Background: Switching from the acute early initiation to late adaptation response after TLR4 stimulation depends on SirT1. Results: Switching from glucose to fatty acid oxidation between initiation and adaptation responses requires SirT6 and SirT1. Conclusion: Bioenergy integrates metabolism and acute inflammation. Significance: Understanding bioenergy shifts during inflammation may enable development of new therapies.

Gene-selective epigenetic reprogramming and shifts in cellular bioenergetics develop when Toll-like receptors (TLR) recognize and respond to systemic life-threatening infections. Using a human monocyte cell model of endotoxin tolerance and human leukocytes from acute systemic inflammation with sepsis, we report that energy sensor sirtuin 1 (SIRT1) coordinates the epigenetic and bioenergy shifts. After TLR4 signaling, SIRT1 rapidly accumulated at the promoters of TNF-␣ and IL-1␤, but not IB␣; SIRT1 promoter binding was dependent on its co-factor, NAD ؉ . During this initial process, SIRT1 deacetylated RelA/ p65 lysine 310 and nucleosomal histone H4 lysine 16 to promote termination of NFB-dependent transcription. SIRT1 then remained promoter bound and recruited de novo induced RelB, which directed assembly of the mature transcription repressor complex that generates endotoxin tolerance. SIRT1 also promoted de novo expression of RelB. During sustained endotoxin tolerance, nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for endogenous production of NAD ؉ , and SIRT1 expression increased. The elevation of SIRT1 required protein stabilization and enhanced translation. To support the coordination of bioenergetics in human sepsis, we observed elevated NAD ؉ levels concomitant with SIRT1 and RelB accumulation at the TNF-␣ promoter of endotoxin tolerant sepsis blood leukocytes. We conclude that TLR4 stimulation and human sepsis activate pathways that couple NAD ؉ and its sensor SIRT1 with epigenetic reprogramming.Two cellular processes predictably accompany TLR-mediated acute systemic inflammation caused by sepsis, a highly destructive and often lethal process. The first process occurs when epigenetic alterations reprogram distinct functional sets of genes to both activate and repress transcription of hundreds of genes (1, 2); this transcriptome reprogramming generates the phenomenon known as endotoxin tolerance (3). Endotoxin tolerance requires TLR 3 receptor signaling of NFB master regulator, which first induces and then represses rapid response and potentially autotoxic proinflammatory TNF-␣ and IL-1␤. To control the initial recognition and response phases induced by TLR, gene-specific reprogramming selectively modifies chromatin structure and shifts nucleosomes on responsive euchromatin to form silent heterochromatin at acute proinflammatory genes; in contrast, genes encoding anti-inflammatory and antimicrobial mediators maintain responsive euchromatin (4, 5). This gene set-selective reprogramming generates a clinically relevant phenotypic transition from the hyperinflammatory to the hypoinflammatory endotoxin tolerant state, which may last hours, days, or weeks, depending on the strength of the initial TLR response (4, 5). The physiologic importance of endotoxin tolerance is still incompletely understood, but likely reflects an attempt to recover homeostasis (3).Others and we (6 -9) reported how temporal transitions in epigenetic programming alter the course of acute inflammation. NFB master ...

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.

Sirtuins (SIRT), first discovered in yeast as NAD+ dependent epigenetic and metabolic regulators, have comparable activities in human physiology and disease. Mounting evidence supports that the seven-member mammalian sirtuin family (SIRT1–7) guard homeostasis by sensing bioenergy needs and responding by making alterations in the cell nutrients. Sirtuins play a critical role in restoring homeostasis during stress responses. Inflammation is designed to “defend and mend” against the invading organisms. Emerging evidence supports that metabolism and bioenergy reprogramming direct the sequential course of inflammation; failure of homeostasis retrieval results in many chronic and acute inflammatory diseases. Anabolic glycolysis quickly induced (compared to oxidative phosphorylation) for ROS and ATP generation is needed for immune activation to “defend” against invading microorganisms. Lipolysis/fatty acid oxidation, essential for cellular protection/hibernation and cell survival in order to “mend,” leads to immune repression. Acute/chronic inflammations are linked to altered glycolysis and fatty acid oxidation, at least in part, by NAD+ dependent function of sirtuins. Therapeutically targeting sirtuins may provide a new class of inflammation and immune regulators. This review discusses how sirtuins integrate metabolism, bioenergetics, and immunity during inflammation and how sirtuin-directed treatment improves outcome in chronic inflammatory diseases and in the extreme stress response of sepsis.

Mechanism-based sepsis treatments are unavailable, and their incidence is rising worldwide. Deaths occur during the early acute phase of hyperinflammation or subsequent postacute hypoinflammatory phase with sustained organ failure. The acute sepsis phase shifts rapidly, and multiple attempts to treat early excessive inflammation have uniformly failed. We reported in a sepsis cell model and human sepsis blood leukocytes that nuclear NAD+ sensor SIRT1 deacetylase remodels chromatin at specific gene sets to switch the acute-phase proinflammatory response to hypoinflammatory. Importantly, SIRT1 chromatin reprogramming is reversible, suggesting that inhibition of SIRT1 might reverse postacute-phase hypoinflammation. We tested this concept in septic mice, using the highly specific SIRT1 inhibitor EX-527, a small molecule that closes the NAD+ binding site of SIRT1. Strikingly, when administered 24 h after sepsis, all treated animals survived, whereas only 40% of untreated mice survived. EX-527 treatment reversed the inability of leukocytes to adhere at the small intestine MVI, reversed in vivo endotoxin tolerance, increased leukocyte accumulation in peritoneum, and improved peritoneal bacterial clearance. Mechanistically, the SIRT1 inhibitor restored repressed endothelial E-selectin and ICAM-1 expression and PSGL-1 expression on the neutrophils. Systemic benefits of EX-527 treatment included stabilized blood pressure, improved microvascular blood flow, and a shift toward proimmune macrophages in spleen and bone marrow. Our findings reveal that modifying the SIRT1 NAD+ axis may provide a novel way to treat sepsis in its hypoinflammatory phase.

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...

The NF-κB family plays a crucial role in the pathogenesis of highly lethal septicemia by modulating transcription of many innate and adaptive immunity genes. Two phases of NF-κB activation occur: cytosolic activation and nuclear transactivation. Septicemia with multiorgan failure is associated with chronic activation of cytosolic NF-κB with translocation and accumulation of increased levels of nuclear p65 in blood leukocytes. Paradoxically, NF-κB-dependent transcription of many proinflammatory genes responding to bacterial LPS endotoxin (LPS) is persistently repressed during septicemia; this phenomenon of LPS tolerance is associated with immunosuppression and poor prognosis. This report suggests an explanation for this paradox. Using an in vitro human leukocyte model and chromatin immunoprecipitation assays, we find that both the cytosolic activation and nuclear transactivation phases of NF-κB occur in LPS responsive THP-1 promonocytes with recruitment and binding of NF-κB p65 at the IL-1β promoter. However, transcriptionally repressed LPS-tolerant THP-1 cells do not bind NF-κB p65 at the IL-1β promoter, despite cytosolic activation and accumulation of p65 in the nucleus. In contrast, NF-κB p50, which also accumulates in the nucleus, constitutively binds to the IL-1β promoter NF-κB site in both LPS-responsive and LPS-tolerant cells. The level of p65 binding correlates with a binary shift in nucleosome remodeling between histone H3 phosphorylation at serine 10 and methylation of histone H3 at lysine 9. We conclude that LPS tolerance disrupts the transactivating stage of NF-κB p65 and altered nucleosome remodeling at the IL-1β promoter in human leukocytes.

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 ...