| TNF is an inflammatory cytokine that plays a critical role in the acute phase response to infection, and its dysregulation has been implicated in the pathology of several inflammatory and autoimmune disorders. TNF gene expression is regulated in a cell type- and inducer-specific manner that involves chromatin alterations at both the TNF promoter and distal DNase I hypersensitive (DH) sites within the TNF/LT locus. While the mechanisms underlying TNF gene activation in monocytes/macrophages and T cells have been studied intensively, the mechanisms of enhanced, repressed, and restored TNF gene expression in the context of classical macrophage activation and endotoxin tolerance remain largely unknown. We set out to understand how TNF gene expression is modulated during these biological processes by characterizing the chromatin environment of the TNF/LT locus. In the context of classical macrophage activation, IFN-gamma primes both circulating monocytes and tissue-resident macrophages so that, upon microbial recognition, there is an enhanced anti-microbial response; importantly, this results in enhanced TNF transcription. The mechanisms involved in priming are unclear; the IFN-gamma-inducible transcription factors IRF1 and STAT1 have been implicated but functional roles for these factors have not been determined. Here, we show in primary human monocyte-derived macrophages (MDMs) that IFN-gamma poises the TNF/LT locus for enhanced TNF transcription by exposing the distal enhancer element hHS-8, and that the ability of this element to promote TNF gene expression in response to priming depends on its recruitment of IRF1. We also demonstrate that IFN-gamma poises hHS-8 for enhancer function by promoting H3K27me3 enrichment, and that subsequent LPS stimulation triggers hHS-8 enhancer activation through demethylation of H3K27me3 followed by acetylation of H3K27. These experiments provide a mechanistic explanation of how IFN-gamma poises the TNF/LT locus for enhanced TNF transcription upon LPS stimulation, while at the same time providing potential targets for selective manipulation of TNF expression in primed macrophages. In the context of endotoxin tolerance, repeated or prolonged exposure to LPS results in the reprogramming of monocytes and macrophages such that inflammatory responses, especially induction of TNF, are down-regulated; in this regard, the cell is considered tolerant or immunosuppressed. Notably, IFN-gamma priming has been shown to abrogate endotoxin tolerance, thereby restoring responses to LPS stimulation. The mechanism of TNF gene repression during endotoxin tolerance and its restoration during IFN-gamma-mediated abrogation of endotoxin tolerance remains unknown. Here, we have characterized the TNF/LT locus in endotoxin-tolerant monocytes and macrophages and found the TNF promoter to be nuclease accessible and enriched for H3K27me3 during repressed transcription. We have also found that IFN-gamma priming restores TNF gene expression in an IRF1-independent manner, suggesting that IFN-gamma primes non-tolerant and endotoxin-tolerant monocytes and macrophages by two distinct mechanisms; furthermore, we observed enrichment of H3K27ac at the TNF promoter and the appearance of a DH site ~4kb upstream of the TNF TSS in response to IFN-gamma treatment of endotoxin-tolerant cells. These results suggest that chromatin modifications at the TNF promoter and distal DNA elements play an important role in IFN-gamma-mediated abrogation of endotoxin tolerance. |
