Sirtuins and Immuno-Metabolism of Sepsis.

Sepsis and septic shock are the leading causes of death in non-coronary intensive care units worldwide. During sepsis-associated immune dysfunction, the early/hyper-inflammatory phase transitions to a late/hypo-inflammatory phase as sepsis progresses. The majority of sepsis-related deaths occur during the hypo-inflammatory phase. There are no phase-specific therapies currently available for clinical use in sepsis. Metabolic rewiring directs the transition from hyper-inflammatory to hypo-inflammatory immune responses to protect homeostasis during sepsis inflammation, but the mechanisms underlying this immuno-metabolic network are unclear. Here, we review the roles of NAD+ sensing Sirtuin (SIRT) family members in controlling immunometabolic rewiring during the acute systemic inflammatory response associated with sepsis. We discuss individual contributions among family members SIRT 1, 2, 3, 4 and 6 in regulating the metabolic switch between carbohydrate-fueled hyper-inflammation to lipid-fueled hypo-inflammation. We further highlight the role of SIRT1 and SIRT2 as potential "druggable" targets for promoting immunometabolic homeostasis and increasing sepsis survival.

NAD+-dependent sirtuin 1 and 6 proteins coordinate a switch from glucose to fatty acid oxidation during the acute inflammatory response.

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and ß to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

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-1ß, 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.

Sepsis, pyruvate, and mitochondria energy supply chain shortage.

Balancing high energy-consuming danger resistance and low energy supply of disease tolerance is a universal survival principle that often fails during sepsis. Our research supports the concept that sepsis phosphorylates and deactivates mitochondrial pyruvate dehydrogenase complex control over the tricarboxylic cycle and the electron transport chain. StimulatIng mitochondrial energetics in septic mice and human sepsis cell models can be achieved by inhibiting pyruvate dehydrogenase kinases with the pyruvate structural analog dichloroacetate. Stimulating the pyruvate dehydrogenase complex by dichloroacetate reverses a disruption in the tricarboxylic cycle that induces itaconate, a key mediator of the disease tolerance pathway. Dichloroacetate treatment increases mitochondrial respiration and ATP synthesis, decreases oxidant stress, overcomes metabolic paralysis, regenerates tissue, organ, and innate and adaptive immune cells, and doubles the survival rate in a murine model of sepsis.

Dynamic and selective nucleosome repositioning during endotoxin tolerance.

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-kappaB 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 alpha (TNFalpha) 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-kappaB 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-kappaB 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-kappaB 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 TNFalpha 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 TNFalpha transcription associated with sepsis.

Facultative heterochromatin formation at the IL-1 beta promoter in LPS tolerance and sepsis.

The clinical phenotype in sepsis that is observed as LPS tolerance is determined by silencing of pro-inflammatory genes like IL-1 beta (IL-1ß). This study shows that facultative heterochromatin (fHC) silences IL-1ß expression during sepsis, where we find dephosphorylated histone H3 serine 10 and increased binding of heterochromatin protein-1 (HP-1) to the promoter. In both human sepsis blood leukocytes and an LPS tolerant human THP-1 cell model, we show that IκBα and v-rel reticuloendotheliosis viral oncogene homolog B (RelB) function as dominant labile mediators of fHC formation at the IL-1ß promoter. Protein synthesis inhibition decreases levels of IκBα and RelB, converts silent fHC to euchromatin, and restores IL-1ß transcription. We further show TLR dependent NFκB p65 and histone H3 serine 10 phosphorylation binding at the promoter. We conclude that the resolution phase of sepsis, which correlates with survival in humans, may depend on the plasticity of chromatin structure as found in fHC.

The NF-kappaB 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-kappaB 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-1beta 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.

SIRT1 Mediates Septic Cardiomyopathy in a Murine Model of Polymicrobial Sepsis.

