Infection Of Bronchial Epithelial Cells Research Articles

Interferon response to respiratory syncytial virus by bronchial epithelium from children with asthma is inversely correlated with pulmonary function

Respiratory viral infection in early childhood, including that from respiratory syncytial virus (RSV), has been previously associated with the development of asthma. We aimed to determine whether exvivo RSV infection of bronchial epithelial cells (BECs) from children with asthma would induce specific gene expression patterns and whether such patterns were associated with lung function among BEC donors. Primary BECs from carefully characterized children with asthma (n=18) and matched healthy children without asthma (n=8) were differentiated at an air-liquid interface for 21days. Air-liquid interface cultures were infected with RSV for 96hours and RNA was subsequently isolated from BECs. In each case, we analyzed gene expression using RNA sequencing and assessed differences between conditions by linear modeling of the data. BEC donors completed spirometry to measure lung function. RSV infection of BECs from subjects with asthma, compared with uninfected BECs from subjects with asthma, led to a significant increase in expression of 6199 genes. There was significantly greater expression of 195 genes in BECs from children with asthma and airway obstruction (FEV1/forced vital capacity<0.85 and FEV1<100% predicted) than in BECs from children with asthma without obstruction, or in BECs from healthy children. These specific genes were found to be highly enriched for viral response genes induced in parallel with types I and III interferons. BECs from children with asthma and with obstructive physiology exhibit greater expression of types I and III interferons and interferon-stimulated genes than do cells from children with normal lung function, and expression of interferon-associated genes correlates with the degree of airway obstruction. These findings suggest that an exaggerated interferon response to viral infection by airway epithelial cells may be a mechanism leading to lung function decline in a subset of children with asthma.

Upregulation of miRNA-4776 in Influenza Virus Infected Bronchial Epithelial Cells Is Associated with Downregulation of NFKBIB and Increased Viral Survival.

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3’ UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB.

Role of the Mycoplasma pneumoniae/Interleukin-8/Neutrophil Axis in the Pathogenesis of Pneumonia.

Neutrophil infiltration is the characteristic pathological feature of M. pneumoniae pneumonia (MPP). This study aimed to explore the associations among neutrophil activity, clinical presentation, and role of the M. pneumoniae/interleukin-8 (IL-8)/neutrophil axis in the pathogenesis of MPP. A total of 42 patients with MPP were prospectively enrolled in the study. Neutrophil activity, including matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO), and neutrophil elastase (NE), were measured. Clinical information was collected for all patients and control group. In vitro, IL-8 production was measured at different time points after M. pneumoniae infection of bronchial epithelial cells, and neutrophil activity was analyzed after IL-8 stimulation. The percentage of neutrophil in the bronchoalveolar lavage fluid was higher in the group of patients with high levels of M. pneumoniae DNA than in those with low levels of M. pneumoniae DNA (P < 0.05). IL-8, MMP-9, and NE in patients with MPP significantly increased compared with controls and decreased after treatment (P < 0.05). MPO and MMP-9 were associated with duration of fever (r = 0.332, P < 0.05) and length of stay (r = 0.342, P < 0.05), respectively. In vitro, M. pneumoniae induced IL-8 production by bronchial epithelial cells in a time dependent manner. MPO, MMP-9 and NE production by neutrophils significantly increased compared with medium controls after IL-8 stimulation. In summary, the M. pneumoniae/IL-8/neutrophil axis likely plays a vital role in the pathogenesis of MPP.

Characterising the Mechanism of Airway Smooth Muscle β2 Adrenoceptor Desensitization by Rhinovirus Infected Bronchial Epithelial Cells

Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.

