Rommy Koetzler

Human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling

BACKGROUND Childhood human rhinovirus (HRV) infections are associated with an increased risk of asthma. We reasoned that HRV infections might be important in the pathogenesis of airway remodeling, thereby providing a mechanism by which these children are at risk of asthma. OBJECTIVE We sought to determine whether HRV infection of airway epithelial cells regulates production of growth factors associated with airway remodeling and to determine whether vascular endothelial growth factor (VEGF) was upregulated in airways during HRV-induced natural colds. METHODS Cultured human airway epithelial cells were infected with HRV. Amphiregulin, activin A, and VEGF protein levels were assayed by means of ELISA, and VEGF mRNA was quantified by using real-time RT-PCR. Pharmacologic inhibitors were used to assess the role of mitogen-activated protein kinase and nuclear factor kappaB pathways. Nasal lavage samples from subjects with confirmed natural HRV infections were assayed for VEGF protein and compared with baseline levels and with control levels. RESULTS HRV infection upregulated amphiregulin, activin A, and VEGF protein levels compared with control media (P < .05). VEGF gene expression was maximally induced 3 hours after infection. HRV-induced generation of VEGF was regulated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways but did not depend on nuclear factor kappaB activation. In subjects with HRV infections, VEGF levels during peak cold symptoms were significantly higher than at baseline (P = .005) or in control subjects (P < .01). CONCLUSION HRV-16 infection upregulates amphiregulin, activin A, and VEGF in airway epithelial cells, and HRV infections in vivo upregulate airway VEGF production.

Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells

BACKGROUND Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease. Nitric oxide (NO) inhibits HRV replication in human airway epithelial cells and suppresses HRV-induced epithelial production of several cytokines and chemokines. OBJECTIVE We sought to delineate the mechanisms by which NO inhibits HRV-induced epithelial production of CXCL10, a chemoattractant for type 1 T cells and natural killer cells. METHODS Primary human bronchial epithelial cells or cells of the BEAS-2B human bronchial epithelial cell line were exposed to HRV-16 in the presence or absence of the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate). A cGMP analogue and an inhibitor of soluble guanylyl cyclase were used to examine the role of the cyclic guanosine monophosphate (cGMP) pathway in the actions of NO. BEAS-2B cells were transfected with CXCL10 promoter-luciferase constructs and the effects of PAPA NONOate were examined to study mechanisms of transcriptional regulation. Electrophoretic mobility shift assays were also used. RESULTS PAPA NONOate inhibited HRV-16-induced increases in CXCL10 mRNA and protein. Inhibition of CXCL10 production occurred through a cGMP-independent pathway. PAPA NONOate inhibited HRV-16-induced CXCL10 transcription by blocking nuclear translocation, binding, or both of both nuclear factor kappaB and IFN response factors (IRFs) to their respective recognition elements in the CXCL10 promoter. CONCLUSIONS NO inhibits HRV-16-induced production of CXCL10 by inhibiting viral activation of nuclear factor kappaB and of IRFs, including IRF-1, through a cGMP-independent pathway. The broad-ranging inhibition of HRV-induced epithelial cytokine and chemokine production by NO suggests a potential therapeutic utility of NO donors in viral exacerbations of asthma and chronic obstructive pulmonary disease.

Selective Transcriptional Down-Regulation of Human Rhinovirus-Induced Production of CXCL10 from Airway Epithelial Cells via the MEK1 Pathway

Human rhinovirus (HRV) infections can trigger exacerbations of lower airway diseases. Infection of airway epithelial cells induces production of a number of proinflammatory chemokines that may exacerbate airway inflammation, including CXCL10, a chemoattractant for type 1 lymphocytes and NK cells. Primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line were used to examine the role of MAPK pathways in HRV-16-induced production of CXCL10. Surprisingly, PD98059 and U0126, two inhibitors of the MEK1/2-ERK MAPK pathway, significantly enhanced HRV-16-induced CXCL10 mRNA and protein. This enhancement was not seen with IFN-β-induced production of CXCL10. Studies using small interfering RNA revealed that knockdown of MEK1, but not MEK2, was associated with enhanced HRV-induced CXCL10 production. Promoter construct studies revealed that PD98059 and U0126 enhanced HRV-16-induced transcriptional activation of CXCL10. HRV-16-induced promoter activation was regulated by two NF-κB binding sites, κB1 and κB2, and by an IFN-stimulated response element. Inhibitors of the MEK1/2-ERK pathway did not alter HRV-16-induced activation of tandem repeat κB1 or κB2 constructs, nor did they alter HRV-16-induced nuclear translocation/binding of NF-κB to either κB1 or κB2 recognition sequences. Furthermore, PD98059 and U0126 did not alter phosphorylation or degradation of IκBα. In contrast, inhibitors of the MEK1/2-ERK pathway, and small interfering RNA knockdown of MEK1, enhanced nuclear translocation/binding of IFN regulatory factor (IRF)-1 to the IFN-stimulated response element recognition sequence in HRV-16 infected cells. We conclude that activation of MEK1 selectively down-regulates HRV-16-induced expression of CXCL10 via modulation of IRF-1 interactions with the gene promoter in human airway epithelial cells.

Nitric oxide inhibits IFN regulatory factor 1 and nuclear factor-κB pathways in rhinovirus-infected epithelial cells

BACKGROUND Nitric oxide (NO) has previously been shown to inhibit human rhinovirus (HRV) replication in airway epithelial cells and to inhibit rhinovirus-induced epithelial cytokine and chemokine production independently of its effects on viral replication by modulating nuclear translocation and binding of transcription factors. OBJECTIVE To define the molecular mechanisms by which NO inhibits HRV-16-induced epithelial production of CXCL10 by affecting nuclear translocation and binding of nuclear factor-kappaB (NF-kappaB) and IFN regulatory factor 1 (IRF-1). METHODS Cultured human airway epithelial cells were infected with HRV-16 in the absence or presence of a NO donor, or were preincubated with 2 highly selective inhibitors of inhibitor of kappaB kinase (IKK)beta and then infected with HRV-16. Effects on the NF-kappaB and IRF-1 pathways were examined by using electrophoretic mobility shift assays, Western blotting, and real-time RT-PCR. RESULTS Nitric oxide directly inhibited the binding of both recombinant NF-kappaB p50 protein and recombinant IRF-1 to their recognition sequences from the CXCL10 promoter. NO also inhibited phosphorylation of the NF-kappaB inhibitor, IkappaBalpha, in HRV-16-infected cells. In addition, both NO and inhibitors of IKKbeta inhibited viral induction of IRF-1 mRNA and protein. CONCLUSIONS Nitric oxide blocks rhinovirus-mediated activation and nuclear translocation of both NF-kappaB and IRF-1. NO also directly inhibits the binding of each of these transcription factors to their respective recognition sites in the CXCL10 promoter. In addition, the ability of HRV-16 to induce epithelial expression of IRF-1 is dependent, at least in part, on viral activation of NF-kappaB.