Interferon-γ Inhibits Ebola Virus Infection.

Ebola virus outbreaks, such as the 2014 Makona epidemic in West Africa, are episodic and deadly. Filovirus antivirals are currently not clinically available. Our findings suggest interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option. Using mouse-adapted Ebola virus, we found that murine interferon gamma administered 24 hours before or after infection robustly protects lethally-challenged mice and reduces morbidity and serum viral titers. Furthermore, we demonstrated that interferon gamma profoundly inhibits Ebola virus infection of macrophages, an early cellular target of infection. As early as six hours following in vitro infection, Ebola virus RNA levels in interferon gamma-treated macrophages were lower than in infected, untreated cells. Addition of the protein synthesis inhibitor, cycloheximide, to interferon gamma-treated macrophages did not further reduce viral RNA levels, suggesting that interferon gamma blocks life cycle events that require protein synthesis such as virus replication. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited Ebola virus infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit negative strand RNA viruses and specifically Ebola virus infection. As treatment of interferon gamma robustly protects mice from lethal Ebola virus infection, we propose that interferon gamma should be further evaluated for its efficacy as a prophylactic and/or therapeutic strategy against filoviruses. Use of this FDA-approved drug could rapidly be deployed during future outbreaks.

Effects of prenatal inhalation exposure to copper nanoparticles on murine dams and offspring.

BACKGROUND: Increasing numbers of individuals may be exposed to nanomaterials during pregnancy. The overarching goal of this investigation was to determine if prenatal inhalation exposure to copper nanoparticles (Cu NPs) has an effect on dams and offspring, including an analysis of inflammatory markers (Th1/Th2 cytokine profiles). METHODS: Physicochemical characterization of Cu NPs was performed. Pregnant and non-pregnant mice (C57Bl/6 J) were exposed to Cu NPs or laboratory air in the whole-body chamber for 4 hrs/day on gestation days (GD) 3-19 (3.5 mg/m(3)). Animals were euthanized on GD 19 (0 week) or 7 weeks later. Bronchoalveolar lavage (BAL) fluid was analyzed for total and differential cells. Cytokine/chemokine concentrations were determined in the BAL fluid and the plasma of dams/non-pregnant mice and pups. Cu content was determined in the lungs and the blood of dams/non-pregnant mice and pups, in the placentas as well as in the whole bodies of pups immediately after delivery. Lungs and placentas were evaluated for histopathological changes. Gene expression of the Th1/Th2 profiles were analyzed in spleens of pups. RESULTS: The survival rate of 7 week old pups exposed to Cu NPs was significantly lower than control pups (73 vs. 97 %). The average litter size, male/female ratio, body weight and lenght at birth were not different between Cu NP-exposed and control mice. Both pregnant and non-pregnant mice exposed to Cu NPs had significant pulmonary inflammation with increased number of neutrophils in the BAL fluid compared to controls. Perivascular lymphoplasmacytic cuffing was found in the lungs of exposed mice and was more pronounced in the non-pregnant group. Similarly, levels of inflammatory cytokines/chemokines IL-12(p40), G-CSF, GM-CSF, KC, MCP-1, MIP-1α, MIP-1ß, RANTES and TNF-α in BAL fluid were significantly higher in non-pregnant than pregnant exposed mice. Histopathology evaluation of placentas did not identify any pathological changes. No translocation of Cu into the placenta or the fetus was found by inductively coupled plasma-mass spectroscopy. Expression of several Th1/Th2 or other immune response genes in pups' spleens were found to be significantly up- or down-regulated. CONCLUSIONS: Prenatal exposure to Cu NPs caused a profound pulmonary inflammation in dams and strong immunomodulatory effects in offspring. There was no clear polarization of genes expressed in pups' spleens towards Th1 or Th2 type of response.

Acrolein-exposed normal human lung fibroblasts in vitro: cellular senescence, enhanced telomere erosion, and degradation of Werner's syndrome protein.

