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The reprogramming of metabolic pathways and processes in immune cells has emerged as an important aspect of the immune response. Metabolic intermediates accumulate as a result of metabolic adaptations and mediate functions outside of metabolism in the regulation of immunity and inflammation. In macrophages, there has been a major focus on 3 metabolites linked to the Krebs cycle, itaconate, succinate, and fumarate, which have been shown to regulate multiple processes. Here, we discuss recent progress on these 3 metabolites with regard to their effect on macrophages in host defense and inflammatory diseases. We also consider the therapeutic opportunities presented from the mimicry of these metabolites or by targeting the enzymes that make or metabolize them in order to leverage the body's own anti-inflammatory response.
The protein NLRP3 has emerged as a central regulator in the inflammatory process, being implicated directly in hereditary cryopyrinopathies, and indirectly in diseases such as gout, Type 2 diabetes and atherosclerosis. NLRP3 is an important regulator of caspase-1, the enzyme that processes the immature form of IL-1β into the active protein. The control of NLRP3 has therefore become a focus of research with evidence for redox regulation, ubiquitination and regulation by miRNA-223, kinases and calcium all emerging as controllers of NLRP3. As our knowledge expands the prospect for precise pharmacological targeting of NLRP3 will improve and could lead to substantial clinical utility.
Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor κB (NFκB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFκB-dependent reporter gene expression but not IκBα degradation. Transient transfection of HEK293 cells with Btk had no effect on NFκB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IκBα degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFκB. Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor κB (NFκB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFκB-dependent reporter gene expression but not IκBα degradation. Transient transfection of HEK293 cells with Btk had no effect on NFκB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IκBα degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFκB.
No abstract is provided for this article.
No abstract is provided for this article.
The extensively studied cytokine IL-1β is an important mediator of the inflammatory response. However, dysregulated release of IL-1β can be detrimental and is attributed to the progression and pathogenesis of multiple inflammatory diseases including, rhuematoid arthritis (RA), atherosclerosis, type 2 diabetes (T2D), Alzheimers disease and gout. IL-1β is encoded as a pro-protein. A multi-protein molecular scaffold termed the "Inflammasome" is responsible for the tightly controlled and coordinated processing of pro-IL-1β. The activation of several NLR (nucleotide-binding oligomerization domain (NOD)-like receptor) family members and PYHIN (pyrin and HIN domain) proteins can drive the formation of inflammasomes. However, the exact biochemical mechanisms governing their activation have been the subject of much research. Different inflammasomes have been demonstrated to respond to the same pathogen inducing a cooperative immune response accountable for the clearance of infection. Here, we review current knowledge surrounding the biochemical regulation of the NLRP1, NLRP3, NLRC4, AIM2 and IFI16 inflammasomes.
No abstract is provided for this article.
No abstract is provided for this article.
The cytoplasm of an activated macrophage is a dangerous place due to the accumulation of reactive oxygen species (ROS), which can perturb cytoplasmic oxidative balance. Macrophage activation through monocyte recruitment in tissues occurs in a highly regulated manner upon detection of microbial pathogen-associated molecular patterns (PAMPs), such as the Gram-negative bacterial cell wall component lipopolysaccharide (LPS), stimulating ROS production. ROS can be generated from many cellular processes, either directly or as a result of incomplete reduction of free radicals (1). One of the main sources of ROS is the NADPH oxidase (NOX), a specialized transmembrane protein complex that generates superoxide (O2–), which has been implicated in several inflammatory diseases (2). The generation of ROS can also stem from the mitochondrial electron transport chain, due to insufficient reduction of superoxide anions. Another source is the inducible form of nitric oxide synthase 2 (iNOS). iNOS produces nitric oxide from L-arginine and is known to play key roles in macrophage function (3,4).
No abstract is provided for this article.