IL-32, a Negative Regulator of Angiogenesis in Chronic Airway Diseases

Chronic asthmatic inflammation in the airways induces angiogenesis demonstrated by several studies in murine models [1] and in human airways [2]. Among the many pro-angiogenic factors involved in asthmatic inflammation in the airways, VEGF-A seems to be the most important one. VEGF-A is mainly produced and secreted by many tissue and immune cells like alveolar epithelial cells, bronchial epithelial cells, smooth muscle cells, fibroblasts, and basophiles [3,4]. VEGF-A plays an important role during asthma inflammation: Increased VEGF-A levels are found during asthma attacks already in children [5] and VEGF levels in induced sputum from asthma patients correlate with asthma severity and airflow obstruction [6]. Most importantly, VEGF-A is up regulated by Th2 cytokines like IL-4, IL-5 and IL-13 involved in the pathogenesis of asthma [7]. In addition, the VEGF release by human primary bronchial epithelial cells is increased by rhinovirus infection [7]. In this context, we demonstrated that IL-32 is induced by rhinovirus infection in bronchial epithelial cells and decreases VEGF production and secretion [8]. IL-32 knockdown leads to an up-regulation of VEGF secretion by primary bronchial epithelial cells suggesting that IL-32 might have an inhibitory role in angiogenesis and might be an important factor counter-regulating angiogenesis in rhinovirus-triggered asthma. In addition, IL-32 is also present in sinus epithelial cells after stimulation with TNF-α and IFN-γ [9]. However, the role of IL-32 in angiogenesis during chronic rhinitis has not yet been investigated. Interestingly, IL-32 could be detected in the sera of asthma patients and correlates with significant lung function improvement after 3 weeks of intensive asthma treatment suggesting that IL-32 may distinguish between asthma subtypes with or without fixed airflow limitations. Although the IL-32–mediated decrease in VEGF production during asthmatic airway inflammation suggests an anti-inflammatory function of IL-32, other studies could demonstrate that IL-32 acts as a pro-inflammatory cytokine inducing TNF-α, IL-8, and macrophage inflammatory protein 2 in monocytes and macrophages [10]. Therefore, more studies investigating IL-32 and its significance for angiogenesis in chronic inflammatory diseases are necessary in the future.

Understanding the effects of IL-18 and IL-1β in a complex pro-inflammatory milieu. (44.11)

Abstract Disease association studies suggest that the cytokines IL1 and IL18 play a role in exacerbations of Chronic Obstructive Pulmonary Disease. A classical role of IL18 is to stimulate NK and T-Cells to produce IFNγ, a cytokine associated with viral exacerbations of COPD. IL1β, which is processed by the same mechanism as IL18, can have a similar effect on IFNγ. We sought to understand their respective role in IFNγ secretion in situations where they both could be released. Treatment of PBMCs with IL18 or IL1β (in the presence of IL12) was able to induce IFNγ release. This response was most sensitive to IL1β but IL18 was able to stimulate a greater maximal response. When LPS was used as a trigger of IFNγ release, a large amount of IL1β (11ng/mL) and a low amount of IL18 (27pg/mL) could be detected in the supernatant. Co-incubation with an IL18 neutralizing antibody, or the IL1 receptor antagonist anakinra, was able to inhibit the IFNγ response by 50% in both cases. In a second system, relevant to COPD exacerbations, the infection of bronchial epithelial cells with a human Rhinovirus induced the release of both IL18 (2.5ng/mL) and IL1β (435pg/mL). Transfer of the supernatant to PBMCs or NK cells induced IFNγ production which was largely IL1-dependent; inhibition of IL18 only had a small effect on IFNγ. This data showed that while they could induce IFNγ following an inflammatory stimulus, it is the context of their release that defines a predominant role for either IL18 or IL1β.

Rhinovirus-Induced IL-1β Release from Bronchial Epithelial Cells Is Independent of Functional P2X7

Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor-dependent inflammatory responses, largely through P2X(7). Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X(7) to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X(7) messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X(7) was observed in BECs, and IL-1β was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1β after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2'-3'-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1β release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1β release further. BEC processing of pro-IL-1β to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X(7) in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing.

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection.

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.