BACKGROUND: Acrolein is a ubiquitous environmental hazard to human health. Acrolein has been reported to activate the DNA damage response and induce apoptosis. However, little is known about the effects of acrolein on cellular senescence. OBJECTIVES: We examined whether acrolein induces cellular senescence in cultured normal human lung fibroblasts (NHLF). METHODS: We cultured NHLF in the presence or absence of acrolein and determined the effects of acrolein on cell proliferative capacity, senescence-associated ß-galactosidase activity, the known senescence-inducing pathways (e.g., p53, p21), and telomere length. RESULTS: We found that acrolein induced cellular senescence by increasing both p53 and p21. The knockdown of p53 mediated by small interfering RNA (siRNA) attenuated acrolein-induced cellular senescence. Acrolein decreased Werner's syndrome protein (WRN), a member of the RecQ helicase family involved in DNA repair and telomere maintenance. Acrolein-induced down-regulation of WRN protein was rescued by p53 knockdown or proteasome inhibition. Finally, we found that acrolein accelerated p53-mediated telomere shortening. CONCLUSIONS: These results suggest that acrolein induces p53-mediated cellular senescence accompanied by enhanced telomere attrition and WRN protein down-regulation.

A microRNA processing defect in smokers' macrophages is linked to SUMOylation of the endonuclease DICER.

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Effects of vitamin D supplementation on alveolar macrophage gene expression: preliminary results of a randomized, controlled trial.

BACKGROUND: Vitamin D deficiency has been implicated as a factor in a number of infectious and inflammatory lung diseases. In the lung, alveolar macrophages play a key role in inflammation and defense of infection, but there are little data exploring the immunomodulatory effects of vitamin D on innate lung immunity in humans. The objective of this study was to determine the effects of vitamin D supplementation on gene expression of alveolar macrophages. METHODS: We performed a parallel, double-blind, placebo-controlled, randomized trial to determine the effects of vitamin D on alveolar macrophage gene expression. Vitamin D3 (1000 international units/day) or placebo was administered to adults for three months. Bronchoscopy was performed pre- and post-intervention to obtain alveolar macrophages. Messenger RNA was isolated from the macrophages and subjected to whole genome exon array analysis. The primary outcome was differential gene expression of the alveolar macrophage in response to vitamin D supplementation. Specific genes underwent validation by polymerase chain reaction methods. RESULTS: Fifty-eight subjects were randomized to vitamin D (n = 28) or placebo (n = 30). There was a marginal overall difference between treatment group and placebo group in the change of 25-hydroxyvitaminD levels (4.43 ng/ml vs. 0.2 ng/ml, p = 0.10). Whole genome exon array analysis revealed differential gene expression associated with change in serum vitamin D levels in the treated group. CCL8/MCP-2 was the top-regulated cytokine gene and was further validated. CONCLUSIONS: Although only a non-significant increased trend was seen in serum vitamin D levels, subjects treated with vitamin D supplementation had immune-related differential gene expression in alveolar macrophages. TRIAL REGISTRATION: ClinicalTrials.org: NCT01967628.

Inositol-requiring enzyme 1 inhibits respiratory syncytial virus replication.

Despite being a major health problem, respiratory syncytial virus (RSV) infections remain without specific therapy. Identification of novel host cellular responses that play a role in the pathogenesis of RSV infection is needed for therapeutic development. The endoplasmic reticulum (ER) stress response is an evolutionarily conserved cellular signaling cascade that has been implicated in multiple biological phenomena, including the pathogenesis of some viral infections. In this study, we investigate the role of the ER stress response in RSV infection using an in vitro A549 cell culture model. We found that RSV infection induces a non-canonical ER stress response with preferential activation of the inositol-requiring enzyme 1 (IRE1) and activated transcription factor 6 (ATF6) pathways with no concomitant significant activation of the protein kinase R-like ER kinase (PERK) pathway. Furthermore, we discovered that IRE1 has an inhibitory effect on RSV replication. Our data characterize, for the first time, the nature of the ER stress response in the setting of RSV infection and identify the IRE1 stress pathway as a novel cellular anti-RSV defense mechanism.

Epigenetic silencing of the human NOS2 gene: rethinking the role of nitric oxide in human macrophage inflammatory responses.

Macrophages, including alveolar macrophages, are primary phagocytic cells of the innate immune system. Many studies of macrophages and inflammation have been done in mouse models, in which inducible NO synthase (NOS2) and NO are important components of the inflammatory response. Human macrophages, in contrast to mouse macrophages, express little detectable NOS2 and generate little NO in response to potent inflammatory stimuli. The human NOS2 gene is highly methylated around the NOS2 transcription start site. In contrast, mouse macrophages contain unmethylated cytosine-phosphate-guanine (CpG) dinucleotides proximal to the NOS2 transcription start site. Further analysis of chromatin accessibility and histone modifications demonstrated a closed conformation at the human NOS2 locus and an open conformation at the murine NOS2 locus. In examining the potential for CpG demethylation at the NOS2 locus, we found that the human NOS2 gene was resistant to the effects of demethylation agents both in vitro and in vivo. Our data demonstrate that epigenetic modifications in human macrophages are associated with CpG methylation, chromatin compaction, and histone modifications that effectively silence the NOS2 gene. Taken together, our findings suggest there are significant and underappreciated differences in how murine and human macrophages respond to inflammatory stimuli.

