Mohamed El Gazzar

NAD+-dependent SIRT1 Deacetylase Participates in Epigenetic Reprogramming during Endotoxin Tolerance

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-α and IL-1β, but not IκBα; 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 NFκB-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-α 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.

G9a and HP1 Couple Histone and DNA Methylation to TNFα Transcription Silencing during Endotoxin Tolerance

TNFα 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 TNFα 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 TNFα promoter is hypermethylated during endotoxin tolerance and that H3K9 methylation and DNA methylation interact to silence TNFα expression. Chromatin immunoprecipitation and RNA interference analysis demonstrated that, in tolerant cells, TNFα 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 TNFα transcription, but had no effect on H3K9 dimethylation. In contrast, G9a knockdown-disrupted promoter silencing and restored TNFα 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 TNFα expression during endotoxin tolerance and may have implication for proinflammatory gene silencing associated with severe systemic inflammation.

Epigenetic Silencing of Tumor Necrosis Factor α during Endotoxin Tolerance

Sustained silencing of potentially autotoxic acute proinflammatory genes like tumor necrosis factor α (TNFα) 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 TNFα proximal promoter nucleosomes and transcription factors adapt to an activation-specific profile by developing characteristic chromatin-based silencing marks. We found increased TNFα mRNA levels in endotoxin-responsive cells that was preceded by dissociation of heterochromatin-binding protein 1α, demethylation of nucleosomal histone H3 lysine 9 (H3(Lys9)), increased phosphorylation of the adjacent serine 10 (H3(Ser10)), and recruitment of NF-κB RelA/p65 to the TNFα promoter. In contrast, endotoxintolerant cells repressed production of TNFα mRNA, retained binding of heterochromatin-binding protein 1α, sustained methylation of H3(Lys9), reduced phosphorylation of H3(Ser10), and showed diminished binding of NF-κB RelA/p65 to the TNFα promoter. Similar levels of NF-κB p50 occurred at the TNFα promoter in the basal state, during active transcription, and in the silenced phenotype. RelB, which acts as a repressor of TNFα 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.

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

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-κB 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-1β 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.

MicroRNAs Distinguish Translational from Transcriptional Silencing during Endotoxin Tolerance

We reported that gene-selective formation of facultative heterochromatin silences transcription of acute inflammatory genes during endotoxin (LPS) tolerance, according to function. We discovered that reversal of the epigenetically silenced transcription restored mRNA levels but not protein synthesis. Here, we find that translation repression of tumor necrosis factor-α (TNFα) occurs independent of transcription silencing during LPS tolerance. The process required to disrupt protein synthesis followed Toll-like receptor 4 (TLR4)-dependent induction of microRNA (miR)-221, miR-579, and miR-125b, which coupled with RNA-binding proteins TTP, AUF1, and TIAR at the 3′-untranslated region to arrest protein synthesis. TTP and AUF1 proteins linked to miR-221, whereas TIAR coupled with miR-579 and miR-125b. Functional inhibition of miR-221 prevented TNFα mRNA degradation, and blocking miR-579 and miR-125b precluded translation arrest. The functional specificity of the TNFα 3′-untranslated region was demonstrated using luciferase reporter with mutations in the three putative miRNA binding sites. Post-transcriptional silencing was gene-specific, because it did not affect production of the IκBα anti-inflammatory protein. These results suggest that TLR4-dependent reprogramming of inflammatory genes is regulated at two separate and distinct levels. The first level of control is mediated by epigenetic modifications at the promoters that control transcription. The second and previously unrecognized level of control is mediated by TLR4-dependent differential expression of miRNAs that exert post-transcriptional controls. The concept of distinct regulation of transcription and translation was confirmed in murine sepsis. We conclude that transcription- and translation-repressive events combine to tightly regulate pro-inflammatory genes during LPS tolerance, a common feature of severe systemic inflammation.

Chromatin-specific remodeling by HMGB1 and linker histone H1 silences proinflammatory genes during endotoxin tolerance.

ABSTRACT Epigenetic silencing of tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) transcription occurs in blood leukocytes of animals and humans after the initiation of severe systemic inflammation (SSI). We previously reported that the epigenetic signature requires induction of NF-κB factor RelB, which directs histone H3K9 dimethylation, disrupts assembly of transcription activator NF-κB p65, and induces a sustained switch from the euchromatin to heterochromatin. Here, we report the novel findings that intracellular high mobility group box 1 protein (HMGB1) and nucleosome linker histone H1 protein are necessary components of endotoxin-mediated silencing of TNF-α in THP-1 human promonocytes. HMGB1 binds the TNF-α promoter during transcription silencing and promotes assembly of the repressor RelB. Depletion of HMGB1 by small interfering RNA results in dissociation of RelB from the promoter and partially restores TNF-α transcription. Histone H1, which typically displaces HMGB1 from nucleosomal DNA, also binds concomitantly with HMGB1 to the heterochromatin of the silenced TNF-α promoter. Combined knockdown of HMGB1 and H1 restores binding of the transcriptionally active NF-κB p65 and reestablishes TNF-α mRNA levels. Chromatin reimmunoprecipitation experiments demonstrate that HMGB1 and H1 are likely recruited to TNF-α sequences independently and that their binding correlates with histone H3K9 dimethylation, as inhibition of histone methylation blocks HMGB1 and H1 binding. Moreover, HMGB1- and H1-mediated chromatin modifications are gene specific during endotoxin silencing in that they also bind and repress acute proinflammatory IL-1β, while no binding nor repression of antiinflammatory IκBα is observed. Finally, we find that H1 and HMGB1 bind to the TNF-α a promoter in human leukocytes obtained from patients with SSI. We conclude proinflammatory HMGB1 and structural nucleosome linker H1 couple as a component of the epigenetic complex that silences acute proinflammatory TNF-α during the assembly of heterochromatin in the SSI phenotype.

Fueling the flame: bioenergy couples metabolism and inflammation

We review the emerging concept that changes in cellular bioenergetics concomitantly reprogram inflammatory and metabolic responses. The molecular pathways of this integrative process modify innate and adaptive immune reactions associated with inflammation, as well as influencing the physiology of adjacent tissue and organs. The initiating proinflammatory phase of inflammation is anabolic and requires glucose as the primary fuel, whereas the opposing adaptation phase is catabolic and requires fatty acid oxidation. The fuel switch to fatty acid oxidation depends on the sensing of AMP and NAD+ by AMPK and the SirT family of deacetylases (e.g., SirT1, ‐6, and ‐3), respectively, which couple inflammation and metabolism by chromatin and protein reprogramming. The AMP‐AMPK/NAD+‐SirT axis proceeds sequentially during acute systemic inflammation associated with sepsis but ceases during chronic inflammation associated with diabetes, obesity, and atherosclerosis. Rebalancing bioenergetics resolves inflammation. Manipulating cellular bioenergetics is identifying new ways to treat inflammatory and immune diseases.

MicroRNA-146a regulates both transcription silencing and translation disruption of TNF-α during TLR4-induced gene reprogramming.

Following the TLR‐dependent initiation phase of acute systemic proinflammatory responses such as sepsis, an adaptive phase represses or activates a specific pattern of gene expression until the inflammation resolves. Here, we used the THP‐1 sepsis cell model of bacterial LPS/endotoxin tolerance to show that TLR4‐induced miR‐146a supports the feed‐forward adaptive processes that silence transcription and disrupt translation of acute proinflammatory genes. First, we found that miR‐146a regulates a pathway that promotes the binding of transcription repressor RelB to the TNF‐α promoter, a step known to precede histone and DNA modifications, which generate facultative heterochromatin to silence acute proinflammatory genes. However, once RelB binding occurred, miR‐146a inhibition could not reverse compacted chromatin, and endotoxin tolerance persisted. Second, we observed that miR‐146a regulates a pathway that supports assembly of the translation repressor complex of TNF‐α by preventing the interaction of the RNA‐binding protein effector Ago2 and RBM4. We also determined that once endotoxin tolerance is established, and specific genes have been reprogrammed, transcription and translation disruption can be reversed only by simultaneously depleting RelB and inhibiting miR‐146a. Thus, miR‐146a induction supports the TLR4‐dependent shift from initiation to gene‐specific repression at two levels. Our results also imply that therapies designed to reverse endotoxin tolerance as potential therapies for sepsis should be directed at the transcription and translation pathways of reprogramming.

Myeloid-derived suppressor cells evolve during sepsis and can enhance or attenuate the systemic inflammatory response.

ABSTRACT Myeloid-derived suppressor cells (MDSCs) are a heterogeneous Gr1+ CD11b+ population of immature cells containing granulocytic and monocytic progenitors, which expand under nearly all inflammatory conditions and are potent repressors of T-cell responses. Studies of MDSCs during inflammatory responses, including sepsis, suggest they can protect or injure. Here, we investigated MDSCs during early and late sepsis. To do this, we used our published murine model of cecal ligation and puncture (CLP)-induced polymicrobial sepsis, which transitions from an early proinflammatory phase to a late anti-inflammatory and immunosuppressive phase. We confirmed that Gr1+ CD11b+ MDSCs gradually increase after CLP, reaching ∼88% of the bone marrow myeloid series in late sepsis. Adoptive transfer of early (day 3) MDSCs from septic mice into naive mice after CLP increased proinflammatory cytokine production, decreased peritoneal bacterial growth, and increased early mortality. Conversely, transfer of late (day 12) MDSCs from septic mice had the opposite effects. Early and late MDSCs studied ex vivo also differed in their inflammatory phenotypes. Early MDSCs expressed nitric oxide and proinflammatory cytokines, whereas late MDSCs expressed arginase activity and anti-inflammatory interleukin 10 (IL-10) and transforming growth factor β (TGF-β). Late MDSCs had more immature CD31+ myeloid progenitors and, when treated ex vivo with granulocyte-macrophage colony-stimulating factor (GM-CSF), generated fewer macrophages and dendritic cells than early MDSCs. We conclude that as the sepsis inflammatory process progresses, the heterogeneous MDSCs shift to a more immature state and from being proinflammatory to anti-inflammatory.

Epigenetics, bioenergetics, and microRNA coordinate gene-specific reprogramming during acute systemic inflammation

Acute systemic inflammation from infectious and noninfectious etiologies has stereotypic features that progress through an initiation (proinflammatory) phase, an adaptive (anti‐inflammatory) phase, and a resolution (restoration of homeostasis) phase. These phase‐shifts are accompanied by profound and predictable changes in gene expression and metabolism. Here, we review the emerging concept that the temporal phases of acute systemic inflammation are controlled by an integrated bioenergy and epigenetic bridge that guides the timing of transcriptional and post‐transcriptional processes of specific gene sets. This unifying connection depends, at least in part, on redox sensor NAD+‐dependent deacetylase, Sirt1, and a NF‐κB‐dependent p65 and RelB feed‐forward and gene‐specific pathway that generates silent facultative heterochromatin and active euchromatin. An additional level of regulation for gene‐specific reprogramming is generated by differential expression of miRNA that directly and indirectly disrupts translation of inflammatory genes. These molecular reprogramming circuits generate a dynamic chromatin landscape that temporally defines the course of acute inflammation.