Sebastian Weiterer

Sepsis Induces Specific Changes in Histone Modification Patterns in Human Monocytes

Background Sepsis is a global burden and the primary cause of death in intensive care units worldwide. The pathophysiological changes induced by the host’s systemic inflammatory response to infection are not yet fully understood. During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes of their basal state of responsiveness. Epigenetic mechanisms like histone modifications are known to participate in the control of immune reactions, but so far the situation during sepsis is unknown. Methods and Findings In a pilot approach, we performed combined chromatin immunoprecipitation followed by high-throughput sequencing to assess the genome-wide distribution of the chromatin modifications histone 3 lysine 4 and 27 trimethylation and lysine 9 acetylation in monocytes isolated from healthy donors (n = 4) and patients with sepsis (n = 2). Despite different underlying causes for sepsis, a comparison over promoter regions shows a high correlation between the patients for all chromatin marks. These findings hold true also when comparing patients to healthy controls. Despite the global similarity, differential analysis reveals a set of distinct promoters with significant enrichment or depletion of histone marks. Further analysis of overrepresented GO terms show an enrichment of genes involved in immune function. To the most prominent ones belong different members of the HLA family located within the MHC cluster together with the gene coding for the major regulator of this locus—CIITA. Conclusions We are able to show for the first time that sepsis in humans induces selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes, shedding light on basal regulatory mechanisms that might be contributing to the functional changes occurring in monocytes.

Plasmatic isoforms of cytokeratin 18 and RAGE after severe trauma: a longitudinal cohort study.

BACKGROUND Life-threatening traumatic injuries lead to a complex inflammation-driven pathophysiology. Receptor of advanced glycation end product (RAGE) is a multiligand receptor of several endogenous alarmins, while cytokeratin 18 is a structural component of the filament of epithelial cells. Both proteins can be frequently found in plasma of patients with different diseases, whereby they have distinct underlying mechanism of formation. In this prospective observational study, we wanted to shed light on the kinetic of plasmatic RAGE and cytokeratin 18 isoforms after severe trauma, thereby also addressing the association of these markers with inflammation and their potential use as biomarkers. METHODS Plasma samples of 77 patients with severe multiple trauma as defined by an Injury Severity Score (ISS) 16 or greater were obtained from a local repository and levels of soluble RAGE, endogenous secretory RAGE, cytokeratin 18, cleaved cytokeratin 18, and interleukin 6 by enzyme-linked immunosorbent assay. Demographic and routine parameters of the cohort were extracted from an electronic patient data management system. RESULTS Both RAGE isoforms were transiently increased in plasma within 24 hours after trauma, while cytokeratin 18 levels were unchanged. Moreover, soluble RAGE concentrations in patients with thoracic injuries were higher compared with patients without injury, and both isoforms of RAGE discriminated between patients with most severe adult respiratory distress syndrome and patients with milder forms. In addition, cleaved and total cytokeratin 18 levels differ between patients with hepatic dysfunction and normal function, without possessing discriminatory power. RAGE and cytokeratin 18 isoforms correlated significantly but to a low extent with interleukin 6, while the isoforms of both parameters correlated to a high extent with one another. CONCLUSION The release of RAGE (but not cytokeratin 18) isoforms occurs early and transiently after trauma and is associated with the extent of injury and inflammatory response. RAGE and cytokeratin 18 isoforms have the potential to act as diagnostic or prognostic biomarkers of lung and hepatic dysfunction. LEVEL OF EVIDENCE Epidemiologic/prognostic study, level IV.

