Inhalable siRNA: potential as a therapeutic agent in the lungs.

RNA interference (RNAi) is gaining increasing popularity both as a molecular biology tool and as a potential therapeutic agent. RNAi is a naturally occurring gene regulatory mechanism, which has a number of advantages over other gene/antisense therapies including specificity of inhibition, potency, the small size of the molecules and the diminished risk of toxic effects, e.g., immune responses. Targeted, local delivery of RNAi to the lungs via inhalation offers a unique opportunity to treat a range of previously untreatable or poorly controlled respiratory conditions. In this timely review we look at the potential applications of RNAi in the lungs for the treatment of a range of diseases including inflammatory and immune conditions, cystic fibrosis, infectious disease and cancer. In 2006 Alnylam initiated the first phase 1 clinical study of an inhaled siRNA for the treatment of respiratory syncytial virus. If its potential as a therapeutic is to be realized, then safe and efficient means of targeted delivery of small interfering RNA (siRNA) to the lungs must be developed. Therefore in this review we also present the latest developments in siRNA delivery to airway cells in vitro and the work to date on in vivo delivery of siRNA to the lungs for the treatment of a range of diseases.

Eosinophil-mediated cholinergic nerve remodeling.

Eosinophils are observed to localize to cholinergic nerves in a variety of inflammatory conditions such as asthma, rhinitis, eosinophilic gastroenteritis, and inflammatory bowel disease, where they are also responsible for the induction of cell signaling. We hypothesized that a consequence of eosinophil localization to cholinergic nerves would involve a neural remodeling process. Eosinophil co-culture with cholinergic IMR32 cells led to increased expression of the M2 muscarinic receptor, with this induction being mediated via an adhesion-dependent release of eosinophil proteins, including major basic protein and nerve growth factor. Studies on the promoter sequence of the M2 receptor indicated that this induction was initiated at a transcription start site 145 kb upstream of the gene-coding region. This promoter site contains binding sites for a variety of transcription factors including SP1, AP1, and AP2. Eosinophils also induced the expression of several cholinergic genes involved in the synthesis, storage, and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase, and acetylcholinesterase. The observed eosinophil-induced changes in enzyme content were associated with a reduction in intracellular neural acetylcholine but an increase in choline content, suggesting increased acetylcholine turnover and a reduction in acetylcholinesterase activity, in turn suggesting reduced catabolism of acetylcholine. Together these data suggest that eosinophil localization to cholinergic nerves induces neural remodeling, promoting a cholinergic phenotype.

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells.

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.

Diverse effects of eosinophil cationic granule proteins on IMR-32 nerve cell signaling and survival.

Activated eosinophils release potentially toxic cationic granular proteins, including the major basic proteins (MBP) and eosinophil-derived neurotoxin (EDN). However, in inflammatory conditions including asthma and inflammatory bowel disease, localization of eosinophils to nerves is associated with nerve plasticity, specifically remodeling. In previous in vitro studies, we have shown that eosinophil adhesion to IMR-32 nerve cells, via nerve cell intercellular adhesion molecule-1, results in an adhesion-dependent release of granule proteins. We hypothesized that released eosinophil granule proteins may affect nerve cell signaling and survival, leading to nerve cell remodeling. Culture in serum-deprived media induced apoptosis in IMR-32 cells that was dose-dependently abolished by inclusion of MBP1 but not by EDN. Both MBP1 and EDN induced phosphorylation of Akt, but with divergent time courses and intensities, and survival was independent of Akt. MBP1 induced activation of neural nuclear factor (NF)-kappaB, from 10 min to 12 h, declining by 24 h, whereas EDN induced a short-lived activation of NF-kappaB. MBP1-induced protection was dependent on phosphorylation of ERK 1/2 and was related to a phospho-ERK-dependent upregulation of the NF-kappaB-activated anti-apoptotic gene, Bfl-1. This signaling pathway was not activated by EDN. Thus, MBP1 released from eosinophils at inflammatory sites may regulate peripheral nerve plasticity by inhibiting apoptosis.

Eosinophil-induced release of acetylcholine from differentiated cholinergic nerve cells.

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 +/- 2% of control at 10(-7) M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 +/- 23% of control at 10(-5) M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 +/- 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 +/- 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.

Eosinophil adhesion to cholinergic IMR-32 cells protects against induced neuronal apoptosis.

Eosinophils release a number of mediators that are potentially toxic to nerve cells. However, in a number of inflammatory conditions, such as asthma and inflammatory bowel disease, it has been shown that eosinophils localize to nerves, and this is associated with enhanced nerve activity. In in vitro studies, we have shown that eosinophil adhesion via neuronal ICAM-1 leads to activation of neuronal NF-kappaB via an ERK1/2-dependent pathway. In this study, we tested the hypothesis that eosinophil adhesion to nerves promotes neural survival by protection from inflammation-associated apoptosis. Exposure of differentiated IMR-32 cholinergic nerve cells to IL-1beta, TNF-alpha, and IFN-gamma, or culture in serum-deprived medium, induced neuronal apoptosis, as detected by annexin V staining, caspase-3 activation, and DNA laddering. Addition of human eosinophils to IMR-32 nerve cells completely prevented all these features of apoptosis. The mechanism of protection by eosinophils was by an adhesion-dependent activation of ERK1/2, which led to the induced expression of the antiapoptotic gene bfl-1. Adhesion to nerve cells did not influence the expression of the related genes bax and bad. Thus, prevention of apoptosis by eosinophils may be a mechanism by which these cells regulate neural plasticity in the peripheral nervous system.

Effect of eosinophil adhesion on intracellular signaling in cholinergic nerve cells.

Eosinophil localization to cholinergic nerves occurs in a variety of inflammatory conditions, including asthma. This localization is mediated by interactions between eosinophil integrins and neuronal vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Eosinophil-nerve cell interactions lead to generation of neuronal reactive oxygen species and release of eosinophil proteins. The effects of eosinophil adhesion on neuronal intracellular signaling pathways were investigated. Eosinophil adhesion to IMR32 cholinergic nerves led to a rapid and sustained activation of the nuclear transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1 in the nerve cells. Eosinophil binding to neuronal ICAM-1 led to a rapid activation of ERK1/2 in nerve cells. Inhibition of ERK1/2 prevented NF-kappaB activation. Eosinophil adhesion to VCAM-1 resulted in AP-1 activation, mediated partially by rapid activation of the p38 mitogen-activated protein kinase. These data show that adhesion of eosinophils induces mitogen-activated protein kinase-dependent activation of the transcription factors NF-kappaB and AP-1 in nerve cells, indicating that eosinophil adhesion may control nerve growth and phenotype.