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The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.
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Antibacterianos , Klebsiella pneumoniae , Polimixina B , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , Antibacterianos/farmacologia , Animais , Polimixina B/farmacologia , Membrana Externa Bacteriana/metabolismo , Polimixinas/farmacologia , Vesículas Extracelulares/metabolismo , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/metabolismo , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacosRESUMO
Extracellular vesicles (EVs) are nanosized particles released by nearly every cell type across all kingdoms of life. As a result, EVs are ubiquitously present in various human body fluids. Composed of a lipid bilayer, EVs encapsulate proteins, nucleic acids, and metabolites, thus playing a crucial role in immunity, for example, by enabling intercellular communication. More recently, there has been increasing evidence that EVs can also act as key regulators of allergic immune responses. Their ability to facilitate cell-to-cell contact and to transport a variety of different biomolecules enables active modulation of both innate and adaptive immune processes associated with allergic reactions. A comprehensive understanding of the intricate mechanisms underlying the interactions among allergens, immune cells, and EVs is imperative to develop innovative strategies for controlling allergic responses. This review highlights the recent roles of host cell- and bacteria-derived EVs in allergic diseases, presenting experimental and clinical evidence that underscores their significance. Additionally, the therapeutic potential of EVs in allergy management is outlined, along with the challenges associated with targeted delivery and cargo stability for clinical use. Optimization of EV composition and targeting strategies holds promise for advancing translational applications and establishing EVs as biomarkers or safe therapeutics for assessing allergic reactions. For these reasons, EVs represent a promising avenue for advancing both our understanding and management of allergic immune processes.
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Vesículas Extracelulares , Hipersensibilidade , Humanos , Vesículas Extracelulares/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Animais , Comunicação Celular/imunologia , Imunidade Inata/imunologia , Alérgenos/imunologia , Imunidade Adaptativa/imunologia , BiomarcadoresRESUMO
Bioactive peptides are key molecules in health and medicine. Deep learning holds a big promise for the discovery and design of bioactive peptides. Yet, suitable experimental approaches are required to validate candidates in high throughput and at low cost. Here, we established a cell-free protein synthesis (CFPS) pipeline for the rapid and inexpensive production of antimicrobial peptides (AMPs) directly from DNA templates. To validate our platform, we used deep learning to design thousands of AMPs de novo. Using computational methods, we prioritized 500 candidates that we produced and screened with our CFPS pipeline. We identified 30 functional AMPs, which we characterized further through molecular dynamics simulations, antimicrobial activity and toxicity. Notably, six de novo-AMPs feature broad-spectrum activity against multidrug-resistant pathogens and do not develop bacterial resistance. Our work demonstrates the potential of CFPS for high throughput and low-cost production and testing of bioactive peptides within less than 24 h.
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Peptídeos Antimicrobianos , Aprendizado Profundo , Replicação do DNA , Simulação de Dinâmica Molecular , Biossíntese de ProteínasRESUMO
Lower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD+ salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD+. Knockdown of NAD+ salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD+ treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD+ production increased NAD+ levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD+ treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD+. Thus, we identified the NAD+ salvage pathway as an antibacterial pathway in Spn infections, predicting an antibacterial mechanism of NAD+.
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Infecções Bacterianas , Nicotinamida-Nucleotídeo Adenililtransferase , Infecções Respiratórias , Humanos , NAD/metabolismo , Proteômica , Citocinas/metabolismo , Linhagem Celular , Trifosfato de Adenosina , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismoRESUMO
Introduction: Community-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. Methods: A 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. Results: In 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. Discussion: Taken 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.
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Many viruses require proteolytic activation of their envelope proteins for infectivity, and relevant host proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of influenza A virus (IAV) and various coronaviruses (CoV). Increased TMPRSS2 expression has been associated with a higher risk of severe influenza infection and enhanced susceptibility to SARS-CoV-2. Here, we found that Legionella pneumophila stimulates the increased expression of TMPRSS2-mRNA in Calu-3 human airway cells. We identified flagellin as the dominant structural component inducing TMPRSS2 expression. The flagellin-induced increase was not observed at this magnitude for other virus-activating host proteases. TMPRSS2-mRNA expression was also significantly increased by LPS, Pam3Cys, and Streptococcus pneumoniae, although less pronounced. Multicycle replication of H1N1pdm and H3N2 IAV but not SARS-CoV-2 and SARS-CoV was enhanced by flagellin treatment. Our data suggest that bacteria, particularly flagellated bacteria, up-regulate the expression of TMPRSS2 in human airway cells and, thereby, may support enhanced activation and replication of IAV upon co-infections. In addition, our data indicate a physiological role of TMPRSS2 in antimicrobial host response.