BACKGROUND: Cardiac dysfunction, a common complication from severe sepsis, is associated with increased morbidity and mortality. However, the molecular mechanisms of septic cardiac dysfunction are poorly understood. SIRT1, a member of the sirtuin family of NAD+-dependent protein deacetylases, is an important immunometabolic regulator of sepsis, and sustained SIRT1 elevation is associated with worse outcomes and organ dysfunction in severe sepsis. Herein, we explore the role of SIRT1 in septic cardiac dysfunction using a murine model of sepsis. METHODS: An in vitro model of inflammation in isolated H9c2 cardiomyocytes was used to confirm SIRT1 response to stimulation with lipopolysaccharide (LPS), followed by a murine model of cecal ligation and puncture (CLP) to investigate the molecular and echocardiographic response to sepsis. A selective SIRT1 inhibitor, EX-527, was employed to test for SIRT1 participation in septic cardiac dysfunction. RESULTS: SIRT1 mRNA and protein levels in cultured H9c2 cardiomyocytes were significantly elevated at later time points after stimulation with LPS. Similarly, cardiac tissue harvested from C57BL/6 mice 36 h after CLP demonstrated increased expression of SIRT1 mRNA and protein compared with sham controls. Administration of EX-527 18 h after CLP reduced SIRT1 protein expression in cardiac tissue at 36 h. Moreover, treatment with EX-527 improved cardiac performance with increased global longitudinal strain and longitudinal strain rate. CONCLUSIONS: Our findings reveal that SIRT1 expression increases in isolated cardiomyocytes and cardiac tissue after sepsis inflammation. Moreover, rebalancing SIRT1 excess in late sepsis improves cardiac performance, suggesting that SIRT1 may serve as a therapeutic target for septic cardiomyopathy.

Stimulating pyruvate dehydrogenase complex reduces itaconate levels and enhances TCA cycle anabolic bioenergetics in acutely inflamed monocytes.

The pyruvate dehydrogenase complex (PDC)/pyruvate dehydrogenase kinase (PDK) axis directs the universal survival principles of immune resistance and tolerance in monocytes by controlling anabolic and catabolic energetics. Immune resistance shifts to immune tolerance during inflammatory shock syndromes when inactivation of PDC by increased PDK activity disrupts the tricarboxylic acid (TCA) cycle support of anabolic pathways. The transition from immune resistance to tolerance also diverts the TCA cycle from citrate-derived cis-aconitate to itaconate, a recently discovered catabolic mediator that separates the TCA cycle at isocitrate and succinate dehydrogenase (SDH). Itaconate inhibits succinate dehydrogenase and its anabolic role in mitochondrial ATP generation. We previously reported that inhibiting PDK in septic mice with dichloroacetate (DCA) increased TCA cycle activity, reversed septic shock, restored innate and adaptive immune and organ function, and increased survival. Here, using unbiased metabolomics in a monocyte culture model of severe acute inflammation that simulates sepsis reprogramming, we show that DCA-induced activation of PDC restored anabolic energetics in inflammatory monocytes while increasing TCA cycle intermediates, decreasing itaconate, and increasing amino acid anaplerotic catabolism of branched-chain amino acids (BCAAs). Our study provides new mechanistic insight that the DCA-stimulated PDC homeostat reconfigures the TCA cycle and promotes anabolic energetics in monocytes by reducing levels of the catabolic mediator itaconate. It further supports the theory that PDC is an energy sensing and signaling homeostat that restores metabolic and energy fitness during acute inflammation.

Live Quality Assurance: Using a Multimedia Messaging Service Group Chat to Instantly Grade Intraoperative Images.

BACKGROUND: The technique for attaining photographic evidence of the critical view of safety (CVS) in laparoscopic cholecystectomy (LC) has previously been defined; however, the consistency, accuracy, and feasibility of CVS in practice is unknown. The aim of this study was to use an already established image sharing and grading system to determine the feasibility of timely feedback after sharing intraoperative images of the CVS and to evaluate if and how cholecystitis affects the ability to attain a CVS. STUDY DESIGN: We studied 193 laparoscopic cholecystectomies performed by 14 surgeons between August 2017 and January 2019. Anterior and posterior intraoperative CVS images were shared using a standard multimedia messaging system (MMS). Images were graded remotely by members of the group using an established scoring system, and their times to response and scores were recorded. Response data were analyzed for the ability to attain timely and consistent CVS scores. RESULTS: There were 74 urgent laparoscopic cholecystectomies for acute cholecystitis and 119 nonurgent cholecystectomies performed during the study period. Scoring of shared images occurred in less than 5 minutes, and peer review (mean 3 responses) showed agreement that was not significantly different. In patients with acute cholecystitis, a small but significant difference was observed between anterior and posterior image scoring agreement. CONCLUSIONS: An established image sharing and grading system for CVS can be used for real-time intraoperative feedback without increasing operative time or compromising private health information. The CVS is almost always attainable; however, decreases in CVS quality and grading agreement are observed in patients with acute cholecystitis.

Pulmonary contusion primes systemic innate immunity responses.