Rhinovirus‐induced basic fibroblast growth factor release mediates airway remodeling features

BackgroundHuman rhinoviruses, major precipitants of asthma exacerbations, induce lower airway inflammation and mediate angiogenesis. The purpose of this study was to assess the possibility that rhinoviruses may also contribute to the fibrotic component of airway remodeling.MethodsLevels of basic fibroblast growth factor (bFGF) mRNA and protein were measured following rhinovirus infection of bronchial epithelial cells. The profibrotic effect of epithelial products was assessed by DNA synthesis and matrix metalloproteinase activity assays. Moreover, epithelial cells were exposed to supernatants from cultured peripheral blood mononuclear cells, obtained from healthy donors or atopic asthmatic subjects and subsequently infected by rhinovirus and bFGF release was estimated. bFGF was also measured in respiratory secretions from atopic asthmatic patients before and during rhinovirus-induced asthma exacerbations.ResultsRhinovirus epithelial infection stimulated mRNA expression and release of bFGF, the latter being positively correlated with cell death under conditions promoting rhinovirus-induced cytotoxicity. Supernatants from infected cultures induced lung fibroblast proliferation, which was inhibited by anti-bFGF antibody, and demonstrated increased matrix metalloproteinase activity. Rhinovirus-mediated bFGF release was significantly higher in an in vitro simulation of atopic asthmatic environment and, importantly, during rhinovirus-associated asthma exacerbations.ConclusionsRhinovirus infection induces bFGF release by airway epithelium, and stimulates stroma cell proliferation contributing to airway remodeling in asthma. Repeated rhinovirus infections may promote asthma persistence, particularly in the context of atopy; prevention of such infections may influence the natural history of asthma.

Lack of an exaggerated inflammatory response on virus infection in cystic fibrosis

Respiratory virus infections play an important role in cystic fibrosis (CF) exacerbations, but underlying pathophysiological mechanisms are poorly understood. We aimed to assess whether an exaggerated inflammatory response of the airway epithelium on virus infection could explain the increased susceptibility of CF patients towards respiratory viruses. We used primary bronchial and nasal epithelial cells obtained from 24 healthy control subjects and 18 CF patients. IL-6, IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and GRO-α/CXCL1 levels in supernatants and mRNA expression in cell lysates were measured before and after infection with rhinoviruses (RV-16 and RV-1B) and RSV. Cytotoxicity was assessed by lactate dehydrogenate assay and flow cytometry. All viruses induced strong cytokine release in both control and CF cells. The inflammatory response on virus infection was heterogeneous and depended on cell type and virus used, but was not increased in CF compared with control cells. On the contrary, there was a marked trend towards lower cytokine production associated with increased cell death in CF cells. An exaggerated inflammatory response to virus infection in bronchial epithelial cells does not explain the increased respiratory morbidity after virus infection in CF patients.

Rhinovirus infection modulates MHC class I-related chain A and B molecules in human respiratory epithelial cells (133.37)

Abstract Cell surface and soluble MICA and MICB molecules induced by stress/viruses could activate, respectively impair, cytotoxic activity of NKG2D+ NK and CD8 T cells. We hypothesised that rhinovirus (RV) infection of bronchial epithelial cells alters the balance between activatory (surface) and inhibitory (soluble) MICA and MICB molecule. MIC molecules were investigated in BEAS-2B cells (a bronchial epithelial cell line) exposed in vitro to RV1B MOI 1 ± IL-4 10ng/ml up to 72 hours by flow cytometry (intracellular and cell surface) and ELISA (soluble molecules in supernatants). RV1B infection of BEAS-2B cells increased the levels of surface MICA in a dose- and replication-dependent manner, peaking at 24h and gradually decreased during 72h. RV1B-induced soluble MICA molecule levels increased in a time-dependent manner, peaking at 72h. IL-4 presence during RV1B infection increased RV1B-iduced surface MICA (MFI: 72±9 versus 45±10, p&amp;lt;0.05) and soluble MICA levels (12±8 versus 9±7, p=0.3). Intracellular and surface MICB molecules were decreased by RV1B infection and IL-4 further decreased intracellular levels. To conclude, RV1B infection in BEAS-2B cells in the presence of IL-4 up-regulated the surface MICA levels but also increased soluble MICA levels which could inhibit in vivo the activation of NKG2D+ cells and by doing this delaying virus clearance and prolonging the airway inflammation in virus-induced asthma.

