scholarly journals Loss of versican and production of hyaluronan in lung epithelial cells are associated with airway inflammation during RSV infection

2020 ◽  
pp. jbc.RA120.016196
Author(s):  
Gerald G. Kellar ◽  
Kaitlyn A. Barrow ◽  
Lucille M. Rich ◽  
Jason S. Debley ◽  
Thomas N. Wight ◽  
...  
Keyword(s):  
Airway Inflammation ◽  
Ex Vivo ◽  
Lung Epithelial Cells ◽  
In Vivo Studies ◽  
Critical Feature ◽  
Human Lung Fibroblast ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix (ECM) changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however, whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production, we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) co-culture model. RSV infection of BEC/HLF co-cultures led to decreased hyaluronidase expression by HLFs, increased accumulation of HA, and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however, BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared to control mice which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared to SPC-Cre(-) RSV-infected littermates. Taken together, these data demonstrate that altered ECM accumulation of HA occurs following RSV infection and may contribute to airway inflammation. Additionally, loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican’s role in inflammatory regulation is complex and dependent on the microenvironment.

scholarly journals Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection

2020 ◽  
Vol 11 ◽  
Author(s):  
Sandra Axberg Pålsson ◽  
Aleksandra Dondalska ◽  
Joseph Bergenstråhle ◽  
Caroline Rolfes ◽  
Albin Björk ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Activity ◽  
Antiviral Treatment ◽  
Lung Epithelial Cells ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections ◽  
Single Stranded Oligonucleotide

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON’s antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.

scholarly journals RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration

2021 ◽  
Author(s):  
Debra T Linfield ◽  
Nannan Gao ◽  
Andjela Raduka ◽  
Terri J Harford ◽  
Giovanni Piedimonte ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Wound Repair ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

The airway epithelium's ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract's normal functions. Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions (FA). Inhibition of Rho kinase (ROCK), a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain (pMLC2) following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed re-epithelialization. There were increased concentrations of pMLC2 in day 7 naphthalene plus RSV animals which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

scholarly journals Alveolar Macrophages Can Control Respiratory Syncytial Virus Infection in the Absence of Type I Interferons

2016 ◽  
Vol 8 (5) ◽  
pp. 452-463 ◽  
Author(s):  
Spyridon Makris ◽  
Monika Bajorek ◽  
Fiona J. Culley ◽  
Michelle Goritzka ◽  
Cecilia Johansson
Keyword(s):  
Respiratory Syncytial Virus ◽  
Viral Replication ◽  
Alveolar Macrophages ◽  
Ex Vivo ◽  
Type I Interferons ◽  
Type I ◽  
Proinflammatory Mediators ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections. Immunity to RSV is initiated upon detection of the virus by pattern recognition receptors, such as RIG-I-like receptors. RIG-I-like receptors signal via MAVS to induce the synthesis of proinflammatory mediators, including type I interferons (IFNs), which trigger and shape antiviral responses and protect cells from infection. Alveolar macrophages (AMs) are amongst the first cells to encounter invading viruses and the ones producing type I IFNs. However, it is unclear whether IFNs act to prevent AMs from serving as vehicles for viral replication. In this study, primary AMs from MAVS (Mavs-/-)- or type I IFN receptor (Ifnar1-/-)-deficient mice were exposed to RSV ex vivo. Wild-type (wt) AMs but not Mavs-/- and Ifnar1-/- AMs produced inflammatory mediators in response to RSV. Furthermore, Mavs-/- and Ifnar1-/- AMs accumulated more RSV proteins than wt AMs, but the infection was abortive. Thus, RIG-I-like receptor-MAVS and IFNAR signalling are important for the induction of proinflammatory mediators from AMs upon RSV infection, but this signalling is not central for controlling viral replication. The ability to restrict viral replication makes AMs ideal sensors of RSV infection and important initiators of immune responses in the lung.

scholarly journals BRD4 Couples NF-κB/RelA with Airway Inflammation and the IRF-RIG-I Amplification Loop in Respiratory Syncytial Virus Infection

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Bing Tian ◽  
Jun Yang ◽  
Yingxin Zhao ◽  
Teodora Ivanciuc ◽  
Hong Sun ◽  
...  
Keyword(s):  
Airway Inflammation ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Innate Response ◽  
Pol Ii ◽  
Rsv Infection ◽  
Amplification Loop ◽  
Syncytial Virus ◽  
Brd4 Inhibition

