Human Metapneumovirus Impairs Apoptosis of Nasal Epithelial Cells in Asthma via HSP70.
ABSTRACT: Asthmatics are highly susceptible to respiratory viral infections, possibly due to impaired innate immunity. However, the exact mechanisms of susceptibility are likely to differ amongst viruses. Therefore, we infected primary nasal epithelial cells (NECs) from adults with mild-to-moderate asthma, with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV) in vitro and investigated the antiviral response. NECs from these asthmatics supported elevated hMPV but not RSV infection, compared to non-asthmatic controls. This correlated with reduced apoptosis and reduced activation of caspase-9 and caspase-3/7 in response to hMPV, but not RSV. The expression of heat shock protein 70 (HSP70), a known inhibitor of caspase activation and subsequent apoptosis, was amplified in response to hMPV infection. Chemical inhibition of HSP70 function restored caspase activation and reduced hMPV infection in NECs from asthmatic subjects. There was no impairment in the production of IFN by NECs from asthmatics in response to either hMPV or RSV, demonstrating that increased infection of asthmatic airway cells by hMPV is IFN-independent. This study demonstrates, for the first time, a mechanism for elevated hMPV infection in airway epithelial cells from adult asthmatics and identifies HSP70 as a potential target for antiviral and asthma therapies.
Project description:BACKGROUND: Airway epithelial cells (AEC) from patients with asthma, appear to have an impaired interferon (IFN)-? and -? response to infection with rhinovirus. OBJECTIVES: To determine if impaired IFN responses can be identified in young children at risk of developing asthma due to atopy and/or early life wheeze, and if the site of infection or the infecting virus influence the antiviral response. METHODS: Nasal (N) and tracheal (T) epithelial cells (EC) were collected from children categorised with atopy and/or wheeze based on specific IgE to locally common aeroallergens and a questionnaire concerning respiratory health. Submerged primary cultures were infected with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV), and IFN production, inflammatory cytokine expression and viral replication quantified. RESULTS: Nasal epithelial cells (NEC), but not tracheal epithelial cells (TEC), from children with wheeze and/or atopy produced less IFN-?, but not IFN-?, in response to RSV infection; this was associated with higher viral shedding. However, IFN-regulated factors IRF-7, Mx-1 and CXCL-10, and inflammatory cytokines were not differentially regulated. NECs and TECs from children with wheeze and/or atopy demonstrated no impairment of the IFN response (? or ?) to hMPV infection. Despite this, more hMPV was shed from these cells. CONCLUSIONS: AECs from children with wheeze and/or atopy do not have an intrinsic defect in the production of IFN-? or -?, however, this response is influenced by the infecting virus. Higher viral load is associated with atopy and wheeze suggesting an impaired antiviral response to RSV and hMPV that is not influenced by production of IFNs.
Project description:Background: Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. Objective: To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro. Methods: Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450K microarray respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing was used to confirm gene expression and DNA methylation respectively. Results: Global methylation was increased in Asthmatics during infection. Disease status and virus infection influenced the methylation of 389 loci. Healthy and asthmatic NECs were characterized predominately by methylation profiles and patterns in loci that were not influenced by virus infection. However, both groups also exhibited distinct DNA methylation profiles in response to infection. Despite these differences, we found that the methylation of small nucleolar RNA, H/ACA box 12 (SNORA12) was common during infection. Further analysis indicated a relationship existed between SNORA12 DNA methylation and gene expression in response to infection. Conclusions: Findings from our study indicate that in addition to the well described phenotypic and genomic differences, the airway epithelium of asthmatics is characterized by a distinct methylome and that epigenetic mechanism may contribute to the development of virus-induced asthma exacerbations. Bisulphite converted DNA from matched mock and human rhinovirus-16 infected nasal epithelial cells from Healthy (n=3) and Asthmatics (n=6) adults were hybridized to the Illumina DNA methylation 450K Bead Array.
Project description:BACKGROUND:Severity of human metapneumovirus (hMPV) lower respiratory illness (LRTI) is considered similar to that observed for respiratory syncytial virus (RSV). However, differences in severity between both pathogens have been noted, suggesting degree of illness may vary in different populations. Moreover, a potential association between hMPV and asthma also suggests that hMPV may preferentially affect asthmatic subjects. METHODS:A population-based surveillance study in children <2 years admitted for severe LRTI in Argentina. Nasopharyngeal aspirates were tested by RT-PCR for hMPV, RSV, influenza A and human rhinovirus. RESULTS:Of 3,947 children, 383 (10%) were infected with hMPV. The hospitalization rate for hMPV LRTI was 2.26 per 1,000 children (95% CI, 2.04-2.49). Thirty-nine (10.2%) patients infected with hMPV experienced life-threatening disease (LTD) [0.23 per 1,000 children (95% CI 0.16-0.31/1,000)], and two died [infant mortality rate 0.024 per 1,000 (95% CI 0.003-0.086)]. In hMPV infected children birth to an asthmatic mother there was an increased risk for LTD (OR=4,72 95% CI, 1.39-16.01). We observed a specific interaction between maternal asthma and hMPV infection affecting risk for LTD. CONCLUSIONS:Maternal asthma increases the risk for LTD in children <2 years old hospitalized for severe hMPV LRTI.
