Dorothee Viemann

Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 Expression

The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome

BACKGROUND The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells. OBJECTIVE To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. METHODS We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. RESULTS In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells. CONCLUSION We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.

Staphylococcus aureus Small-Colony Variants Are Adapted Phenotypes for Intracellular Persistence

BACKGROUND Staphylococcus aureus is an important human pathogen of endovascular diseases, which can take a chronic course with a high relapse rate despite antimicrobial treatment. Thus far, persistent and antibiotic-refractory infections have been largely associated with a subpopulation of S. aureus, the small-colony variants (SCVs). METHODS In this work, we used endothelial cells to investigate infection with the highly virulent wild-type isolate (6850), 2 stable isogenic SCV phenotypes (hemB mutant IIb13 and JB1), and the complemented mutant. RESULTS All strains were highly invasive in endothelial cells but largely differed in host response induction. Microarray analysis showed that wild-type phenotypes up-regulated a large number of endothelial genes (including genes involved in innate immunity), whereas the SCVs did not cause these dramatic changes. The inflammatory response and cytotoxicity were strongest shortly after infection and largely decreased within the following days, which was accompanied by a fast elimination of intracellular wild-type bacteria. By contrast, SCVs survived within endothelial cells at high numbers. CONCLUSION S. aureus intracellular persistence via the development of an adapted subpopulation of SCVs most likely represents an important strategy of S. aureus to hide within the host cells, which could be a reservoir for chronic infections.

TNF induces distinct gene expression programs in microvascular and macrovascular human endothelial cells

The relevance of the diversity of endothelial cells (ECs) for the response to inflammatory stimuli is currently not well defined. Using oligonucleotide microarray technique, we systematically analyzed the tumor necrosis factor (TNF)‐induced expression profile in human microvascular ECs (HMEC) and macrovascular human umbilical vein ECs (HUVEC), analyzing 13,000 human genes by microarray analysis. Using strict inclusion and exclusion criteria, microarray analysis revealed that about half of the TNF‐induced genes were specific for HMEC‐1 or HUVEC. The microarray data could widely be confirmed by quantitative reverse transcriptase‐polymerase chain reaction and at the protein level. It is interesting that the majority of those genes regulated depending on the cell type encoded for chemokines, cytokines, and cell surface molecules. Our results argue for a more careful consideration of specific effects restricted to distinct subtypes of ECs. The establishment of EC type‐specific expression patterns may thus provide the basis for a selective manipulation of specific endothelial subtypes in different inflammatory diseases.

Erk5 Activation Elicits a Vasoprotective Endothelial Phenotype via Induction of Krüppel-like Factor 4 (KLF4)

The MEK5/Erk5 MAPK cascade has recently been implicated in the regulation of endothelial integrity and represents a candidate pathway mediating the beneficial effects of laminar flow, a major factor preventing vascular dysfunction and disease. Here we expressed a constitutively active mutant of MEK5 (MEK5D) to study the transcriptional and functional responses to Erk5 activation in human primary endothelial cells. We provide evidence that constitutive Erk5 activation elicits an overall protective phenotype characterized by increased apoptosis resistance and a decreased angiogenic, migratory, and inflammatory potential. This is supported by bioinformatic microarray analysis, which uncovered a statistical overrepresentation of corresponding functional clusters as well as a significant induction of anti-thrombotic, hemostatic, and vasodilatory genes. We identify KLF4 as a novel Erk5 target and demonstrate a critical role of this transcription factor downstream of Erk5. We show that KLF4 expression largely reproduces the protective phenotype in endothelial cells, whereas KLF4 siRNA suppresses expression of various Erk5 targets. Additionally, we show that vasoprotective statins potently induce KLF4 and KLF4-dependent gene expression via activation of Erk5. Our data underscore a major protective function of the MEK5/Erk5/KLF4 module in ECs and implicate agonistic Erk5 activation as potential strategy for treatment of vascular diseases.

Neutrophil-derived S100A12 in acute lung injury and respiratory distress syndrome

Objective:Both persistent accumulation and activation of neutrophils may contribute to the most severe form of acute lung injury, acute respiratory distress syndrome. We analyzed the expression of neutrophil-derived S100A12 and the proinflammatory receptor for advanced glycation end products (RAGE) in patients with acute respiratory distress syndrome. Additional in vivo and in vitro experiments were performed to further analyze the contribution of S100A12 to pulmonary inflammation. Subjects:We included 14 patients with acute respiratory distress syndrome and eight controls. In addition, 16 healthy subjects were included in an experimental lipopolysaccharide challenge model. Interventions:Concentrations of S100A12 and soluble RAGE were analyzed in bronchoalveolar lavage fluid. The expression of S100A12 and RAGE in lung biopsies from patients was analyzed by immunohistochemistry. S100A12 was also analyzed in bronchoalveolar lavage fluid from eight healthy subjects after challenge with lipopolysaccharide and compared with eight controls who received placebo inhalation. Effects of S100A12 on endothelial cells were analyzed in vitro. Main Results:Patients with acute respiratory distress syndrome had significantly enhanced pulmonary S100A12 expression and higher S100A12 protein concentrations in bronchoalveolar lavage fluid than controls. Levels of soluble RAGE were not significantly elevated in acute respiratory distress syndrome. S100A12 concentrations decreased with time from disease onset. In healthy volunteers, S100A12 was elevated in bronchoalveolar lavage fluid after lipopolysaccharide inhalation. In vitro experiments confirmed strong proinflammatory effects of human S100A12. Conclusions:S100A12 and its receptor RAGE are found at high concentrations in pulmonary tissue and bronchoalveolar lavage fluid in acute lung injury. S100A12 expression may reflect neutrophil activation during lung inflammation and contribute to pulmonary inflammation and endothelial activation via binding to RAGE.