Peripheral blood monocytes (PBMCs) display innate antiviral response to rhinovirus (RV) infected bronchial epithelial cells (133.44)

Abstract Rhinovirus (RV) infection is an important trigger of acute asthma and COPD. Study objectives; were to characterise the response of PBMCs and CD8 lymphocytes when exposed to RV infected bronchial epithelial cells. Methods; Human Calu3 cells were used as a model for bronchial epithelial cells and infected with RV-43 and RV-1B. PBMCs were obtained from healthy individuals and separated by Ficoll-Paque centrifugation. CD8 lymphocytes were identified as CD3+, CD8+ by flow cytometry. PBMCs were exposed to infected Calu3 cells, cells exposed to UV inactive RV, PHA and TLR3 agonist PolyI:C. Response was measured by ELISA for release of interferon(IFN)-γ and TNF-α. CD8 activation with expression of perforin or proliferation measured by carboxyfluorescein succinimidyl ester assay. Results: Exposure of PBMCs to Calu3 cells infected with both RV led to significant release of IFN-γ and TNF-α, smaller release was seen in response to UV inactive RV. Exposure to PolyI:C also led to a similar release. CD8+ cells were found to be the primary source of IFN-γ release. CD8+ cells did not show an increase in perforin expression or evidence of proliferation. Conclusions; exposure of PBMCs and CD8+ cells to an RV infected bronchial epithelium alone results in an innate immune response mediated by TLR3 without evidence of a CD8 adaptive immune response.

Respiratory virus induction of alpha‐, beta‐ and lambda‐interferons in bronchial epithelial cells and peripheral blood mononuclear cells

Respiratory viruses, predominantly rhinoviruses are the major cause of asthma exacerbations. Impaired production of interferon-beta in rhinovirus infected bronchial epithelial cells (BECs) and of the newly discovered interferon-lambdas in both BECs and bronchoalveolar lavage cells, is implicated in asthma exacerbation pathogenesis. Thus replacement of deficient interferon is a candidate new therapy for asthma exacerbations. Rhinoviruses and other respiratory viruses infect both BECs and macrophages, but their relative capacities for alpha-, beta- and lambda-interferon production are unknown. To provide guidance regarding which interferon type is the best candidate for development for treatment/prevention of asthma exacerbations we investigated respiratory virus induction of alpha-, beta- and lambda-interferons in BECs and peripheral blood mononuclear cells (PBMCs) by reverse transferase-polymerase chain reaction and enzyme-linked immunosorbent assay. Rhinovirus infection of BEAS-2B BECs induced interferon-alpha mRNA expression transiently at 8 h and interferon-beta later at 24 h while induction of interferon-lambda was strongly induced at both time points. At 24 h, interferon-alpha protein was not detected, interferon-beta was weakly induced while interferon-lambda was strongly induced. Similar patterns of mRNA induction were observed in primary BECs, in response to both rhinovirus and influenza A virus infection, though protein levels were below assay detection limits. In PBMCs interferon-alpha, interferon-beta and interferon-lambda mRNAs were all strongly induced by rhinovirus at both 8 and 24 h and proteins were induced: interferon-alpha>-beta>-lambda. Thus respiratory viruses induced expression of alpha-, beta- and lambda-interferons in BECs and PBMCs. In PBMCs interferon-alpha>-beta>-lambda while in BECs, interferon-lambda>-beta>-alpha. We conclude that interferon-lambdas are likely the principal interferons produced during innate responses to respiratory viruses in BECs and interferon-alphas in PBMCs, while interferon-beta is produced by both cell types.

The Potential Role of Lung Epithelial Cells and β‐defensins in Experimental Latent Tuberculosis

Mycobacterium tuberculosis is a facultative intracellular pathogen capable of producing both progressive disease and latent infection. Latent infection is clinically asymptomatic and is manifested only by a positive tuberculin test or a chest radiograph that shows scars or calcified nodules indicative of resolved primary tuberculosis infection. In this study, we used a well-characterized model of latent tuberculosis infection in B6D2F1 mice to compare the production of beta-defensin-3 by infected bronchial epithelial cells and macrophages. We demonstrated by immunolectronmicroscopy that M. tuberculosis can actually infect epithelial cells and induce significant higher production of beta-defensin-3 associated to mycobacteria than infected macrophages. These results demonstrate that lung epithelium harbour mycobacteria during experimental chronic infection; being a possible reservoir of latent mycobacteria in vivo, beta-defensins might participate in bacilli killing or dormancy induction.

