Charles E. McCall

Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells.

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-1β 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.

Tolerance to endotoxin-induced expression of the interleukin-1 beta gene in blood neutrophils of humans with the sepsis syndrome.

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 beta. 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 beta gene, as determined by decreased synthesis of the IL-1 beta protein and reductions in IL-1 beta mRNA. This down-regulation of the IL-1 beta 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 beta 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-1 beta 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.

Lipopolysaccharide- and Lipoteichoic Acid-Induced Tolerance and Cross-Tolerance: Distinct Alterations in IL-1 Receptor-Associated Kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1β and TNF-α are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1β/TNF-α production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1β/TNF-α production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1β/TNF-α upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.

Complete Deficiency of Leukocyte Glucose-6-Phosphate Dehydrogenase with Defective Bactericidal Activity

A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H(2)O(2)-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H(2)O(2) were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H(2)O(2); F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H(2)O(2) production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H(2)O(2) in human granulocytes.

Role of protein kinases in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists. Differential effects of a novel protein kinase inhibitor.

Isoquinoline sulfonamides have recently been shown to exert novel inhibitory effects on mammalian protein kinases by competitively binding to the ATP substrate site (Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki, 1984, Biochemistry, 23: 5036-5041). We synthesized a unique analog of the previously reported compounds, 1-(5-isoquinolinesulfonyl) piperazine (C-I), in order to assess the role of protein kinases in modulating the agonist-stimulated oxidative burst of human polymorphonuclear leukocytes (PMN). Compound C-I, at micromolar concentration, markedly inhibited the release of superoxide anion from human PMN stimulated with phorbol myristate acetate or the synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol. These data are consonant with previously reported data which indicate that the calcium and phospholipid-dependent protein kinase, protein kinase C, serves as the intracellular receptor for these agonists. In contrast, superoxide anion production stimulated by the complement anaphylatoxin peptide C5a or the synthetic chemotaxin formyl-methionyl-leucyl-phenylalanine were not inhibited by C-I. These data suggest that parallel pathways exist for the agonist-stimulated respiratory burst of human neutrophils, only one of which utilizes the calcium and phospholipid-dependent protein kinase.

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

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. 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 β 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.

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.

Endotoxin Tolerance Disrupts Chromatin Remodeling and NF-κB Transactivation at the IL-1β Promoter

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.

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.

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.