Innate tolerance is mediated by inhibition of specific signaling pathways rather than by epigenetic silencing of effector genes

Macrophages activated through a pattern-recognition receptor (PRR) enter a transient state of tolerance characterized by hyporesponsiveness to restimulation of the same receptor. Signaling-based and epigenetic mechanisms are invoked to explain tolerance. The epigenetic scenario predicts that: (i) activation of a PRR should cross-tolerize to agonists of unrelated PRRs; (ii) some genes become hyperresponsive to homologous rechallenge due to distinct chromatin modifications. We show that in human macrophages tolerized by TLR4 or NOD1 agonists, signaling and transcriptional responses to homologous stimuli are inhibited partially or completely. Many genes are upregulated due to primary stimulation, but are unresponsive to homologous restimulation. Gene hyperresponsiveness to homologous rechallenge is a rare and inconsistent phenomenon. However, most genes that have become unresponsive to homologous stimuli show unchanged or elevated responses to agonists of PRRs signaling via distinct pathways. Thus, inhibition of specific signaling pathways rather than epigenetic silencing is the dominant mechanism of innate tolerance. Human macrophages were generated by culturing blood monocytes from a healthy donor with GM-CSF (40 ng/ml) for 6 days. To induce tolerance, macrophages were treated for 24 h with a NOD1 agonist (M-triDAP), a TLR4 agonist (LPS) or medium as a control. After 24 h, cells were washed and recultured with medium, M-triDAP or LPS for 1 h or 4 h, at which points total RNA samples were collected. Two identically designed independent experiments using macrophages generated from the same donor at two different occasions were performed (two biological replicates)