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Serina Endopeptidases , Humanos , Flagelina/farmacologia , Vírus da Influenza A/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Lipopolissacarídeos/farmacologia , RNA Mensageiro , SARS-CoV-2 , Serina Endopeptidases/genéticaRESUMO
BACKGROUND: Mental disorders (MDs) are one of the leading causes for workforce sickness absence and disability worldwide. The burden, costs and challenges are enormous for the individuals concerned, employers and society at large. Although most MDs are characterised by a high risk of relapse after treatment or by chronic courses, interventions that link medical-psychotherapeutic approaches with work-directed components to facilitate a sustainable return to work (RTW) are rare. This protocol describes the design of a study to evaluate the (cost-)effectiveness and implementation process of a multimodal, clinical and work-directed intervention, called RTW-PIA, aimed at employees with MDs to achieve sustainable RTW in Germany. METHODS: The study consists of an effectiveness, a health-economic and a process evaluation, designed as a two-armed, multicentre, randomised controlled trial, conducted in German psychiatric outpatient clinics. Sick-listed employees with MDs will receive either the 18-month RTW-PIA treatment in conjunction with care as usual, or care as usual only. RTW-PIA consists of a face-to-face individual RTW support, RTW aftercare group meetings, and web-based aftercare. Assessments will be conducted at baseline and 6, 12, 18 and 24 months after completion of baseline survey. The primary outcome is the employees´ achievement of sustainable RTW, defined as reporting less than six weeks of working days missed out due to sickness absence within 12 months after first RTW. Secondary outcomes include health-related quality of life, mental functioning, RTW self-efficacy, overall job satisfaction, severity of mental illness and work ability. The health-economic evaluation will be conducted from a societal and public health care perspective, as well as from the employer's perspective in a cost-benefit analysis. The design will be supplemented by a qualitative effect evaluation using pre- and post-interviews, and a multimethod process evaluation examining various predefined key process indicators from different stakeholder perspectives. DISCUSSION: By applying a comprehensive, multimethodological evaluation design, this study captures various facets of RTW-PIA. In case of promising results for sustainable RTW, RTW-PIA may be integrated into standard care within German psychiatric outpatient clinics. TRIAL REGISTRATION: The study was prospectively registered with the German Clinical Trials Register ( DRKS00026232 , 1 September 2021).
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Transtornos Mentais , Retorno ao Trabalho , Humanos , Qualidade de Vida , Transtornos Mentais/terapia , Satisfação no Emprego , Análise Custo-Benefício , Licença Médica , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Multicêntricos como AssuntoRESUMO
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.
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Vesículas Extracelulares , Sepse , Humanos , Células Endoteliais/metabolismo , Ribonucleases/metabolismo , Receptor 4 Toll-Like/metabolismo , Fatores de Proteção , Pulmão/metabolismo , RNA Mensageiro/metabolismo , Bactérias , Sepse/metabolismoRESUMO
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).
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Vesículas Extracelulares , Malária Falciparum , Malária , Animais , Humanos , Proteoma/metabolismo , Osteopontina/metabolismo , Malária/diagnóstico , Biomarcadores , Malária Falciparum/diagnóstico , Vesículas Extracelulares/metabolismoRESUMO
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.
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Vesículas Extracelulares , Receptor 4 Toll-Like , Humanos , Proteínas Adaptadoras de Transporte Vesicular , Escherichia coli , Macrófagos , Replicação ViralRESUMO
Legionella pneumophila (L.p.) is a bacterial pathogen which is a common causative agent of pneumonia. In humans, it infects alveolar macrophages and transfers hundreds of virulence factors that interfere with cellular signalling pathways and the transcriptomic landscape to sustain its own replication. By this interaction, it has acquired eukaryote-like protein motifs by gene transfer events that partake in the pathogenicity of Legionella. In a computational screening approach for eukaryotic motifs in the transcriptome of Legionella, we identified the L.p. strain Corby protein ABQ55614 as putative histone-deacetylase and named it "suppressing modifier of histones 1" (Smh1). During infection, Smh1 is translocated from the Legionella vacuole into the host cytosol. When expressed in human macrophage THP-1 cells, Smh1 was localized predominantly in the nucleus, leading to broad histone H3 and H4 deacetylation, blunted expression of a large number of genes (e.g. IL-1ß and IL-8), and fostered intracellular bacterial replication. L.p. with a Smh1 knockdown grew normally in media but showed a slight growth defect inside the host cell. Furthermore, Smh1 showed a very potent histone deacetylation activity in vitro, e.g. at H3K14, that could be inhibited by targeted mutation of the putative catalytic center inferred by analogy with eukaryotic HDAC8, and with the deacetylase inhibitor trichostatin A. In summary, Smh1 displays functional homology with class I/II type HDACs. We identified Smh1 as a new Legionella virulence factor with a eukaryote-like histone-deacetylase activity that moderates host gene expression and might pave the way for further histone modifications.IMPORTANCELegionella pneumophila (L.p.) is a prominent bacterial pathogen, which is a common causative agent of pneumonia. In order to survive inside the host cell, the human macrophage, it profoundly interacts with host cell processes to advance its own replication. In this study, we identify a bacterial factor, Smh1, with yet unknown function as a host histone deacetylase. The activity of this factor in the host cell leads to attenuated gene expression and increased intracellular bacterial replication.