INTRODUCTION: Traumatic injury may result in an exaggerated response to subsequent immune stimuli such as nosocomial infection. This "second hit" phenomenon and molecular mechanism(s) of immune priming by traumatic lung injury, specifically, pulmonary contusion, remain unknown. We used an animal model of pulmonary contusion to determine whether the injury resulted in priming of the innate immune response and to test the hypothesis that resuscitation fluids could attenuate the primed response to a second hit. METHODS: Male, 8 to 9 weeks, C57/BL6 mice with a pulmonary contusion were challenged by a second hit of intratracheal administration of the Toll-like receptor 4 agonist, lipopolysaccharide (LPS, 50 microg) 24 hours after injury (injury + LPS). Other experimental groups were injury + vehicle or LPS alone. A separate group was injured and resuscitated by 4 cc/kg of hypertonic saline (HTS) or Lactated Ringer's (LR) resuscitation before LPS challenge. Mice were killed 4 hours after LPS challenge and blood, bronchoalveolar lavage, and tissue were isolated and analyzed. Data were analyzed using one-way analysis of variance with Bonferroni multiple comparison posttest for significant differences (*p < or = 0.05). RESULTS: Injury + LPS showed immune priming observed by lung injury histology and increased bronchoalveolar lavage neutrophilia, lung myeloperoxidase and serum IL-6, CXCL1, and MIP-2 levels when compared with injury + vehicle or LPS alone. After injury, resuscitation with HTS, but not Lactated Ringer's was more effective in attenuating the primed response to a second hit. CONCLUSION: Pulmonary contusion primes innate immunity for an exaggerated response to a second hit with the Toll-like receptor 4 agonist, LPS. We observed synergistic increases in inflammatory mediator expression in the blood and a more severe lung injury in injured animals challenged with LPS. This priming effect was reduced when HTS was used to resuscitate the animal after lung contusion.

Cysteine thiol oxidation on SIRT2 regulates inflammation in obese mice with sepsis.

Obesity increases morbidity and mortality in acute illnesses such as sepsis and septic shock. We showed previously that the early/hyper-inflammatory phase of sepsis is exaggerated in obese mice with sepsis; sirtuin 2 (SIRT2) modulates sepsis inflammation in obesity. Evidence suggests that obesity with sepsis is associated with increased oxidative stress. It is unknown whether exaggerated hyper-inflammation of obesity with sepsis modulates the SIRT2 function in return. We showed recently that SIRT6 oxidation during hyper-inflammation of sepsis modulates its glycolytic function. This study tested the hypothesis that increased oxidative stress and direct SIRT2 oxidation exaggerate hyper-inflammation in obesity with sepsis. Using spleen and liver tissue from mice with diet-induced obesity (DIO) we studied oxidized vs. total SIRT2 expression during hyper- and hypo-inflammation of sepsis. To elucidate the mechanism of SIRT2 oxidation (specific modifications of redox-sensitive cysteines) and its effect on inflammation, we performed site-directed mutations of redox-sensitive cysteines Cys221 and Cys224 on SIRT2 to serine (C221S and C224S), transfected HEK293 cells with mutants or WT SIRT2, and studied SIRT2 enzymatic activity and NFĸBp65 deacetylation. Finally, we studied the effect of SIRT2 mutation on LPS-induced inflammation using RAW 264.7 macrophages. In an inverse relationship, total SIRT2 decreased while oxidized SIRT2 expression increased during hyper-inflammation and SIRT2 was unable to deacetylate NFĸBp65 with increased oxidative stress of obesity with sepsis. Mechanistically, both the mutants (C221S and C224S) show decreased (1) SIRT2 enzymatic activity, (2) deacetylation of NFĸBp65, and (3) anti-inflammatory activity in response to LPS vs. WT SIRT2. Direct oxidation modulates SIRT2 function during hyper-inflammatory phase of obesity with sepsis via redox sensitive cysteines.

Frontline Science: Monocytes sequentially rewire metabolism and bioenergetics during an acute inflammatory response.