Conference Article| May 01 1995 Depolarising levels of KCl inhibit endothelin-1-mediated release of arachidonic acid in rat C6 glioma cells D.J. Dunican; D.J. Dunican 1Biochemistry Department, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar R. Griffiths; R. Griffiths 1Biochemistry Department, Trinity College, Dublin 2, Ireland*Division of Biochemistry, Irvine Building, North St., St. Andrews KY16 9AI, UK Search for other works by this author on: This Site PubMed Google Scholar L.A.J. O'Neill; L.A.J. O'Neill 1Biochemistry Department, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar D.C. Williams D.C. Williams 1Biochemistry Department, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (2): 285S. https://doi.org/10.1042/bst023285s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation D.J. Dunican, R. Griffiths, L.A.J. O'Neill, D.C. Williams; Depolarising levels of KCl inhibit endothelin-1-mediated release of arachidonic acid in rat C6 glioma cells. Biochem Soc Trans 1 May 1995; 23 (2): 285S. doi: https://doi.org/10.1042/bst023285s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CNS, central nervous system, Et-1, endothelin, AA, arachidonate, PKC, protein kinase C, PLA2, phospholipase A2 This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.
A model for NFϰB activation involving reactive oxygen intermediates has recently been proposed. We have explored this model in three cell lines, Jurkat T cells, EL4.NOB-1 T cells and KB epidermal cells using hydrogen peroxide and two physiological activators of NFϰB, interleukin-1 (IL1) and tumor necrosis factor (TNF) as stimuli. In agreement with earlier studies hydrogen peroxide activated NFϰB in Jurkat, although only at much higher concentrations (10 mM) than those previously reported. However, hydrogen peroxide failed to activate in the two other cell lines under a range of conditions. Similarly, N-acetylcysteine only proved inhibitory in hydrogen peroxide and TNF treated Jurkat and failed to inhibit IL1 and TNF-activated NFϰB in EL4.NOB-1 and KB cells respectively. N-Acetylcysteine inhibited IL1-induced interleukin-2 in EL4, however, demonstrating that N-acetylcysteine was biologically active. These results suggest that the reactive oxygen model of NFϰB activation may be cell-type restricted. In contrast to the results with N-acetylcysteine, the antioxidant and metal chelator, pyrolidine dithiocarbamate (PDTC) inhibited NFϰB activation, although these effects may be unrelated to any antioxidant properties. PDTC also inhibited IL1-induced interleukin-2. Finally, studies with the pro-oxidant diamide showed that this could not activate NFϰB in any of the cells and in contrast proved inhibitory. The results from this study therefore suggest that the reactive oxygen model of NFϰB activation may be restricted to certain cell types and that the presence of such a system is not required for the activation of NFϰB by ILI and TNF.
Chromatin immunoprecipitation (ChIP) is widely used in many fields to analyze the distribution of specific proteins, or their modified isoforms, across defined DNA domains. ChIP procedures fall into two main categories, namely, those that use native chromatin prepared by nuclease digestion (designated NChIP), and those that use chromatin in which DNA and proteins are crosslinked, either chemically or with UV light (designated XChIP). Each procedure has its own advantages and drawbacks. Here, we outline the methods currently in use in our laboratory to isolate and immunoprecipitate native chromatin from cultured cells, and to isolate and analyze immunoprecipitated protein and DNA.
No abstract is provided for this article.
