Genomic Responses of Human Immune and Non-Immune Cells to Glucocorticoids

Glucocorticoids are among the most frequently prescribed immunosuppressive and anti-inflammatory medications worldwide. Long-term use, however, is complicated by serious non-immunologic side effects. Ongoing in vitro experiments with human primary cells in our laboratory suggest that there are indeed fundamental differences in the genomic response of immune and non-immune cells to glucocorticoids. These and other aspects of drug action at the genomic level have not been completely characterized. This study will attempt to generate a list of human genes and non-coding RNAs that are differentially expressed and regulated in response to glucocorticoids between immune and non-immune cells. These data will be used to identify transcripts, their corresponding proteins, and the molecular pathways that are best candidates for targeted intervention. Potential targets could be validated with small interfering RNA (siRNA) libraries, with the long-term goal of developing small-molecule or nanoparticle-facilitated RNA interference (RNAi) interventions that reproduce the therapeutic action of glucocorticoids in immune cells while avoiding their harmful side effects on other tissues.

Healthy volunteers will undergo baseline blood collection prior to receiving a single intravenous dose of 250 milligrams of methylprednisolone sodium succinate. Blood will be collected in one of two regimens: 1 and 2 hours or 2 and 4 hours after the start of the infusion. A skin punch biopsy may be obtained before healthy volunteers receive IV methylprednisolone. If so, topical methylprednisolone will be applied to a limited area of skin, contralateral to the site of the baseline skin biopsy, and an additional skin biopsy will be obtained 4 hours after drug administration, from the area where topical methylprednisolone was applied. Follow-up phone calls 1 day and 1 week after discharge will document any adverse effects related to the drug or skin biopsy. Total length of individual study participation is 1-5 weeks.

Blood samples will be processed for isolation of hematopoietic cell sub-population (neutrophils, B cells, CD4+ T cells, CD8+ T cells, monocytes, and natural killer [NK] cells). Laboratory studies will be performed in the purified cells, with the goal of understanding the human response to glucocorticoids in vivo at the level of RNA (e.g., RNA sequencing, small -RNA-sequencing, real-time PCR), DNA (e.g., ChIP-seq, methylation analysis, DNA sequencing, genotyping, and protein (e.g., flow cytometry, mass spectrometry). At each time point, serum methylprednisolone levels will be measured and flow cytometry for standard lineage markers will be performed. Skin biopsies will be subjected to RNA isolation for RNA sequencing and small-RNA sequencing. A fragment of each skin biopsy will undergo fibroblast isolation and culture for in vitro exposure to glucocorticoids and for the generation of induced pluripotent stem (iPS) cells.

• INCLUSION CRITERIA:

• Age 18 to 64 years
• Willingness to have samples stored for future research
• Willingness to undergo genetic testing

Exclusion criteria

• Body Mass Index less than 18 or greater than 35
• Difficult peripheral venous access (as determined by study staff at screening)
• History of severe allergic reaction to glucocorticoids
• History of autoimmune or autoinflammatory disease
• Active solid or hematologic malignancy
• History of a skin condition (such as psoriasis, pemphigus, or atopic dermatitis) that could affect the results of the transcriptional analysis of the skin biopsy samples
• Diabetes mellitus
• Cancer chemotherapy within the past 5 years
• Surgery within the past 8 weeks
• History of recent (within the past 30 days) infection
• A positive test for human immunodeficiency virus, or hepatitis A, B or C virus infection (viral markers hepatitis screen, which includes HBsAg, anti-HCV IgG, anti-HAV IgM).
• A positive or indeterminate test for latent tuberculosis (interferon gamma release assay)
• History of parasitic, amebic, fungal or mycobacterial infections, or other possible latent infections
• Coagulation test (PT and PTT) results outside of normal range
• History of a bleeding disorder
• Use of a glucocorticoid (including topical or inhaled), a nonsteroidal anti-inflammatory drug (including aspirin), an anti-epileptic drug, an anticoagulant, a statin, fluoxetine, diltiazem, amiodarone, clarithromycin, ketoconazole, or St. John s wort, within the past 30 days
• Vaccination within the past 30 days
• Receipt of an immunosuppressant or immunomodulatory drug within the past 30 days
• Pregnancy, current or within the past 90 days, or trying to become pregnant during the study
• Current breastfeeding
• Complete blood count (CBC) and/or acute care panel values are both outside of the NIH Department of Laboratory Medicine normal reference range and deemed clinically significant by the principal investigator
• Any Electrocardiogram (ECG) abnormality that is clinically significant
• Any condition that, in the investigator s opinion, may put the participant at undue risk or compromise the study s scientific objectives
• Participation in a clinical protocol which includes an intervention that, in the opinion of the investigator, may affect the results of the current study