Integrative genomic analysis of the human response to glucocorticoids

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

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Abstract

Below is a summary of the key scientific contributions made by our laboratory over the past year: 1. Neutrophils are a key target of glucocorticoids: During the past year, we have studied the response of human neutrophils to glucocorticoids. Through an in vivo study in which we administered the glucocorticoid methylprednisolone to a group of 20 healthy volunteers, then sampled individual immune cells serially from peripheral blood, we were able to study the transcriptional response to glucocorticoids in highly pure neutrophils. Surprisingly, neutrophils appear to be the most transcriptionally responsive cell type to glucocorticoid administration. A series of follow-up in vitro and in vivo studies have been performed over the past year to reveal the molecular basis for this strong transcriptional response. In addition, we have formed a research collaboration involving six laboratories across four NIH institutes, to perform live-tracking of rhesus macaque neutrophils by PET-CT imaging and in vivo flow cytometry, before and after glucocorticoid administration. These experiments will allow us to study the effects of glucocorticoids on neutrophil migration, and to contribute to long-standing questions regarding neutrophil kinetics. 2. Human neutrophils are more heterogeneous than previously thought: In collaboration with Dr. Mariana Kaplan's lab at NIAMS, we have identified and overcome a key obstacle to the analysis of human neutrophils by single-cell RNA sequencing. The resulting analyses have shed light on neutrophil diversity. Neutrophils were long believed to be transcriptionally inactive and homogeneous cells. Instead, we have found that circulating human neutrophils exist in distinct transcriptional states. 3. 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 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 advanced a Research Collaborative Agreement with the National Center for Advancing Translational Research (NCATS) to test its utility in a drug-discovery program. A manuscript reporting this method has been submitted and reviewed, and is undergoing revisions. 4. Generation of a web tool for the study of the cellular response to glucocorticoids: Over the past year, our laboratory developed a bioinformatic tool that allows scientists to investigate the transcriptional response of different cell types to glucocorticoids. The tool, called GCgx, will be publicly available from the NIAID Bioinformatics Portal. A manuscript describing the tool has been submitted and reviewed, and is undergoing revisions.