Effects of Eyjafjallajökull volcanic ash on innate immune system responses and bacterial growth in vitro.

BACKGROUND: On 20 March 2010, the Icelandic volcano Eyjafjallajökull erupted for the first time in 190 years. Despite many epidemiological reports showing effects of volcanic ash on the respiratory system, there are limited data evaluating cellular mechanisms involved in the response to ash. Epidemiological studies have observed an increase in respiratory infections in subjects and populations exposed to volcanic eruptions. METHODS: We physicochemically characterized volcanic ash, finding various sizes of particles, as well as the presence of several transition metals, including iron. We examined the effect of Eyjafjallajökull ash on primary rat alveolar epithelial cells and human airway epithelial cells (20-100 µg/cm(2)), primary rat and human alveolar macrophages (5-20 µg/cm(2)), and Pseudomonas aeruginosa (PAO1) growth (3 µg/104 bacteria). RESULTS: Volcanic ash had minimal effect on alveolar and airway epithelial cell integrity. In alveolar macrophages, volcanic ash disrupted pathogen-killing and inflammatory responses. In in vitro bacterial growth models, volcanic ash increased bacterial replication and decreased bacterial killing by antimicrobial peptides. CONCLUSIONS: These results provide potential biological plausibility for epidemiological data that show an association between air pollution exposure and the development of respiratory infections. These data suggest that volcanic ash exposure, while not seriously compromising lung cell function, may be able to impair innate immunity responses in exposed individuals.

Cigarette smoking decreases global microRNA expression in human alveolar macrophages.

Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs) could control, in part, the unique messenger RNA (mRNA) expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory) and M2 (anti-inflammatory) polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an "inverse" M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages.

Coordinated DNA methylation and gene expression changes in smoker alveolar macrophages: specific effects on VEGF receptor 1 expression.

Cigarette smoking is implicated in numerous diseases, including emphysema and lung cancer. The clinical expression of lung disease in smokers is not well explained by currently defined variations in gene expression or simple differences in smoking exposure. Alveolar macrophages play a critical role in the inflammation and remodeling of the lung parenchyma in smoking-related lung disease. Significant gene expression changes in alveolar macrophages from smokers have been identified. However, the mechanism for these changes remains unknown. One potential mechanism for smoking-altered gene expression is via changes in cytosine methylation in DNA regions proximal to gene-coding sequences. In this study, alveolar macrophage DNA from heavy smokers and never smokers was isolated and methylation status at 25,000 loci determined. We found differential methylation in genes from immune-system and inflammatory pathways. Analysis of matching gene expression data demonstrated a parallel enrichment for changes in immune-system and inflammatory pathways. A significant number of genes with smoking-altered mRNA expression had inverse changes in methylation status. One gene highlighted by this data was the FLT1, and further studies found particular up-regulation of a splice variant encoding a soluble inhibitory form of the receptor. In conclusion, chronic cigarette smoke exposure altered DNA methylation in specific gene promoter regions in human alveolar macrophages.

Coordinated changes in AHRR methylation in lymphoblasts and pulmonary macrophages from smokers.

Smoking is associated with a wide variety of adverse health outcomes including cancer, chronic obstructive pulmonary disease, diabetes, depression, and heart disease. Unfortunately, the molecular mechanisms through which these effects are conveyed are not clearly understood. To examine the potential role of epigenetic factors in these processes, we examined the relationship of smoking to genome wide methylation and gene expression using biomaterial from two independent samples, lymphoblast DNA and RNA (n = 119) and lung alveolar macrophage DNA (n = 19). We found that in both samples current smoking status was associated with significant changes in DNA methylation, in particular at the aryl hydrocarbon receptor repressor (AHRR), a known tumor suppressor. Both baseline DNA methylation and smoker associated DNA methylation signatures at AHRR were highly correlated (r = 0.94 and 0.45, respectively). DNA methylation at the most differentially methylated AHRR CpG residue in both samples, cg0557592, was significantly associated with AHRR gene expression. Pathway analysis of lymphoblast data (genes with most significant methylation changes) demonstrated enrichment in protein kinase C pathways and in TGF beta signaling pathways. For alveolar macrophages, pathway analysis demonstrated alterations in inflammation-related processes. We conclude that smoking is associated with functionally significant genome wide changes in DNA methylation in both lymphoblasts and pulmonary macrophages and that further integrated investigations of these epigenetic effects of smoking on carcinogenesis and other related co-morbidities are indicated.

