Histone deacetylase 2-mediated deacetylation of the Ribonuclease 1 promoter in inflamed human endothelial cells

Endothelial cells (ECs) function as protective barrier to separate the blood from the surrounding tissue by conducting crucial roles in regulation and maintenance of vascular homeostasis, such as control of vessel permeability or coagulation. Therefore, dysfunction of the EC barrier due to inflammation, infection or injury can cause a variety of vascular pathologies, such as thrombosis or atherosclerosis. In this context, the circulating extracellular endonuclease Ribonuclease 1 (RNase1) was identified as a vessel- and tissue-protective enzyme and a potent regulator of vascular homeostasis. Upon acute inflammation, RNase1 functions as a natural counterpart to extracellular RNA (eRNA), a damage-associated molecular pattern, via degradation to protect the EC cell layer from excessive inflammation. However, long-term inflammation disrupts the RNase1-eRNA system. Thereby, eRNA accumulates in the extracellular space to induce massive proinflammatory cytokine release from circulating inflammatory cells, such as tumor necrosis factor alpha (TNF-α) or interleukin 1 beta (IL-1β). These cytokines negatively affect the EC layer by downregulation of RNase1 presumably through activation of histone deacetylases (HDACs). In this regard, this study investigated whether inflammation-mediated deacetylase function of HDACs suppresses RNase1 expression in human ECs through modulation of chromatin modifications. Proinflammatory stimulation with TNF-α or IL-1β of human umbilical vein endothelial cells significantly reduced RNase1 expression. Thus, identification of the RNASE1 promoter region and analysis of its chromatin state revealed the association of RNASE1 repression with deacetylation of histone 3 at lysine 27 and histone 4. The important role of HDACs in this process was further confirmed by administration of the specific class I HDAC1-3 inhibitor MS275 that successfully restored RNASE1 promoter acetylation and mRNA abundance upon TNF-α or IL-1β treatment. These results indicate an essential impact of HDAC1-3 in RNase1 regulation. Additionally, identification of specific HDACs involved in RNase1 regulation was obtained by chromatin immunoprecipitation kinetics confirming significant accumulation of HDAC2 at the RNASE1 promoter upon TNF-α stimulation. These findings were further validated by siRNA double knockdown of HDAC2 and its redundant enzyme HDAC1, which also recovered RNase1 mRNA abundance upon proinflammatory stimulation. In conclusion, our data identified HDAC2 as a crucial factor in RNase1 regulation in human ECs. HDAC2 is recruited to the RNASE1 promoter site to attenuate histone acetylation and suppress subsequent gene repression. This effect can be blocked by the specific HDAC inhibitor MS275 implicating the potential of HDAC inhibitors as novel therapeutic strategy to promote vascular integrity by preventing RNase1 downregulation in EC inflammation

RNA species in the host-pathogen dynamics during Legionella infection of human macrophages