ABSTRACT The airway mucosa expresses protective interferon (IFN) and inflammatory cytokines in response to respiratory syncytial virus (RSV) infection. In this study, we examine the role of bromodomain containing 4 (BRD4) in mediating this innate immune response in human small airway epithelial cells. We observe that RSV induces BRD4 to complex with NF-κB/RelA. BRD4 is functionally required for expression of the NF-κB-dependent inflammatory gene regulatory network (GRN), including the IFN response factor 1 (IRF1) and IRF7, which mediate a cross talk pathway for RIG-I upregulation. Mechanistically, BRD4 is required for cyclin-dependent kinase 9 (CDK9) recruitment and phospho-Ser 2 carboxy-terminal domain (CTD) RNA polymerase (Pol) II formation on the promoters of IRF1, IRF7, and RIG-I, producing their enhanced expression by transcriptional elongation. We also find that BRD4 independently regulates CDK9/phospho-Ser 2 CTD RNA Pol II recruitment to the IRF3-dependent IFN-stimulated genes (ISGs). In vivo, poly(I·C)-induced neutrophilia and mucosal chemokine production are blocked by a small-molecule BRD4 bromodomain inhibitor. Similarly, BRD4 inhibition reduces RSV-induced neutrophilia, mucosal CXC chemokine expression, activation of the IRF7-RIG-I autoamplification loop, mucosal IFN expression, and airway obstruction. RSV infection activates BRD4 acetyltransferase activity on histone H3 Lys (K) 122, demonstrating that RSV infection activates BRD4 in vivo. These data validate BRD4 as a major effector of RSV-induced inflammation and disease. BRD4 is required for coupling NF-κB to expression of inflammatory genes and the IRF-RIG-I autoamplification pathway and independently facilitates antiviral ISG expression. BRD4 inhibition may be a strategy to reduce exuberant virus-induced mucosal airway inflammation. IMPORTANCE In the United States, 2.1 million children annually require medical attention for RSV infections. A first line of defense is the expression of the innate gene network by infected epithelial cells. Expression of the innate response requires the recruitment of transcriptional elongation factors to rapidly induce innate response genes through an unknown mechanism. We discovered that RSV infection induces a complex of bromodomain containing 4 (BRD4) with NF-κB and cyclin-dependent kinase 9 (CDK9). BRD4 is required for stable CDK9 binding, phospho-Ser 2 RNA Pol II formation, and histone acetyltransferase activity. Inhibition of BRD4 blocks Toll-like receptor 3 (TLR3)-dependent neutrophilia and RSV-induced inflammation, demonstrating its importance in the mucosal innate response in vivo. Our study shows that BRD4 plays a central role in inflammation and activation of the IRF7-RIG-I amplification loop vital for mucosal interferon expression. BRD4 inhibition may be a strategy for modulating exuberant mucosal airway inflammation.

scholarly journals Development of surface engineered antigenic exosomes as vaccines for respiratory syncytial virus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suyeon Hong ◽  
Shaobo Ruan ◽  
Zachary Greenberg ◽  
Mei He ◽  
Jodi L. McGill
Keyword(s):  
Respiratory Syncytial Virus ◽  
Immune Responses ◽  
Ex Vivo ◽  
Subcutaneous Administration ◽  
Antigenic Peptides ◽  
Vaccine Platform ◽  
Mhc I ◽  
Syncytial Virus ◽  
Tract Infections

AbstractRespiratory syncytial virus (RSV) is one of the main pathogens associated with lower respiratory tract infections in infants and young children worldwide. Exosomes secreted by antigen presenting cells (APCs) can elicit immune responses by carrying major histocompatibility complex (MHC) class I molecules complexed with antigenic peptides and other co-stimulating factors. Therefore, we developed novel immunomagnetic nanographene particles to sequentially isolate, surface engineer, and release intact dendritic cell (DC) exosomes for use as a potential vaccine platform against RSV. The H-2Db-restricted, immunodominant peptides from RSV (M187–195 and NS161–75) were introduced to MHC-I on DC-derived exosomes to express peptide/MHC-I (pMHC-I) complexes. A mouse model of RSV infection was used to define the immunogenicity of surface engineered exosomes for activating virus-specific immune responses. Ex vivo assays demonstrated that engineered exosomes carrying RSV-specific peptides can elicit interferon-gamma (IFN-γ) production by virus-specific CD8+ T cells isolated from RSV-infected C57BL/6 mice. In vivo assays demonstrated that subcutaneous administration of both M187–195 and NS161–75 engineered exosomes to mice, with or without additional adjuvant, appeared safe and well tolerated, however, did not prime antigen-specific CD8+ T cell responses. Surface engineered exosomes are immunogenic and promising for further development as a vaccine platform.