Protein kinase R, IκB kinase-β and NF-κB are required for human rhinovirus induced pro-inflammatory cytokine production in bronchial epithelial cells

Rhinovirus infections cause the majority of acute exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease, with increased pro-inflammatory cytokine production by infected bronchial epithelial cells contributing to disease pathogenesis. Theses diseases are a huge cause of morbidity worldwide, and contribute a major economic burden to healthcare costs. Current steroid based treatments are only partially efficient at controlling virus induced inflammation, which remains an unmet therapeutic goal. Although NF-κB has been implicated, the precise mechanisms of rhinovirus induction of pro-inflammatory gene expression in bronchial epithelial cells are unclear. We hypothesised that rhinovirus replication and generation of dsRNA was an important process of pro-inflammatory cytokine induction. Using pharmalogical (2-aminopurine and a new small molecule inhibitor) and genetic inhibition of the dsRNA binding kinase protein kinase R, striking inhibition of dsRNA (polyrIC) and rhinovirus induced CCL5, CXCL8 and IL-6 protein was observed. Using confocal microscopy, rhinovirus induced protein kinase R phosphorylation co-located with NF-κB p65 nuclear translocation. Focusing on CXCL8, both rhinovirus infection and dsRNA treatment required IκB kinase-β for induction of CXCL8. Analysis of cis-acting sites in the CXCL8 promoter revealed that both rhinovirus infection and dsRNA treatment upregulated CXCL8 promoter activation via NF-κB and NF-IL6 binding sites. Together, the results demonstrate the importance of dsRNA in induction of pro-inflammatory cytokines by rhinoviruses, and suggest that protein kinase R is involved in NF-κB mediated gene transcription of pro-inflammatory cytokines via IκB kinase-β. These molecules regulating rhinovirus induction of inflammation represent therapeutic targets.

Moraxella catarrhalisinduces inflammatory response of bronchial epithelial cells via MAPK and NF-κB activation and histone deacetylase activity reduction

Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease (COPD) and may also contribute to the pathogenesis of COPD. Little is known about M. catarrhalis-bronchial epithelium interaction. We investigated activation of M. catarrhalis infected bronchial epithelial cells and characterized the signal transduction pathways. Moreover, we tested the hypothesis that the M. catarrhalis-induced cytokine expression is regulated by acetylation of histone residues and controlled by histone deacetylase activity (HDAC). We demonstrated that M. catarrhalis induced a strong time- and dose-dependent inflammatory response in the bronchial epithelial cell line (BEAS-2B), characterized by the release of IL-8 and GM-CSF. For this cytokine liberation activation of the ERK and p38 mitogen-activated protein (MAP) kinases and transcription factor NF-kappaB was required. Furthermore, M. catarrhalis-infected bronchial epithelial cells showed an enhanced acetylation of histone H3 and H4 globally and at the promoter of the il8 gene. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A augmented the M. catarrhalis-induced IL-8 response. After exposure to M. catarrhalis, we found a decrease in global histone deacetylase expression and activity. Our findings suggest that M. catarrhalis-induced activation of il8 gene transcription was caused by interference with epigenetic mechanisms regulating il8 gene accessibility. Our findings provide insight into important molecular and cellular mechanisms of M. catarrhalis-induced activation of human bronchial epithelium.

Cross-talk between ERK-1/2 and STAT-1-alpha pathways in respiratory syncytial virus-induced early gene expression in human epithelial cells

