In vitro mucin degradation and paracellular permeability by fecal water from Crohn's disease patients.

Aim: This study aimed to examine the impact of fecal water (FW) of active and remissive Crohn's disease (CD) patients on mucin degradation and epithelial barrier function. Methods: FW and bacterial membrane vesicles (MVs) were isolated from fresh fecal samples of six healthy controls (HCs) and 12 CD patients. Bacterial composition was determined by 16S rRNA gene amplicon sequencing. Results: In vitro FW-induced mucin degradation was higher in CD samples versus HC (p < 0.01), but not associated with specific bacterial genera. FW of three remissive samples decreased transepithelial electrical resistance in Caco-2 cells by 78-87% (p < 0.001). MVs did not induce barrier alterations. Conclusion: The higher mucin-degradation capacity of CD-derived FW might suggest contributions of microbial products to CD pathophysiology.

Metagenomic Profiling of Fecal-Derived Bacterial Membrane Vesicles in Crohn's Disease Patients.

BACKGROUND: In the past, many studies suggested a crucial role for dysbiosis of the gut microbiota in the etiology of Crohn's disease (CD). However, despite being important players in host-bacteria interaction, the role of bacterial membrane vesicles (MV) has been largely overlooked in the pathogenesis of CD. In this study, we addressed the composition of the bacterial and MV composition in fecal samples of CD patients and compared this to the composition in healthy individuals. METHODS: Fecal samples from six healthy subjects (HC) in addition to twelve CD patients (six active, six remission) were analyzed in this study. Fecal bacterial membrane vesicles (fMVs) were isolated by a combination of ultrafiltration and size exclusion chromatography. DNA was obtained from the fMV fraction, the pellet of dissolved feces as bacterial DNA (bDNA), or directly from feces as fecal DNA (fDNA). The fMVs were characterized by nanoparticle tracking analysis and cryo-electron microscopy. Amplicon sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess microbial composition of all fractions. RESULTS: Beta-diversity analysis showed that the microbial community structure of the fMVs was significantly different from the microbial profiles of the fDNA and bDNA. However, no differences were observed in microbial composition between fDNA and bDNA. The microbial richness of fMVs was significantly decreased in CD patients compared to HC, and even lower in active patients. Profiling of fDNA and bDNA demonstrated that Firmicutes was the most dominant phylum in these fractions, while in fMVs Bacteroidetes was dominant. In fMV, several families and genera belonging to Firmicutes and Proteobacteria were significantly altered in CD patients when compared to HC. CONCLUSION: The microbial alterations of MVs in CD patients particularly in Firmicutes and Proteobacteria suggest a possible role of MVs in host-microbe symbiosis and induction or progression of inflammation in CD pathogenesis. Yet, the exact role for these fMV in the pathogenesis of the disease needs to be elucidated in future studies.

Non-steroidal anti-inflammatory drugs increase urinary neutrophil gelatinase-associated lipocalin in recreational runners.

OBJECTIVES: To study the effects of running with/without the use of pain killers on urinary neutrophil gelatinase-associated lipocalin (uNGAL) and other parameters of kidney function in recreational runners. METHODS: Participants of the 10- and 21.1-km Weir Venloop race were enrolled and their urine samples collected before and after the run. Urine dipstick and other conventional tests used to assess kidney function were performed. The presence of ibuprofen, diclofenac, naproxen, and/or paracetamol was assessed by LC-MS/MS. uNGAL was measured with a two-step chemiluminescent immunoassay. RESULTS: NSAIDs/analgesics were detected in urine of 5 (14.4%) 10-km runners and 13 (28.9%) 21.1-km runners. Only half-marathon participants showed significant increases in uNGAL (pre: 11.7 [7.1-34.3] ng/mL; post: 33.4 [17.4-50.4] ng/mL; P = .0038). There was a significant effect of NSAID/analgesic use on uNGAL increase (F2, 76  = 4.210, P = .004). Post hoc tests revealed that uNGAL increased significantly in runners who tested positive for ibuprofen/naproxen compared to runners who did not use any medications (P = .045) or those who tested positive for paracetamol (P = .033). Running distance had a significant influence on the increase in uNGAL (F1, 53  = 4.741, P < .05), specific gravity (F1, 60  = 9.231, P < .01), urinary creatinine (F1, 61  = 10.574, P < .01), albumin (F1, 59  = 4.888, P < .05), and development of hematuria (χ2 (4) = 18.44, P = .001). CONCLUSIONS: Running distance and use of ibuprofen/naproxen were identified as risk factors for uNGAL increase in recreational runners.

Cell Type- and Exposure-Specific Modulation of CD63/CD81-Positive and Tissue Factor-Positive Extracellular Vesicle Release in response to Respiratory Toxicants.