Metabolism directs the severe acute inflammatory reaction of monocytes to guard homeostasis. This occurs by sequentially activating anabolic immune effector mechanisms, switching to immune deactivation mechanisms and then restoring immunometabolic homeostasis. Nuclear sirtuin 1 and mitochondrial pyruvate dehydrogenase kinase metabolically drive this dynamic and are druggable targets that promote immunometabolic resolution in septic mice and increase survival. We used unbiased metabolomics and a validated monocyte culture model of activation, deactivation, and partial resolution of acute inflammation to sequentially track metabolic rewiring. Increases in glycogenolysis, hexosamine, glycolysis, and pentose phosphate pathways were aligned with anabolic activation. Activation transitioned to combined lipid, protein, amino acid, and nucleotide catabolism during deactivation, and partially subsided during early resolution. Lipid metabolic rewiring signatures aligned with deactivation included elevated n-3 and n-6 polyunsaturated fatty acids and increased levels of fatty acid acylcarnitines. Increased methionine to homocysteine cycling increased levels of s-adenosylmethionine rate-limiting transmethylation mediator, and homocysteine and cysteine transsulfuration preceded increases in glutathione. Increased tryptophan catabolism led to elevated kynurenine and de novo biosynthesis of nicotinamide adenine dinucleotide from quinolinic acid. Increased branched-chain amino acid catabolism paralleled increases in succinyl-CoA. A rise in the Krebs cycle cis-aconitate-derived itaconate and succinate with decreased fumarate and acetyl-CoA levels occurred concomitant with deactivation and subsided during early resolution. The data suggest that rewiring of metabolic and mitochondrial bioenergetics by monocytes sequentially activates, deactivates, and resolves acute inflammation.

Sirtuin1 Targeting Reverses Innate and Adaptive Immune Tolerance in Septic Mice.

Resistance and tolerance to infection are two universal fitness and survival strategies used by inflammation and immunity in organisms and cells to guard homeostasis. During sepsis, however, both strategies fail, and animal and human victims often die from combined innate and adaptive immune suppression with persistent bacterial and viral infections. NAD+-sensing nuclear sirtuin1 (SIRT1) epigenetically guards immune and metabolic homeostasis during sepsis. Pharmacologically inhibiting SIRT1 deacetylase activity in septic mice reverses monocyte immune tolerance, clears infection, rebalances glycolysis and glucose oxidation, resolves organ dysfunction, and prevents most septic deaths. Whether SIRT1 inhibition during sepsis treatment concomitantly reverses innate and T cell antigen-specific immune tolerance is unknown. Here, we show that treating septic mice with a SIRT1 selective inhibitor concordantly reverses immune tolerance splenic dendritic and antigen-specific tolerance of splenic CD4+ and CD8+ T cells. SIRT1 inhibition also increases the ratio of IL12 p40+ and TNFα proinflammatory/immune to IL10 and TGFß anti-inflammatory/immune cytokines and decreases the ratio of CD4+ TReg repressor to CD4+ activator T cells. These findings support the unifying concept that nuclear NAD+ sensor SIRT1 broadly coordinates innate and adaptive immune reprogramming during sepsis and is a druggable immunometabolic enhancement target.

Antioxidant treatment after injury suppresses second hit immune priming.

BACKGROUND: Pulmonary contusion (PC) is a common injury that often results in priming for exaggerated inflammatory responses to a second hit. Previous studies used a mouse model of pulmonary contusion and showed an early and sustained reduction of SIRT1 protein and activity in the lung and bronchoalveolar lavage (BAL) cells of injured mice. Sustained decrease in SIRT1 was associated with a primed phenotype in injured mice challenged with an inflammatory stimulus. This study tests the hypothesis that pulmonary contusion induces oxidant production that modifies and decreases SIRT1 and primes the lung for the second-hit response. METHODS: A mouse model of pulmonary contusion was used to investigate injury-induced oxidant changes in SIRT1. Second-hit responses were evaluated by infection (Streptococcus pneumoniae) and inflammatory challenge using bacterial lipopolysaccharide. BAL, lung tissue, and blood were collected and used to evaluate inflammatory responses and SIRT1 levels, oxidant modification, and activity. Levels of NO in the BAL from mice and patients with PC were also assessed. RESULTS: We found that oxidants produced as a result of pulmonary contusion resulted in modification of SIRT1. S-Nitrosylation was observed and correlated with increased inducible nitric oxide synthase expression after injury. Anti-oxidant treatment of injured mice preserved SIRT1 activity, decreased second hit responses and improved lung function. Elevated NO levels in the BAL of PC patients was associated with acute respiratory distress syndrome or diagnosis of pneumonia. CONCLUSIONS: We conclude that oxidative stress in the lung after injury induces redox modification of SIRT1 and contributes to priming of the lung for a second-hit response. Antioxidant treatment suggests that SIRT1 activity after injury may be beneficial in suppressing second-hit responses.

Pyruvate dehydrogenase complex stimulation promotes immunometabolic homeostasis and sepsis survival.

