Lymphocyte senescence in COPD is associated with decreased histone deacetylase 2 expression by pro-inflammatory lymphocytes

Abstract

Background: Histone acetyltransferases (HAT) and histone deacetylases (HDAC) are enzymes that upregulate and down-regulate pro-inflammatory gene transcription respectively. HDAC2 is required by corticosteroids to switch off activated inflammatory genes and is reduced in lung macrophages in COPD. We have shown that COPD patients have increased steroid resistant CD28null (senescent) pro-inflammatory T and NKT-like peripheral blood cells (particularly CD8+ subsets) and we hypothesized that these changes would be associated with a loss of HDAC2 from these senescent pro-inflammatory lymphocytes.

Methods: Blood was collected from 10 COPD and 10 aged-matched controls. Intracellular pro-inflammatory cytokines, IFNγ and TNFα, and expression of CD28, HDAC2 and HAT, were determined in lymphocyte subsets in the presence of ± 5 mg/ml theophylline (HDAC2 activator), 10 μM prednisolone and 2.5 ng/ml cyclosporine A (immunosuppressant), using flow cytometry.

Results: There was a loss of HDAC2 from CD28null CD8+ T and NKT-like cells in COPD. There was a significant negative correlation between HDAC2 expression and the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -.763, p < 0.001 for T-cell IFNγ). There was a synergistic upregulation of HDAC2 and associated decrease in pro-inflammatory cytokine production in CD28nullCD8+ T and NKT-like cells in the presence of 5 mg/L theophylline + 10(-6) M prednisolone or 2.5 ng/mL cyclosporine A (CsA).

Conclusions: Lymphocyte senescence in COPD is associated with loss of HDAC2 in CD28nullCD8+ T and NKT-like cells. Alternative treatment options such as combined theophylline with low-dose CsA, that inhibit these pro-inflammatory cells, may reduce systemic inflammation in COPD.

Fig. 1

The percentage of CD28+ and CD28null (CD28-) CD8+ and CD8- T cells (clear bars) and NKT-like cells (grey bars) in patients with COPD. Data presented as box plots. There was a significant decrease in the percentage of CD28nullCD8+ T and NKT-like cells expressing HDAC2 compared with CD28+CD8- and CD28+CD8+ Tand NKT-like cells

Fig. 2

The percentage of CD28+ and CD28null (CD28-) CD8+ and CD8- T cells (clear bars) and NKT-like cells (grey bars) producing IFNγ in patients with COPD. Data presented as box plots. There was a significant decrease in the percentage of CD28nullCD8+ T and NKT-like cells expressing IFNγ compared with CD28+CD8- and CD28+CD8+ T and NKT-like cells

Fig. 3

There was a significant negative correlation between the percentage of CD28nullCD8+ T cells expressing HDAC2 and producing IFNγ in COPD subjects

Fig. 4

a There was a significant correlation between the percentage of CD28nullCD8+ T cells co-expressing HAT.IFNγ and IFNγ. b A representative dot plot showing an increased percentage of HAT+ CD28nullCD8+ T cells co-expressing IFNγ + (23 %) in a COPD patient compared with HAT + CD28+ CD8+ T cells (9 %)

Fig. 5

a Representative laser confocal images of HDAC2 staining (red) in FACS-sorted CD28null (right) and CD28+ T cells (left). Blue was DAPI counterstaining. Scale bar = 8um. The bar graph depicts results of quantitative analysis by ImageJ. Experiments were repeated 3 times, showing similar results. *** p < 0.05. b Representative Western Blot of equal numbers of sorted CD28+ and CD28null T cells, stained for HDAC2 expression. There was a decrease in the 55kD band corresponding to the HDAC2 in CD28null T cells compared with CD28+ T cells. Bar graph showing HDAC2 expression relative to β-actin from CD28 null (CD28-) and CD28+ T cells (mean ± sem from 3 experiments)

Fig. 6

Correlation between HDAC2 expression by CD28nullCD8+ T cells with FEV1 (% predicted) in COPD subjects

Fig. 7

a Graphs showing the effect of 5 mg/ml theophylline (Th) ± 10⁻⁶ M prednisolone (MP) or 2.5 ng/mL cyclosporine A (CsA) or combination on the upregulation of HDAC2 (a) and inhibition of IFNγ production by CD28nullCD8+ T cells is shown in (b). There was no effect on HDAC2 or IFNγ in the presence of theophylline alone. There was significant increase in the percentage of CD28nullCD8 + T cells expressing HDAC2 in the presence of MP, CsA or combination (p < 0.05 for all). There was a synergistic increase in the percentage of CD28nullCD8 + T cells expressing HDAC2 in the presence of theophylline and MP or CsA or a combination. c Representative dot plots showing the combined effect of 5 mg/mL theophylline (Theo) and 2.5 ng/mL cyclosporine A (CsA) on the percentage of CD28null CD8+ T (top plots) and NKT-like cells (bottom plots) expressing HDAC2 and producing IFNγ. Note the significant increase in HDAC2 and significant decrease in IFNγ in both CD28null subsets in the presence of theophylline and cyclosporine A