The clinical role of host and bacterial-derived extracellular vesicles in pneumonia.

Pneumonia is among the leading causes of morbidity and mortality worldwide. Due to constant evolution of respiratory bacteria and viruses, development of drug resistance and emerging pathogens, it constitutes a considerable health care threat. To enable development of novel strategies to control pneumonia, a better understanding of the complex mechanisms of interaction between host cells and infecting pathogens is vital. Here, we review the roles of host cell and bacterial-derived extracellular vesicles (EVs) in these interactions. We discuss clinical and experimental as well as pathogen-overarching and pathogen-specific evidence for common viral and bacterial elicitors of community- and hospital-acquired pneumonia. Finally, we highlight the potential of EVs for improved management of pneumonia patients and discuss the translational steps to be taken before they can be safely exploited as novel vaccines, biomarkers, or therapeutics in clinical practice.

p38 and Casein Kinase 2 Mediate Ribonuclease 1 Repression in Inflamed Human Endothelial Cells via Promoter Remodeling Through Nucleosome Remodeling and Deacetylase Complex.

Vascular pathologies, such as thrombosis or atherosclerosis, are leading causes of death worldwide and are strongly associated with the dysfunction of vascular endothelial cells. In this context, the extracellular endonuclease Ribonuclease 1 (RNase1) acts as an essential protective factor in regulation and maintenance of vascular homeostasis. However, long-term inflammation causes strong repression of RNase1 expression, thereby promoting endothelial cell dysfunction. This inflammation-mediated downregulation of RNase1 in human endothelial cells is facilitated via histone deacetylase (HDAC) 2, although the underlying molecular mechanisms are still unknown. Here, we report that inhibition of c-Jun N-terminal kinase by small chemical compounds in primary human endothelial cells decreased physiological RNase1 mRNA abundance, while p38 kinase inhibition restored repressed RNase1 expression upon proinflammatory stimulation with tumor necrosis factor alpha (TNF-α) and poly I:C. Moreover, blocking of the p38 kinase- and HDAC2-associated kinase casein kinase 2 (CK2) by inhibitor as well as small interfering RNA (siRNA)-knockdown restored RNase1 expression upon inflammation of human endothelial cells. Further downstream, siRNA-knockdown of chromodomain helicase DNA binding protein (CHD) 3 and 4 of the nucleosome remodeling and deacetylase (NuRD) complex restored RNase1 repression in TNF-α treated endothelial cells implicating its role in the HDAC2-containing repressor complex involved in RNase1 repression. Finally, chromatin immunoprecipitation in primary human endothelial cells confirmed recruitment of the CHD4-containing NuRD complex and subsequent promoter remodeling via histone deacetylation at the RNASE1 promoter in a p38-dependent manner upon human endothelial cell inflammation. Altogether, our results suggest that endothelial RNase1 repression in chronic vascular inflammation is regulated by a p38 kinase-, CK2-, and NuRD complex-dependent pathway resulting in complex recruitment to the RNASE1 promoter and subsequent promoter remodeling.

Endothelial Ribonuclease 1 in Cardiovascular and Systemic Inflammation.

The vascular endothelial cell layer forms the inner lining of all blood vessels to maintain proper functioning of the vascular system. However, dysfunction of the endothelium depicts a major issue in context of vascular pathologies, such as atherosclerosis or thrombosis that cause several million deaths per year worldwide. In recent years, the endothelial extracellular endonuclease Ribonuclease 1 (RNase1) was described as a key player in regulation of vascular homeostasis by protecting endothelial cells from detrimental effects of the damage-associated molecular pattern extracellular RNA upon acute inflammation. Despite this protective function, massive dysregulation of RNase1 was observed during prolonged endothelial cell inflammation resulting in progression of several vascular diseases. For the first time, this review article outlines the current knowledge on endothelial RNase1 and its role in function and dysfunction of the endothelium, thereby focusing on the intensive research from recent years: Uncovering the underlying mechanisms of RNase1 function and regulation in response to acute as well as long-term inflammation, the role of RNase1 in context of vascular, inflammatory and infectious diseases and the potential to develop novel therapeutic options to treat these pathologies against the background of RNase1 function in endothelial cells.

