Interleukin-1 receptor-associated kinase (IRAK), a signal transducer for interleukin-1, has also been suggested to participate in the Toll-like receptor-mediated innate immune response to bacterial endotoxin lipopolysaccharide (LPS). Using the human promonocytic THP-1 cell line, we demonstrated that the endogenous IRAK is quickly activated in response to bacterial LPS stimulation, as measured by its in vitro kinase activity toward myelin basic protein. LPS also triggers the association of IRAK with MyD88, the adaptor protein linking IRAK to the Toll-like receptor/interleukin-1␤ receptor intracellular domain. Macrophage cells with prolonged LPS treatment become tolerant to additional dose of LPS and no longer express inflammatory cytokines. Endotoxin tolerance is a common phenomenon observed in blood from sepsis patients. We observed for the first time that the quantity of IRAK is greatly reduced in LPS-tolerant THP-1 cells, and its activity no longer responds to further LPS challenge. In addition, IRAK does not associate with MyD88 in the tolerant cells. Furthermore, application of AG126, a putative tyrosine kinase inhibitor, can substantially alleviate the LPS-induced cytokine gene expression and can also decrease IRAK level and activity. Our study indicates that IRAK is essential for LPS-mediated signaling and that cells may develop endotoxin tolerance by down-regulating IRAK.

TNF␣ gene expression is silenced in the endotoxin tolerant phenotype that develops in blood leukocytes after the initial activation phase of severe systemic inflammation or sepsis. The silencing phase can be mimicked in vitro by LPS stimulation. We reported that the TNF␣ transcription is disrupted in endotoxin tolerant THP-1 human promonocyte due to changes in transcription factor binding and enrichment with histone H3 dimethylated on lysine 9 (H3K9). Here we show that the TNF␣ promoter is hypermethylated during endotoxin tolerance and that H3K9 methylation and DNA methylation interact to silence TNF␣ expression. Chromatin immunoprecipitation and RNA interference analysis demonstrated that, in tolerant cells, TNF␣ promoter is bound by the H3K9 histone methyltransferase G9a which dimethylates H3K9 and creates a platform for HP1 binding, leading to the recruitment of the DNA methyltransferase Dnmt3a/b and an increase in promoter CpG methylation. Knockdown of HP1 resulted in a decreased Dnmt3a/b binding, sustained G9a binding, and a modest increase in TNF␣ transcription, but had no effect on H3K9 dimethylation. In contrast, G9a knockdown-disrupted promoter silencing and restored TNF␣ transcription in tolerant cells. This correlated with a near loss of H3K9 dimethylation, a significant decrease in HP1 and Dnmt3a/b binding and promoter CpG methylation. Our results demonstrate a central role for G9a in this process and suggest that histone methylation and DNA methylation cooperatively interact via HP1 to silence TNF␣ expression during endotoxin tolerance and may have implication for proinflammatory gene silencing associated with severe systemic inflammation.Epigenetic mechanisms generate heritable marks on DNA and N-terminal tails of histones that maintain stable patterns of gene expression and are crucial in regulating gene activity as they impact chromatin structure and dynamics. These chromatin-based modifications control the recruitment of specific transcription factors and/or chromatin effectors, thereby providing a mechanism by which histones and DNA modifications regulate gene transcription (reviewed in Refs. 1-3). Methylation of histone H3 on lysine 9 (H3K9) and DNA on 5-cytosine bases, within the context of CpG dinucleotides, are two epigenetic marks whose increased levels are associated with heterochromatin formation and transcriptional silencing of several gene promoters (4).H3K9 can exist in mono-, di-, or trimethylated state. Mono-and dimethylation are catalyzed by the histone methyltransferase G9a, whereas trimethylation is catalyzed by the methyltransferase SUV39h and is predominant in pericentric (constitutive) heterochromatin domains (1, 5). While G9a can also trimethylate H3K9 in vitro (6), it is a major histone methyltransferase for mono-and dimethylation of H3K9 in euchromatic (regulated) domains (7). Methylated H3K9 serves as a docking site for chromatin modifiers such as the heterochromatin-binding protein 1 (HP1), 2 which mediates heterochromatin formation and is implicated in gene silencing (4,...

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 ...

Using a THP-1 human promonocyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype, we previously showed that tolerant cells remain responsive to LPS endotoxin with degradation of IκB in the cytosol and nuclear translocation and accumulation of p50 and p65 NF-κB transcription factors. Despite this, endotoxin-inducible NF-κB-dependent innate immunity genes, like IL-1β, remained transcriptionally unresponsive in the tolerant phenotype, similar to the endotoxin tolerance observed in sepsis patients. In this study, we examined this paradox and found that RelB, another member of the NF-κB family, is induced during the establishment of tolerance. RelB expression correlated with IL-1β repression, and sepsis patients showed increased RelB when compared with normal controls. Transient expression of RelB inhibited IL-1β in endotoxin-responsive cells. In the inverse experiment, small inhibitory RNAs decreased RelB expression in tolerant cells and restored endotoxin induction of IL-1β. When we examined tolerant cell extracts, we found transcriptionally inactive NF-κB p65/RelB heterodimers. Taken together, our findings demonstrate that RelB can repress proinflammatory gene expression, and suggest that RelB expression in sepsis patient blood leukocytes may play a role in the endotoxin-tolerant phenotype.

IL-1R-associated kinase (IRAK) plays a pivotal role in IL-1R/Toll-like receptor (TLR)-mediated signaling and NF-κB activation. IRAK from leukocytes undergoes rapid activation and inactivation/degradation following IL-1 or LPS stimulation. The rapid degradation of IRAK may serve as a negative feedback mechanism of down-regulating IL-1R/TLR-mediated signaling and cytokine gene transcription. Although IL-1/IL-1R-triggered IRAK degradation has been studied in detail, the mechanism of LPS-induced IRAK activation and degradation is not clearly defined. In this study, we demonstrate that the IRAK N-terminal 186-aa region is required for LPS-induced degradation. The N-terminally truncated IRAK protein expressed in human monocytic THP-1 cells remains stable upon LPS challenge. In comparison, IRAK as well as the IRAK mutant with C-terminal truncation undergo degradation with LPS stimulation. We demonstrate that pretreatment with protein kinase C inhibitor calphostin inhibits LPS-induced IRAK degradation. Furthermore, we observe coimmunoprecipitation of endogenous IRAK and protein kinase C-ζ protein. We show that functional TLR4 is required for LPS-mediated IRAK degradation. IRAK protein in the murine GG2EE cells harboring a mutated TLR4 gene does not undergo degradation upon LPS treatment. In sharp contrast, we observe that the IRAK homolog, IRAK2, does not undergo degradation upon prolonged LPS treatment, suggesting complex regulation of the innate immunity network upon microbial challenge.