Chronic exposure to respiratory stressors increases the risk for pulmonary and cardiovascular diseases. Previously, we have shown that cigarette smoke extract (CSE) triggers the release of CD63+CD81+ and tissue factor (TF)+ procoagulant extracellular vesicles (EVs) by bronchial epithelial cells via depletion of cell surface thiols. Here, we hypothesized that this represents a universal response for different pulmonary cell types and respiratory exposures. Using bead-based flow cytometry, we found that bronchial epithelial cells and pulmonary fibroblasts, but not pulmonary microvascular endothelial cells or macrophages, release CD63+CD81+ and TF+ EVs in response to CSE. Cell surface thiols decreased in all cell types upon CSE exposure, whereas depletion of cell surface thiols using bacitracin only triggered EV release by epithelial cells and fibroblasts. The thiol-antioxidant NAC prevented the EV induction by CSE in epithelial cells and fibroblasts. Exposure of epithelial cells to occupational silica nanoparticles and particulate matter (PM) from outdoor air pollution also enhanced EV release. Cell surface thiols were mildly decreased and NAC partly prevented the EV induction for PM10, but not for silica and PM2.5. Taken together, induction of procoagulant EVs is a cell type-specific response to CSE. Moreover, induction of CD63+CD81+ and TF+ EVs in bronchial epithelial cells appears to be a universal response to various respiratory stressors. TF+ EVs may serve as biomarkers of exposure and/or risk in response to respiratory exposures and may help to guide preventive treatment decisions.

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles.

Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease.

Extracellular vesicles (EV) are secreted signaling entities that enhance various pathological processes when released in response to cellular stresses. Respiratory exposures such as cigarette smoke and air pollution exert cellular stresses and are associated with an increased risk of several chronic diseases. The aim of this review was to examine the evidence that modifications in EV contribute to respiratory exposure-associated diseases. Publications were searched using PubMed and Google Scholar with the search terms (cigarette smoke OR tobacco smoke OR air pollution OR particulate matter) AND (extracellular vesicles OR exosomes OR microvesicles OR microparticles OR ectosomes). All original research articles were included and reviewed. Fifty articles were identified, most of which investigated the effect of respiratory exposures on EV release in vitro (25) and/or on circulating EV in human plasma (24). The majority of studies based their main observations on the relatively insensitive scatter-based flow cytometry of EV (29). EV induced by respiratory exposures were found to modulate inflammation (19), thrombosis (13), endothelial dysfunction (11), tissue remodeling (6), and angiogenesis (3). By influencing these processes, EV may play a key role in the development of cardiovascular diseases and chronic obstructive pulmonary disease and possibly lung cancer and allergic asthma. The current findings warrant additional research with improved methodologies to evaluate the contribution of respiratory exposure-induced EV to disease etiology, as well as their potential as biomarkers of exposure or risk and as novel targets for preventive or therapeutic strategies.

Redox-dependent thiol modifications: implications for the release of extracellular vesicles.

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell's redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

BACKGROUND: During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. RESULTS: Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. CONCLUSIONS: Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells.

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.

Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection.

Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected.

Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections.

Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants.

INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment.

Viral-bacterial interactions in the respiratory tract.

In the respiratory tract, viruses and bacteria can interact on multiple levels. It is well known that respiratory viruses, particularly influenza viruses, increase the susceptibility to secondary bacterial infections. Numerous mechanisms, including compromised physical and immunological barriers, and changes in the microenvironment have hereby been shown to contribute to the development of secondary bacterial infections. In contrast, our understanding of how bacteria shape a response to subsequent viral infection is still limited. There is emerging evidence that persistent infection (or colonization) of the lower respiratory tract (LRT) with potential pathogenic bacteria, as observed in diseases like chronic obstructive pulmonary disease or cystic fibrosis, modulates subsequent viral infections by increasing viral entry receptors and modulating the inflammatory response. Moreover, recent studies suggest that even healthy lungs are not, as had long been assumed, sterile. The composition of the lung microbiome may thus modulate responses to viral infections. Here we summarize the current knowledge on the co-pathogenesis between viruses and bacteria in LRT infections.

Exposure to common respiratory bacteria alters the airway epithelial response to subsequent viral infection.

