Effects of Systemically Administered Hydrocortisone on the Human Immunome

Abstract

Corticosteroids have been used for decades to modulate inflammation therapeutically, yet there is a paucity of data on their effects in humans. We examined the changes in cellular and molecular immune system parameters, or "immunome", in healthy humans after systemic corticosteroid administration. We used multiplexed techniques to query the immunome in 20 volunteers at baseline, and after intravenous hydrocortisone (HC) administered at moderate (250 mg) and low (50 mg) doses, to provide insight into how corticosteroids exert their effects. We performed comprehensive phenotyping of 120 lymphocyte subsets by high dimensional flow cytometry, and observed a decline in circulating specific B and T cell subsets, which reached their nadir 4-8 hours after administration of HC. However, B and T cells rebounded above baseline 24 hours after HC infusion, while NK cell numbers remained stable. Whole transcriptome profiling revealed down regulation of NF-κB signaling, apoptosis, and cell death signaling transcripts that preceded lymphocyte population changes, with activation of NK cell and glucocorticoid receptor signaling transcripts. Our study is the first to systematically characterize the effects of corticosteroids on the human immunome, and we demonstrate that HC exerts differential effects on B and T lymphocytes and natural killer cells in humans.

Figure 1. Effects of systemically administered hydrocortisone (HC) on circulating peripheral blood cells in humans.

Healthy volunteers had phlebotomy at baseline and after they were administered 50 mg (a,c) or 250 mg (b,d) intravenous HC. (a,b): absolute neutrophil counts, absolute lymphocyte counts, and absolute monocyte counts were determined by complete blood counts. (c,d): Quantitation of CD45+CD3+ T cells, CD45+CD3+CD8-CD4+ T cells, CD45+CD3+CD4-CD8+ T cells, CD45+CD3-CD19+ B cells, and CD45+CD3-CD16+CD56+ NK cells were determined by clinical flow cytometry as described in the Materials and Methods. Each panel depicts data from up to ten individuals treated with either 50 mg or 250 mg HC.Data for a single patient are shown as a single thin line. Think line represents median across patients with error bars indicating 25-th and 75-th quantile. Time points with significant changes in cell counts comparing with baseline are marked with *for p < 0.05, and **for p < 0.01 (Wilcoxon signed-rank test).

Figure 2. Staining patterns and gating strategies for T lineage.

PBMCs were stained according to the Methods section. Debris was excluded from analysis by gating on FSC vs. SSC and (a) live mononuclear cells were selected by gating on the cells that excluded the viability dye. Doublets of cells were eliminated using width measurements. T cells were identified by the expression of CD45 and CD3 (b), and T cell subsets were identified by expression of CD4 and/or CD8. Both CD4+ and CD8+ subsets were examined for expression of surface markers allowing definition of memory and naïve cells subpopulations (CD27, CD45RA-RO, CCR7, panels c and d). Markers associated with the populations of interest (e.g. Treg, Th17, Th22) included FOXp3, CD25, CD39, CD103, CD146, and cytokines (IL17a, TNFa) staining were used to gate and characterize these cells (e).

Figure 3. Coherently changed cell populations following HC treatment.

Cell frequencies at 4 hours compared to baseline and at 24 hours comparing to four hours (pairwise t-test’s FDR corrected p-values less than 0.1 and absolute log-fold-change (of percent of parent population) higher than 0.1 for at least one comparison) for subjects receiving 50 mg (left) or 250 mg (right) of steroid. Populations are grouped by tubes (T, B and NK cells) and sorted by change in direction (indicated by trend line on the left) and meta p-value across all comparisons). Bars are colored by log10 (p-value), with blue and red colors denoting decrease or increase in percent of parent population, respectively. Asterisks are added if FDR corrected p-value is <0.05 (*) or <0.01 (**).

Figure 4. Post-treatment coherent changes in gene transcription.

(a) Number of differentially expressed genes (FDR <0.05 and absolute log-fold-change >0.5) for subjects received 50 mg (left) or 250 mg (right) of HC between one and 24 hours comparing to baseline (0 hour). (b) Top differentially expressed genes (88) with FDR <0.05 and absolute log-fold-change >1 at least for one comparison.

Figure 5. Blood transcriptome modules and cell specific gene sets enriched in coherently changing genes after systemic administration of 50 mg and 250 mg of HC.

The enrichment analysis was performed separately for up-regulated (enrichment p-values are represented by red colors) and down-regulated (blue colors) genes. The Transcription modules (a) and cell population specific gene sets (b) with FDR corrected enrichment p-values less than 1 × 10⁻⁵ at least at one time points and one HC dosage were included. For each module/timepoint cell color in lower left corner illustrates p-value for 50 mg dosage, and in upper right corner – for 250 mg.

Figure 6. Natural killer cell gene signature changes after HC administration.

Gene expression profiles of top genes from the Cytotoxic/NK Cell blood transcriptome module (labeled M3.6 on Fig. 5a) with largest change from baseline to 4 hours. Each line represents an individual patient with blue and red color indicating patients receiving 50 or 250 mg of hydrocortisone respectively.

Figure 7. Cytokine levels as measured by Luminex assay after HC administration.

Results are shown as the percentage changes from baseline concentration for each person. Thin lines indicate individual patients received HC dosage represented by color. Median changes for each dosage as well as for all the patients are shown as thick lines. Significance of changes from the baseline were estimated by nonparametric analyses with Wilcoxon matched pairs (signed-rank) test for all patients together. For each cytokine time points with p values lower than 0.05 are marked with*, and those with Benjamini-Hochberg FDR lower than 0.05 are marked with**.