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
nanoparticlefacilitated 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 affitional 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:

1. Age 18 to 64 years

2. Willingness to have samples stored for future research

3. Willingness to undergo genetic testing

EXCLUSION CRITERIA:

1. Body Mass Index less than 18 or greater than 35

2. Difficult peripheral venous access (as determined by study staff at screening)

3. History of severe allergic reaction to glucocorticoids

4. History of autoimmune or autoinflammatory disease

5. Active solid or hematologic malgnancy

6. 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

7. Diabetes mellitus

8. Cancer chemotherapy within the past 5 years

9. Surgery within the past 8 weeks

10. History of recent (within the past 30 days) infection

11. 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).

12. A positive or indeterminate test for latent tuberculosis (interferon gamma release
assay)

13. History of parasitic, amebic, fungal or mycobacterial infections, or other possible
latent infections

14. Coagulation test (PT and PTT) results outside of normal range

15. History of a bleeding disorder

16. 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

17. Vaccination within the past 30 days

18. Receipt of an immunosuppressant or immunomodulatory drug within the past 30 days

19. Pregnancy, current or within the past 90 days

20. Current breastfeeding

21. 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 (PI)

22. Any Electrocardiogram (ECG) abnormality that is clinically significant

23. Any condition that, in the investigator s opinion, may put the participant at undue
risk or compromise the study s scientific objectives

24. Participation in a clinical protocol which includes an intervention that, in the
opinion of the investigator, may affect the results of the current study