Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae.

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Alpha-1-antitrypsin-deficiency is associated with lower cardiovascular risk: an approach based on federated learning.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory multisystemic disease caused by environmental exposures and/or genetic factors. Inherited alpha-1-antitrypsin deficiency (AATD) is one of the best recognized genetic factors increasing the risk for an early onset COPD with emphysema. The aim of this study was to gain a better understanding of the associations between comorbidities and specific biomarkers in COPD patients with and without AATD to enable future investigations aimed, for example, at identifying risk factors or improving care. METHODS: We focused on cardiovascular comorbidities, blood high sensitivity troponin (hs-troponin) and lipid profiles in COPD patients with and without AATD. We used clinical data from six German University Medical Centres of the MIRACUM (Medical Informatics Initiative in Research and Medicine) consortium. The codes for the international classification of diseases (ICD) were used for COPD as a main diagnosis and for comorbidities and blood laboratory data were obtained. Data analyses were based on the DataSHIELD framework. RESULTS: Out of 112,852 visits complete information was available for 43,057 COPD patients. According to our findings, 746 patients with AATD (1.73%) showed significantly lower total blood cholesterol levels and less cardiovascular comorbidities than non-AATD COPD patients. Moreover, after adjusting for the confounder factors, such as age, gender, and nicotine abuse, we confirmed that hs-troponin is a suitable predictor of overall mortality in COPD patients. The comorbidities associated with AATD in the current study differ from other studies, which may reflect geographic and population-based differences as well as the heterogeneous characteristics of AATD. CONCLUSION: The concept of MIRACUM is suitable for the analysis of a large healthcare database. This study provided evidence that COPD patients with AATD have a lower cardiovascular risk and revealed that hs-troponin is a predictor for hospital mortality in individuals with COPD.

Lactate induces metabolic and epigenetic reprogramming of pro-inflammatory Th17 cells.

Increased lactate levels in the tissue microenvironment are a well-known feature of chronic inflammation. However, the role of lactate in regulating T cell function remains controversial. Here, we demonstrate that extracellular lactate predominantly induces deregulation of the Th17-specific gene expression program by modulating the metabolic and epigenetic status of Th17 cells. Following lactate treatment, Th17 cells significantly reduced their IL-17A production and upregulated Foxp3 expression through ROS-driven IL-2 secretion. Moreover, we observed increased levels of genome-wide histone H3K18 lactylation, a recently described marker for active chromatin in macrophages, in lactate-treated Th17 cells. In addition, we show that high lactate concentrations suppress Th17 pathogenicity during intestinal inflammation in mice. These results indicate that lactate is capable of reprogramming pro-inflammatory T cell phenotypes into regulatory T cells.

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner.

BACKGROUND: Lung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host-pathogen interactions during pneumococcal infections. METHODS: Following in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid. RESULTS: In vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment. CONCLUSION: Streptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms. Video Abstract.

Bacterial extracellular vesicles repress the vascular protective factor RNase1 in human lung endothelial cells.

BACKGROUND: Sepsis is one of the leading causes of death worldwide and characterized by blood stream infections associated with a dysregulated host response and endothelial cell (EC) dysfunction. Ribonuclease 1 (RNase1) acts as a protective factor of vascular homeostasis and is known to be repressed by massive and persistent inflammation, associated to the development of vascular pathologies. Bacterial extracellular vesicles (bEVs) are released upon infection and may interact with ECs to mediate EC barrier dysfunction. Here, we investigated the impact of bEVs of sepsis-related pathogens on human EC RNase1 regulation. METHODS: bEVs from sepsis-associated bacteria were isolated via ultrafiltration and size exclusion chromatography and used for stimulation of human lung microvascular ECs combined with and without signaling pathway inhibitor treatments. RESULTS: bEVs from Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium significantly reduced RNase1 mRNA and protein expression and activated ECs, while TLR2-inducing bEVs from Streptococcus pneumoniae did not. These effects were mediated via LPS-dependent TLR4 signaling cascades as they could be blocked by Polymyxin B. Additionally, LPS-free ClearColi™ had no impact on RNase1. Further characterization of TLR4 downstream pathways involving NF-кB and p38, as well as JAK1/STAT1 signaling, revealed that RNase1 mRNA regulation is mediated via a p38-dependent mechanism. CONCLUSION: Blood stream bEVs from gram-negative, sepsis-associated bacteria reduce the vascular protective factor RNase1, opening new avenues for therapeutical intervention of EC dysfunction via promotion of RNase1 integrity. Video Abstract.

