Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
The Health Information Technology for Economic and Clinical Health Act of 2009 and the Centers for Medicare & Medicaid Services “meaningful use” incentive programs, in tandem with the boundless additional requirements for detailed reporting of quality metrics, have galvanized hospital efforts to implement hospital-based electronic health records. As such, emergency department information systems (EDISs) are an important and unique component of most hospitals’ electronic health records. System functionality varies greatly and affects physician decisionmaking, clinician workflow, communication, and, ultimately, the overall quality of care and patient safety. This article is a joint effort by members of the Quality Improvement and Patient Safety Section and the Informatics Section of the American College of Emergency Physicians. The aim of this effort is to examine the benefits and potential threats to quality and patient safety that could result from the choice of a particular EDIS, its implementation and optimization, and the hospital’s or physician group’s approach to continuous improvement of the EDIS. Specifically, we explored the following areas of potential EDIS safety concerns: communication failure, wrong order–wrong patient errors, poor data display, and alert fatigue. Case studies are presented that illustrate the potential harm that could befall patients from an inferior EDIS product or suboptimal execution of such a product in the clinical environment. The authors have developed 7 recommendations to improve patient safety with respect to the deployment of EDISs. These include ensuring that emergency providers actively participate in selection of the EDIS product, in the design of processes related to EDIS implementation and optimization, and in the monitoring of the system’s ongoing success or failure. Our recommendations apply to emergency departments using any type of EDIS: custom-developed systems, best-of-breed vendor systems, or enterprise systems.
Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.
The importance of NF-κB activation and deficient anti-viral interferon induction in the pathogenesis of rhinovirus-induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF-κB p65 nuclear expression, NF-κB p65 DNA binding in lung tissue and NF-κB-regulated airway inflammation. In vitro inhibition of NF-κB reduced rhinovirus-induced pro-inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus-infected p65-deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF-κB p65 is required for pro-inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1−/− mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF-κB p65 (to suppress inflammation but preserve anti-viral immunity) and type I IFN signalling (to enhance deficient anti-viral immunity) to treat rhinovirus-induced exacerbations of airway diseases.
asthma; inflammation; interferon; NF-kappaB; rhinovirus
Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.
Rhinoviral infection is an important trigger of acute inflammatory exacerbations in patients with underlying airway disease. We have previously established that interleukin-1β (IL-1β) is central in the communication between epithelial cells and monocytes during the initiation of inflammation. In this study we explored the roles of IL-1β and its signaling pathways in the responses of airway cells to rhinovirus-1B (RV-1B) and further determined how responses to RV-1B were modified in a model of bacterial coinfection. Our results revealed that IL-1β dramatically potentiated RV-1B-induced proinflammatory responses, and while monocytes did not directly amplify responses to RV-1B alone, they played an important role in the responses observed with our coinfection model. MyD88 is the essential signaling adapter for IL-1β and most Toll-like receptors. To examine the role of MyD88 in more detail, we created stable MyD88 knockdown epithelial cells using short hairpin RNA (shRNA) targeted to MyD88. We determined that IL-1β/MyD88 plays a role in regulating RV-1B replication and the inflammatory response to viral infection of airway cells. These results identify central roles for IL-1β and its signaling pathways in the production of CXCL8, a potent neutrophil chemoattractant, in viral infection. Thus, IL-1β is a viable target for controlling the neutrophilia that is often found in inflammatory airway disease and is exacerbated by viral infection of the airways.
Summary: The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.
Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8+ T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8+ T cells, we demonstrated that RSV-infected BECs increased CD8+ T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8+ T cells enhanced CD8+ T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.
Rationale: Respiratory virus infections are associated with chronic obstructive pulmonary disease (COPD) exacerbations, but a causative relationship has not been proven. Studies of naturally occurring exacerbations are difficult and the mechanisms linking virus infection to exacerbations are poorly understood. We hypothesized that experimental rhinovirus infection in subjects with COPD would reproduce the features of naturally occurring COPD exacerbations and is a valid model of COPD exacerbations.
Objectives: To evaluate experimental rhinovirus infection as a model of COPD exacerbation and to investigate the mechanisms of virus-induced exacerbations.
Methods: We used experimental rhinovirus infection in 13 subjects with COPD and 13 nonobstructed control subjects to investigate clinical, physiologic, pathologic, and antiviral responses and relationships between virus load and these outcomes.
Measurements and Main Results: Clinical data; inflammatory mediators in blood, sputum, and bronchoalveolar lavage; and viral load in nasal lavage, sputum, and bronchoalveolar lavage were measured at baseline and after infection with rhinovirus 16. After rhinovirus infection subjects with COPD developed lower respiratory symptoms, airflow obstruction, and systemic and airway inflammation that were greater and more prolonged compared with the control group. Neutrophil markers in sputum related to clinical outcomes and virus load correlated with inflammatory markers. Virus load was higher and IFN production by bronchoalveolar lavage cells was impaired in the subjects with COPD.