Balic et al. describe a new role for STAT3 in TLR4 signalling in macrophages, linking LPS mediated activation of this innate immune receptor to phosphorylation of mitochondrial STAT3, resulting in distinct metabolic reprogramming. In this article, we discuss a new study from the laboratory of Ashley Mansell that successfully links innate immune receptor signaling to metabolic changes in macrophages, establishing for the first time an uninterrupted pathway from ligand binding to mitochondrial reprogramming and subsequential effector function modulation in this cell type.1 Macrophages were first described by Ilya Metchnikoff as early as 1882 and have since been identified as critical players in innate immunity. The main function of macrophages is the clearance of microbes, cancer cells, cellular debris and other foreign components in a process called phagocytosis, in which the macrophage engulfs and digests its target. Macrophages detect foreign substances through an array of pattern-recognition receptors (PRRs), one of which is the lipopolysaccharide (LPS) sensing receptor Toll-like receptor 4 (TLR4). Activation of TLR4 on macrophages induces distinct metabolic changes, characterized by increased glycolysis, accumulation of the tricarboxylic acid (TCA) cycle metabolite succinate2 and decreased oxidative phosphorylation (OXPHOS).3 These changes facilitate the increased demand for energy and biomaterials and are crucial for macrophage cytokine production and effector function.4 A direct result of succinate accumulation in macrophages is the stabilization of hypoxia-inducible factor-1α (HIF-1α), a key mediator in the expression of pro-glycolytic genes and the production of pro-inflammatory cytokines such as interleukin-1β (IL-1β).5 Precise mechanistic details on how TLR4 shapes metabolic reprogramming, however, are lacking. TLR4 drives phosphorylation of the well-known TLR activated kinases TANK-binding kinase 1 (TBK-1) and IκB kinase-ε (IKKε)6 and promotes glycolysis.7 In an elegant study reported in this issue, Balic et al. link these kinases to the signal transducer and activator of transcription 3 (STAT3), which was known to impact mitochondrial metabolism8 and to induce the generation of reactive oxygen species (ROS).9 The study proposes a new non-canonical role for STAT3 in TLR-mediated signaling. Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a well-known mediator of signaling events downstream of TLR activation.10 Due to its structure, Balic et al. expected TRAF6 to be a binding partner of activated STAT3, a hypothesis they were able to confirm by identifying TRAF6 binding motifs on STAT3, and subsequently pulling down STAT3 together with TRAF6 in LPS-activated macrophages, thus presenting the first link between STAT3 activation and TLR signaling. STAT3 phosphorylation on Tyr705 is known to induce translocation to the nucleus and to activate the expression of several genes; however, TLR4 stimulation induced phosphorylation at a different site, Ser727, rapidly enough to be considered part of direct downstream signaling events, rather than an autocrine secondary effect. The group and others had previously shown that phosphorylation at this particular site translocates STAT3 to the mitochondria. Here, STAT3 alters mitochondrial metabolism by driving the activity of complex I and II of the electron transport chain, ultimately enhancing mitochondrial respiration.8 STAT3 phosphorylation was not detected in the myeloid differentiation primary response 88 (MyD88) deficient cells. As MyD88 is a well-known adaptor protein downstream of TLR signaling, these results integrate STAT3 into TLR signaling for the first time (Figure 1). At this point, the relationship between Ser727 and Tyr705 phosphorylation of STAT3 should be mentioned, as these events are not subject to two completely independent functioning pathways. Both phosphorylation events seem to influence each other under certain conditions; however, details are subject to debate. Regardless, the authors next aimed to investigate whether STAT3 Ser727 phosphorylation was indeed critical for TLR-induced metabolic reprogramming. Mutating the relevant serine in mouse peritoneal macrophages decreased both resting glycolysis and the glycolytic burst upon TLR4 stimulation, confirming phosphorylation of Ser727 to be a critical step in the pathway. The mutant cells exhibited a drastically reduced oxygen consumption rate (OCR) and maximal respiratory capacity compared with wild type cells. These cells were viable, but unable to respond to LPS stimulation, as respiratory capacity could not be upregulated above basal levels. Other than changes in the respiratory chain, LPS-stimulated macrophages also exhibit characteristic breaks in the TCA cycle, leading to accumulation of the pro-inflammatory metabolite succinate. Succinate production not only deprives the respiratory chain of its substrate, it is also required for ROS pro-inflammatory IL-1 production.5 The authors found that in the above mentioned STAT3 mutants, LPS-induced