A labile transcriptional repressor modulates endotoxin tolerance.

Microbial components such as bacterial endotoxin lipopolysaccharide (LPS) can trigger highly lethal septic shock. The cardinal features of septic leukocytes include the repressed production of inflammatory cytokines, such as interleukin-1 beta (IL-1␤), and elevated production of anti-inflammatory cytokines, such as secretory interleukin-1 receptor antagonist (sIL-1RA). Pro-and anti-inflammatory cytokine gene transcriptions are equally repressed in septic leukocytes due to disruption of the LPS signaling pathway at the level of interleukin-1 receptor-associated kinase. The selective elevation of sIL-1RA protein in septic blood is caused by efficient translation of residual sIL-1RA message. In this study, we report that the LPS-inducible phosphatidylinositol 3-kinase (PI3-kinase)-dependent signaling pathway contributes to the elevated translation of sIL-1RA in septic/LPS-adapted leukocytes. We also observe that this pathway is gene specific and does not affect the production of proinflammatory IL-1␤ protein.

Section: Sil-1ra and Il-1␤ Proteins Are Produced Differentially Insupporting

confidence: 60%

The Phosphatidylinositol 3-Kinase Pathway Selectively Controls sIL-1RA Not Interleukin-1β Production in the Septic Leukocytes

Learn

Boger

et al. 2001

“…LPS-mediated tolerance that mimics the SSI phenotype in THP-1 cells was previously described (23). Briefly, LPS tolerance is generated by an initial stimulation with LPS (0111:B4; 1 g/ml) for 16 h followed by re-stimulation with 1.0 g/ml LPS for 3 h. This LPS acts exclusively through TLR4 receptor as determined in cells lacking TLR4.…”

Section: Methodsmentioning

confidence: 99%

The NF-κB Factor RelB and Histone H3 Lysine Methyltransferase G9a Directly Interact to Generate Epigenetic Silencing in Endotoxin Tolerance

Chen

Gazzar

Yoza

et al. 2009

181

177

The interplay of transcription factors, histone modifiers, and DNA modification can alter chromatin structure that epigenetically controls gene transcription. During severe systemic inflammatory (SSI), the generation of facultative heterochromatin from euchromatin reversibly silences transcription of a set of acute proinflammatory genes. This gene-specific silencing is a salient feature of the endotoxin tolerant phenotype that is found in blood leukocytes of SSI patients and in a human THP-1 cell model of SSI. We previously reported that de novo induction of the NF-B transcription factor RelB by endotoxin activation is necessary and sufficient for silencing transcription of acute proinflammatory genes in the endotoxin tolerant SSI phenotype. Here, we examined how RelB silences gene expression and found that RelB induces facultative heterochromatin formation by directly interacting with the histone H3 lysine 9 methyltransferase G9a. We found that heterochromatin protein 1 (HP1) and G9a formed a complex at the interleukin-1␤ promoter that is dependent on the Rel homology domain (RHD) of RelB. RelB knockdown disassociated the complex and reversed transcription silencing. We also observed that whereas RelB chromatin binding was independent of G9a, RelB transcriptional silencing required G9a accumulation at the silenced promoter. Binding between RelB and G9a was confirmed by glutathione S-transferase pulldown in vitro and coimmunoprecipitation in vivo. These data provide novel insight into how RelB is required to initiate silencing in the phenotype associated with severe systemic inflammation in humans, a disease with major morbidity and mortality.Inflammation is an evolutionarily conserved stereotypic stress response primarily orchestrated by temporal alterations in gene expression, with important contributions from complement, coagulation, and neurogenic processes (1, 2). The genetic information encoded to generate inflammation regulates distinct functional sets of genes, including pro-and anti-inflammatory modifiers, directors of cell death, and mediators of cell respiration and metabolism (3). The initiating stage of virtually all inflammation depends on sensory receptors coupled to intracellular signals that activate the immunity master regulator NF-B to generate p65 and p50 transactivating heterodimers at euchromatin promoters of a set of early response proinflammatory genes. When spread throughout the circulation, this early stage may precipitate the extreme stress response of severe systemic inflammation (SSI). 4 Later stages of inflammation reprogramming often require protein synthesis and induce expression of distinct sets of genes with anti-inflammatory, survival, and energy regulation (4, 5). Recent data in humans from our laboratory and in animals from others highlight the role of epigenetics in regulating gene expression in the SSI phenotype (6 -11). These epigenetic events provide specificity and plasticity among distinct sets of genes and depend on varied chromatin structure and modifications rather t...

“…For induction of LPS tolerance (29), cells were incubated overnight with 1 g/ml Gram-negative bacterial LPS from Escherichia coli 0111:B4 (Sigma). LPS-tolerant and LPS-responsive (normal) cells were washed with incomplete medium and cultured at 0.5-1.0 ϫ 10 6 cells/ml and stimulated with 1 g/ml LPS for the indicated times.…”

Section: Methodsmentioning

confidence: 99%

Dynamic and Selective Nucleosome Repositioning during Endotoxin Tolerance

Gazzar

Liu

Yoza

et al. 2010

Sepsis is encoded by a sequel of transcription activation and repression events that initiate, sustain, and resolve severe systemic inflammation. The repression/silencing phase occurs in blood leukocytes of animals and humans following the initiation of systemic inflammation due to developing endotoxin tolerance. We previously reported that NF-B transcription factor RelB and histone H3 lysine methyltransferase G9a directly interact to induce facultative heterochromatin assembly and regulate epigenetic silencing during endotoxin tolerance, which is a major feature of sepsis. The general objective of this study was to assess whether dynamic temporal, structural, and positional changes of nucleosomes influence the sepsis phenotype. We used the THP-1 sepsis cell model to isolate mononucleosomes by rapid cell permeabilization and digestion of chromatin with micrococcal nuclease and then compared tumor necrosis factor ␣ (TNF␣) proximal promoter nucleosome alignment in endotoxin-responsive and -tolerant phenotypes. We found differential and dynamic repositioning of nucleosomes from permissive to repressive locations during the activation and silencing phases of transcription reprogramming and identified the following mechanisms that may participate in the process. 1) Two proximal nucleosomes repositioned to expose the primary NF-B DNA binding site in endotoxin-responsive cells, and this "promoter opening" required the ATP-independent chaperone NAP1 to replace the core histone H2A with the H2A.Z variant. 2) During RelB-dependent endotoxin tolerance, the two nucleosomes repositioned and masked the primary NF-B DNA binding site. 3) Small interfering RNA-mediated inhibition of RelB expression prevented repressive nucleosome repositioning and tolerance induction, but the "open" promoter required endotoxin-induced NF-B p65 promoter binding to initiate transcription, supporting the known requirement of p65 posttranslational modifications for transactivation. 4) Sustaining the permissive promoter state after RelB knockdown required ATP-dependent nucleosome remodeler BAF complex. Moreover, we found that forced expression of RelB in responsive cells induced repressive nucleosome positioning and silenced TNF␣ transcription, demonstrating the plasticity of nucleosome remodeling and its dependence on RelB. Our data suggest that nucleosome repositioning controls both the induction and epigenetic silencing phases of TNF␣ transcription associated with sepsis.