Conclusions: We have developed a new model of COPD exacerbation that strongly supports a causal relationship between rhinovirus infection and COPD exacerbations. Impaired IFN production and neutrophilic inflammation may be important mechanisms in virus-induced COPD exacerbations.
disease exacerbation; respiratory tract infections; COPD; rhinovirus
Rhinovirus infections are the major cause of asthma exacerbations. We hypothesised that IL-15, a cytokine implicated in innate and acquired antiviral immunity, may be deficient in asthma and important in the pathogenesis of asthma exacerbations. We investigated regulation of IL-15 induction by rhinovirus in human macrophages in vitro, IL-15 levels in bronchoalveolar lavage (BAL) fluid and IL-15 induction by rhinovirus in BAL macrophages from asthmatic and control subjects, and related these to outcomes of infection in vivo. Rhinovirus induced IL-15 in macrophages was replication-, NF-κB- and α/β interferon-dependent. BAL macrophage IL-15 induction by rhinovirus was impaired in asthmatics and inversely related to lower respiratory symptom severity during experimental rhinovirus infection. IL-15 levels in BAL fluid were also decreased in asthmatics and inversely related with airway hyperresponsiveness and with virus load during in vivo rhinovirus infection. Deficient IL-15 production in asthma may be important in the pathogenesis of asthma exacerbations.
We previously reported deficiency in interferon production in asthma, which correlated with disease severity and viral load during experimental rhinovirus infection. Here we show that macrophages produce IL-15 upon rhinovirus infection and that IFN-β plays an important role in IL-15 production. In asthmatic subjects, there is a deficiency in rhinovirus-induced production of IL-15 by macrophages, which indicates immunodeficiency in asthma is surprisingly broad, also involving IL-15, an important cytokine that bridges innate and acquired immunity. These results show that IFN-β therapy in asthma exacerbations could be effective not only due to direct anti-viral effects of IFN-β, but also by inducing IL-15 production. We also show induction of IFN-β and IL-15 to be NF-kB dependent, an important finding which has implications for NF-kB inhibitor drug development programmes as these drugs have potential to worsen rather than improve asthma exacerbation severity, by further enhancing deficiencies of IL-15 and IFN-β. This study investigating the role of IL-15 in rhinovirus infection and asthma has also major implications in other diseases, for example pandemic influenza, where asthma is a major risk factor for severe disease and death, and COPD and cystic fibrosis where IFN-β deficiency is also present.
allergy; asthma; interferon; mouse model; Th1/Th2
The relative roles of the endosomal TLR3/7/8 versus the intracellular RNA helicases RIG-I and MDA5 in viral infection is much debated. We investigated the roles of each pattern recognition receptor in rhinovirus infection using primary bronchial epithelial cells. TLR3 was constitutively expressed; however, RIG-I and MDA5 were inducible by 8–12 h following rhinovirus infection. Bronchial epithelial tissue from normal volunteers challenged with rhinovirus in vivo exhibited low levels of RIG-I and MDA5 that were increased at day 4 post infection. Inhibition of TLR3, RIG-I and MDA5 by siRNA reduced innate cytokine mRNA, and increased rhinovirus replication. Inhibition of TLR3 and TRIF using siRNA reduced rhinovirus induced RNA helicases. Furthermore, IFNAR1 deficient mice exhibited RIG-I and MDA5 induction early during RV1B infection in an interferon independent manner. Hence anti-viral defense within bronchial epithelium requires co-ordinated recognition of rhinovirus infection, initially via TLR3/TRIF and later via inducible RNA helicases.
Host-pathogen interactions are mediated by pattern recognition receptors that identify conserved structures of micro-organisms that are distinct from self. During a viral infection, important pattern recognition receptors include the endosomal Toll-like receptors (TLRs), and a second set of cytoplasmic pattern recognition receptors known as the RNA helicases. Many studies have highlighted the importance of TLR3, TLR7/8 and the RNA helicases in providing robust anti-viral immunity via interferon induction and inflammation. Both endosomal TLR and cytoplasmic RNA helicase mediated pathways are believed to exist as separate yet non-redundant entities; however, little thought is given to why both systems exist, and few studies also consider how both pathways together contribute to anti-viral immunity. Using models of rhinovirus infection in primary bronchial epithelial cell culture in vitro and experimental infection in mouse and human models in vivo, we show that the RNA helicases are preferentially induced early in the infection cycle via TLR3 mediated signaling events, and work in a co-ordinated, systematic manner. The results help understand the complex events that determine effective innate immunity to rhinovirus infection and how these processes contribute to virus induced exacerbations of asthma and chronic obstructive pulmonary disease.
Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication in macrophages has not been reported. Tumor necrosis factor alpha (TNF-α) is implicated in the pathogenesis of acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. We hypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocation of NF-κB, leading to TNF-α production. Using macrophages derived from the human monocytic cell line THP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive in THP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived but deficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cells supported complete virus replication. We demonstrated RV-induced activation of NF-κB and colocalization of p65/NF-κB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-α release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely (THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we established the requirement for NF-κB but not p38 mitogen-activated protein kinase in induction of TNF-α. These data suggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated in the pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-κB as a promising target for development of new therapeutic intervention strategies.
Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.
Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K+ concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K+ concentrations and was inhibited when K+ and/or Cl− was absent from the medium. The results suggested that the well-documented Na+-K+-2Cl− cotransport system was responsible for the K+ influx activated during the RVI. However, inhibitors of Na+-K+-2Cl− cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K+ influx in T. foetus.