Project description:Aims: Asthma, characterized by airway obstruction and hyper-responsiveness, is more severe and less responsive to treatment in obese subjects. While alterations in mitochondrial function and redox signaling have been implicated in asthma pathogenesis, it is unclear whether these mechanisms differ in lean versus obese asthmatics. In addition, we previously demonstrated that circulating platelets from asthmatic individuals have altered bioenergetics; however, it is unknown whether platelet mitochondrial changes reflect those observed in airway epithelial cells. Herein we hypothesized that lean and obese asthmatics show differential bioenergetics and redox signaling in airway cells and that these alterations could be measured in platelets from the same individual. Results: Using freshly isolated bronchial airway epithelial cells and platelets from lean and obese asthmatics and healthy individuals, we show that both cell types from obese asthmatics have significantly increased glycolysis, basal and maximal respiration, and oxidative stress compared with lean asthmatics and healthy controls. This increased respiration was associated with enhanced arginine metabolism by arginase, which has previously been shown to drive respiration. Inducible nitric oxide synthase (iNOS) was also upregulated in cells from all asthmatics. However, due to nitric oxide synthase uncoupling in obese asthmatics, overall nitric oxide (NO) bioavailability was decreased, preventing NO-dependent inhibition in obese asthmatic cells that was observed in lean asthmatics. Innovation and Conclusion: These data demonstrate bioenergetic differences between lean and obese asthmatics that are, in part, due to differences in NO signaling. They also suggest that the platelet may serve as a useful surrogate to understand redox, oxidative stress and bioenergetic changes in the asthmatic airway.
Project description:The airway epithelium forms the interface between the inhaled environment and the lung. The airway epithelium is dysfunctional in asthma and epigenetic mechanisms are considered a contributory factor. We hypothesised that the DNA methylation profiles of cultured primary airway epithelial cells (AECs) would differ between cells isolated from individuals with asthma (n = 17) versus those without asthma (n = 16). AECs were isolated from patients by two different isolation techniques; pronase digestion (9 non-asthmatic, 8 asthmatic) and bronchial brushings (7 non-asthmatic and 9 asthmatic). DNA methylation was assessed using an Illumina Infinium HumanMethylation450 BeadChip array. DNA methylation of AECs clustered by isolation technique and linear regression identified 111 CpG sites differentially methylated between isolation techniques in healthy individuals. As a consequence, the effect of asthmatic status on DNA methylation was assessed within AEC samples isolated using the same technique. In pronase isolated AECs, 15 DNA regions were differentially methylated between asthmatics and non-asthmatics. In bronchial brush isolated AECs, 849 differentially methylated DNA regions were identified with no overlap to pronase regions. In conclusion, regardless of cell isolation technique, differential DNA methylation was associated with asthmatic status in AECs, providing further evidence for aberrant DNA methylation as a signature of epithelial dysfunction in asthma.
Project description:BACKGROUND:Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES:To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS:Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS:We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS:Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.
Project description:Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis and asthma exacerbation.The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection.Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air-liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection.Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-?B. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology.These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.
Project description:BACKGROUND:Some investigators find a deficiency in IFN production from airway epithelial cells infected with human rhinovirus in asthma, but whether this abnormality occurs with other respiratory viruses is uncertain. OBJECTIVE:To assess the effect of influenza A virus (IAV) and respiratory syncytial virus (RSV) infection on IFN production and viral level in human bronchial epithelial cells (hBECs) from subjects with and without asthma. METHODS:Primary-culture hBECs from subjects with mild to severe asthma (n = 11) and controls without asthma (hBECs; n = 7) were infected with live or ultraviolet-inactivated IAV (WS/33 strain), RSV (Long strain), or RSV (A/2001/2-20 strain) with multiplicity of infection 0.01 to 1. Levels of virus along with IFN-? and IFN-? and IFN-stimulated gene expression (tracked by 2'-5'-oligoadenylate synthetase 1 and myxovirus (influenza virus) resistance 1 mRNA) were determined up to 72 hours postinoculation. RESULTS:After IAV infection, viral levels were increased 2-fold in hBECs from asthmatic subjects compared with nonasthmatic control subjects (P < .05) and this increase occurred in concert with increased IFN-?1 levels and no significant difference in IFNB1, 2'-5'-oligoadenylate synthetase 1, or myxovirus (influenza virus) resistance 1mRNA levels. After RSV infections, viral levels were not significantly increased in hBECs from asthmatic versus nonasthmatic subjects and the only significant difference between groups was a decrease in IFN-? levels (P < .05) that correlated with a decrease in viral titer. All these differences were found only at isolated time points and were not sustained throughout the 72-hour infection period. CONCLUSIONS:The results indicate that IAV and RSV control and IFN response to these viruses in airway epithelial cells is remarkably similar between subjects with and without asthma.
Project description:Asthma is a common disorder of the airways characterized by airway inflammation and by decline in lung function and airway remodeling in a subset of asthmatics. Airway remodeling is characterized by structural changes which include airway smooth muscle hypertrophy/hyperplasia, subepithelial fibrosis due to thickening of the reticular basement membrane, mucus metaplasia of the epithelium, and angiogenesis. Epidemiologic studies suggest that both genetic and environmental factors may contribute to decline in lung function and airway remodeling in a subset of asthmatics. Environmental factors include respiratory viral infection-triggered asthma exacerbations, and tobacco smoke. There is also evidence that several asthma candidate genes may contribute to decline in lung function, including ADAM33, PLAUR, VEGF, IL13, CHI3L1, TSLP, GSDMB, TGFB1, POSTN, ESR1 and ARG2. In addition, mediators or cytokines, including cysteinyl leukotrienes, matrix metallopeptidase-9, interleukin-33 and eosinophil expression of transforming growth factor-?, may contribute to airway remodeling in asthma. Although increased airway smooth muscle is associated with reduced lung function (i.e. forced expiratory volume in 1 second) in asthma, there have been few long-term studies to determine how individual pathologic features of airway remodeling contribute to decline in lung function in asthma. Clinical studies with inhibitors of individual gene products, cytokines or mediators are needed in asthmatic patients to identify their individual role in decline in lung function and/or airway remodeling.
Project description: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.