of the key scientific contributions made by our laboratory over the past year: 1. The human response to glucocorticoids is highly cell-type-dependent: 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. We also began our analysis of data obtained from our clinical study of the in vivo response to glucocorticoid administration in six immune cell types (B lymphocytes, CD4+ and CD8+ T lymphocytes, monocytes, neutrophils, and NK cells) and one primarily non-immune tissue (skin) obtained from 20 healthy human volunteers studied at the NIH Clinical Center. Our results suggest that the genomic response to glucocorticoids is highly cell-type-dependent. We have found that not only the magnitude but also the direction of transcriptional regulation by glucocorticoids can be different across cell types. These results have important implications. For the past seven decades, the field of glucocorticoid biology has relied heavily on immortalized and cancer cell lines, animal models, and highly mixed populations of primary human cells. While this has provided valuable insight into the molecular biology of glucocorticoid signaling, our results suggest that additional information at the level of individual cell types is needed to develop a more accurate understanding of the effects of glucocorticoids on the immune system, as observations made in one cell type are unlikely to apply to others. Our work broadens the range of human cells in which glucocorticoid responses have been studied and focuses on highly purified populations of human primary cells, which are more likely to yield medically relevant information. Over the past year, this work has been published. 2. Glucocorticoids impair B cell receptor signaling and intracellular toll-like receptor (TLR) signaling in primary human B cells: Our initial analysis of the transcriptional response to glucocorticoids in primary human cells revealed a significant effect on genes involved in B cell receptor signaling and TLR signaling. In collaboration with two other NIAID investigators, Susan Moir and Iain Fraser, we performed additional analyses of human B cells exposed to glucocorticoids and demonstrated a functional effect of this class of drugs on both signaling pathways. Cross-talk between these two signaling pathways has been implicated in the pathogenesis of several autoimmune diseases in which glucocorticoids are commonly used. Our results suggest that this may be an important component of the mechanism of action of glucocorticoids in autoimmune diseases and could open the path to targeted interventions that may mimic the clinical effects of glucocorticoids in these conditions, without the associated toxicity. Over the past year, this work has been published. 3. New insight into the mechanism of glucocorticoid-induced eosinopenia: Glucocorticoids are extensively used to treat human diseases that involve increased numbers or activity of eosinophils. It has been known for nearly seven decades that glucocorticoid administration leads to a rapid and profound decrease in circulating human eosinophils, and this is believed to play a role in their clinical effect in eosinophil disorders. However, the underlying mechanism remains unknown. In collaboration with Drs. Amy Klion (NIAID), Cynthia Dunbar (NHLBI), and Noriko Sato (NCI), we have addressed this knowledge gap employing a combination of genomics approaches and live tracking of radiolabeled eosinophils. We have established that glucocorticoids induce a reproducible pattern of eosinophil migration out of the circulation, and that this migration is dependent on upregulation of a specific chemokine receptor gene. Pharmacologic blockade of this chemokine receptor prevents glucocorticoid-induced eosinopenia, which suggests that our findings are mechanistically important. Over the past year, we have presented this work at national and international meetings, and a second manuscript is in preparation. 4. Development of a platform for the identification of compounds that mimic the immune-relevant actions of glucocorticoids: Our work on the genomic effects of glucocorticoids in human primary cells is identifying previously unrecognized actions of this class of drugs that are likely to be important for their clinical effects. One of the long-term goals of this work is to identify compounds with a more targeted range of actions and fewer side effects which can mimic the immune-relevant actions of glucocorticoids in specific disease states. In collaboration with Gianluca Pegoraro (NCI) and Iain Fraser (NIAID), we have over the past year developed a high-throughput screening method that could eventually be used for this purpose. Unlike existing methodologies, our platform uses single-molecule RNA FISH to provide single-cell targeted gene expression data and can be used in primary cells. Over the past year, we have made improvements to this methodology and have established a Research Collaborative Agreement with the National Center for Advancing Translational Research (NCATS) to test its utility in a drug-discovery program.
| 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 |