Use of biomarkers in sepsis. Update and perspectives

Sepsis and related complications are a challenge for intensive care medicine. Despite many advances in antibiotic therapy sepsis remains one of the most common diseases of patients in intensive care units and is designated as the main cause of death in critically ill patients. Persisting sepsis leads to impaired immunity, resulting in immunosuppression. Unspecific predictive signs complicate an early diagnosis; however, an early initiation of adequate therapy is of crucial importance for the prognosis. Scoring systems can be applied for the initial evaluation but are controversially discussed concerning the monitoring of disease progression and therapy as well as outcome prediction. Biomarkers are considered as a complementary approach.ZusammenfassungDie Sepsis und damit verbundene Komplikationen sind eine große Herausforderung für die Intensivmedizin. Trotz vieler Fortschritte in der antibiotischen Therapie gehört die Sepsis zu den häufigsten Krankheitsentitäten auf Intensivstationen und ist als Haupttodesursache kritisch kranker Patienten beschrieben. Persistiert die Sepsis, kommt es zu einer Störung der Immunität bis zur Immunsuppression. Unspezifische Frühzeichen erschweren oftmals eine schnelle Diagnose. Der rasche Beginn einer adäquaten Therapie ist allerdings für die Prognose von besonderer Bedeutung. Scoring-Systeme dienen der initialen Evaluation, werden aber in Bezug auf Verlaufs- und Therapie-Monitoring sowie Vorhersagekraft für die Sterblichkeit kontrovers diskutiert. Biomarker gelten hierfür als ergänzender Ansatz.AbstractSepsis and related complications are a challenge for intensive care medicine. Despite many advances in antibiotic therapy sepsis remains one of the most common diseases of patients in intensive care units and is designated as the main cause of death in critically ill patients. Persisting sepsis leads to impaired immunity, resulting in immunosuppression. Unspecific predictive signs complicate an early diagnosis; however, an early initiation of adequate therapy is of crucial importance for the prognosis. Scoring systems can be applied for the initial evaluation but are controversially discussed concerning the monitoring of disease progression and therapy as well as outcome prediction. Biomarkers are considered as a complementary approach.

Tumor Necrosis Factor Alpha Induces a Serotonin Dependent Early Increase in Ciliary Beat Frequency and Epithelial Transport Velocity in Murine Tracheae

The tracheal epithelium prevents via its highly effective clearance mechanism the contamination of the lower airways by pathogens. This mechanism is driven by ciliary bearing cells which are not only in contact with the gas phase; in addition they are also influenced by inflammatory mediators. These mediators can alter the protective function of the epithelium. Since the pro-inflammatoric cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role within the inflammatory cascade, we investigated its effect onto the tracheal epithelium measured by its ciliary beat frequency and the particle transport velocity. In organ explant experiments the ciliary beat frequency and the particle transport velocity were measured under the application of TNF-α using tracheae from male C57BL6J mice. We observed a dose dependent TNF-α induced increase of both particle transport velocity and ciliary beat frequency. Knock out mice experiments made evident that the increase was depended on the expression of tumor necrosis factor receptor 1 (TNF-R1). The increases in ciliary beat frequency as well as the accelerated particle transport velocity were either inhibited by the unspecific serotonin antagonist methysergide or by cyproheptadine a specific 5-HT2 receptor antagonist. Thus, acetylcholine antagonists or nitric oxide synthase (NOS) inhibitors failed to inhibit the TNF-α induced activation. In conclusion, TNF-α may play a pivotal role in the protection of lower airways by inducing ciliary activity and increase in particle transport velocity via TNF-R1 and 5-HT2 receptor.

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner. Conclusion: The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

Einsatz von Biomarkern in der Sepsis

Sepsis and related complications are a challenge for intensive care medicine. Despite many advances in antibiotic therapy sepsis remains one of the most common diseases of patients in intensive care units and is designated as the main cause of death in critically ill patients. Persisting sepsis leads to impaired immunity, resulting in immunosuppression. Unspecific predictive signs complicate an early diagnosis; however, an early initiation of adequate therapy is of crucial importance for the prognosis. Scoring systems can be applied for the initial evaluation but are controversially discussed concerning the monitoring of disease progression and therapy as well as outcome prediction. Biomarkers are considered as a complementary approach.ZusammenfassungDie Sepsis und damit verbundene Komplikationen sind eine große Herausforderung für die Intensivmedizin. Trotz vieler Fortschritte in der antibiotischen Therapie gehört die Sepsis zu den häufigsten Krankheitsentitäten auf Intensivstationen und ist als Haupttodesursache kritisch kranker Patienten beschrieben. Persistiert die Sepsis, kommt es zu einer Störung der Immunität bis zur Immunsuppression. Unspezifische Frühzeichen erschweren oftmals eine schnelle Diagnose. Der rasche Beginn einer adäquaten Therapie ist allerdings für die Prognose von besonderer Bedeutung. Scoring-Systeme dienen der initialen Evaluation, werden aber in Bezug auf Verlaufs- und Therapie-Monitoring sowie Vorhersagekraft für die Sterblichkeit kontrovers diskutiert. Biomarker gelten hierfür als ergänzender Ansatz.AbstractSepsis and related complications are a challenge for intensive care medicine. Despite many advances in antibiotic therapy sepsis remains one of the most common diseases of patients in intensive care units and is designated as the main cause of death in critically ill patients. Persisting sepsis leads to impaired immunity, resulting in immunosuppression. Unspecific predictive signs complicate an early diagnosis; however, an early initiation of adequate therapy is of crucial importance for the prognosis. Scoring systems can be applied for the initial evaluation but are controversially discussed concerning the monitoring of disease progression and therapy as well as outcome prediction. Biomarkers are considered as a complementary approach.