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.

NF-κB-mediated inhibition of microRNA-149-5p regulates Chitinase-3-like 1 expression in human airway epithelial cells.

Lower respiratory tract infections are among the most common causes of death worldwide. Main pathogens leading to these severe infections are viruses and gram-positive bacteria that activate toll-like receptor (TLR)-mediated immune responses via pathogen-associated molecular patterns. One protective factor induced during infection is Chitinase-3-like 1 (CHI3L1), which exerts various functions, e.g. in host cell proliferation and bacterial counteraction, and has been proposed as a biomarker in several acute and chronic inflammatory conditions. MicroRNAs (miR) have become important regulators of inflammation and infection and are considered therapeutic targets in recent years. However, it is not known whether microRNAs play a role in the regulation of CHI3L1 expression in TLR-mediated respiratory epithelial cell inflammation. In this study, we analysed the pre- and post-transcriptional regulation of CHI3L1 by TLRs in bronchial epithelial cells. Therefore, we stimulated BEAS-2B cells with the bacterial TLR2-ligand lipoteichoic acid or the viral dsRNA analogue poly(I:C). We observed an increase in the expression of CHI3L1, which was dependent on TNF-α-mediated NF-κB activation in TLR2- and TLR3-activated cells. Moreover, TLR2 and - 3 stimulation caused downregulation of the microRNA miR-149-5p, an effect that could be suppressed by inhibiting NF-κB translocation into the nucleus. Luciferase reporter assays identified a direct interaction of miR-149-5p with the CHI3L1 3´untranslated region. This interaction was confirmed by inhibition and overexpression of miR-149-5p in BEAS-2B cells, which altered the expression levels of CHI3L1 mRNA. In summary, miR-149-5p directly regulates CHI3L1 in context of TLR-mediated airway epithelial cell inflammation and may be a potential therapeutic target in inflammation and other diseases.

Inflammation-mediated deacetylation of the ribonuclease 1 promoter via histone deacetylase 2 in endothelial cells.

Ribonuclease 1 (RNase1) is a circulating extracellular endonuclease that regulates the vascular homeostasis of extracellular RNA and acts as a vessel- and tissue-protective enzyme. Upon long-term inflammation, high amounts of proinflammatory cytokines affect endothelial cell (EC) function by down-regulation of RNase1. Here, we investigated the transcriptional regulation of RNase1 upon inflammation in HUVECs. TNF-α or IL-1ß stimulation reduced the expression of RNase1 relative to the acetylation state of histone 3 at lysine 27 and histone 4 of the RNASE1 promoter. Inhibition of histone deacetylase (HDAC) 1, 2, and 3 by the specific class I HDAC inhibitor MS275 abolished the TNF-α- or IL-1ß-mediated effect on the mRNA and chromatin levels of RNase1. Moreover, chromatin immunoprecipitation kinetics revealed that HDAC2 accumulates at the RNASE1 promoter upon TNF-α stimulation, indicating an essential role for HDAC2 in regulating RNase1 expression. Thus, proinflammatory stimulation induced recruitment of HDAC2 to attenuate histone acetylation at the RNASE1 promoter site. Consequently, treatment with HDAC inhibitors may provide a new therapeutic strategy to stabilize vascular homeostasis in the context of inflammation by preventing RNase1 down-regulation in ECs.-Bedenbender, K., Scheller, N., Fischer, S., Leiting, S., Preissner, K. T., Schmeck, B. T., Vollmeister, E. Inflammation-mediated deacetylation of the ribonuclease 1 promoter via histone deacetylase 2 in endothelial cells.