IL-10 is a key regulator of the immune system that critically determines health and disease. Its expression is finely tuned both at the transcriptional and posttranscriptional levels. Although the importance of posttranscriptional regulation of IL-10 has been previously shown, understanding the underlying mechanisms is still in its infancy. In this study, using a combination of bioinformatics and molecular approaches, we report that microRNA (hsamiR-106a) regulates IL-10 expression. The hsa-miR-106a binding site in the 3 UTR of IL10 has been identified by site-directed mutagenesis studies. Also, the involvement of transcription factors, Sp1 and Egr1, in the regulation of hsa-miR-106a expression and concomitant decrease in the IL-10 expression, has also been demonstrated. In summary, our results showed that IL-10 expression may be regulated by miR-106a, which is in turn transcriptionally regulated by Egr1 and Sp1.Egr1 ͉ IL-10 ͉ microRNA ͉ SP1 ͉ miR-106a promoter

Airway epithelial injury is the hallmark of various respiratory diseases, but its mechanisms remain poorly understood. While 13-S-hydroxyoctadecadienoic acid (13-S-HODE) is produced in high concentration during mitochondrial degradation in reticulocytes little is known about its role in asthma pathogenesis. Here, we show that extracellular 13-S-HODE induces mitochondrial dysfunction and airway epithelial apoptosis. This is associated with features of severe airway obstruction, lung remodeling, increase in epithelial stress related proinflammatory cytokines and drastic airway neutrophilia in mouse. Further, 13-S-HODE induced features are attenuated by inhibiting Transient Receptor Potential Cation Channel, Vanilloid-type 1 (TRPV1) both in mouse model and human bronchial epithelial cells. These findings are relevant to human asthma, as 13-S-HODE levels are increased in human asthmatic airways. Blocking of 13-S-HODE activity or disruption of TRPV1 activity attenuated airway injury and asthma mimicking features in murine allergic airway inflammation. These findings indicate that 13-S-HODE induces mitochondrial dysfunction and airway epithelial injury.

MicroRNAs (miRs) regulate immunological pathways in health and disease, and a number of miRs have been shown to be altered in mouse models of asthma. The secretion of interleukin-10 (IL-10), an anti-inflammatory cytokine, has been shown to be defective in many inflammatory diseases including asthma. We recently demonstrated that miR-106a inhibits IL-10 in a post-transcriptional manner. In this study, we investigated the effect of inhibition of mmu-miR106a in asthmatic condition to find its possible role as a therapeutic target. Our in vitro experiments with mouse macrophage, RAW264.7, revealed that mmu-miR-106a potentially decreased IL-10 along with increase in proinflammatory cytokine. Furthermore, administration of mmu-miR-106a to naive mice reduced IL-10 levels in lungs in a dose-dependent manner without altering lung histology. Most interestingly, knockdown of mmu-miR-106a in an established allergic airway inflammation has significantly alleviated most of the features of asthma such as airway hyperresponsiveness, airway inflammation, increased Th2 response, goblet cell metaplasia, and subepithelial fibrosis along with increase in IL-10 levels in lung. This represents the first in vivo proof of a miRNA-mediated regulation of IL-10 with a potential to reverse an established asthmatic condition.

Altered arginine metabolism, the uncoupling of nitric oxide synthase (NOS) by asymmetric dimethyl-arginine (ADMA), increased oxo-nitrosative stress, and cellular injury were reported in airway epithelial cells in asthma. Statins improve vascular endothelial dysfunction by reducing ADMA and increasing endothelial NOS (eNOS), thereby reducing oxo-nitrosative stress in cardiovascular diseases. Whether statin therapy leads to similar beneficial effects in lung epithelium in asthma is unknown. The effects of simvastatin therapy after sensitization (40 mg/kg, intraperitoneally) on markers of arginine and NO metabolism and features of asthma were ascertained in a murine model of allergic asthma. The effects of simvastatin on the expression of NOS in A549 lung epithelial cells were studied in vitro. Simvastatin induced eNOS in lung epithelial cells in vitro. In acute and chronic models of asthma, simvastatin therapy was associated with significantly reduced airway inflammation, airway hyperresponsiveness, and airway remodeling. ADMA and inducible nitric oxide synthase were reduced by simvastatin, but eNOS was increased. A marked reduction of nitrotyrosine, a marker of oxo-nitrosative stress, was evident in airway epithelium. Cell injury markers such as cytosolic cytochrome c, caspases 3 and 9 and apoptotic protease activating factor 1 (Apaf-1) were also reduced. Simvastatin improves dysfunctional nitric oxide metabolism in allergically inflamed lungs. Important pleiotropic mechanisms may be responsible for the statin-induced reduction of airway inflammation, epithelial injury, and airway hyperresponsiveness.

We showed recently that IL-4 causes mitochondrial dysfunction in allergic asthma. IL-4 is also known to induce 12/15-lipoxygenase (12/15-LOX), a potent candidate molecule in asthma. Because vitamin E (Vit-E) reduces IL-4 and inhibits 12/15-LOX in vitro, here we tested the hypothesis that Vit-E may be effective in restoring key mitochondrial dysfunctions, thus alleviating asthma features in an experimental allergic murine model. Ovalbumin (OVA)-sensitized and challenged male BALB/c mice showed the characteristic features of asthma such as airway hyperresponsiveness (AHR), airway inflammation, and airway remodeling. In addition, these mice showed increase in the expression and metabolites of 12/15-LOX, reduction in the activity and expression of the third subunit of mitochondrial cytochrome-c oxidase, and increased cytochrome c in lung cytosol, which indicate that OVA sensitization and challenge causes mitochondrial dysfunction. Vit-E was administered orally to these mice, and 12/15-LOX expression, key mitochondrial functions, ultrastructural changes of mitochondria in bronchial epithelia, and asthmatic parameters were determined. Vit-E treatment reduced AHR, Th2 response including IL-4, IL-5, IL-13, and OVA-specific IgE, eotaxin, transforming growth factor-beta1, airway inflammation, expression and metabolites of 12/15-LOX in lung cytosol, lipid peroxidation, and nitric oxide metabolites in the lung, restored the activity and expression of the third subunit of cytochrome-c oxidase in lung mitochondria and bronchial epithelia, respectively, reduced the appearance of cytochrome c in lung cytosol, and also restored mitochondrial ultrastructural changes of bronchial epithelia. In summary, these findings show that Vit-E reduces key mitochondrial dysfunctions and alleviates asthmatic features.