This was the third year of the project. During this year: 1. We carried out a clinical study (16-I-0126) aimed at understanding the genomic response to glucocorticoids in human immune and non-immune cells in vivo. This is the first study ever performed to evaluate the genomic response to glucocorticoids in highly pure immune cell sub-populations in vivo. This year, we recruited an initial cohort of healthy volunteers for the study. The study was performed at the NIH Clinical Center Day Hospital. Volunteers first received a thorough clinical evaluation, to ensure their eligibility for the study. Those who were eligible were brought back to the NIH Clinical Center Day Hospital for an infusion visit. During the infusion visit, each volunteer received a single intravenous dose of the glucocorticoid methylprednisolone. We then performed serial sampling (before the infusion, and 2 and 4 hours after) to study the in vivo genomic response to the glucocorticoid in six immune cell types (B lymphocytes, CD4+ and CD8+ T lymphocytes, monocytes, neutrophils, and NK cells) and one primarily non-immune tissue (skin). We also banked serial samples of serum and peripheral blood mononuclear cells from each individual. Finally, we obtained fibroblast cultures for long-term storage. The study was completed successfully. Sampling was completed as planned. There were no serious adverse events or serious protocol deviations. To ensure the availability and reproducibility of the study, all clinical data was collected in a structured format, using custom templates in the Clinical Research Information Management System of NIAID (CRIMSON). Similarly, all samples banked were de-identified, barcode-labeled, and tracked using the Biological Specimen Inventory (BSI) system. We expect the results of this initial human study to complement the in vitro work of the past two years, advancing towards our goal of generating a detailed understanding of the genes and pathways that are responsible for the anti-inflammatory and immunosuppressive effects of glucocorticoids, and how these differ from the effects of this group of drugs on non-immune cells. 2. We advanced our analysis of the genomic response of five primary human immune cells (B lymphocytes, CD4+ and CD8+ T lymphocytes, monocytes, and neutrophils) and six primary human non-immune cells (fibroblasts, endothelial cells, myoblasts, osteoblasts, pre-adipocytes, and mature adipocytes) exposed in vitro to the glucocorticoid methylprednisolone and sampled serially after the exposure. Through this work, we have identified the genes and pathways that respond to the glucocorticoid stimulus in each cell type. We have also identified genes with known immune function that respond to the glucocorticoid stimulus in each immune cell type, but not in the non-immune cell types. Furthermore, we have identified genes that respond to the glucocorticoid stimulus in immune cells, but are not expressed at all in the non-immune cells studied. These genes, and the molecular pathways in which they are involved, represent an initial set of potential targets for future therapeutic interventions aimed at reproducing the rapid and potent anti-inflammatory and immunosuppressive actions of glucocorticoids, without the extensive side effects that glucocorticoids have on non-immune tissues. 4. We developed a method for reliably and reproducibly evaluating the response of unstimulated and stimulated human whole blood to glucocorticoids in vitro. This method will enhance our ability to preform pre-clinical assessments of the response to glucocorticoids under diverse conditions, at the RNA and protein levels. 3. In collaboration with Drs. Amy Klion (NIAID), Cynthia Dunbar (NHLBI), and Peter Choyke (NCI), we continued our study of the effects of glucocorticoids on human eosinophils. Glucocorticoids are extensively used to treat human diseases that involve increased numbers or activity of eosinophils, but similar to other indications their exact mechanism of action in patients with eosinophilia remains unknown. We began this work by recruiting an initial cohort of five patients with benign hypereosinophilia, a condition in which circulating levels of eosinophils are elevated. We administered a single dose of the glucocorticoid prednisone and described the kinetics of the observed drop in circulating eosinophils, finding a consistent pattern of decline between two and three hours after administration. It remains unclear whether this drop occurs in response to molecular changes within the eosinophils or in other cell types (endothelial or reticuloendothelial cells, for example). To understand the molecular changes that occur within eosinophils in the two hours preceding the observed decline, we performed sequencing of total RNA in eosinophils from these five subjects, after serial sampling. This analysis revealed an interesting set of candidate genes, which are involved in leukocyte trafficking and change significantly in eosinophils after exposure to the glucocorticoid. Our data do not suggest that eosinophils undergo apoptosis before they leave the peripheral circulation, and the question of where eosinophils go once they leave the circulating compartment after the glucocorticoid dose remains unanswered. Before we address this in humans, we need an appropriate animal model. Mouse eosinophils, unfortunately, do not show a similar response to glucocorticoids compared to humans. We have documented, however, that rhesus macaque eosinophils do. As part of this collaboration, we have developed a method for purification and radiolabeling of macaque eosinophils, and we are preparing a live cell-tracking experiment in rhesus macaques exposed in vivo to a single dose of prednisone. We expect this combination of genomic and live-imaging data to give us an unprecedented view of the action of glucocorticoids on eosinophils.
| Khoury, P; Stokes, K; Gadkari, M et al. (2018) Glucocorticoid-induced eosinopenia in humans can be linked to early transcriptional events. Allergy 73:2076-2079 |
| Magoulas, Pilar L; Shchelochkov, Oleg A; Bainbridge, Matthew N et al. (2018) Syndromic congenital myelofibrosis associated with a loss-of-function variant in RBSN. Blood 132:658-662 |