Galactomannan and Zymosan Block the Epinephrine-Induced Particle Transport in Tracheal Epithelium.

Background Ciliary beating by respiratory epithelial cells continuously purges pathogens from the lower airways. Here we investigated the effect of the fungal cell wall polysaccharides Galactomannan (GM) and Zymosan (Zym) on the adrenergic activated particle transport velocity (PTV) of tracheal epithelium. Methods Experiments were performed using tracheae isolated from male C57BL/6J mice. Transport velocity of the cilia bearing epithelial cells was measured by analysing recorded image sequences. Generation of reactive oxygen species (ROS) were determined using Amplex Red reagents. PCR experiments were performed on isolated tracheal epithelium to identify adrenergic receptor mRNA. Results The adrenergic receptors α1D, α2A, β1 and β2 have been identified in isolated tracheal epithelium. We found epinephrine responsible for an increase in PTV, which could only be reduced by selective β-receptor-inhibition. In addition, either GM or Zym prevented the epinephrine induced PTV increase. Furthermore, we observed a strong ROS generation evoked by GM or Zym. However, epinephrine induced increase in PTV recovered in the presence of GM and Zym after application of ROS scavengers. Conclusion Both GM or Zym trigger reversible ROS generation in tracheal tissue leading to inhibition of the β-adrenergic increase in PTV.

[Epigenetic regulation in sepsis : current state of knowledge].

ZusammenfassungDie Sepsis ist das Krankheitsbild, das aus einer schweren systemischen Immunreaktion des Körpers auf eine Infektion unterschiedlicher Ursache resultiert. Initial reagiert das Immunsystem mit einer überschießenden Aktivierung von Entzündungszellen und der Ausschüttung proinflammatorischer Zytokine. Gleichzeitig wirken körpereigene Mechanismen durch antiinflammatorische Mediatoren und Immunzellen dieser generalisierten Entzündungsreaktion als Gegenregulation entgegen. Auch diese kompensatorische antiinflammatorische Immunantwort kann entsprechend der proinflammatorischen Reaktion übersteigert sein und resultiert dann in einer prolongierten sepsisinduzierten Immunsuppression. Die Gründe für eine solche persistierende antiinflammatorische Reaktion und die daraus folgende Vulnerabilität sind unklar. Allerdings gibt es Hinweise, dass ein septisches Ereignis die Grundeigenschaften der Immunzellen durch epigenetische Modifikation verändert. Veränderungen von Histonmodifikationen und Änderungen der Aktivierungsmechanismen von Transkriptionsfaktoren scheinen dabei in vielen Zellen des Immunsystems, wie Makrophagen, wichtige Rollen zu spielen sowie dadurch die Genregulation und Transkriptionsmechanismen der Zelle zu beeinflussen. Dieser Beitrag gibt einen Überblick über den aktuellen Stand der epigenetischen Sepsisforschung und über bisherige Erkenntnisse zu den langfristigen Auswirkungen der Sepsis auf das Immunsystem.AbstractSepsis is known to be a severe systemic immune reaction based on an infection of various origins. The initial immune response is accompanied by excess activation of immune cells and release of proinflammatory cytokines. Simultaneously initiated compensatory mechanisms lead to high levels of anti-inflammatory mediators to counterbalance the generalized inflammatory reaction; however, the compensatory immunoreaction itself equally overreacts and results in a prolonged sepsis-induced immunosuppression. The underlying mechanisms for these exaggerated immune responses and the resulting global immunosuppression that increase the risk for secondary infection are still unknown. Recent findings indicate that epigenetic mechanisms change basic properties of important immune cells by mechanisms leading to changes in gene expression. Dynamic exchanges of histone modifications result in a variation of transcription and seem to play a key role in cell function of macrophages and other immune cells. This article provides a current overview of epigenetic sepsis research and the sepsis-induced effects on the immune system.