BACKGROUND: Colonization of the airways with potential pathogenic bacteria is observed in a number of chronic respiratory diseases, such as COPD or cystic fibrosis. Infections with respiratory viruses are known triggers of exacerbations of these diseases. We here investigated if pre-exposure to bacteria alters the response of lung epithelial cells to subsequent viral infection. METHODS: Bronchial epithelial cells (BEAS-2B cells and primary bronchial epithelial cells) were exposed to heat-inactivated Haemophilus influenzae, Pseudomonas aeruginosa or Streptococcus pneumoniae and subsequently infected with respiratory syncytial virus (RSV), type 2 human adenovirus or influenza B. Levels of pro-inflammatory cytokines, viral replication and expression of pattern recognition receptors were determined in culture supernatants and/or cell lysates. RESULTS: Exposure of BEAS-2B cells to H. influenzae before and during RSV-infection synergistically increased the release of IL-6 (increase above calculated additive effect at 72 h: 56 % ± 3 %, mean ± SEM) and IL-8 (53 % ± 12 %). This effect was sustained even when bacteria were washed away before viral infection and was neither associated with enhanced viral replication, nor linked to increased expression of key pattern recognition receptors. P. aeruginosa enhanced the release of inflammatory cytokines to a similar extent, yet only if bacteria were also present during viral infection. S. pneumoniae did not enhance RSV-induced cytokine release. Surprisingly, adenovirus infection significantly reduced IL-6 release in cells exposed to either of the three tested bacterial strains by on average more than 50 %. Infection with influenza B on the other hand did not affect cytokine production in BEAS-2B cells exposed to the different bacterial strains. CONCLUSION: Pre-exposure of epithelial cells to bacteria alters the response to subsequent viral infection depending on the types of pathogen involved. These findings highlight the complexity of microbiome interactions in the airways, possibly contributing to the susceptibility to exacerbations and the natural course of airway diseases.

Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells.

Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (ß-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells.

Haemophilus influenzae increases the susceptibility and inflammatory response of airway epithelial cells to viral infections.

Nontypeable Haemophilus influenzae (NTHI), a common colonizer of lungs of patients with chronic obstructive pulmonary disease (COPD), can enhance expression of the cellular receptor intercellular adhesion molecule 1 (ICAM-1), which in turn can be used by major group human rhinoviruses (HRVs) for attachment. Here, we evaluated the effect of NTHI-induced up-regulation of ICAM-1 on viral replication and inflammatory responses toward different respiratory viruses. Therefore, human bronchial epithelial cells were pretreated with heat-inactivated NTHI (hi-NTHI) and subsequently infected with either HRV16 (major group), HRV1B (minor group), or respiratory syncytial virus (RSV). Pretreatment with hi-NTHI significantly up-regulated ICAM-1 in BEAS-2B cells and primary bronchial epithelial cells. Concomitantly, release of infectious HRV16 particles was increased in cells pretreated with hi-NTHI. Pretreatment with hi-NTHI also caused a significant increase in HRV16 RNA, whereas replication of HRV1B and RSV were increased to a far lesser extent and only at later time points. Interestingly, release of IL-6 and IL-8 after RSV, but not HRV, infection was synergistically increased in hi-NTHI-pretreated BEAS-2B cells. In summary, exposure to hi-NTHI significantly enhanced sensitivity toward HRV16 but not HRV1B or RSV, probably through ICAM-1 up-regulation. Furthermore, hi-NTHI pretreatment may enhance the inflammatory response to RSV infection, suggesting that preexisting bacterial infections might exaggerate inflammation during secondary viral infection.

Efficacy of IFN-λ1 to protect human airway epithelial cells against human rhinovirus 1B infection.

Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-λ1 and compared it with the previously established protecting effect of IFN-ß. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-ß (500 IU/ml) or IFN-λ1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-λ1 and IFN-ß treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-ß induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-λ1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-λ1 is comparable to IFN-ß. Yet, the long-lasting induction of ISGs by IFN-λ1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-ß.

The host immune response contributes to Haemophilus influenzae virulence.

BACKGROUND: There is compelling evidence that infections with non-typeable Haemophilus influenzae (NTHi) are associated with exacerbations in COPD patients. However, NTHi has also been isolated frequently during clinically stable disease. In this study we tested the hypothesis that genetically distinct NTHi isolates obtained from COPD patients differ in virulence which could account for dissimilarities in the final outcome of an infection (stable vs. exacerbation). RESULTS: NTHi isolates (n = 32) were obtained from stable COPD patients, or during exacerbations. Genetically divergent NTHi isolates were selected and induction of inflammation was assessed as an indicator of virulence using different in vitro models. Despite marked genomic differences among NTHi isolates, in vitro studies could not distinguish between NTHi isolates based on their inflammatory capacities. Alternatively, when using a whole blood assay results demonstrated marked inter-, but not intra-individual differences in cytokine release between healthy volunteers irrespective of the origin of the NTHi isolate used. CONCLUSION: Results suggest that the individual immune reactivity might be an important predictor for the clinical outcome (exacerbation vs. no exacerbation) following NTHi infection.

Macrophage microRNA-155 promotes cardiac hypertrophy and failure.

BACKGROUND: Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. METHODS AND RESULTS: Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. CONCLUSIONS: Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.