of Thoroughbred stallions during the last 200 years to refine autochthonous breeds has strongly affected the distribution of Y-chromosomal variation in modern horse breeds and has led to the replacement of autochthonous Y chromosomes. Only a few northern European breeds bear unique variants at high frequencies or fixed within but not shared among breeds. Our Y-chromosomal data complement the well established mtDNA lineages and document the male side of the genetic history of modern horse breeds and breeding practices.

Allopolyploid speciation and ongoing backcrossing between diploid progenitor and tetraploid progeny lineages in the Achillea millefolium species complex: analyses of single-copy nuclear genes and genomic AFLP

BackgroundIn the flowering plants, many polyploid species complexes display evolutionary radiation. This could be facilitated by gene flow between otherwise separate evolutionary lineages in contact zones. Achillea collina is a widespread tetraploid species within the Achillea millefolium polyploid complex (Asteraceae-Anthemideae). It is morphologically intermediate between the relic diploids, A. setacea-2x in xeric and A. asplenifolia-2x in humid habitats, and often grows in close contact with either of them. By analyzing DNA sequences of two single-copy nuclear genes and the genomic AFLP data, we assess the allopolyploid origin of A. collina-4x from ancestors corresponding to A. setacea-2x and A. asplenifolia-2x, and the ongoing backcross introgression between these diploid progenitor and tetraploid progeny lineages.ResultsIn both the ncpGS and the PgiC gene tree, haplotype sequences of the diploid A. setacea-2x and A. asplenifolia-2x group into two clades corresponding to the two species, though lineage sorting seems incomplete for the PgiC gene. In contrast, A. collina-4x and its suspected backcross plants show homeologous gene copies: sequences from the same tetraploid individual plant are placed in both diploid clades. Semi-congruent splits of an AFLP Neighbor Net link not only A. collina-4x to both diploid species, but some 4x individuals in a polymorphic population with mixed ploidy levels to A. setacea-2x on one hand and to A. collina-4x on the other, indicating allopolyploid speciation as well as hybridization across ploidal levels.ConclusionsThe findings of this study clearly demonstrate the hybrid origin of Achillea collina-4x, the ongoing backcrossing between the diploid progenitor and their tetraploid progeny lineages. Such repeated hybridizations are likely the cause of the great genetic and phenotypic variation and ecological differentiation of the polyploid taxa in Achillea millefolium agg.

Nuclear and plastid haplotypes suggest rapid diploid and polyploid speciation in the N Hemisphere Achillea millefolium complex (Asteraceae)

BackgroundSpecies complexes or aggregates consist of a set of closely related species often of different ploidy levels, whose relationships are difficult to reconstruct. The N Hemisphere Achillea millefolium aggregate exhibits complex morphological and genetic variation and a broad ecological amplitude. To understand its evolutionary history, we study sequence variation at two nuclear genes and three plastid loci across the natural distribution of this species complex and compare the patterns of such variations to the species tree inferred earlier from AFLP data.ResultsAmong the diploid species of A. millefolium agg., gene trees of the two nuclear loci, ncpGS and SBP, and the combined plastid fragments are incongruent with each other and with the AFLP tree likely due to incomplete lineage sorting or secondary introgression. In spite of the large distributional range, no isolation by distance is found. Furthermore, there is evidence for intragenic recombination in the ncpGS gene. An analysis using a probabilistic model for population demographic history indicates large ancestral effective population sizes and short intervals between speciation events. Such a scenario explains the incongruence of the gene trees and species tree we observe. The relationships are particularly complex in the polyploid members of A. millefolium agg.ConclusionsThe present study indicates that the diploid members of A. millefolium agg. share a large part of their molecular genetic variation. The findings of little lineage sorting and lack of isolation by distance is likely due to short intervals between speciation events and close proximity of ancestral populations. While previous AFLP data provide species trees congruent with earlier morphological classification and phylogeographic considerations, the present sequence data are not suited to recover the relationships of diploid species in A. millefolium agg. For the polyploid taxa many hybrid links and introgression from the diploids are suggested.

Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation‐associated mRNA decay remains to be established. Using time‐resolved high‐resolution RNA binding analysis of the mRNA‐destabilizing protein tristetraprolin (TTP), an inflammation‐limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non‐destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP‐controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA‐stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co‐regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome‐wide atlas (http://ttp-atlas.univie.ac.at) of cis‐acting elements controlling mRNA decay in inflammation.

STAT1β is not dominant negative and is capable of contributing to gamma interferon-dependent innate immunity.

ABSTRACT The transcription factor STAT1 is essential for interferon (IFN)-mediated immunity in humans and mice. STAT1 function is tightly regulated, and both loss- and gain-of-function mutations result in severe immune diseases. The two alternatively spliced isoforms, STAT1α and STAT1β, differ with regard to a C-terminal transactivation domain, which is absent in STAT1β. STAT1β is considered to be transcriptionally inactive and to be a competitive inhibitor of STAT1α. To investigate the functions of the STAT1 isoforms in vivo, we generated mice deficient for either STAT1α or STAT1β. As expected, the functions of STAT1α and STAT1β in IFN-α/β- and IFN-λ-dependent antiviral activity are largely redundant. In contrast to the current dogma, however, we found that STAT1β is transcriptionally active in response to IFN-γ. In the absence of STAT1α, STAT1β shows more prolonged IFN-γ-induced phosphorylation and promoter binding. Both isoforms mediate protective, IFN-γ-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiencies. Our data shed new light on the potential contributions of the individual STAT1 isoforms to STAT1-dependent immune responses. Knowledge of STAT1βs function will help fine-tune diagnostic approaches and help design more specific strategies to interfere with STAT1 activity.