CXCR2 regulates respiratory syncytial virus-induced airway hyperreactivity and mucus overproduction

Severe inflammation and mucus overproduction are partially responsible for respiratory syncytial virus (RSV)-induced disease in infants. Using a murine model, we characterized the virally induced chemokine receptors responsible for mediating the pathophysiological response to RSV infection, we found that CXCR2 mRNA was induced at 4 days after RSV infection. Immunohistochemical staining demonstrated that CXCR2 protein was expressed on alveolar macrophages. Immunoneutralization of CXCR2 resulted in decreased airway hyperreactivity relative to the RSV-infected controls. In addition, there was decreased mucus in the bronchoalveolar lavage fluid, decreased periodic-acid Schiff staining, and significantly less mucus-associated gob-5 mRNA and protein in anti-CXCR2-treated mice. The effects of anti-CXCR2 treatment were not a result of differences in viral clearance or neutrophil influx, as these parameters were comparable in both groups of animals. To confirm our immunoneutralization studies, we performed experiments in CXCR2(-/-) mice. Results in CXCR2(-/-) mice recapitulated results from our immunoneutralization studies. However, CXCR2(-/-) mice also showed a statistically significant decrease in muc5ac, relative to RSV-infected wild-type animals. Thus, CXCR2 may be a relevant target in the pathogenesis of RSV bronchiolitis, since it contributes to mucus production and airway hyperreactivity in our model of RSV infection.     

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D₃ is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D₃ on RSV pathogenies. Here, we investigated the impact of vitamin D₃ on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)₂D₃, the active form of vitamin D₃, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)₂D₃ induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)₂D₃ exposure, indicating that vitamin D₃ triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)₂D₃ in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D₃ against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D₃ as a prophylactic intervention to protect the respiratory epithelium during RSV infections.     

CD8 T cells are essential in the development of respiratory syncytial virus-induced lung eosinophilia and airway hyperresponsiveness

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways. The immune cell requirements for these responses to RSV infection are not well defined. To delineate the role of CD8 T cells in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice depleted of CD8 T cells to develop these symptoms of RSV infection. BALB/c mice were depleted of CD8 T cells using anti-CD8 Ab treatment before intranasal administration of infectious RSV. Six days postinfection, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and levels of IFN-gamma, IL-4, and IL-5 in bronchoalveolar lavage fluid were monitored. RSV infection resulted in airway eosinophilia and AHR in control mice, but not in CD8-depleted animals. Further, whereas RSV-infected mice secreted increased amounts of IL-5 into the airways as compared with noninfected controls, no IL-5 was detectable in both bronchoalveolar lavage fluid and culture supernatants from CD8-depleted animals. Treatment of CD8-depleted mice with IL-5 fully restored both lung eosinophilia and AHR. We conclude that CD8 T cells are essential for the influx of eosinophils into the lung and the development of AHR in response to RSV infection.     

Differential role for TLR3 in respiratory syncytial virus-induced chemokine expression

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young infants worldwide. Previous studies have reported that the induction of interleukin-8/CXCL8 and RANTES/CCL5 correlates with disease severity in humans. The production of these chemokines is elicited by viral replication and is NF-kappaB dependent. RSV, a negative-sense single-stranded RNA virus, requires full-length positive-sense RNA for synthesis of new viral RNA. The aim of our studies was to investigate whether active viral replication by RSV could evoke chemokine production through TLR3-mediated signaling pathways. In TLR3-transfected HEK 293 cells, live RSV preferentially activated chemokines in both a time- and dose-dependent manner compared to vector controls. RSV was also shown to upregulate TLR3 in human lung fibroblasts and epithelial cells (MRC-5 and A549). Targeting the expression of TLR3 with small interfering RNA decreased synthesis of IP-10/CXCL10 and CCL5 but did not significantly reduce levels of CXCL8. Blocking the expression of the adapter protein MyD88 established a role for MyD88 in CXCL8 production, whereas CCL5 synthesis was found to be MyD88 independent. Production of CCL5 by RSV was induced directly through TLR3 signaling pathways and did not require interferon (IFN) signaling through the IFN-alpha/beta receptor. TLR3 did not affect viral replication, since equivalent viral loads were recovered from RSV-infected cells despite altered TLR3 expression. Taken together, our studies indicate that TLR3 mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells.     