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Eucariotos , Legionella pneumophila , Humanos , Histonas/genética , Legionella pneumophila/genética , Células Eucarióticas , Pesquisa , Fatores de Virulência/genética , Histona Desacetilases , Proteínas RepressorasRESUMO
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.
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Vesículas Extracelulares/metabolismo , Pneumonia Bacteriana/microbiologia , Pneumonia Viral/microbiologia , Animais , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/terapia , Resistência Microbiana a Medicamentos , Pneumonia Associada a Assistência à Saúde/microbiologia , Pneumonia Associada a Assistência à Saúde/terapia , Interações entre Hospedeiro e Microrganismos , Humanos , Pneumonia Bacteriana/terapia , Pneumonia Viral/terapiaRESUMO
Pneumonia causes the highest mortality of all infectious diseases worldwide. The most common pathogens are bacteria but there are also epidemic or pandemic lung infections caused by influenza or coronaviruses, such as the current pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to the occurrence of antibiotic resistance and immune pathologies, such as in sepsis, important challenges lie in considering the susceptibility of individual patients. Here, age, medication and comorbidities are considered; however, there is also clear evidence of genetic influences on the individual risk of developing pneumonia or developing a severe course of the disease. This article discusses the genetic influences on pneumonia and the clinical significance.
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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.
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Vesículas Extracelulares/metabolismo , Pneumonia/sangue , Doença Pulmonar Obstrutiva Crônica/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos de Coortes , Diagnóstico Diferencial , Progressão da Doença , Humanos , Pneumonia/diagnóstico , Proteoma/metabolismo , Doença Pulmonar Obstrutiva Crônica/diagnósticoRESUMO
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.
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Líquido da Lavagem Broncoalveolar/química , Vesículas Extracelulares/química , Influenza Humana/patologia , MicroRNAs/análise , Síndrome do Desconforto Respiratório/patologia , Células A549 , Adulto , Idoso , Células Epiteliais Alveolares/química , Células Epiteliais Alveolares/virologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/imunologia , Masculino , Pessoa de Meia-Idade , Orthomyxoviridae , Adulto JovemRESUMO
Immune response in the lung has to protect the huge alveolar surface against pathogens while securing the delicate lung structure. Macrophages and alveolar epithelial cells constitute the first line of defense and together orchestrate the initial steps of host defense. In this study, we analysed the influence of macrophages on type II alveolar epithelial cells during Legionella pneumophila-infection by a systems biology approach combining experimental work and mathematical modelling. We found that L. pneumophila-infected THP-1-derived macrophages provoke a pro-inflammatory activation of neighboring lung epithelial cells, but in addition render them hypo-responsive to direct infection with the same pathogen. We generated a kinetic mathematical model of macrophage activation and identified a paracrine mechanism of macrophage-secreted IL-1ß inducing a prolonged IRAK-1 degradation in lung epithelial cells. This intercellular crosstalk may help to avoid an overwhelming inflammatory response by preventing excessive local secretion of pro-inflammatory cytokines and thereby negatively regulating the recruitment of immune cells to the site of infection. This suggests an important but ambivalent immunomodulatory role of macrophages in lung infection.
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Células Epiteliais Alveolares/imunologia , Legionella pneumophila/imunologia , Doença dos Legionários/patologia , Macrófagos/imunologia , Células A549 , Técnicas de Cocultura , Humanos , Modelos Teóricos , Biologia de Sistemas , Células THP-1RESUMO
Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1ß in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or - partially - RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.
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Efeito Espectador , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Monócitos/metabolismo , Células A549 , Exossomos/microbiologia , Vesículas Extracelulares/microbiologia , Humanos , Doença dos Legionários/microbiologia , Doença dos Legionários/patologia , Monócitos/microbiologia , Células THP-1RESUMO
BACKGROUND: Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction. METHODS: WT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot. RESULTS: L. pneumophila infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In MyD88-/- cells, induction of inflammatory markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in MyD88-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot. CONCLUSION: Taken together, we characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host cell interaction, potentially relevant for new treatment options.
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Amidoidrolases/metabolismo , Legionella pneumophila/fisiologia , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Regiões 3' não Traduzidas , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Animais , Sequência de Bases , Quimiocina CXCL1/análise , Genótipo , Interleucina-6/análise , Interleucina-6/genética , Interleucina-6/metabolismo , Doença dos Legionários/genética , Doença dos Legionários/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Fator 88 de Diferenciação Mieloide/deficiência , Células RAW 264.7 , Alinhamento de Sequência , Transdução de Sinais , TranscriptomaRESUMO
Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.