Glucocorticoids are steroid hormones that are secreted upon stress. Their effects are mediated by the glucocorticoid receptor, which acts as a transcription factor. Because the antiinflammatory... Show moreGlucocorticoids are steroid hormones that are secreted upon stress. Their effects are mediated by the glucocorticoid receptor, which acts as a transcription factor. Because the antiinflammatory activity of glucocorticoids has been well established, they are widely used clinically to treat many inflammatory and immune-related diseases. However, the exact specificity, mechanisms, and level of regulation of different inflammatory pathways have not been fully elucidated. In the present study, a tail fin amputation assay was used in 3-day-old zebrafish larvae to study the immunomodulatory effects of the synthetic glucocorticoid beclomethasone. First, a transcriptome analysis was performed, which showed that upon amputation mainly immune-related genes are regulated. This regulation was inhibited by beclomethasone for 86% of regulated genes. For two immune-related genes, tlr4bb and alox5ap, the amputation-induced increase was not attenuated by beclomethasone. Alox5ap is involved in eicosanoid biosynthesis, but the increase in leukotriene B4 concentration upon amputation was abolished, and lipoxin A4 levels were unaffected by beclomethasone. Furthermore, we studied the migration of neutrophils and macrophages toward the wound site. Our results show that amputation induced migration of both types of leukocytes and that this migration was dependent on de novo protein synthesis. Beclomethasone treatment attenuated the migratory behavior of neutrophils in a glucocorticoid receptor-dependent manner but left the migration of macrophages unaffected. In conclusion, beclomethasone has a dramatic inhibitory effect on the amputation-induced proinflammatory gene regulation, and this is reflected in an inhibition of the neutrophil migration but not the migration of macrophages, which are likely to be involved in inflammation resolution. Show less
Glucocorticoid receptor (GR) signaling is versatile and plays an essential role in the survival and well-being of organisms. Glucocorticoids are also widely used clinically in order to combat... Show moreGlucocorticoid receptor (GR) signaling is versatile and plays an essential role in the survival and well-being of organisms. Glucocorticoids are also widely used clinically in order to combat inflammatory medical conditions. In the present thesis we aimed at studying the GR pathway by means of stimulation with synthetic GCs and genetic manipulation. Since the GR is a transcription factor, our main readout for GR function in most of our experimental settings was transcriptome analysis. By gathering whole transcriptome information, we aimed at unraveling the molecular pathways affected by GR signaling in different physiological conditions, thus exploring its functional role. As a model organism we employed the zebrafish, since it allows fine genetic, molecular and cellular experimental approaches and its GR pathway closely resembles that of humans. Our aim was also to further characterize the function of this versatile signaling cascade in zebrafish, in order to establish this animal model as a valid system for detailed as well as high throughput research on GR, enabling us to test hypotheses and complement results obtained from other well-established experimental animal models such as rodents. Show less