Legionella pneumophila (L.p.) is a gram-negative, intracellular pathogen and a common cause of severe community-acquired pneumonia. In humans, L.p. replicates primarily within alveolar macrophages. It manipulates vital host cell functions such as vesicle trafficking and gene expression by the secretion of over 300 effector proteins into the host cell cytosol. Thus, L.p. modifies its host cell to promote its own replication. An unbiased and global analysis of the molecular changes and biological processes that are associated with bacterial infections of human cells can provide new insights into host-pathogen interactions. Therefore, one goal of this study was to characterize expression changes of different RNA species in response to infection with L.p. in human primary blood-derived macrophages (BDMs) or differentiated THP-1 cells. This work is structured into two parts: (1) a functional study on how miRNA manipulations can alter L.p. replication in macrophages and (2) an in depth analysis of transcriptomic events in host and pathogen during infection. (1) In the last few decades, miRNAs have been established as critical modulators of immune function. Therefore, one aim of this study was to identify the miRNA profile of L.p.-infected macrophages and to determine the functional impact of a miRNA manipulation on L.p. replication. BDMs of healthy donors were infected with L.p. strain Corby. Small RNA sequencing revealed the miRNA profile in BDMs following L.p. infection. An upregulation of miR-146a and miR-155, as well as downregulation of miR-221 and miR-125b was validated by qPCR in macrophages. miRNA regulation in response to infection seems to be due to transcriptional regulation of miRNA promoters, since the histone acetylation levels at the promoter and the pri-miR expression correlated with the miRNA expression upon L.p.- infection. Overexpression and knock down experiments of miR-125b, miR-221 and miR-579 in combination were performed for functional characterization and showed an influence of all three miRNAs on bacterial replication. A SILAC approach revealed the protein MX1 as downregulated following simultaneous overexpression of all three miRNAs. MX1 is an interferon-induced GTP-binding protein important for antiviral defence. As shown by validation experiments, MX1 knockdown in macrophages led to an increased replication of L.p., as seen following overexpression of the miRNAs. Since in silico analysis predicted no binding sites for either miRNA in the 3’UTR of MX1, Ingenuity pathway analysis was performed to find the linking molecules. DDX58 (RIG-I), a sensor for cytosolic RNA, was validated as a target for miR-221, while the tumour suppressor TP53 was shown to be targeted by miR-125b via luciferase reporter assays. An siRNA-mediated knockdown of both, TP53 and DDX58, respectively, led to an enhanced replication of L.p. in macrophages. Thus, DDX58 and TP53 were validated as linking molecules between the three miRNAs and MX1. Additionally, the aforementioned SILAC approach revealed a downregulation of LGALS8 which was later validated as a target of miR-579. LGALS8 is a cytosolic lectin which binds carbohydrates and localizes to damaged vesicles. Knockdown of LGALS8 enhanced intracellular replication in macrophages. Thus, MX1 and LGALS8 were identified as targets of the three miRNAs (miR-125b, miR-221, miR-579) and to be responsible for the restriction of L.p. replication within human macrophages. (2) The transcriptional profile of L.p. during the course of infection in human macrophages was next to be established. Dual RNA-Sequencing was performed to determine the regulation of coding and non-coding RNA species during the course of infection of both, host and pathogen, simultaneously. After adaptation and optimization of existing protocols, macrophages were infected using a GFP-expressing L.p. strain Corby. To separate infected cells (gfp+) from the non-invaded bystander cells (gfp-), flow cytometry sorting was performed. Furthermore, Pam3CSK4 was used to generate TLR2-activated cells. RNA from all different samples, and also RNA from cultivated Legionella, was sequenced. Differential gene expression analysis was performed using DESeq2 resulting in 4,144 differentially expressed human genes (across multiple conditions) and 2,707 differentially expressed Legionella genes (across two time points). The DESeq analysis of the separated RNA fractions from host cells revealed differentially expressed mRNAs (3,504), lncRNAs (495), and miRNAs (145). 1,128 differentially expressed genes were exclusively significantly regulated in invaded cells (gfp+ at 8 and 16 h). Some of these were validated via qPCR including BCL10, SOD1, IRS1, CYR61, ATG5, RND3 and JUN. In addition, the simultaneous upregulation of the genes ZFAND2A and HSPA1 in the bystander and in Legionella-invaded cells was validated. The analysis of the bacterial mRNAs revealed a switch of gene usage, i.e. inverse regulation at 8 and 16 h post infection. This switch included genes which are involved in iron metabolism, stress response, glycolysis and lipid biosynthesis. Hence, differentially expressed genes within different growth phases of the infection cycle were identified. This dataset is the first of its kind to cover a respiratory pathogen. The dual RNA-Sequencing performed in this study provides data to encapsulate the RNA landscape of coding and non-coding RNAs in pathogen and host. In summary, the results have deepened our insight into the infection process and the molecular interaction of L.p. and its host cells and will help to understand the complex interplay between host and pathogen by allowing for the in silico re-construction of an RNA interaction network. Furthermore, the present study will help to establish potential new candidates for diagnosis and therapy

Novel protein biomarkers for pneumonia and acute exacerbations in COPD: a pilot study