Epigenetische Regulation in der Sepsis@@@Epigenetic regulation in sepsis: Aktueller Wissensstand@@@Current state of knowledge

ZusammenfassungDie Sepsis ist das Krankheitsbild, das aus einer schweren systemischen Immunreaktion des Körpers auf eine Infektion unterschiedlicher Ursache resultiert. Initial reagiert das Immunsystem mit einer überschießenden Aktivierung von Entzündungszellen und der Ausschüttung proinflammatorischer Zytokine. Gleichzeitig wirken körpereigene Mechanismen durch antiinflammatorische Mediatoren und Immunzellen dieser generalisierten Entzündungsreaktion als Gegenregulation entgegen. Auch diese kompensatorische antiinflammatorische Immunantwort kann entsprechend der proinflammatorischen Reaktion übersteigert sein und resultiert dann in einer prolongierten sepsisinduzierten Immunsuppression. Die Gründe für eine solche persistierende antiinflammatorische Reaktion und die daraus folgende Vulnerabilität sind unklar. Allerdings gibt es Hinweise, dass ein septisches Ereignis die Grundeigenschaften der Immunzellen durch epigenetische Modifikation verändert. Veränderungen von Histonmodifikationen und Änderungen der Aktivierungsmechanismen von Transkriptionsfaktoren scheinen dabei in vielen Zellen des Immunsystems, wie Makrophagen, wichtige Rollen zu spielen sowie dadurch die Genregulation und Transkriptionsmechanismen der Zelle zu beeinflussen. Dieser Beitrag gibt einen Überblick über den aktuellen Stand der epigenetischen Sepsisforschung und über bisherige Erkenntnisse zu den langfristigen Auswirkungen der Sepsis auf das Immunsystem.AbstractSepsis is known to be a severe systemic immune reaction based on an infection of various origins. The initial immune response is accompanied by excess activation of immune cells and release of proinflammatory cytokines. Simultaneously initiated compensatory mechanisms lead to high levels of anti-inflammatory mediators to counterbalance the generalized inflammatory reaction; however, the compensatory immunoreaction itself equally overreacts and results in a prolonged sepsis-induced immunosuppression. The underlying mechanisms for these exaggerated immune responses and the resulting global immunosuppression that increase the risk for secondary infection are still unknown. Recent findings indicate that epigenetic mechanisms change basic properties of important immune cells by mechanisms leading to changes in gene expression. Dynamic exchanges of histone modifications result in a variation of transcription and seem to play a key role in cell function of macrophages and other immune cells. This article provides a current overview of epigenetic sepsis research and the sepsis-induced effects on the immune system.

Epigenetische Regulation in der Sepsis

ZusammenfassungDie Sepsis ist das Krankheitsbild, das aus einer schweren systemischen Immunreaktion des Körpers auf eine Infektion unterschiedlicher Ursache resultiert. Initial reagiert das Immunsystem mit einer überschießenden Aktivierung von Entzündungszellen und der Ausschüttung proinflammatorischer Zytokine. Gleichzeitig wirken körpereigene Mechanismen durch antiinflammatorische Mediatoren und Immunzellen dieser generalisierten Entzündungsreaktion als Gegenregulation entgegen. Auch diese kompensatorische antiinflammatorische Immunantwort kann entsprechend der proinflammatorischen Reaktion übersteigert sein und resultiert dann in einer prolongierten sepsisinduzierten Immunsuppression. Die Gründe für eine solche persistierende antiinflammatorische Reaktion und die daraus folgende Vulnerabilität sind unklar. Allerdings gibt es Hinweise, dass ein septisches Ereignis die Grundeigenschaften der Immunzellen durch epigenetische Modifikation verändert. Veränderungen von Histonmodifikationen und Änderungen der Aktivierungsmechanismen von Transkriptionsfaktoren scheinen dabei in vielen Zellen des Immunsystems, wie Makrophagen, wichtige Rollen zu spielen sowie dadurch die Genregulation und Transkriptionsmechanismen der Zelle zu beeinflussen. Dieser Beitrag gibt einen Überblick über den aktuellen Stand der epigenetischen Sepsisforschung und über bisherige Erkenntnisse zu den langfristigen Auswirkungen der Sepsis auf das Immunsystem.AbstractSepsis is known to be a severe systemic immune reaction based on an infection of various origins. The initial immune response is accompanied by excess activation of immune cells and release of proinflammatory cytokines. Simultaneously initiated compensatory mechanisms lead to high levels of anti-inflammatory mediators to counterbalance the generalized inflammatory reaction; however, the compensatory immunoreaction itself equally overreacts and results in a prolonged sepsis-induced immunosuppression. The underlying mechanisms for these exaggerated immune responses and the resulting global immunosuppression that increase the risk for secondary infection are still unknown. Recent findings indicate that epigenetic mechanisms change basic properties of important immune cells by mechanisms leading to changes in gene expression. Dynamic exchanges of histone modifications result in a variation of transcription and seem to play a key role in cell function of macrophages and other immune cells. This article provides a current overview of epigenetic sepsis research and the sepsis-induced effects on the immune system.