Expression of Toll-Like Receptors in Neonatal Sepsis

Despite recent identification of specific pattern recognition receptors (PRR) for distinct microbial structures, data indicating their relevance in human infectious diseases are limited. We determined the expression levels of the Toll-like receptor (TLR)2 and TLR4 by flow cytometry on granulocytes and monocytes of healthy neonates compared with healthy adults. The basal expression of TLR2 was only slightly lower in neonatal phagocytes, whereas no differences could be detected for TLR4. Analyzing neonates with sepsis, we found an impressive up-regulation of TLR2 on blood phagocytes already at initial presentation of symptoms. Comparison with C-reactive protein, IL-8, and IL-6 suggested that TLR2 expression on monocytes is comparably valuable as an early sepsis marker. TLR2 was differentially regulated during neonatal sepsis, showing a constant up-regulation on monocytes but only a transient increase on granulocytes. Surprisingly, TLR4 showed no remarkable changes. Our results revealed a mild deficiency of TLR2 expression in newborns and demonstrated a differential expression of TLR2 but not TLR4 in the course of neonatal sepsis, which could reflect specific inflammatory responses to distinct pathogens. The definition of TLR expression patterns might open a new field of therapeutic targets for neonatal sepsis.

Essential Impact of NF-κB Signaling on the H5N1 Influenza A Virus-Induced Transcriptome

Systemic infections of humans and birds with highly pathogenic avian influenza A viruses of the H5N1 subtype are characterized by inner bleedings and a massive overproduction of cytokines known as cytokine storm. Growing evidence supports the role of endothelial cells in these processes. The aim of this study was to elucidate determinants of this strong response in endothelial cells with a focus on the transcription factor NF-κB. This factor is known as a major regulator of inflammatory response; however, its role in influenza virus replication and virus-induced immune responses is controversially discussed. By global mRNA profiling of infected cells in the presence or absence of a dominant negative mutant of IκB kinase 2 that specifically blocks the pathway, we could show that almost all H5N1 virus-induced genes depend on functional NF-κB signaling. In particular, activation of NF-κB is a bottleneck for the expression of IFN-β and thus influences the expression of IFN-dependent genes indirectly in the primary innate immune response against H5N1 influenza virus. Control experiments with a low pathogenic influenza strain revealed a much weaker and less NF-κB-dependent host cell response.

Proinflammatory S100A12 Can Activate Human Monocytes via Toll-like Receptor 4

RATIONALE S100A12 is overexpressed during inflammation and is a marker of inflammatory disease. Furthermore, it has been ascribed to the group of damage-associated molecular pattern molecules that promote inflammation. However, the exact role of human S100A12 during early steps of immune activation and sepsis is only partially described thus far. OBJECTIVES We analyzed the activation of human monocytes by granulocyte-derived S100A12 as a key function of early inflammatory processes and the development of sepsis. METHODS Circulating S100A12 was determined in patients with sepsis and in healthy subjects with experimental endotoxemia. The release of human S100A12 from granulocytes as well as the promotion of inflammation by activation of human monocytes after specific receptor interaction was investigated by a series of in vitro experiments. MEASUREMENTS AND MAIN RESULTS S100A12 rises during sepsis, and its expression and release from granulocytes is rapidly induced in vitro and in vivo by inflammatory challenge. A global gene expression analysis of S100A12-activated monocytes revealed that human S100A12 induces inflammatory gene expression. These effects are triggered by an interaction of S100A12 with Toll-like receptor 4 (TLR4). Blocking S100A12 binding to TLR4 on monocytes or TLR4 expressing cell lines (HEK-TCM) abrogates the respective inflammatory signal. On the contrary, blocking S100A12 binding to its second proposed receptor (receptor for advanced glycation end products [RAGE]) has no significant effect on inflammatory signaling in monocytes and RAGE-expressing HEK293 cells. CONCLUSIONS Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis.

The Contact Allergen Nickel Triggers a Unique Inflammatory and Proangiogenic Gene Expression Pattern via Activation of NF-κB and Hypoxia-Inducible Factor-1α

Nickel compounds are prime inducers of contact allergy reactions in humans. To identify the signal transduction pathways mediating the cellular responses to nickel and to elucidate their hierarchy, we performed Affymetrix gene profiling using human primary endothelial cells, which strongly respond to nickel stimulation. Overall, we found 258 significantly modulated transcripts, comprising 140 up-regulated and 118 down-regulated genes. The bulk of those genes were identified as targets of two distinct signaling cascades, the IKK2/NF-κB pathway and a proangiogenic pathway mediated by HIF-1α, which accumulates upon exposure to nickel. Using dominant-interfering mutants and retroviral RNA interference technology, we demonstrate that both pathways act independently to regulate expression of nonoverlapping gene pools. NF-κB activation mediates most of the proinflammatory responses to nickel. Nickel-dependent HIF-1α activation primarily modulates expression of genes involved in proliferation, survival, metabolism, and signaling, albeit the induction of some proinflammatory nickel-response genes, most prominently IL-6, which we identified as novel bona fide HIF-1α target in this study, is also critically dependent on this pathway. Furthermore, we provide evidence that transactivation of both transcription factors partially depends on p38 MAPK activation that contributes to the intensity of at least some target genes. Taken together, our data provide mechanistic insight into the complex network of nickel-induced cellular events and identify IKK2/NF-κB and HIF-1α as important pathways involved in processes such as delivery of “second signals” in contact hypersensitivity reactions to nickel.