succinate production was reduced compared with wild type cells. Conversely, the mutants exhibited increased the production of lactate, suggesting that due to defective mitochondrial metabolism, they rely more on aerobic glycolysis than wild type macrophages. These data suggest that Ser727 phosphorylation of STAT3 is not only required for induction of OXPHOS, but also for LPS-induced accumulation of succinate. The authors now asked what kinases might phosphorylate STAT3? As the group had previously shown mitochondrial STAT3 to be responsible for the generation of ROS,11 mitochondrial ROS concentrations were used as a readout while screening a large library of kinase inhibitors. They pointed to TBK-1, which was then shown to interact with both TRAF6 and STAT3 upon LPS stimulation. Deletion of TBK-1 confirmed this kinase to be responsible for Ser727 phosphorylation of STAT3. The related kinase IKKε was subsequently shown to be required for full STAT3 phosphorylation as well. Both kinases were previously mainly known for being involved in TLR-induced IRF3 phosphorylation and induction of IFNβ, another pro-inflammatory cytokine.12 TBK-1 dysregulation has been found to be involved in multiple inflammatory diseases such as colitis, rheumatoid arthritis, hepatitis, and atherosclerosis,13 while an interesting study published this year has implicated IKKε in tumor formation in breast cancer.14 These data elegantly explain the immunomodulatory properties of these kinases by revealing a new substrate and completing the signaling pathway below. Furthermore, this discovery reveals differences between metabolic reprogramming in myeloid cell lines. While dendritic cells for example rely on phosphoinositide 3-kinase (PI3K) and Akt activity for TBK-1/IKKε induced glycolysis, macrophages increase glycolytic activity independent of these mediators. Lastly, it only remained to be investigated whether the metabolic changes associated with TLR4-induced STAT3 signaling result in actual functional consequences in macrophages. A key effector function in LPS-stimulated macrophages is the production of IL-1β. Generation of this pro-inflammatory cytokine is critically dependent on TCA cycle remodeling and the subsequent succinate accumulation and should therefore be mediated by the pathway described in this study. When stimulated with LPS, macrophages with mutated STAT3 were impaired in IL-1β mRNA generation and protein production compared with wild type cells. The production of IL-6, IL-10 and tumor necrosis factor (TNF) upon LPS challenge was also reduced in the mutants, while basal IL-10 expression was slightly increased. STAT3 mutant mice were also analyzed in vivo in a model of LPS-induced sepsis, and, in line with the in vitro experiments, the production of IL-1β and IL-6, as well as chemokine (C-C motif) ligand 2 (Ccl2) were decreased in the serum of mutant mice following LPS challenge, while TNF production was unchanged. This is surprising as TNF dependent macrophage functions have been postulated to require mitochondrial STAT3,15 suggesting the possibility of surrogate pathways in physiologically relevant conditions. The roles of TNF and also of the anti-inflammatory cytokine IL-10 in this pathway could indeed be a subject for further investigation. Together, these findings demonstrate a critical role of STAT3 Ser727 phosphorylation in LPS-induced cytokine production in macrophages and therefore effector function. The authors have unraveled a new process in LPS-induced metabolic reprogramming in macrophages. LPS induces TRAF6-mediated phosphorylation of STAT3 by TBK-1, resulting in localization of STAT3 to the mitochondria, shaping metabolic remodelling and subsequently effector functions. One interesting question for further exploration is precisely how STAT3 regulates mitochondrial functions. It is well established that mitochondrial STAT3 can regulate ATP synthesis, increase mitochondrial Ca2+ influx and decrease ROS release in T and B lymphocytes. Here, STAT3 associates with various components of the electron transport chain (ETC), enhancing ETC complex activity.16 Interestingly, a recent study has found that mitochondrial STAT3 phosphorylated at Ser727 induced autophagy in human gastric epithelial cells when infected with Helicobacter pylori,17 expanding the list of the potential functions of mitochondrial STAT3. As a well-described transcription factor, STAT3 could possibly also directly regulate mitochondrial gene expression. The study presented in this article elegantly pieces together established pathways to increase our understanding of immune cells to explain cell fate and effector function. It is a great example of how expanding one’s horizon and interpreting previous observations can lead to important new discoveries. The authors declare no conflict of interest. Hauke J Weiss: Conceptualization; Writing-original draft; Writing-review & editing. Luke O'Neill: Conceptualization; Writing-original draft; Writing-review & editing.
No abstract is provided for this article.