Rationale NFkappaB and STATs are activated in epithelial cells following exposure to respiratory syncytial virus (RSV) (Kong et al, BBRC, 2003). However, the roles of other signaling pathways such as ERK-1/2 are poorly understood. The roles of ERK-1/2 in activation of RSV infection and activating other signaling pathways were examined in this study using human epithelial cells. Methods The activation of ERK-1/2 was analyzed by Western blotting. Inhibitors and dominant negative (DN) mutant constructs were used to evaluate cross-talk. Viral replication was measured by quantifying the percentage of infected cells and by plaque assay. Results RSV exposure induced phosphorylation of ERK-1/2 in A549 cells within 5 min, which persisted for 2 h. Heparin, an inhibitor of RSV attachment, abolished the activation of ERK-1/2. Also, A549 cells transfected with the DN-MEK-1 showed a >60% decrease in the number of infected cells compared to the wild type construct (P<0.05). Treatment of cells with a MEK-1 inhibitor, PD98059, significantly decreased the phosphorylation of both STAT-1-alpha and IkappaB suggesting cross-talk between the ERK-1/2 and STAT-1 pathways. Furthermore, exposure of normal human bronchial epithelial cells to PD98059 (MEK-1 inhibitor), AG490 (JAK-2 inhibitor), or Ro318220 (PKC inhibitor) caused a significant reduction in the number of infected cells suggesting that ERK-1/2, JAK-2 and PKC are required for successful RSV infection in bronchial epithelial cells. Conclusions These results demonstrate that ERK-1/2 activation is required in RSV-induced early gene expression and that cross-talk between ERK-1/2 and STAT-1 is critical to RSV-induced expression of proinflammatory molecules and potential exacerbation of asthma.

Rhinovirus infection up-regulates eotaxin and eotaxin-2 expression in bronchial epithelial cells.

Human rhinoviruses (RVs) are the most common precipitants of asthma exacerbations. RV infection of bronchial epithelium results in local airway inflammation inducing eosinophil recruitment and activation. Induction of eosinophil chemoattractants could represent a central mechanism, as well as a prime target for intervention. To assess the effect of RV infection on mRNA expression and production of eosinophil chemoattractants by bronchial epithelial cells in-vitro. BEAS-2B cells were infected with major and minor RVs and the mRNA expression of IL-8, RANTES, MIP-1alpha, eotaxin, eotaxin-2, MCP-2, MCP-3 and MCP-4 was assessed by reverse transcription PCR. In cases where mRNA induction was observed, a fluoroimmunoassay was used to confirm protein production. To assess the virus-specificity of the observed reactions, cells were also exposed to inactivated RVs. RV infection was able to up-regulate mRNA expression of IL-8, RANTES, MIP-1alpha, eotaxin and eotaxin-2, did not affect MCP-4, while MCP-2 and MCP-3 were not expressed either at baseline or after virus infection. Protein production was confirmed for IL-8, RANTES and eotaxin, but not for MIP-1alpha. When RVs were inactivated cytokine up-regulation was almost completely lost. Infection of bronchial epithelial cells with RVs results in the production of a wide array of mediators that are able to chemoattract eosinophils. These include the eosinophil-specific molecules eotaxin and eotaxin-2, in addition to IL-8 and RANTES, which are the most abundant. Eosinophil recruitment after RV infection of bronchial epithelium could represent a central event in the pathogenesis of virus-induced asthma exacerbations.

Expression of cytokines on human bronchial epithelial cells induced by influenza virus A.

Bronchial epithelial cells play an important role in the pathogenesis of some inflammatory diseases of bronchial mucosa. Epithelial-cell-derived cytokines are important in the elucidation of the mechanism by which airway inflammation occurs, especially in respiratory virus infection, because these cells are the primary sites of viral infection. We infected bronchial epithelial cells, NCI-H292, with influenza virus A (H3N2) and examined the concentrations of cytokines, interleukin-6 (IL-6), IL-8 and regulated on activation, normal T cells, expressed and secreted (RANTES), in the culture media of infected cells using the enzyme-linked immunosorbent assay system and gene expression of RANTES on epithelial cells by the reverse-transcriptase-polymerase chain reaction method. We found that significant amounts of IL-6, IL-8 and RANTES were released. RANTES mRNA was also detected in infected bronchial epithelial cells. It is suggested that cytokine production in human bronchial epithelial cells may contribute to the pathogenesis of airway inflammatory disorders.

Infection of cultured human airway epithelial cells by influenza a virus

The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway. We report in vitro infection of human airway epithelial cells by influenza A virus. Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H 1N 1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection. All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H 1N 1 virus. No hemadsorption was detected with the WSN virus. One of two transformed cell lines showed a 5–10% hemadsorption to cells after H 1N 1 exposure and none following exposure to WSN. Immunoflourescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells. Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins. Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant. Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time. The data indicate that influenze A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.