Bacterial vesicles block viral replication in macrophages via TLR4-TRIF-axis.

Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract.

Surface proteome of plasma extracellular vesicles as mechanistic and clinical biomarkers for malaria.

PURPOSE: Malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum (P. falciparum) is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling. CONCLUSION: sEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers. TRIAL REGISTRATION: The trial was registered in the Deutsches Register Klinischer Studien (DRKS-ID: DRKS00012518).

Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Influenza virus-mediated suppression of bronchial Chitinase-3-like 1 secretion promotes secondary pneumococcal infection.

Infections of the lung are among the leading causes of death worldwide. Despite the preactivation of innate defense programs during viral infection, secondary bacterial infection substantially elevates morbidity and mortality rates. Particularly problematic are co-infections with influenza A virus (IAV) and the major bacterial pathogen Streptococcus pneumoniae. However, the molecular processes underlying the severe course of such co-infections are not fully understood. Previously, the absence of secreted glycoprotein Chitinase-3-like 1 (CHI3L1) was shown to increase pneumococcal replication in mice. We therefore hypothesized that an IAV preinfection decreases CHI3L1 levels to promote pneumococcal infection. Indeed, in an air-liquid interface model of primary human bronchial epithelial cells (hBECs), IAV preinfection interfered with apical but not basolateral CHI3L1 release. Confocal time-lapse microscopy revealed that the gradual loss of apical CHI3L1 localization during co-infection with influenza and S. pneumoniae coincided with the disappearance of goblet as well as ciliated cells and increased S. pneumoniae replication. Importantly, extracellular restoration of CHI3L1 levels using recombinant protein significantly reduced bacterial load in influenza preinfected bronchial models. Thus, recombinant CHI3L1 may provide a novel therapeutic means to lower morbidity and mortality associated with post-influenza pneumococcal infections.

Detection and segmentation of morphologically complex eukaryotic cells in fluorescence microscopy images via feature pyramid fusion.

Detection and segmentation of macrophage cells in fluorescence microscopy images is a challenging problem, mainly due to crowded cells, variation in shapes, and morphological complexity. We present a new deep learning approach for cell detection and segmentation that incorporates previously learned nucleus features. A novel fusion of feature pyramids for nucleus detection and segmentation with feature pyramids for cell detection and segmentation is used to improve performance on a microscopic image dataset created by us and provided for public use, containing both nucleus and cell signals. Our experimental results indicate that cell detection and segmentation performance significantly benefit from the fusion of previously learned nucleus features. The proposed feature pyramid fusion architecture clearly outperforms a state-of-the-art Mask R-CNN approach for cell detection and segmentation with relative mean average precision improvements of up to 23.88% and 23.17%, respectively.

Efficient antisense inhibition reveals microRNA-155 to restrain a late-myeloid inflammatory programme in primary human phagocytes.

A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells. The classical CPP octaarginine (R8) enabled antisense peptide-nucleic-acid (PNA) oligomer delivery into nearly 100% of human blood-derived macrophages without apparent cytotoxicity even up to micromolar concentrations. In a proof-of-principle experiment, we successfully de-repressed the global microRNA-155 regulome in primary human macrophages using a PNA-R8 oligomer, which phenocopies a CRISPR-Cas9 induced gene knockout. Interestingly, although it is often believed that fairly high concentrations (µM) are needed to achieve antisense activity, our PNA-R8 was effective at 200 nM. RNA-seq characterized microRNA-155 as a broad-acting riboregulator, feedback restraining a late myeloid differentiation-induced pro-inflammatory network, comprising MyD88-signalling and ubiquitin-proteasome components. Our results highlight the important role of the microRNA machinery in fine-control of blood-derived human phagocyte immunity and open the door for further studies on regulatory RNAs in difficult-to-transfect primary human immune cells.

Surface Proteome of Plasma Extracellular Vesicles as Biomarkers for Pneumonia and Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Community-acquired pneumonia (CAP) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) represent a major burden of disease and death and their differential diagnosis is critical. A potential source of relevant accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an extracellular vesicle array to find proteins on plasma sEVs that are differentially expressed and possibly allow the differential diagnosis between CAP and AECOPD. Plasma samples were analyzed from 21 healthy controls, 24 patients with CAP, and 10 with AECOPD . The array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy controls and patients with CAP or AECOPD. We found CD45 and CD28 to be the best discrimination markers between CAP and AECOPD in receiver operating characteristic analyses, with an area under the curve >0.92. Additional ensemble feature selection revealed the possibility to distinguish between CAP and AECOPD even if the patient with CAP had COPD, with a panel of CD45, CD28, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor necrosis factor-R-II, and CD16. CONCLUSION: The discrimination of sEV-associated proteins is a minimally invasive method with potential to discriminate between CAP and AECOPD.