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway.

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.

Induction of inflammasome-dependent pyroptosis by carbon black nanoparticles.

Inhalation of nanoparticles has been implicated in respiratory morbidity and mortality. In particular, carbon black nanoparticles are found in many different environmental exposures. Macrophages take up inhaled nanoparticles and respond via release of inflammatory mediators and in some cases cell death. Based on new data, we propose that exposure of macrophages (both a macrophage cell line and primary human alveolar macrophages) to carbon black nanoparticles induces pyroptosis, an inflammasome-dependent form of cell death. Exposure of macrophages to carbon black nanoparticles resulted in inflammasome activation as defined by cleavage of caspase 1 to its active form and downstream IL-1ß release. The cell death that occurred with carbon black nanoparticle exposure was identified as pyroptosis by the protective effect of a caspase 1 inhibitor and a pyroptosis inhibitor. These data demonstrate that carbon black nanoparticle exposure activates caspase 1, increases IL-1ß release after LPS priming, and induces the proinflammatory cell death, pyroptosis. The identification of pyroptosis as a cellular response to carbon nanoparticle exposure is novel and relates to environmental and health impacts of carbon-based particulates.

Vitamin D effects on lung immunity and respiratory diseases.

Our understanding of vitamin D metabolism and biological effects has grown exponentially in recent years and it has become clear that vitamin D has extensive immunomodulatory effects. The active vitamin D generating enzyme, 1α-hydroxylase, is expressed by the airway epithelium, alveolar macrophages, dendritic cells, and lymphocytes indicating that active vitamin D can be produced locally within the lungs. Vitamin D generated in tissues is responsible for many of the immunomodulatory actions of vitamin D. The effects of vitamin D within the lungs include increased secretion of the antimicrobial peptide cathelicidin, decreased chemokine production, inhibition of dendritic cell activation, and alteration of T-cell activation. These cellular effects are important for host responses against infection and the development of allergic lung diseases like asthma. Epidemiological studies do suggest that vitamin D deficiency predisposes to viral respiratory tract infections and mycobacterial infections and that vitamin D may play a role in the development and treatment of asthma. Randomized, placebo-controlled trials are lacking but ongoing.

Identification of an autophagy defect in smokers' alveolar macrophages.

Alveolar macrophages are essential for clearing bacteria from the alveolar surface and preventing microbe-induced infections. It is well documented that smokers have an increased incidence of infections, in particular lung infections. Alveolar macrophages accumulate in smokers' lungs, but they have a functional immune deficit. In this study, we identify an autophagy defect in smokers' alveolar macrophages. Smokers' alveolar macrophages accumulate both autophagosomes and p62, a marker of autophagic flux. The decrease in the process of autophagy leads to impaired protein aggregate clearance, dysfunctional mitochondria, and defective delivery of bacteria to lysosomes. This study identifies the autophagy pathway as a potential target for interventions designed to decrease infection rates in smokers and possibly in individuals with high environmental particulate exposure.

Respiratory syncytial virus limits alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) phosphorylation to maintain translation and viral replication.

The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR), a cellular kinase relevant to limiting viral replication (Groskreutz, D. J., Monick, M. M., Powers, L. S., Yarovinsky, T. O., Look, D. C., and Hunninghake, G. W. (2006) J. Immunol. 176, 1733-1740). It is activated by autophosphorylation, likely triggered by a double-stranded RNA intermediate during replication of the virus. In most instances, ph-PKR targets the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) protein via phosphorylation, leading to an inhibition of translation of cellular and viral protein. However, we found that although ph-PKR increases in RSV infection, significant eIF2alpha phosphorylation is not observed, and inhibition of protein translation does not occur. RSV infection attenuates eIF2alpha phosphorylation by favoring phosphatase rather than kinase activity. Although PKR is activated, RSV sequesters PKR away from eIF2alpha by binding of the kinase to the RSV N protein. This occurs in conjunction with an increase in the association of the phosphatase, PP2A, with eIF2alpha following PKR activation. The result is limited phosphorylation of eIF2alpha and continued translation of cellular and viral proteins.

Vitamin D decreases respiratory syncytial virus induction of NF-kappaB-linked chemokines and cytokines in airway epithelium while maintaining the antiviral state.

