Glucocorticoid dysregulation of natural killer cell function through epigenetic modification

Abstract

It is well-established that psychological distress reduces natural killer cell activity (NKCA) and dysregulates cytokine balance. This may be mediated by stress-induced release of glucocorticoids, which have broad effects on the immune system, including the suppression of NKCA and alteration of cytokine production. The purpose of this study was to evaluate epigenetic mechanisms that may underlie the effect of glucocorticoids on NK cells, using the human NK cell line, NK92. Treatment of NK92 cells with the synthetic glucocorticoid, dexamethasone, at a concentration of 10⁻⁷M, produced a significant reduction in NKCA. Glucocorticoid inhibition was a consequence of not only a reduced capacity of the NK cells to bind to tumor targets but also a reduced production of granule constituents (perforin and granzyme B) with no detectable effect on granule exocytosis. Glucocorticoids also reduced the constitutive and the stimulated production of the cytokines, IL-6, TNF alpha and IFN gamma, and reduced the surface expression of LFA-1. Glucocorticoid treatment also reduced global histone acetylation, the acetylation of histone 4 lysine position 8, and the accessibility of the proximal promoters of perforin, interferon gamma and granzyme B. Histone acetylation was recovered by treatment of the NK cells with a histone deacetylase inhibitor, which also restored NKCA and IFN gamma production. These results demonstrate glucocorticoids to dysregulate NK cell function at least in part through an epigenetic mechanism, which reduces promoter accessibility through modification of histone acetylation status. This epigenetic modification decreases the expression of effector proteins necessary to the full functional activity of NK cells.

Figure 1

The effect of dexamethasone on Natural Killer Cell Lytic Activity is illustrated. NK92 cells were treated with varying amounts of dexamethasone for 24 hrs. Percent (%) inhibition was calculated by: [(Lytic Units No Treatment)-(Lytic Units Dexamethasone Treatment)]/(Lytic Units No Treatment) × 100. Values are presented as the mean and standard error of the mean (SEM). N = at least three independent experiments. *, P < 0.05, Treatment with Dexamethasone vs. No Treatment (NT).

Figure 2

The effect of dexamethasone on constitutive production of cytokines and perforin is illustrated. NK92 cells were treated for 24 hr with dexamethasone (10⁻⁷ M). Supernatants were collected and assayed for (a) IFN-gamma, (b) IL-6 and (c) perforin by ELISA. Results are expressed as mean ± SEM; N = at least three independent experiments. NT=No Treatment. p values represent statistically significant difference, No Treatment (NT) vs. Dexamethasone at indicated concentration,*, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3

The effect of dexamethasone on induced production of intracellular cytokines is illustrated. NK92 cells were treated with dexamethasone (Dex, 10⁻⁷ M) for 24 hr and then stimulated with leukocyte activation cocktail for 4 hrs. Intracellular cytokine staining was performed using anti-human IFN-gamma (a) and anti-human TNF-alpha (b) antibodies and assessed by flow cytometry. Representative results are depicted. NT=No Treatment.

Figure 4

The effect of dexamethasone and the histone deacetylase inhibitor (TSA) on acetylation status of H4-K8. (a) Representative western Blot analysis of nuclear extracts of NK92 cells probed with antibodies specific for the acetylated form of H4-K8. (b) Blots (n= 3) were standardized to total H3 protein (blots not shown) and blot density quantified using Image J software. NT = No Treatment, Dex= Dexamethasone, TSA = Trichostatin A (100 nM). % Change= [(Density Dex or Dex +TSA)/(Density NT)] × 100. Results are expressed as mean ± SEM (n = 3). Statistically significant difference, ***p <0.001, No Treatment (NT) vs. Dexamethasone (10⁻⁷), +++ p <0.001, Dexamethasone vs. Dexamethasone + TSA.

Figure 5

Promoter specific changes in acetylation status and glucocorticoid receptor recruitment is shown. NK92 cells were treated with 10⁻⁷ M dexamethasone for 24 hr. Cells were collected, cross-linked, sheared, and lysed. Total sheared lysates were isolated with antibodies specific for (a) H4-K8 Ac or (b) glucocorticoid receptor (GR). Fold change in acetylation and GR recruitment at the granzyme B (GZMB), perforin, IFN-gamma, and TNF-alpha promoters as compared to beta globin and calculated using the Pfaffl Method. Results are expressed as mean ± SEM (N= 4). Statistically significant difference ***p <0.001, No Treatment vs. Dexamethasone (10⁻⁷M), ++ p<0.01, +++ p <0.001, Dexamethasone vs. Dexamethasone + TSA.