The cellular mechanisms that directly regulate the inflammatory response after Toll-like receptor (TLR) stimulation are unresolved at present. Here we report that glycogen synthase kinase 3 (GSK3) differentially regulates TLR-mediated production of pro-and anti-inflammatory cytokines. Stimulation of monocytes or peripheral blood mononuclear cells with TLR2, TLR4, TLR5 or TLR9 agonists induced substantial increases in interleukin 10 production while suppressing the release of proinflammatory cytokines after GSK3 inhibition. GSK3 regulated the inflammatory response by differentially affecting the nuclear amounts of transcription factors NF-κB subunit p65 and CREB interacting with the coactivator CBP. Administration of a GSK3 inhibitor potently suppressed the proinflammatory response in mice receiving lipopolysaccharide and mediated protection from endotoxin shock. These findings demonstrate a regulatory function for GSK3 in modulating the inflammatory response.The ability of the innate immune system to recognize and respond to microbial components has been largely attributed to the family of type I transmembrane receptors called Toll-like receptors (TLRs) 1,2 . TLRs are expressed mainly on antigen-presenting cells such as monocytes-macrophages and dendritic cells and show an ability to discriminate among distinct molecular patterns associated with microbial components. Recognition of microbial products by TLRs leads to a variety of signal transduction pathways that regulate the nature, magnitude and duration of the inflammatory response 3 . Although the production of proinflammatory cytokines is important for mediating the initial host defense against invading pathogens 4 , an inability to regulate the nature or duration of the host's inflammatory response can be detrimental, as in chronic inflammatory diseases. The underlying cellular mechanisms that directly control pro-versus anti-inflammatory cytokine production after TLR stimulation are unresolved, but studies have shown the TLR signaling pathway can activate phosphatidylinositol 3-OH kinase (PI(3)K) 5 to limit production of tumor necrosis factor (TNF) and interleukin 12 (IL-12) 6-8 . Moreover, TLR2-induced activation of the PI(3)K pathway enhances IL-10 production, whereas IL-12 production is reduced 9 . Those studies suggested critical involvement of the PI(3)K pathway in differentially controlling pro-and antiinflammatory cytokine production, but it is unclear at present if this pathway is limited to TLR2 signaling and how production of other inflammatory mediators induced by other TLR ligands is affected. Here we have characterized how intracellular TLR signaling affected 'downstream' effector molecules of the PI(3)K pathway to determine if a central effector molecule is responsible for mediating the ability of this pathway to differentially dictate the host's inflammatory response. Inhibition of glycogen synthase kinase 3 (GSK3), the constitutively active downstream kinase of the PI(3)K pathway, mediated the ability of this pathway to selectively...

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.Lipopolysaccharides (LPS) are among the most potent inflammatory bacterial mediators and have been strongly implicated in the inflammatory response associated with gram-negative sepsis. Most LPS signaling studies have used LPS preparations derived from species within the Enterobacteriaceae, which possess relatively well-conserved lipid A structures (reviewed in reference 36). A convergence of data suggest that these prototypic LPS preparations, when highly purified, elicit LPS responses that are restricted in the use of TLR4 as the principal signal-transducing molecule (reviewed in reference 21), which is strongly supported by the finding that synthetic E. coli lipid A activated Toll-like receptor 4 (TLR4) and not TLR2 transfectants (8). However, the lipid A of nonenterobacterial species, e.g., Porphyromonas gingivalis, which has been implicated in the inflammation associated with chronic periodontitis (reviewed in reference 9), differs both structurally and functionally from enterobacterial lipid A. Specifically, the major species of P. gingivalis lipid A is composed of unique branched fatty acids, with longer carbon chains than in enterobacterial lipid A, the absence of a phosphoryl group at position 4Ј of the nonreducing glucosamine, as well as other modifications ( Fig. 1) (1). Consistent with these structural differences is the finding that P. gingivalis LPS activity is poorly inhibited by polymyxin B (12), which has been postulated to inactivate LPS by binding electrostatically to negatively charged phosphate groups, leading to a subsequent interaction of polymyxin B with the hydrophobic fatty acids (25, 33). Although P. gingivalis-induced signaling was shown some time ago to be CD14 dependent (34), site-specific mutagenesis of CD14 suggests that the substitution of certain charged amino acids differentially affects the abilities of Escherichia coli and P. gingivalis LPS to bind CD14 (4, 5). In addition, binding of P. gingivalis LPS to LPS binding protein has been reported to be 100-fo...

