Epigenetic Silencing of Tumor Necrosis Factor α during Endotoxin Tolerance

Abstract

Sustained silencing of potentially autotoxic acute proinflammatory genes like tumor necrosis factor ␣ (TNF␣) occurs in circulating leukocytes following the early phase of severe systemic inflammation. Aspects of this gene reprogramming suggest the involvement of epigenetic processes. We used THP-1 human promonocytes, which mimic gene silencing when rendered endotoxin-tolerant in vitro, to test whether TNF␣ proximal promoter nucleosomes and transcription factors adapt to an activation-specific profile by developing characteristic chromatinbased silencing marks. We found increased TNF␣ mRNA levels in endotoxin-responsive cells that was preceded by dissociation of heterochromatin-binding protein 1␣, demethylation of nucleosomal histone H3 lysine 9 (H3(Lys 9 )), increased phosphorylation of the adjacent serine 10 (H3(Ser 10 )), and recruitment of NF-B RelA/p65 to the TNF␣ promoter. In contrast, endotoxintolerant cells repressed production of TNF␣ mRNA, retained binding of heterochromatin-binding protein 1␣, sustained methylation of H3(Lys 9 ), reduced phosphorylation of H3(Ser 10 ), and showed diminished binding of NF-B RelA/p65 to the TNF␣ promoter. Similar levels of NF-B p50 occurred at the TNF␣ promoter in the basal state, during active transcription, and in the silenced phenotype. RelB, which acts as a repressor of TNF␣ transcription, remained bound to the promoter during silencing. These results support an immunodeficiency paradigm where epigenetic changes at the promoter of acute proinflammatory genes mediate their repression during the late phase of severe systemic inflammation.Gene reprogramming during severe systemic inflammation generates, among other patterns, silencing of acute proinflammatory genes, such as TNF␣ 2 and IL-1␤, that initiate acute systemic inflammation and damage to multiple organs (1, 2). The silencing of acute proinflammatory genes, which normally follows an initial activation phase (3), is clinically relevant in humans because it participates in generating an acquired state of immunodeficiency that correlates with poor prognosis and increased mortality (4). Gene silencing as a result of disrupted transcription occurs in circulating and tissue leukocytes during severe systemic inflammation in animals and humans (2, 5, 6). The silenced component of gene reprogramming is characterized by a tolerance to endotoxin and can persist for days or even weeks (5). Endotoxin tolerance is defined by the repressed expression of a set of proinflammatory genes in response to the stimulation of the Toll-like receptor 4 by endotoxin. Endotoxin tolerance is constitutively present in blood leukocytes obtained from humans and animals with severe systemic inflammation and can be generated in vitro by using endotoxin as a primary stimulus of macrophages (7).The complex mechanisms responsible for gene silencing are regulated at many levels and continue to emerge. At the level of chromatin, covalent modifications of the NH 2 -terminal tails of the four core histones (H2A, H2B, H3, and H4) play an essential role ...

Dynamic and Selective Nucleosome Repositioning during Endotoxin Tolerance

“…RelB Remodels Nucleosomes and Induces Tolerance in LPSresponsive Cells-We have shown that RelB binds to the TNF␣ and IL-1␤ promoter nucleosomes in tolerant cells only and that RelB knockdown reactivates their transcription (26,27). In addition, our finding that RelB knockdown resulted in nucleosomal occupancy profile similar to that seen in R1 cells (see Fig.…”

“…This process correlates with diminished binding of the active NF-B factor p65 and increased binding of feedback repressor transcription factor RelB to the proximal promoters. RelB is essential initiator of silencing by directly interacting with and recruiting G9a to promote and maintain a silent heterochromatin structure (27,32). Our finding that inhibiting RelB expression in tolerant cells reactivated TNF␣ and IL-1␤ transcription (26,27,31) raised the possibility that nucleosome remodeling physically contributes to transcriptional silencing of proinflammatory genes in SSI.…”

“…After centrifugation for 5 min at 10,000 rpm, chromatin solution was precleared by incubation with protein G-agarose beads and then subjected to immunoprecipitation with antibody against H3K9me2, H3K4me3 (Millipore, Temecula, CA), H2A.Z (Active Motif), H2A, NAP1 (Santa Cruz Biotechnology, Inc.), or BAF47 (BD Biosciences). Chromatin was decrosslinked, and DNA was extracted as described previously (27).…”

“…Promoter-TNF␣ transcription is significantly induced after LPS activation of THP-1 cells but is silenced once cells become LPStolerant despite a second dose of LPS (27,28). Therefore, we sought to determine whether the chromatin structure contributes to TNF␣ induction or silencing by comparing the nucleosomal organization across a 1-kb promoter fragment in the different cellular phenotypes.…”

“…Therefore, we sought to determine whether the chromatin structure contributes to TNF␣ induction or silencing by comparing the nucleosomal organization across a 1-kb promoter fragment in the different cellular phenotypes. LPS-responsive (normal) THP-1 cells were made tolerant (T) by pretreatment with LPS overnight and then washed and restimulated, together with the responsive (R) cells, for 1 h. We chose the 1 h time point because our previous work demonstrated that after 1 h, TNF␣ mRNA is significantly increased in responsive cells but was not expressed in tolerant cells (27). We consider resting, responsive cells (R0) as the base-line response; therefore, nucleosome positions in these cells will be identified as "in native/default positions."…”