Limited understanding of the mechanisms responsible for life-threatening organ and immune failure hampers scientists' ability to design sepsis treatments. Pyruvate dehydrogenase kinase 1 (PDK1) is persistently expressed in immune-tolerant monocytes of septic mice and humans and deactivates mitochondrial pyruvate dehydrogenase complex (PDC), the gate-keeping enzyme for glucose oxidation. Here, we show that targeting PDK with its prototypic inhibitor dichloroacetate (DCA) reactivates PDC; increases mitochondrial oxidative bioenergetics in isolated hepatocytes and splenocytes; promotes vascular, immune, and organ homeostasis; accelerates bacterial clearance; and increases survival. These results indicate that the PDC/PDK axis is a druggable mitochondrial target for promoting immunometabolic and organ homeostasis during sepsis.

Toll-like receptor 2 participates in the response to lung injury in a murine model of pulmonary contusion.

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We report that Toll-like receptor (TLR) 2 participates in the inflammatory response to lung injury. To show this, we use a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans based on histologic, morphologic, and biochemical criteria of acute lung injury. The inflammatory response to pulmonary contusion in our mouse model is characterized by pulmonary edema, neutrophil transepithelial migration, and increased expression of the innate immunity proinflammatory cytokines IL 1beta and IL 6, the adhesion intracellular adhesion molecule 1, and chemokine (CXC motif) ligand 1. Compared with wild-type animals, contused Tlr2(-/-) mice have significantly reduced pulmonary edema and neutrophilia. These findings are associated with decreased levels of circulating chemokine (CXC motif) ligand 1. In contrast, systemic IL 6 levels remain elevated in the TLR2-deficient phenotype. These results show that TLR2 has a primary role in the neutrophil response to acute lung injury. We suggest that an unidentified noninfectious ligand generated by pulmonary contusion acts via TLR2 to generate inflammatory responses.

Sirtuin 2 Regulates Microvascular Inflammation during Sepsis.

Objective. Sepsis and septic shock, the leading causes of death in noncoronary intensive care units, kill more than 200,000/year in the US alone. Circulating cell-endothelial cell interactions are the rate determining factor in sepsis inflammation. Sirtuin, a seven-member family of proteins (SIRT1-7), epigenetically controls inflammation. We have studied the roles of SIRTs 1, 3, and 6 in sepsis previously. In this project, we studied the role of SIRT2 on sepsis-related inflammation. Methods. Sepsis was induced in C57Bl/6 (WT), SIRT2 knockout (SIRT2KO), and SIRT2 overexpressing (SIRT2KI) mice by cecal ligation and puncture (CLP). We studied leukocyte/platelet adhesion using intravital microscopy and E-selectin/ICAM-1 adhesion molecule expression in the small intestine with immunohistochemistry (IHC) six hours post-CLP/sham surgery. We also studied 7-day survival rates in WT, SIRT2KO, and SIRT2KI sepsis mice. Results. Compared to WT mice, SIRT2KO mice show exaggeration while SIRT2KI mice show attenuation of cellular adhesion with sepsis in the small intestine. We also show that the small intestinal E-selectin and ICAM-1 expressions increased in SIRT2KO and decreased in SIRT2KI mice versus those in WT sepsis mice. We show that the 7-day survival rate is decreased in SIRT2KO and increased in SIRT2KI sepsis mice. Conclusion. SIRT2 modulates microvascular inflammation in sepsis and affects survival.

The Oxidative State of Cysteine Thiol 144 Regulates the SIRT6 Glucose Homeostat.

Control of glucose homeostasis plays a critical role in health and lifespan and its dysregulation contributes to inflammation, cancer and aging. NAD + dependent Sirtuin 6 (SIRT6) is a glucose homeostasis regulator in animals and humans and its regulation at the molecular level is unknown. Here, we report that a cysteine thiol redox sensor contributes to the role of SIRT6 in controlling glucose homeostasis. Sulfenylation of SIRT6 occurs in THP1 cells and primary human promonocytes during inflammation and in splenocytes from mice with sepsis. Inhibiting xanthine oxidase, a major reactive oxygen species (ROS) contributor during acute inflammation, reduces sulfenylation of SIRT6, glucose transporter Glut1 expression, glucose uptake, and glycolysis. A block in glycolysis associated with monocyte deactivation by endotoxin, a process contributing to immunometabolic paralysis in human and mouse sepsis monocytes, can be reversed by increasing H2O2 and sulfenylating SIRT6. Mutation analysis of SIRT6 Cys144, which lies in its phylogenetically conserved zinc-associated Cys-X-X-Cys motif near the catalytic domain of the protein, decreases SIRT6 deacetylase activity and promotes glycolysis. These results suggest that direct and reversible cysteine thiol 144 may play a functional role in SIRT6-dependent control over monocyte glycolysis, an important determinant of effector innate immune responses.

Sirtuins Link Inflammation and Metabolism.

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.