TMPRSS2 Is the Major Activating Protease of Influenza A Virus in Primary Human Airway Cells and Influenza B Virus in Human Type II Pneumocytes.

Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.

High eosinophil blood counts are associated with a shorter length of hospital stay in exacerbated COPD patients - a retrospective analysis.

BACKGROUND: In COPD, the course of the disease including morbidity and mortality is strongly associated with severe exacerbations. The current GOLD recommendations emphasize blood eosinophil counts as a marker for responsiveness to inhaled corticosteroids (ICS). Retrospective analyses from randomized clinical trials indicate a favorable response to systemic corticosteroids in exacerbated COPD patients with blood eosinophils > 2%, however data outside clinical trials are scarce. PATIENTS AND METHODS: We retrospectively evaluated data from 1007 cases of patients who were admitted to the University Medical Center Marburg between 01/2013 and 12/2018. All patients had been diagnosed with an acute exacerbation of COPD (ICD-10 J44.0/J44.1). Our analysis was based on a subgroup of 417 patients in whom a full blood cell count was obtained at the day of admission. Patients were predominantly male (63.3%), had a median age of 74 years (IQR 65 years - 83 years) and a median FEV1 of 1.03 l (42.6% predicted). We compared the hospital length of stay and other outcome parameters using established thresholds for the eosinophil blood cell count (100 and 300 eosinophils/µl and 2%). RESULTS: Patients with low eosinophils (< 2%, <100 cells/µl) had a longer median time in hospital (length of hospital stay - LOS) as compared to patients with high eosinophils (< 2%: 9.31 vs. ≥2%:7 days, and < 100/µl: 10 vs. 100-300/µl: 8 vs. > 300/µl: 7 days). The median CRP was higher in patients with low eosinophils as compared to the other groups (< 2%: 22.7 vs. ≥2%: 9 mg/dl and < 100: 25 vs. 100-300: 13.5 vs. > 300: 7.1 mg/dl). Time to re-hospitalization or time to death did not differ between strata of eosinophils. Sensitivity analysis in a subgroup of patients in which pneumonia was excluded by chest x-ray did not significantly alter the results. CONCLUSION: The results support the hypothesis that patients with severe COPD exacerbations and elevated blood eosinophil counts respond better to systemic corticosteroid treatment than patients with a non-eosinophilic exacerbation.

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.

Proviral MicroRNAs Detected in Extracellular Vesicles From Bronchoalveolar Lavage Fluid of Patients With Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EVs) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs (miRNAs). In this study, we found significant changes in the miRNA composition of EVs in the bronchoalveolar lavage fluid from patients with IAV-induced ARDS. Among the 9 significantly deregulated microRNAs, miR-17-5p was upregulated in patients' BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication.

An Interactive Macrophage Signal Transduction Map Facilitates Comparative Analyses of High-Throughput Data.

Macrophages (Mϕs) are key players in the coordination of the lifesaving or detrimental immune response against infections. The mechanistic understanding of the functional modulation of Mϕs by pathogens and pharmaceutical interventions at the signal transduction level is still far from complete. The complexity of pathways and their cross-talk benefits from holistic computational approaches. In the present study, we reconstructed a comprehensive, validated, and annotated map of signal transduction pathways in inflammatory Mϕs based on the current literature. In a second step, we selectively expanded this curated map with database knowledge. We provide both versions to the scientific community via a Web platform that is designed to facilitate exploration and analysis of high-throughput data. The platform comes preloaded with logarithmic fold changes from 44 data sets on Mϕ stimulation. We exploited three of these data sets-human primary Mϕs infected with the common lung pathogens Streptococcus pneumoniae, Legionella pneumophila, or Mycobacterium tuberculosis-in a case study to show how our map can be customized with expression data to pinpoint regulated subnetworks and druggable molecules. From the three infection scenarios, we extracted a regulatory core of 41 factors, including TNF, CCL5, CXCL10, IL-18, and IL-12 p40, and identified 140 drugs targeting 16 of them. Our approach promotes a comprehensive systems biology strategy for the exploitation of high-throughput data in the context of Mϕ signal transduction. In conclusion, we provide a set of tools to help scientists unravel details of Mϕ signaling. The interactive version of our Mϕ signal transduction map is accessible online at https://vcells.net/macrophage.

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.