IntroductionCommunity-acquired pneumonia (CAP) and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) result in high morbidity, mortality, and socio-economic burden. The usage of easily accessible biomarkers informing on disease entity, severity, prognosis, and pathophysiological endotypes is limited in clinical practice. Here, we have analyzed selected plasma markers for their value in differential diagnosis and severity grading in a clinical cohort.MethodsA pilot cohort of hospitalized patients suffering from CAP (n = 27), AECOPD (n = 10), and healthy subjects (n = 22) were characterized clinically. Clinical scores (PSI, CURB, CRB65, GOLD I-IV, and GOLD ABCD) were obtained, and interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-2-receptor (IL-2R), lipopolysaccharide-binding protein (LBP), resistin, thrombospondin-1 (TSP-1), lactotransferrin (LTF), neutrophil gelatinase-associated lipocalin (NGAL), neutrophil-elastase-2 (ELA2), hepatocyte growth factor (HGF), soluble Fas (sFas), as well as TNF-related apoptosis-inducing ligand (TRAIL) were measured in plasma.ResultsIn CAP patients and healthy volunteers, we found significantly different levels of ELA2, HGF, IL-2R, IL-6, IL-8, LBP, resistin, LTF, and TRAIL. The panel of LBP, sFas, and TRAIL could discriminate between uncomplicated and severe CAP. AECOPD patients showed significantly different levels of LTF and TRAIL compared to healthy subjects. Ensemble feature selection revealed that CAP and AECOPD can be discriminated by IL-6, resistin, together with IL-2R. These factors even allow the differentiation between COPD patients suffering from an exacerbation or pneumonia.DiscussionTaken together, we identified immune mediators in patient plasma that provide information on differential diagnosis and disease severity and can therefore serve as biomarkers. Further studies are required for validation in bigger cohorts

Globale Expressionsprofile Pneumokokken-infizierter Bronchialepithelzellen - Einfluss der miRNA-3135b und des Nicotinamidstoffwechselweges auf die bakterielle Replikation

Streptococcus pneumoniae (S. pneumoniae), auch als Pneumococcus bezeichnet, ist ein grampositives Bakterium, welches gewöhnlich als Kommensale asymptomatisch den humanen Nasopharynx besiedelt, jedoch auch schwere Erkrankungen bis hin zur Sepsis oder Meningitis auslösen kann. Pneumokokken sind Hauptverursacher der Pneumonie beim Menschen und fordern jährlich mehrere Millionen Opfer weltweit. Weiterhin können Koinfektionen mit Influenza A Viren die Erkrankung verschlimmern. Die Epithelzellen des humanen Respirationstraktes bilden die erste Verteidigungsbarriere gegen die Infektion. Es sind jedoch viele Aspekte der Interaktion zwischen Epithelzellen und S. pneumoniae noch nicht umfassend geklärt. Um diese Interaktion detailliert zu analysieren, wurde ein Expressionsprofil aus mRNAs, Proteinen und miRNAs von infizierten Bronchialepithelzellen erstellt. Zusätzlich wurde ein Koinfektionsmodell in humanem ex vivo Lungengewebe zu Vergleichszwecken untersucht. Signalweg‐Analysen der infizierten Epithelzellen ergaben eine verstärkte Regulation des Zellzyklus zum späten Zeitpunkt der Infektion (16 h). Eine Vernetzung der Daten mit dem miRNA‐Profil offenbarte wenige, bereits bekannte Verknüpfungen. Dennoch konnten mit Hilfe der miRNA‐Untersuchungen behandlungsabhängige Expressionsmuster detektierten werden, welche S. pneumoniae‐spezifische miRNAs, wie die induzierte miRNA‐3135b, aufzeigten. Bei dieser hypothetischen miRNA könnte es sich tatsächlich um ein t‐RNA‐deriviertes Fragment (tRF) handeln. Eine Überexpression der miRNA‐3135b resultierte in einer signifikanten Reduktion der Pneumokokken‐Last, was auf einen Abwehrmechanismus der Epithelzellen hindeutet. Zudem zeigte die RNA‐Sequenzierung nach miRNA‐3135b‐Überexpression verschiedene putative Ziel‐mRNAs, deren Funktionen bisher nur eingeschränkt bekannt sind. Des Weiteren weisen funktionelle Analysen der mRNAs und Proteine auf eine Regulation des Nicotinamidmetabolismus hin. Die in den Epithelzellen durchgeführte Depletion von NAMPT, dem Schlüsselenzym dieses Stoffwechselweges, führte zu einer verminderten Replikation von S. pneumoniae. Weiterhin bewirkte die Zugabe von Nicotinamid‐Mononukleotid (NMN) eine gesteigerte Replikationsrate der Bakterien. Dies deutet auf NMN als wichtige Nährstoffquelle von Pneumokokken hin. Die Daten dieser Arbeit erweitern die Kenntnisse zur Interaktion von humanen Epithelzellen und Pneumokokken und könnten zur Identifizierung alternativer und neuer Therapiestrategien genutzt werden

ADAM8 signaling drives neutrophil migration and ARDS severity

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8–/– mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain

IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis

IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond