Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation

Abstract

Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4(+)CD25(hi)Foxp3(+) Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1-null mice and Treg-depleted (diphtheria toxin-treated Foxp3(DTR)) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 10(5) DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS.

Keywords: 5-aza-2′-deoxycytidine; DNA methylation; Foxp3; acute lung injury; epigenetics.

Figures

Figure 1.

5-aza-2′-deoxycytidine (DAC) treatment promotes resolution of lung injury in wild-type (WT) mice. (A) Body weight relative to baseline was plotted after injury. (B–D) Bronchoalveolar lavage (BAL) total protein (B), total cell counts (C), and neutrophil counts (D) were determined in WT mice 2 and 5 days after injury with intratracheal (i.t.) LPS. (E) Lung sections 2 and 5 days after injury were stained with hematoxylin and eosin. Original magnification: ×20; ×200 (insets). *P < 0.05; †P < 0.001 (Mann-Whitney U test; n = 8 per group). Values reported are mean ± SEM. N.S., not significant.

Figure 2.

Lung regulatory T cell (Treg) number, activation state, suppressive phenotype, and proliferative capacity increase with DAC treatment after injury. (A) Lung CD4+CD25hiFoxp3+ cells are shown as number in the right lung, frequency of lung cells, and frequency of CD4+ cells 2 and 5 days after injury in WT mice. (B–D) Foxp3 (B); CD44, CD39, and CTLA-4 (C) expression; and the percentage of Ki-67+ Tregs (D) were determined by fluorescence in lung Tregs 5 days after injury. Accompanying bar graphs show summary mean fluorescence intensity (MFI) (B and C) and the percentage of Ki-67+ Tregs (D). *P < 0.05, **P < 0.01, and †P < 0.001 (Mann-Whitney U test for cell numbers/frequencies [A and D] or t test with Holm-Sidak correction for multiple comparisons [mean fluorescence intensities, B and C]; n = 8 per group). Values reported are mean ± SEM.

Figure 3.

Lymphocyte-deficient (Rag-1−/−) mice do not resolve lung injury in response to DAC treatment. (A) Body weight relative to baseline was plotted after injury. (B–D) BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in Rag-1−/− mice 5 days after injury with LPS. (E) Lung sections were stained with hematoxylin and eosin. Original magnification: ×20; ×200 (insets). P > 0.05 (Mann-Whitney U test; n = 5 per group). Values reported are mean ± SEM.

Figure 4.

DAC does not promote resolution in Treg-depleted mice. (A) Body weight relative to baseline was plotted beginning with the first diphtheria toxin dose (2 d before injury). Arrowheads represent diphtheria toxin (DTx) doses. (B–D) BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in DTx-treated Foxp3DTR mice (Foxp3DTR DTx+) 5 days after injury with LPS. (E) Lung sections were stained with hematoxylin and eosin. Original magnification: ×20; ×200 (insets). P > 0.05 (Mann-Whitney U test; n = 7 per group). Values reported are mean ± SEM.

Figure 5.

DAC alters CD4+ T cell phenotype and function in vitro. (A) Splenic WT CD4+CD25− conventional T cells (Tconv) or Tregs were cultured with DAC for 48 hours. Foxp3 fluorescence is plotted for shown DAC concentrations (vehicle, 10 and 100 nM). (B and C) CD4+CD25hiFoxp3+ Treg expression of CD44, CD39, and CTLA-4 (B) and the percentage of Ki-67+ Tregs (C) were determined by fluorescence in cultured Tregs treated with vehicle or 100 nM DAC. Accompanying bar graphs show summary MFIs (A and B) and the percentage of Ki-67+ Tregs (C). (D) T effector cells (Teff) were sorted from WT spleens, labeled with CellTrace Violet, and cultured in the presence of anti-CD3/CD28 and varying Treg ratios. Tregs were previously cultured in the presence of vehicle or 100 nM DAC. (E) Global DNA methylation was measured in Tregs cultured with vehicle or 100 nM DAC and compared with methylated control DNA. Experiments were conducted in triplicate and repeated three times. Proliferation data are representative of three independent experiments. *P < 0.05, **P < 0.01, and †P < 0.001 (t test with Holm-Sidak correction for multiple comparisons [MFI, A and B] or Mann-Whitney U test [C–E]). Values reported are mean ± SEM.

Figure 6.

Adoptive transfer (AT) of DAC-treated Tregs rescues the injury phenotype in Treg-depleted mice (Foxp3DTR DTx+). (A) Body weight relative to baseline was plotted after injury. Arrowheads represent DTx doses. (B–D) BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in Treg-depleted mice 7 days after injury with LPS. (E) Lung sections 7 days after injury were stained with hematoxylin and eosin. Original magnification: ×20; ×200 (insets). (F) Transforming growth factor (TGF)-β concentrations were measured in BAL fluid. (G) Exogenous (CD4+CD25hiAPC+GFP−) lung Tregs are shown as number in the right lung, frequency of lung cells, and frequency of CD4+ cells 7 days after injury. (H and I) Foxp3 (H); CD44, CD39, and CTLA-4 expression; and the percentage of Ki-67+ Tregs (I) were determined by fluorescence in exogenous lung Tregs 7 days after injury. Accompanying bar graphs show summary MFIs (H and I) and the percentage of Ki-67+ Tregs (I, last panel). *P < 0.05 and †P < 0.001 by Mann-Whitney U test (lung injury parameters, TGF-β levels, and cell numbers/frequencies, A–D, F, G, and I [last panel] or t test with Holm-Sidak correction for multiple comparisons (MFI, H and I [first three panels]) (n = 6 per group). Values reported are mean ± SEM.

Figure 7.

DAC rescue treatment has favorable effects in an influenza (flu) model. (A) Body weight relative to baseline is shown 10 days after inoculation with influenza. (B–D) BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in WT mice 10 days after intratracheal inoculation with influenza. (E) Lung sections 10 days after inoculation were stained with hematoxylin and eosin. Original magnification: ×20; ×200 (insets). (F) Lung CD4+CD25hiFoxp3+ cells are shown as number in the right lung, frequency of lung cells, and frequency of CD4+ cells 10 days after inoculation. (G) Foxp3 expression was determined by fluorescence in lung Tregs. The accompanying bar graph shows summary mean fluorescence intensities. *P < 0.05, **P < 0.01, and †P < 0.001 (Mann-Whitney U test; n = 9 per group). Values reported are mean ± SEM.