Epidemiological studies suggest that low vitamin D levels may increase the risk or severity of respiratory viral infections. In this study, we examined the effect of vitamin D on respiratory syncytial virus (RSV)-infected human airway epithelial cells. Airway epithelium converts 25-hydroxyvitamin D3 (storage form) to 1,25-dihydroxyvitamin D3 (active form). Active vitamin D, generated locally in tissues, is important for the nonskeletal actions of vitamin D, including its effects on immune responses. We found that vitamin D induces IkappaBalpha, an NF-kappaB inhibitor, in airway epithelium and decreases RSV induction of NF-kappaB-driven genes such as IFN-beta and CXCL10. We also found that exposing airway epithelial cells to vitamin D reduced induction of IFN-stimulated proteins with important antiviral activity (e.g., myxovirus resistance A and IFN-stimulated protein of 15 kDa). In contrast to RSV-induced gene expression, vitamin D had no effect on IFN signaling, and isolated IFN induced gene expression. Inhibiting NF-kappaB with an adenovirus vector that expressed a nondegradable form of IkappaBalpha mimicked the effects of vitamin D. When the vitamin D receptor was silenced with small interfering RNA, the vitamin D effects were abolished. Most importantly we found that, despite inducing IkappaBalpha and dampening chemokines and IFN-beta, there was no increase in viral mRNA or protein or in viral replication. We conclude that vitamin D decreases the inflammatory response to viral infections in airway epithelium without jeopardizing viral clearance. This suggests that adequate vitamin D levels would contribute to reduced inflammation and less severe disease in RSV-infected individuals.

Cigarette smoke alters respiratory syncytial virus-induced apoptosis and replication.

Individuals exposed to cigarette smoke have a greater number and severity of viral infections, including respiratory syncytial virus (RSV) infections, than do nonsmokers, but the cellular mechanism is unknown. Our objective was to determine the mechanism by which cigarette smoke augments viral infection. We hypothesize that cigarette smoke causes necrosis and prevents virus-induced cellular apoptosis, and that this is associated with increased inflammation and viral replication. Primary airway epithelial cells were exposed to cigarette smoke extract for 2 days, followed by 1 day of RSV exposure. Western blot detection of cleaved caspases 3 and 7 showed less apoptosis when cells were treated with cigarette smoke before viral infection. This finding was confirmed with ELISA and TUNEL detection of apoptosis. Measures of cell viability, including propidium iodide staining, ATP assay, and cell counts, indicated that cigarette smoke causes necrosis rather than virus-induced apoptosis. Using plaque assay and fluorescently-labeled RSV, we showed that although there were less live cells in the cigarette smoke-pretreated group, viral load was increased. The effect was inhibited by pretreatment of cells with N-acetylcysteine and aldehyde dehydrogenase, suggesting that the effect was primarily mediated by reactive aldehydes. Cigarette smoke causes necrosis rather than apoptosis in viral infection, resulting in increased inflammation and enhanced viral replication.

Cigarette smoke induces cellular senescence via Werner's syndrome protein down-regulation.

RATIONALE: Werner's syndrome is a genetic disorder that causes premature aging due to loss-of-function mutations in a gene encoding a member of the RecQ helicase family. Both Werner's syndrome and cigarette smoking accelerate aging. No studies have examined the effect of cigarette smoke on Werner's syndrome protein. OBJECTIVES: To investigate the role of Werner's syndrome protein in cigarette smoke-induced cellular senescence. METHODS: Cellular senescence and amounts of Werner's syndrome protein were measured in fibroblasts isolated from patients with emphysema and compared with age-matched nonsmokers. The in vitro effects of cigarette smoke on amounts of Werner's syndrome protein, function, and senescence were also evaluated in primary human lung fibroblasts and epithelial cells. MEASUREMENTS AND MAIN RESULTS: Cultured lung fibroblasts isolated from patients with emphysema exhibited a senescent phenotype accompanied by a decrease in Werner's syndrome protein. Cigarette smoke extract decreased Werner's syndrome protein in cultured fibroblasts and epithelial cells. Werner's syndrome protein-deficient fibroblasts were more susceptible to cigarette smoke-induced cellular senescence and cell migration impairment. In contrast, exogenous overexpression of Werner's syndrome protein attenuated the cigarette smoke effects. CONCLUSIONS: Cigarette smoke induces cellular senescence and cell migration impairment via Werner's syndrome protein down-regulation. Rescue of Werner's syndrome protein down-regulation may represent a potential therapeutic target for smoking-related diseases.

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.