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.

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 ...

The induction of genes of host cells stimulated by microbial products such as endotoxin and the tolerance of cells to endotoxin excitation play critical roles in the pathogenesis of microbial-induced acute disseminated inflammation with multiorgan failure (the sepsis syndrome). One gene that is induced in phagocytic cells by endotoxin and that appears to play an essential role in the pathogenesis of the sepsis syndrome is IL-1,8. We report here that blood neutrophils (PMN) of patients with the sepsis syndrome (sepsis PMN) are consistently tolerant to endotoxin-induced expression of the IL-1,8 gene, as determined by decreased synthesis of the IL-113 protein and reductions in IL-1,8 mRNA. This down-regulation of the IL-1#j gene in sepsis PMN occurs concomitant with an upregulation in the constitutive expression of the type 2 IL-1 receptor (IL-1R2). These phenotypic changes do not persist in PMN of patients recovering from the sepsis syndrome. Tolerance has stimulus and response specificity since sepsis PMN tolerant to endotoxin can respond normally to Staphylococcus aureus stimulation of IL-1,B production and they normally secrete elastase. Uninfected patients with severe trauma or shock from causes are not tolerant to endotoxin and tolerance is not limited to patients infected with Gram-negative bacteria. The mechanism responsible for tolerance involves pretranslational events and is not due to loss of the CD14 surface protein, a receptor required for endotoxin induction of IL-1B in PMN. The physiological significance of the tolerance to endotoxin and increased expression of IL-1R2 on sepsis PMN is unknown, but may represent an attempt by the host to protect itself from the deleterious effects of disseminated inflammation. (J. Clin. Invest. 1993. 91:853-861.)

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.

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