Role of nicotinamide adenine dinucleotide phosphate oxidase in mediating vesicant-induced interleukin-6 secretion in human airway epithelial cells.

Aerosolized exposure to the chemical warfare vesicant sulfur mustard and its analog nitrogen mustard (HN2) is known to induce airway lesions associated with secretion of proinflammatory cytokines such as IL-6. We have shown recently that HN2 challenge induced IL-6 secretion in human airway epithelial cells, a process mediated via epidermal growth factor receptor (EGFR) signaling. In this study, we evaluated the role of redox signaling in regulating HN2-induced, EGFR-mediated IL-6 secretions in primary cultured normal human bronchial epithelial cells (NHBECs) in the air-liquid interface. HN2-induced EGFR phosphorylation and IL-6 secretion in NHBECs were inhibited by the antioxidant N-acetyl-L-cysteine (NAC) and by the flavoprotein inhibitor diphenyleneiodonium chloride (DPI). These observations suggested that the inflammatory response in NHBECs after HN2 challenge was mediated via oxidative stress. HN2 exposure induced increased reactive oxygen species (ROS) formation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in NHBECs, findings that were inhibited by NAC and DPI treatment. Among NADPH oxidase isoforms, mRNA expression of dual oxidase (DUOX)1 and DUOX2 were up-regulated by HN2. Furthermore, knockdown of DUOX1 or DUOX2 by short hairpin RNA resulted in inhibition of ROS generation, EGFR pathway activation, and IL-6 secretion in NHBECs. These results provide evidence that redox signaling plays a pivotal role in the HN2-induced airway inflammation and underscore the importance of DUOX1 and DUOX2 in vesicant-induced IL-6 secretion in human airway epithelial cells.

Asbestos-induced mesothelioma: is fiber biopersistence really a critical factor?

This Commentary highlights the research by Qi et al detailing the similarities and differences between crocidolite and chrysotile asbestos in terms of their transcriptional effects and transforming actions in human mesothelial cells.

Epidermal growth factor receptor signaling mediates vesicant-induced airway epithelial secretion of interleukin-6 and production of mucin.

The chemical warfare agent sulfur mustard (HD) and its analogue nitrogen mustard (HN2) are highly reactive vesicants that can cause airway epithelial injury. However, little is known about the mechanisms governing vesicant-related airway damage. This study assessed the role of epidermal growth factor receptor (EGFR) signaling in mediating the effects of exposure to vesicants on the secretion of cytokines and production of mucin in human airway epithelial cells. Normal human bronchial epithelial cells (NHBECs) at an air-liquid interface were challenged apically with either 200 µM HN2 or medium alone (mock treatment, MT), and cultures were evaluated for receptor fate, the secretion of IL-6, and the production of both total mucin and Mucin 5AC (MUC5AC). Exposure to HN2 induced the activation of both EGFR and (44/42)mitogen-activated protein kinase ((44/42)MAPK), as well as the ubiquitination and colocalization of EGFR within lysosomal structures. Moreover, challenge with HN2 induced the up-regulation of IL-6 and MUC5AC at the mRNA and protein levels, and stimulated the secretion of total mucin in NHBECs. HN2-related effects on the secretion of IL-6 and the production of total mucin and MUC5AC were reversed by the selective EGFR inhibitor AG1478 and by an EGFR-blocking antibody. The HN2-induced activation of (44/42)MAPK and the up-regulation of IL-6 secretion in NHBECs were also largely reversed by a transforming growth factor-α (TGF-α)-blocking antibody and by the metalloprotease inhibitor GM 6001, suggesting that the HN2-related effects on EGFR signaling were TGF-α-dependent. Collectively, these findings suggest that EGFR signaling may play a significant role in mediating vesicant-induced airway epithelial injury.

A clinically relevant in vitro model for evaluating the effects of aerosolized vesicants.

The chemical warfare vesicant sulfur mustard (HD) is a known toxic agent to the human respiratory tract and the major airways are considered to be a primary target of HD-induced injury. However, there is no consensus regarding which model systems are most appropriate for studying the effects of aerosolized vesicants on human airway epithelium. In this study, we evaluated the consequences of exposure of differentiated human respiratory epithelial cells in air-liquid interface to mechlorethamine (HN2), an HD functional analog. HN2 challenge was administered via the apical (air) interface over a wide dose range (20-400 microM) to differentiated HBE1 cells. Cultures were observed over 1-48 h for evidence of HN2-induced morphologic abnormalities as well as for possible cellular cytotoxicity, apoptotic changes, and induction of cytokine secretion. HN2 at concentrations of > or =200 microM caused disruption and denudation of the airway epithelial architecture within 24h of exposure. Moreover, HN2-induced cytotoxic and apoptotic changes in HBE1 cells in a dose- and time-dependent fashion. HN2 challenge also induced secretion of chemokines and proinflammatory cytokines including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, IL-8, RANTES, MCP-1, IP-10, G-CSF, GM-CSF and IL-15. These observations parallel those described in the lungs of HD-exposed victims and underscore the utility and potential applicability of this model to future mechanistic studies of vesicant-induced pulmonary injury.

Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference.

BACKGROUND: Ebola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs). METHODS: Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid-lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge. RESULTS: Treatment of guinea pigs with a pool of the L gene-specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge. CONCLUSIONS: Further development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats.

RNA interference targeting Akt promotes apoptosis in hypoxia-exposed human neuroblastoma cells.

Overactivation of the PI3 kinase/Akt pathway plays an essential role in the development and progression of various tumors. Akt is a key component of this pathway and hyperactivated in different tumors including neuroblastoma and glioma. In the present study, we tested the therapeutic efficacy of siRNA targeting Akt in inducing apoptotic cell death in NBFL cells (a human neuroblastoma cell line) subjected to anoxia/reoxygenation (A/R), a process that has been shown to modulate growth and progression of malignant tumors. We observed that siRNA targeting Akt effectively induced apoptotic cell death in NBFL cells (as determined by TUNEL assay and activated caspase-3 immunoreactivity) under normoxic conditions, an effect that was greatly enhanced under conditions of A/R. These findings underscore the importance of Akt signaling in promoting survival of neuroblastoma cells and may have potential therapeutic applications.

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression.

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.

Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.

Disseminated intravascular coagulation is a prominent manifestation of Ebola virus (EBOV) infection. Here, we report that tissue factor (TF) plays an important role in triggering the hemorrhagic complications that characterize EBOV infections. Analysis of samples obtained from 25 macaques showed increased levels of TF associated with lymphoid macrophages, whereas analysis of peripheral blood-cell RNA showed increased levels of TF transcripts by day 3. Plasma from macaques contained increased numbers of TF-expressing membrane microparticles. Dysregulation of the fibrinolytic system developed during the course of infection, including a rapid decrease in plasma levels of protein C. Infection of primary human monocytes/macrophages (PHMs) was used to further evaluate the role of TF in EBOV infections. Analysis of PHM RNA at 1-48 h showed increased TF transcripts, whereas levels of TF protein were dramatically increased by day 2. Thus, chemotherapeutic strategies aimed at controlling overexpression of TF may ameliorate the effects of EBOV hemorrhagic fever.

Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.

Ebola virus (EBOV) infection causes a severe and fatal hemorrhagic disease that in many ways appears to be similar in humans and nonhuman primates; however, little is known about the development of EBOV hemorrhagic fever. In the present study, 21 cynomolgus monkeys were experimentally infected with EBOV and examined sequentially over a 6-day period to investigate the pathological events of EBOV infection that lead to death. Importantly, dendritic cells in lymphoid tissues were identified as early and sustained targets of EBOV, implicating their important role in the immunosuppression characteristic of EBOV infections. Bystander lymphocyte apoptosis, previously described in end-stage tissues, occurred early in the disease-course in intravascular and extravascular locations. Of note, apoptosis and loss of NK cells was a prominent finding, suggesting the importance of innate immunity in determining the fate of the host. Analysis of peripheral blood mononuclear cell gene expression showed temporal increases in tumor necrosis factor-related apoptosis-inducing ligand and Fas transcripts, revealing a possible mechanism for the observed bystander apoptosis, while up-regulation of NAIP and cIAP2 mRNA suggest that EBOV has evolved additional mechanisms to resist host defenses by inducing protective transcripts in cells that it infects. The sequence of pathogenetic events identified in this study should provide new targets for rational prophylactic and chemotherapeutic interventions.

Asbestos inhalation induces tyrosine nitration associated with extracellular signal-regulated kinase 1/2 activation in the rat lung.

Nitration of proteins by peroxynitrite (ONOO-) has been shown to critically alter protein function in vitro. We have shown previously that asbestos inhalation induced nitrotyrosine formation, a marker of ONOO- production, in the rat lung. To determine whether asbestos-induced protein nitration may affect mitogen-activated protein kinase (MAPK) signaling pathways, lung lysates from crocidolite and chrysotile asbestos-exposed rats and from sham-exposed rats were immunoprecipitated with anti-nitrotyrosine antibody, and captured proteins were subjected to Western blotting with anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. Both types of asbestos inhalation induced significantly greater phosphorylation of ERK1/2 in rat lung lysates than was noted after sham exposure. Phosphorylated ERK proteins co-immunoprecipitated with nitrotyrosine. Moreover, in MAPK functional assays using Elk-1 substrate, immunoprecipitated phospho-ERK1/2 in lung lysates from both crocidolite-exposed and chrysotile-exposed rats demonstrated significantly greater phosphorylation of Elk-1 than was noted after sham exposure. Asbestos inhalation also induced ERK phosphorylation in bronchoalveolar lavage cells. Lung sections from rats exposed to crocidolite or chrysotile (but not from sham-exposed rats nor from rats exposed to "inert" carbonyl iron particles) demonstrated strong immunoreactivity for nitrotyrosine and phospho-ERK1/2 in alveolar macrophages and bronchiolar epithelium. These findings suggest that asbestos fibers may activate the ERK signaling pathway by generating ONOO- or other nitrating species that induce tyrosine nitration and phosphorylation of critical signaling molecules.