Post-transcriptional regulation of gene expression by microRNAs has been implicated in the regulation of chronic physiological and pathological responses. In this report, we demonstrate that changes in the expression of microRNAs (miRNAs) can also regulate acute inflammatory responses in human lung alveolar epithelial cells. Thus, stimulation with interleukin (IL)-1β results in a rapid time- and concentration-dependent increase in miRNA-146a and, to a lesser extent, miRNA-146b expression although these increases were only observed at high IL-1β concentration. Examination of miRNA function by over-expression and inhibition showed that increased miRNA-146a expression negatively regulated the release of the pro-inflammatory chemokines, IL-8 and RANTES. Subsequent examination of the mechanism demonstrated that the action of miRNA-146a was mediated at the translational level and not through down-regulation of proteins involved in the IL-1β signalling pathway or chemokine transcription or secretion. Overall, these studies indicate that rapid increase in miRNA-146a expression provides a novel mechanism for negative regulation of severe inflammation during the innate immune response.

The therapeutic application of siRNA shows promise as an alternative approach to small molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the non-viral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line, showed that siRNA conjugated to cholesterol, TAT(48-60) and penetratin but not siRNA alone achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localisation within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6hrs. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown whilst penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression whilst the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA mediated p38 Figure S1, dose-dependent percentile p38 MAP kinase mRNA levels in vitro following lipofection with three mouse p38 MAP kinase siRNA or two mismatch controls; Figure S2, analytical HPLC and electrospray mass spectrograms of peptide-and cholesteryl-RNA constructs; Figure S3, analytical gels of CPP and cholesterol conjugate annealing products; Figure S4, cell viability following incubation with siRNA, CPP or siRNA conjugates; Figure S5, MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.