scholarly journals Neuromedin U Induces Pulmonary Group 2 Innate Lymphoid Cell Activation via NMUR1 Pathway During Acute Respiratory Syncytial Virus Infection

2021 ◽  
Author(s):  
Weiwei LIU ◽  
Si Wang ◽  
Jia Wang ◽  
Rui Zheng ◽  
Dalu Wang ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Airway Inflammation ◽  
Cell Activation ◽  
Regulatory Effect ◽  
Key Factor ◽  
Rsv Infection ◽  
Group 2 ◽  
Syncytial Virus ◽  
Underlying Mechanisms

Abstract Background：The activated group 2 innate lymphocytes (ILC2s) play a crucial role in respiratory syncytial virus (RSV)-induced airway inflammation by secreting large amount of type 2 cytokines. Although the classical activator IL-33 is the key factor for ILC2 activation, a regulatory effect of neurotransmitter-neuromedin U (NMU) has also been reported. However, whether and how NMU can be elicited by RSV infection and regulate pulmonary ILC2 activation remains unclear.Methods： The regulatory effect and underlying mechanisms of NMU on ILC2 activation were determined by using RSV-infected wild-type and NMU-knockout mice. The expression of NMU in the lungs and NMUR1 on ILC2s were measured by Real-time PCR and Western blot. Flow cytometry and ELISA were used to detect the proliferation and activation of ILC2s. The type of neurons secreting NMU and its possible secretion mechanism during RSV infection were also analyzed.Results：Acute RSV infection induced the production of NMU in the lungs of mice and up-regulate the expression of NMUR1 on the pulmonary ILC2s. In vivo administration of NMU exacerbated RSV-induced airway inflammation by promoting the proliferation and activation of pulmonary ILC2s via NMUR1 pathway. In this process, PI3K, MEK and NFAT signal proteins might be involved. Furthermore, pulmonary neurons responded to the stimulation of RSV and secreted NMU in TLR4 and TLR7-dependent manners.Conclusion：Our data suggest that NMU is an ILC2 activator other than the classical activator, revealing a novel effect of neurotransmitter on RSV-induced airway inflammation.

scholarly journals 1447. Ex Vivo Human Bladder Tissue Model to Evaluate Lactobacillus-Containing Formulations as Preventative Treatment Against Common Urogenital Pathogens

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S528-S528
Author(s):  
Danielle Nicklas ◽  
Patrick Finnegan ◽  
Zach Siler ◽  
Marnie Peterson ◽  
Shantha Sarangapani
Keyword(s):  
Bacterial Infections ◽  
Ex Vivo ◽  
Multidrug Resistant ◽  
In Vivo Studies ◽  
Antimicrobial Efficacy ◽  
Preventative Treatment ◽  
Human Bladder ◽  
Bladder Tissue ◽  
Tract Infections

Abstract Background Urinary tract infections (UTIs) are common bacterial infections in adults, and catheter-associated UTIs are the most common nosocomial infection. The rise of multidrug-resistant organisms and an increased focus on antibiotic stewardship has influenced the development of novel treatments against such infections, and there is growing interest in the use of probiotics for antimicrobial therapy. We used an ex vivo human bladder tissue (HBT) model to evaluate the antimicrobial efficacy and biocompatibility of lactobacillus-based developmental formulations (created and supplied by ICET, Inc.) for preventative treatment against common UTI pathogens. Methods To assess antimicrobial efficacy, lactobacillus-based formulations (live and attenuated) were spiked with five prevalent UTI organisms (5 × 103 CFU/mL). Ex vivo HBT explants were treated with 300 μL of spiked formulation for 6 and 24 h at 37°C, then processed and plated on selective agars. Biocompatibility studies assessed ex vivo HBT tissue viability and inflammatory response (IL-8) to lactobacillus-containing formulations with MTT assay and ELISA at 2 h post-treatment. Results At 6 h, live lactobacillus-containing formulations (29–124, 29-124C) were bacteriostatic (90.00–99.89% log CFU/mL reduction) against Escherichia coli and Klebsiella pneumoniae and bactericidal (≥99.90% log CFU/mL reduction) against Candida albicans, Enterococcus faecalis, and Proteus mirabilis. By 24 h, live formulations were bactericidal against all five organisms tested. Attenuated formulation 29–125 achieved bacteriostatic efficacy against E. coli, K. pneumoniae, and P. mirabilis and bactericidal efficacy against C. albicans and E. faecalis at 24 h. Biocompatibility assessments following 2 h exposure to lactobacillus-based formulations revealed exposed explants were fully viable, with no significant changes in IL-8 production compared with PBS-treated controls. Conclusion This study suggests lactobacillus-based formulations are effective and safe options for UTI prevention. While this static ex vivo human bladder mucosalmodel does not fully replicate the dynamic and diluting conditions that occur in vivo, we anticipate that our findings will be confirmed by future in vivo studies. Disclosures All authors: No reported disclosures.