Role of Ras in metal-induced EGF receptor signaling and NF-kappaB activation in human airway epithelial cells

We showed previously that epithelial growth factor (EGF) receptor (EGFR) signaling is triggered by metallic compounds associated with ambient air particles. Specifically, we demonstrated that As, Zn, and V activated the EGFR tyrosine kinase and the downstream kinases MEK1/2 and ERK1/2. In this study, we examined the role of Ras in EGFR signaling and the nuclear factor-kappaB (NF-kappaB) activation pathway and the possible interaction between these two signaling pathways in a human airway epithelial cell line (BEAS-2B) exposed to As, V, or Zn ions. Each metal significantly increased Ras activity, and this effect was inhibited by the EGFR tyrosine kinase activity inhibitor PD-153035. Adenoviral-mediated overexpression of a dominant-negative mutant form of Ras(N17) significantly blocked MEK1/2 or ERK1/2 phosphorylation in As-, Zn-, or V-exposed BEAS-2B cells but caused little inhibition of V-, Zn- or EGF-induced EGFR tyrosine phosphorylation. This confirmed Ras as an important intermediate effector in EGFR signaling. Interestingly, V, but not As, Zn, or EGF, induced IkappaBalpha serine phosphorylation, IkappaBalpha breakdown, and NF-kappaB DNA binding. Moreover, PD-153035 and overexpression of Ras(N17) each significantly blocked V-induced IkappaBalpha breakdown and NF-kappaB activation, while inhibition of MEK activity with PD-98059 failed to do so. In summary, exposure to As, Zn, and V initiated EGFR signaling and Ras-dependent activation of MEK1/2 and ERK1/2, but only V induced Ras-dependent NF-kappaB nuclear translocation. EGFR signaling appears to cross talk with NF-kappaB signaling at the level of Ras, but additional signals appear necessary for NF-kappaB activation. Together, these data suggest that, in V-treated BEAS-2B cells, Ras-dependent signaling is essential, but not sufficient, for activation of NF-kappaB.     

Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus. Role in viral-induced interferon regulatory factor activation

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2',7'-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression.     

Advances in understanding respiratory syncytial virus infection in airway epithelial cells and consequential effects on the immune response

This article reviews aspects of respiratory syncytial virus (RSV) infection in airway epithelial cells (AECs), including cytopathogenesis, entry, replication and the induction of immune response to the virus, including a new role for thymic stromal lymphopoietin in RSV immunopathology.     

Dual role of RACK1 in airway epithelial mesenchymal transition and apoptosis

Airway epithelial apoptosis and epithelial mesenchymal transition (EMT) are two crucial components of asthma pathogenesis, concomitantly mediated by TGF-β1. RACK1 is the downstream target gene of TGF-β1 shown to enhancement in asthma mice in our previous study. Balb/c mice were sensitized twice and challenged with OVA every day for 7 days. Transformed human bronchial epithelial cells, BEAS-2B cells were cultured and exposed to recombinant soluble human TGF-β1 to induced apoptosis (30 ng/mL, 72 hours) and EMT (10 ng/mL, 48 hours) in vitro, respectively. siRNA and pharmacological inhibitors were used to evaluate the regulation of RACK1 protein in apoptosis and EMT. Western blotting analysis and immunostaining were used to detect the protein expressions in vivo and in vitro. Our data showed that RACK1 protein levels were significantly increased in OVA-challenged mice, as well as TGF-β1-induced apoptosis and EMT of BEAS-2B cells. Knockdown of RACK1 (siRACK1) significantly inhibited apoptosis and decreased TGF-β1 up-regulated EMT related protein levels (N-cadherin and Snail) in vitro via suppression of JNK and Smad3 activation. Moreover, siSmad3 or siJNK impaired TGF-β1-induced N-cadherin and Snail up-regulation in vitro. Importantly, JNK gene silencing (siERK) also impaired the regulatory effect of TGF-β1 on Smad3 activation. Our present data demonstrate that RACK1 is a concomitant regulator of TGF-β1 induces airway apoptosis and EMT via JNK/Smad/Snail signalling axis. Our findings may provide a new insight into understanding the regulation mechanism of RACK1 in asthma pathogenesis.     

Growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in infants and children. To study RSV replication, we have developed an in vitro model of human nasopharyngeal mucosa, human airway epithelium (HAE). RSV grows to moderate titers in HAE, though they are significantly lower than those in a continuous epithelial cell line, HEp-2. In HAE, RSV spreads over time to form focal collections of infected cells causing minimal cytopathic effect. Unlike HEp-2 cells, in which wild-type and live-attenuated vaccine candidate viruses grow equally well, the vaccine candidates exhibit growth in HAE that parallels their level of attenuation in children.     

NADPH oxidase NOX5-S mediates acid-induced cyclooxygenase-2 expression via activation of NF-kappaB in Barrett's esophageal adenocarcinoma cells

We have shown that the NADPH oxidase NOX5-S may play an important role in the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) by increasing cell proliferation and decreasing apoptosis. However, the mechanism of the acid-induced NOX5-S-mediated increase in cell proliferation is not known. We found that, in SEG1 EA cells, the acid-induced increase in prostaglandin E2 (PGE2) production was mediated by activation of cyclooxygenase-2 (COX2) but not by COX1. Acid treatment increased intracellular Ca2+, and a blockade of intracellular Ca2+ increase inhibited the acid-induced increase in COX2 expression and PGE2 production. Knockdown of NOX5-S or NF-kappaB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE2 production in SEG1 cells. Acid treatment significantly decreased IkappaBalpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappaB in vivo activation reporter plasmid, pNF-kappaB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IkappaBalpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappaB-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H2O2 production, COX2 expression, PGE2 production, and thymidine incorporation. The increase in thymidine incorporation occurring in NOX5-S-overexpressing Barrett's cells or induced by acid treatment in SEG1 EA cells was significantly decreased by COX2 inhibitors or small interfering RNA. We conclude that acid-induced COX2 expression and PGE2 production depend on an increase in cytosolic Ca2+ and sequential activation of NOX5-S and NF-kappaB in SEG1 cells. COX2-derived PGE2 production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.     

Bradykinin B2 receptor mediates NF-kappaB activation and cyclooxygenase-2 expression via the Ras/Raf-1/ERK pathway in human airway epithelial cells

In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-kappaB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-kappaB inhibitor (pyrrolidine dithiocarbate), and an IkappaB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the kappaB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IkappaB kinase alphabeta (IKKalphabeta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-protein complex, and kappaB-luciferase activity. BK-mediated increase in IKKalphabeta activity and formation of the NF-kappaB-specific DNA-protein complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKalphabeta and NF-kappaB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).     

支气管上皮细胞在气道高反应中的作用

气道高反应的发病机制目前仍然不清楚,但人多数人认同是气道的一种慢性炎症。近十年来,上皮缺陷学说逐渐成为解释气道高反应机制的主流观点。气道上皮不再被仅仅看作为单纯的机械屏障,而是机体内环境与外部环境相互作用的界面。气道上皮具有广泛的生理作用,包括抗氧化、内分泌和外分泌、黏液运输、生物代谢、结构性黏附、损伤修复、应激或炎症信号传递、抗原递呈作用等。借助这些生理作用,支气管上皮细胞在气道局部微环境稳态维持中发挥重要作用。有理由相信,气道上皮的结构完整性缺陷或功能紊乱是哮喘和慢性阻塞性肺疾病等气道高反应性疾病的启动环节。     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.     

Incomplete regulation of NF-kappaB by IkappaBalpha during respiratory syncytial virus infection in A549 cells

Respiratory syncytial virus (RSV) infection of airway epithelial cells results in persistent NF-kappaB activation and NF-kappaB-mediated interleukin-8 production. Previous studies in airway epithelial cells demonstrated that tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation is transient due to regulation by IkappaBalpha. However, during RSV infection, IkappaBalpha has only a partial inhibitory effect on NF-kappaB activation. Studies presented here demonstrate that neither increased IkappaBalpha production which occurs as a result of RSV-induced NF-kappaB activation nor inhibition of proteasome-mediated IkappaBalpha degradation results in a reversal of RSV-induced NF-kappaB activation. Thus, while manipulation of IkappaBalpha results in reversal of TNF-alpha-induced NF-kappaB activation, manipulation of IkappaBalpha does not result in a reversal of RSV-induced NF-kappaB activation.     

NF-kappaB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-kappaB (NF-kappaB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-kappaB in cells isolated from five CF (DeltaF508/DeltaF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-kappaB subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-kappaB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-kappaB inhibitor IkappaBalpha were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-kappaB to nuclei of primary CF epithelial cell cultures, but intranuclear IkappaBalpha may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.