In just a few years, glycogen synthase kinase-3 (GSK3) has transformed from an obscure enzyme seldom encountered in the immune literature to one implicated in an improbably large number of roles. GSK3 is a crucial regulator of the balance between pro-and anti-inflammatory cytokine production in both the periphery and the central nervous system, endowing GSK3 inhibitors such as lithium with the capacity to diminish inflammation. T cell proliferation, differentiation, and survival are influenced by GSK3. Many effects stem from GSK3 regulation of critical transcription factors, such as NF-κB, NFAT and STATs. These discoveries led to the rapid application of GSK3 inhibitors to animal models of sepsis, arthritis, colitis, multiple sclerosis, and others that demonstrated their potential for therapeutic interventions. Introductory overviewThe innate and adaptive immune systems are crucial for sustaining life but can also contribute to a host of debilitating diseases. Investigators have wrestled with numerous strategies to maintain or restore a healthy balance in the activities of these systems. During the last few years, the ubiquitous serine/threonine kinase glycogen synthase kinase-3 (GSK3) was identified as a regulator of many components of the immune system, suggesting it may be a plausible therapeutic target in inflammatory and autoimmune diseases. Although unobtrusively named due to its initial identification as an enzyme phosphorylating glycogen synthase, GSK3 has since been found to be a point of convergence of many signaling pathways and to regulate many cellular functions through its capacity to phosphorylate over 50 substrates [1]. The complexity of actions of GSK3 is mirrored by the complex mechanisms that regulate its actions (Box 1). Ironically, GSK3 is inhibited by the cation lithium, the simplest of all drugs used therapeutically in humans [2]. Lithium is the classic therapeutic treatment for bipolar disorder (previously called manic-depression), and exerts a broad range of effects on immune cells (Box 2). The complexities of GSK3 regulation offer multiple strategies to control GSK3, for example by regulating individual kinases that phosphorylate GSK3 or the association of proteins with GSK3 in complexes that are specific for individual signaling pathways, and the availability of an inhibitor approved for human use promises rapid application for new intervention objectives. Here we review current knowledge about the roles of GSK3 in innate and adaptive immunity and summarize preliminary animal testing using GSK3 inhibitors in animal models of a rapidly expanding number of diseases. GSK3 regulates innate immunityThe crucial role of GSK3 in inflammation was established by the finding that active GSK3 is necessary for pro-inflammatory cytokine production following stimulation of TLRs [3]. For example, GSK3 deficiency induced pharmacologically with lithium or other GSK3 inhibitors or by molecular manipulations reduced by 67-90% the production of proinflammatory interleukin-6 (IL-6), IL-1β, IL-12p40...

Objectives The PAREPET (Prediction of ARrhythmic Events with Positron Emission Tomography) study sought to test the hypothesis that quantifying inhomogeneity in myocardial sympathetic innervation could identify patients at highest risk for sudden cardiac arrest (SCA). Background Left ventricular ejection fraction (LVEF) is the only parameter identifying patients at risk of SCA who benefit from an implantable cardiac defibrillator (ICD). Methods We prospectively enrolled 204 subjects with ischemic cardiomyopathy (LVEF ≤35%) eligible for primary prevention ICDs. Positron emission tomography (PET) was used to quantify myocardial sympathetic denervation (11C-meta-hydroxyephedrine [11C-HED]), perfusion (13N-ammonia) and viability (insulin-stimulated 18F-2-deoxyglucose). The primary endpoint was SCA defined as arrhythmic death or ICD discharge for ventricular fibrillation or ventricular tachycardia >240 beats/min. Results After 4.1 years follow-up, cause-specific SCA was 16.2%. Infarct volume (22 ± 7% vs. 19 ± 9% of left ventricle [LV]) and LVEF (24 ± 8% vs. 28 ± 9%) were not predictors of SCA. In contrast, patients developing SCA had greater amounts of sympathetic denervation (33 ± 10% vs. 26 ± 11% of LV; p = 0.001) reflecting viable, denervated myocardium. The lower tertiles of sympathetic denervation had SCA rates of 1.2%/year and 2.2%/year, whereas the highest tertile had a rate of 6.7%/year. Multivariate predictors of SCA were PET sympathetic denervation, left ventricular end-diastolic volume index, creatinine, and no angiotensin inhibition. With optimized cut-points, the absence of all 4 risk factors identified low risk (44% of cohort; SCA <1%/year); whereas ≥2 factors identified high risk (20% of cohort; SCA ~12%/year). Conclusions In ischemic cardiomyopathy, sympathetic denervation assessed using 11C-HED PET predicts cause-specific mortality from SCA independently of LVEF and infarct volume. This may provide an improved approach for the identification of patients most likely to benefit from an ICD. (Prediction of ARrhythmic Events With Positron Emission Tomography [PAREPET]; NCT01400334)

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-κB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-α secretion, IL-1R-associated kinase-1, and IκB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-κB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 “homotolerance,” respectively). Despite inhibited NF-κB DNA binding, increased levels of nuclear NF-κB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-α secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-α. Experiments designed to elucidate novel mechanisms of NF-κB inhibition in tolerized cells revealed the potential contribution of IκBε and IκBξ inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-κB signaling pathway.