Epigenetic Regulation of Tolerance to Toll-Like Receptor Ligands in Alveolar Epithelial Cells

To protect the host against exuberant inflammation and injury responses, cells have the ability to become hyporesponsive or tolerized to repeated stimulation by microbial and nonmicrobial insults. The lung airspace is constantly exposed to a variety of exogenous and endogenous Toll-like receptor (TLR) ligands, yet the ability of alveolar epithelial cells (AECs) to be tolerized has yet to be examined. We hypothesize that type II AECs will develop a tolerance phenotype upon repeated TLR agonist exposure. To test this hypothesis, primary AECs isolated from the lungs of mice and a murine AEC cell line (MLE-12) were stimulated with either a vehicle control or a TLR ligand for 18 hours, washed, then restimulated with either vehicle or TLR ligand for an additional 6 hours. Tolerance was assessed by measurement of TLR ligand-stimulated chemokine production (monocyte chemoattractant protein [MCP]-1/CCL2, keratinocyte chemoattractant [KC]/CXCL1, and macrophage inflammatory protein [MIP]-2/CXCL2). Sequential treatment of primary AECs or MLE-12 cells with TLR agonists resulted in induction of either tolerance or cross-tolerance. The induction of tolerance was not due to expression of specific negative regulators of TLR signaling (interleukin-1 receptor associated kinase [IRAK]-M, Toll-interacting protein [Tollip], single Ig IL-1-related receptor [SIGIRR], or suppressor of cytokine signaling [SOCS]), inhibitory microRNAs (miRs; specifically, miR-155 and miR146a), or secretion of inhibitory or regulatory soluble mediators (prostaglandin E2, IL-10, transforming growth factor-b, or IFN-a/b). Moreover, inhibition of histone demethylation or DNA methylation did not prevent the development of tolerance. However, treatment of AECs with the histone deacetylase inhibitors trichostatin A or suberoylanilide hyrozamine resulted in reversal of the tolerance phenotype. These findings indicate a novel mechanism by which epigenetic modification regulates the induction of tolerance in AECs.