scholarly journals IκB Kinase Is a Critical Regulator of Chemokine Expression and Lung Inflammation in Respiratory Syncytial Virus Infection

2004 ◽  
Vol 78 (5) ◽  
pp. 2232-2241 ◽  
Author(s):  
Helene A. Haeberle ◽  
Antonella Casola ◽  
Zoran Gatalica ◽  
Sharon Petronella ◽  
Hans-Juergen Dieterich ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Airway Inflammation ◽  
Critical Role ◽  
Regulatory Protein ◽  
Mucosal Inflammation ◽  
Specific Cell ◽  
Iκb Kinase ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACT Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IκB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IκBα, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-κB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKβ with the regulatory protein NEMO showed a striking reduction of lung NF-κB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKβ may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.

Ex Vivo Inhibition of Platelet Aggregation in Patients with Severe Limb Arteriopathy Treated with BAY U3405, a Specific IX A2 Receptor Antagonist

1994 ◽  
Vol 72 (05) ◽  
pp. 659-662 ◽  
Author(s):  
S Bellucci ◽  
W Kedra ◽  
H Groussin ◽  
N Jaillet ◽  
P Molho-Sabatier ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Randomized Study ◽  
In Vivo Studies ◽  
Walking Distance ◽  
Peripheral Blood Flow ◽  
Walking Ability ◽  
Double Blind ◽  
Platelet Factor

SummaryA double-blind, placebo-controlled randomized study with BAY U3405, a specific thromboxane A2 (TX A2) receptor blocker, was performed in patients suffering from severe stade II limb arteriopathy. BAY U3405 or placebo was administered in 16 patients at 20 mg four times a day (from day 1 to day 3). Hemostatic studies were done before therapy, and on day 2 and day 3 under therapy. On day 3, BAY U3405 was shown to induce a highly statistically significant decrease of the velocity and the intensity of the aggregations mediated by arachidonic acid (56 ± 37% for the velocity, 58 ± 26% for the intensity) or by U46619 endoperoxide analogue (36 ± 35% for the velocity, 37 ± 27% for the intensity). Similar results were already observed on day 2. By contrast, such a decrease was not noticed with ADP mediated platelet aggregation. Furthermore, plasma levels of betathrombo-globulin and platelet factor 4 remained unchanged. Peripheral hemodynamic parameters were also studied. The peripheral blood flow was measured using a Doppler ultrasound; the pain free walking distance and the total walking ability distance were determined under standardized conditions on a treadmill. These last two parameters show a trend to improvement which nevertheless was not statistically significant. All together these results encourage further in vivo studies using BAY U3405 or related compounds on a long-term administration.

Chemical Permeation Enhancers for Transdermal Delivery of Thiocolchicoside: Assessment of Ex-vivo Skin Flux and In-vivo Pharmacokinetics

2017 ◽  
Vol 10 (5) ◽  
pp. 3827-3835
Author(s):  
Y Madhusudan Rao ◽  
Gayatri P ◽  
Ajitha M ◽  
P. Pavan Kumar ◽  
Kiran kumar
Keyword(s):  
Passive Control ◽  
Ex Vivo ◽  
Skin Permeation ◽  
In Vivo Studies ◽  
Permeation Enhancers ◽  
Casting Technique ◽  
Chemical Permeation Enhancers ◽  
Chemical Permeation ◽  
In Vivo Pharmacokinetics

Present investigation comprises the study of ex-vivo skin flux and in-vivo pharmacokinetics of Thiocolchicoside (THC) from transdermal films. The films were fabricated by solvent casting technique employing combination of hydrophilic and hydrophobic polymers. A flux of 18.08 µg/cm2h and 13.37µg/cm2h was achieved for optimized formulations containing 1, 8-cineole and oleic acid respectively as permeation enhancers. The observed flux values were higher when compared to passive control (8.66 µg/cm2h). Highest skin permeation was observed when 1,8-cineole was used as chemical permeation enhancer and it considerably (2-2.5 fold) improved the THC transport across the rat skin. In vivo studies were performed in rabbits and samples were analysed by LC-MS-MS. The mean area under the curve (AUC) values of transdermal film showed about 2.35 times statistically significant (p<0.05) improvement in bioavailability when compared with the oral administration of THC solution. The developed transdermal therapeutic systems using chemical permeation enhancers were suitable for drugs like THC in effective management of muscular pain.    

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