Foxp3⁺ Regulatory T-Cell DNA Demethylation Accelerates Resolution of Acute Lung Injury

Rationale

Our previous work defined an essential role for CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in resolution of acute lung injury (ALI). As DNA demethylation governs Treg development and function, we tested the hypothesis that DNA methyltransferase inhibition promotes ALI resolution via an effect on Tregs.

Methods

Male C57BL/6 wild-type and Foxp3^DTR mice received intratracheal Escherichia coli LPS. Starting 24 hours later, we administered a daily intraperitoneal injection of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) or vehicle. To assess the direct effect of DNA demethylation on Treg function, ex vivo DAC-treated or control Tregs were adoptively transferred to Treg-depleted Foxp3^DTR mice 1 hour after LPS. Lung injury measures included survival, weight loss, lung histology, and bronchoalveolar lavage (BAL) cell count, differential, and protein measurement. Lung single-cell suspension was stained for flow cytometry.

Measurements and Main Results

Vehicle-treated (control) wild-type mice experienced higher mortality, sustained weight loss, and nonresolving lung injury by Day 5 post-LPS, whereas DAC-treated animals gained weight and exhibited resolving lung histology and decreased BAL neutrophils and protein. DAC-treated wild-type mice displayed lung Tregs with increased suppressive phenotype (higher expression of Foxp3, CD39, and CTLA-4) and increased proliferative capacity (Ki-67). Systemic DAC had no salutary effect on Treg-depleted Foxp3^DTR mice. Adoptive transfer of 2 × 10⁵ DAC-treated Tregs, but not control Tregs, rescued Treg-depleted mice from ongoing inflammation on Day 7 post-LPS.

Conclusions

The DNA methyltransferase inhibitor DAC accelerated resolution of LPS-induced lung injury and required Tregs for its effect. Lung Tregs from DAC-treated mice displayed increased markers of suppressive phenotype and proliferation. Tregs mediated the proresolution action of DNA methyltransferase inhibition.