Marc B. Hershenson

Role of Double-Stranded RNA Pattern Recognition Receptors in Rhinovirus-Induced Airway Epithelial Cell Responses

Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-β (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, 10-kDa IFN-γ-inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.

Tolerance and Dependence in Neonates Sedated with Fentanyl during Extracorporeal Membrane Oxygenation

We undertook a retrospective chart review of 37 neonates who received fentanyl by continuous infusion while undergoing extracorporeal membrane oxygenation (ECMO) between May 1986 and October 1988. We quantified the doses of all sedatives utilized, determined the incidence of neonatal abstinence syndrome (NAS), and identified risk factors associated with NAS. We determined peak fentanyl infusion rate, mean fentanyl infusion rate, total fentanyl dose, and duration of ECMO therapy. NAS was observed in 21 of 37 neonates (57%). In both the NAS and non-NAS neonates, mean infusion rate increased steadily during ECMO therapy, from a mean of 11.6 +/- 6.9 (SD) micrograms.kg-1.h-1 on day 1 to a mean of 52.5 +/- 19.4 (SD) micrograms.kg-1.h-1 by day 8. Total fentanyl dose and duration of ECMO were significantly greater in neonates with NAS. We found that neonates with a total dose greater than 1.6 mg/kg or an ECMO duration greater than 5 days had a significantly greater incidence of NAS (chi-squared test, P less than 0.01 and P less than 0.005; odds ratios = 7.0 and 13.9, respectively). With multiple logistic regression, ECMO duration was found to be the most powerful predictor of the occurrence of NAS. We also measured plasma fentanyl concentrations in a separate group of 5 neonates receiving fentanyl by continuous infusion for sedation. Fentanyl concentrations increased steadily during the period of infusion, suggesting the development of tolerance to the sedating effects. We conclude that continuous administration of fentanyl for sedation is associated with the uniform development of tolerance and a significant incidence of dependence. Alternative approaches to sedation should be investigated.

Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

RATIONALE Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. OBJECTIVES We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. METHODS Primary human airway epithelial cells grown at air-liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (R(T)) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. MEASUREMENTS AND MAIN RESULTS Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in R(T) without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease R(T), suggesting a requirement for viral replication. Reduced R(T) was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, IFN-gamma and IL-1beta reversed corresponding cytokine-induced reductions in R(T) but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. CONCLUSIONS RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function.

Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes

We tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Passage 1 or 2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways emerged. One-sixth of the cells developed an elongated morphology and aligned into bundles. Elongated myocytes contained cables of contractile myofilaments, dense bodies, gap junctions, and membrane caveoli, ultrastructural features of contractile SM in tissue. These cells immunostained intensely for SM alpha-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific actin-binding protein), and Western analysis of culture lysates disclosed 1.8 (SM alpha-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases during serum deprivation. Immunoreactive M3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. ACh (10(-3) M) induced a marked shortening (59.7 +/- 14.4% of original length) in 62% of elongated myocytes made semiadherent by gentle proteolytic digestion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cultured airway myocytes that did not elongate during serum deprivation instead became short and flattened, lost immunoreactivity for contractile proteins, lacked the M3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate that prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.

Extracellular Signal-Regulated Kinase 7 (ERK7), a Novel ERK with a C-Terminal Domain That Regulates Its Activity, Its Cellular Localization, and Cell Growth

ABSTRACT Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function.

Characterization of a Rac1 Signaling Pathway to Cyclin D1 Expression in Airway Smooth Muscle Cells

We examined the importance of the Rho family GTPase Rac1 for cyclin D1 promoter transcriptional activation in bovine tracheal myocytes. Overexpression of active Rac1 induced transcription from the cyclin D1 promoter, whereas platelet-derived growth factor (PDGF)-induced transcription was inhibited by a dominant-negative allele of Rac1, suggesting that Rac1 functions as an upstream activator of cyclin D1 in this system. Rac1 forms part of the NADPH oxidase complex that generates reactive oxygen species such as H2O2. PDGF stimulated a substantial increase in intracellular reactive oxygen species, as measured by the fluorescence of dichlorofluorescein-loaded cells, and this was blocked by the glutathione peroxidase mimetic ebselen. Pretreatment with ebselen, catalase, and the flavoprotein inhibitor diphenylene iodonium each attenuated PDGF- and Rac1-mediated cyclin D1 promoter activation, while having no effect on the induction of cyclin D1 by mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase-1 (MEK1), the upstream activator of ERKs. Antioxidant treatment also inhibited PDGF-induced cyclin D1 protein expression and DNA synthesis. Overexpression of an N-terminal fragment of p67 phox , a component of NADPH oxidase which interacts with Rac1, attenuated PDGF-induced cyclin D1 promoter activity, whereas overexpression of the wild-type p67 did not. Finally, Rac1 was neither required nor sufficient for ERK activation. Taken together, these data suggest a model by which two distinct signaling pathways, the ERK and Rac1 pathways, positively regulate cyclin D1 and smooth muscle growth.

Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03-2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin(-/-)) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin(-/-) mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention.

IL-17–Induced Pulmonary Pathogenesis during Respiratory Viral Infection and Exacerbation of Allergic Disease

Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17(-/-) knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections.

Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor.

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

Increased Cytokine Response of Rhinovirus-infected Airway Epithelial Cells in Chronic Obstructive Pulmonary Disease

RATIONALE Airway inflammation is a central feature of chronic obstructive pulmonary disease (COPD). COPD exacerbations are often triggered by rhinovirus (RV) infection. OBJECTIVES We hypothesized that airway epithelial cells from patients with COPD maintain a proinflammatory phenotype compared with control subjects, leading to greater RV responses. METHODS Cells were isolated from tracheobronchial tissues of 12 patients with COPD and 10 transplant donors. Eight patients with COPD had severe emphysema, three had mild to moderate emphysema, and one had no emphysema. All had moderate to severe airflow obstruction, and six met criteria for chronic bronchitis or had at least one exacerbation the previous year. Cells were grown at air-liquid interface and infected with RV serotype 39. Cytokine and IFN expression was measured by ELISA. Selected genes involved in inflammation, oxidative stress, and proteolysis were assessed by focused gene array and real-time polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS Compared with control subjects, cells from patients with COPD demonstrated increased mRNA expression of genes involved in oxidative stress and the response to viral infection, including NOX1, DUOXA2, MMP12, ICAM1, DDX58/RIG-I, STAT1, and STAT2. COPD cells showed elevated baseline and RV-stimulated protein levels of IL-6, IL-8/CXCL8, and growth-related oncogene-alpha/CXCL1. COPD cells demonstrated increased viral titer and copy number after RV infection, despite increased IL-29/IFN-lambda1, IL-28A/IFN-lambda2, and IFN-inducible protein-10/CXCL10 protein levels. Finally, RV-infected COPD cultures showed increased mRNA expression of IL28A/IFNlambda2, IL29/IFNlambda1, IFIH1/MDA5, DDX58/RIG-I, DUOX1, DUOX2, IRF7, STAT1, and STAT2. CONCLUSIONS Airway epithelial cells from patients with COPD show higher baseline levels of cytokine expression and increased susceptibility to RV infection, despite an increased IFN response.