[show abstract][hide abstract] ABSTRACT: The apoptosis associated speck-like protein containing a CARD (ASC) is an essential component of several inflammasomes, multiprotein complexes that regulate caspase-1 activation and inflammation. We report here an interaction between promyelocytic leukemia protein (PML) and ASC. We observed enhanced formation of ASC dimers in PML- deficient macrophages. These macrophages also display enhanced levels of ASC in the cytosol. Furthermore, IL-1β production was markedly enhanced in these macrophages in response to both NLRP3 and AIM2 inflammasome activation and following BMDM infection with herpes simplex virus-1 (HSV-1) and Salmonella typhimurium. Collectively our data indicate that PML limits ASC function, retaining ASC in the nucleus.
Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: MicroRNA-155 (miR-155) is highly expressed in many cancers such as B cell lymphomas and myeloid leukaemia, and inflammatory disorders such as rheumatoid arthritis, atopic dermatitis and multiple sclerosis. The role of miR-155 as both a promoter of inflammation and an oncogenic agent provides a clear need for miR-155 itself to be stringently regulated. We therefore investigated the transcriptional regulation of miR-155 in response to the respective pro- and anti-inflammatory mediators LPS and IL-10. Bioinformatic analysis revealed Ets binding sites on the miR-155 promoter, and we found that Ets2 is critical for miR-155 induction by LPS. Truncation and mutational analysis of the miR-155 promoter confirmed the role of the Ets2 binding site proximal to the transcription start site for LPS responsiveness. We observed increased binding of Ets2 to the miR-155 promoter and Ets2 deficient mice displayed decreased induction of miR-155 in response to LPS. IL-10 inhibited the induction of Ets2 mRNA and protein by LPS, thereby decreasing Ets2 function on the pri-155 promoter. We have thus identified Ets2 as a key novel regulator in both the positive and negative control of miR-155 in the inflammatory response.
Journal of Biological Chemistry 12/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Succinate is an intermediate of the tricarboxylic acid (TCA) cycle, and plays a crucial role in adenosine triphosphate (ATP) generation in mitochondria. Recently, new roles for succinate outside metabolism have emerged. Succinate stabilizes the transcription factor hypoxia-inducible factor-1α (HIF-1α) in specific tumors and in activated macrophages, and stimulates dendritic cells via its receptor succinate receptor 1. Furthermore, succinate has been shown to post-translationally modify proteins. This expanding repertoire of functions for succinate suggests a broader role in cellular activation. We review the new roles of succinate and draw parallels to other metabolites such as NAD(+) and citrate whose roles have expanded beyond metabolism and into signaling.
Trends in cell biology 12/2013; · 12.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Nlrp3 inflammasome is a key controller of the proinflammatory cytokine IL-1β. Iyer and colleagues (2013) demonstrate that Nlrp3 activators promote an interaction between cardiolipin in mitochondria and Nlrp3. Cardiolipin is thus a hydrophobic danger signal that, upon translocation to the outer mitochondrial membrane, activates Nlrp3, promoting inflammation.
[show abstract][hide abstract] ABSTRACT: Inflammatory immune cells, when activated, display much the same metabolic profile as a glycolytic tumor cell. This involves a shift in metabolism away from oxidative phosphorylation towards aerobic glycolysis, a phenomenon known as the Warburg effect. The result of this change in macrophages is to rapidly provide ATP and metabolic intermediates for the biosynthesis of immune and inflammatory proteins. In addition, a rise in certain tricarboxylic acid cycle intermediates occurs notably in citrate for lipid biosynthesis, and succinate, which activates the transcription factor Hypoxia-inducible factor. In this review we take a look at the emerging evidence for a role for the Warburg effect in the immune and inflammatory responses. The reprogramming of metabolic pathways in macrophages, dendritic cells, and T cells could have relevance in the pathogenesis of inflammatory and metabolic diseases and might provide novel therapeutic strategies.
[show abstract][hide abstract] ABSTRACT: An inducible program of inflammatory gene expression is central to antimicrobial defenses. Signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin modifying factors collaborate to control this response. Here, we identify a long noncoding RNA that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2 mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad acting regulatory component of the circuit that controls the inflammatory response.
[show abstract][hide abstract] ABSTRACT: In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to Poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IKKε and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 which is required for Sp1 gene transcription activated by Poly(I:C). We show that Sp1 is required for IL-15 induction by both Poly(I:C) and Respiratory Syncitial Virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in anti-viral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.
Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The interplay between immunity, inflammation and metabolic changes is a growing field of research. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are families of innate immune receptors and their role in our immune response is well documented. Exciting new evidence is emerging with regard to their role in the regulation of metabolism and the activation of inflammatory pathways during the progression of metabolic disorders such as type 2 diabetes and atherosclerosis. The pro-inflammatory cytokine IL-1β appears to play a central role in these disorders. There is also evidence for metabolites such as NAD+ (acting via deacetylases such as SIRT1 and SIRT2) and succinate (which regulates the transcription factor HIF1α) being signals which regulate innate immunity. In addition the over-production of metabolites extracellularly such as uric acid and cholesterol crystals act as signals sensed by NLRP3, leading to the production of IL-1β. These observations are casting new light on the role of metabolism during host defense and inflammation.
Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: A cyclic dinucleotide comprised of GMP and AMP was previously shown to be a key intermediate during activation of innate immune responses to cytosolic DNA. A report by Patel and Tuschl groups published in Cell reveals the structure of the enzyme involved in the synthesis of this second messenger and identifies this cyclic dinucleotide as a unique compound in metazoan cell signaling.
[show abstract][hide abstract] ABSTRACT: Expression of the microRNA miR-223 is deregulated during influenza or hepatitis B infection and in Crohn's disease, type 2 diabetes, leukaemia and lymphoma. Although this may also be the result of the disease per se, increasing evidence suggests a role for miR-223 in limiting inflammation to prevent collateral damage during infection and in preventing oncogenic myeloid transformation. Validated targets for miR-223 that have effects on inflammation and infection include Granzyme B, IKKα, Roquin and STAT3. With regard to cancer, validated targets include C/EBPβ, E2F1, FOXO1 and NFI-A. The effect of miR-223 on these targets has been documented individually, however it is more likely that miR-223 affects multiple targets simultaneously for key processes where the microRNA is important. Such processes include haematopoietic cell differentiation, particularly towards the granulocyte lineage (where miR-223 is abundant) and as cells progress down the myeloid lineage (where miR-223 expression decreases). NF-κB and the NLRP3 inflammasome are important inflammatory mechanisms that are dampened by miR-223 in these cell types. The miRNA can also directly target viruses such as HIV, leading to synergistic effects during infection. Here we review the recent studies of miR-223 function to show how it modulates inflammation, infection and cancer development. This article is protected by copyright. All rights reserved.
Journal of Internal Medicine 06/2013; · 6.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: The discovery of Toll-like receptors (TLRs) was an important event for immunology research and was recognized as such with the awarding of the 2011 Nobel Prize in Physiology or Medicine to Jules Hoffmann and Bruce Beutler, who, together with Ralph Steinman, the third winner of the 2011 Nobel Prize and the person who discovered the dendritic cell, were pioneers in the field of innate immunity. TLRs have a central role in immunity - in this Timeline article, we describe the landmark findings that gave rise to this important field of research.
[show abstract][hide abstract] ABSTRACT: MyD88 adapter-like (Mal)-deficient mice displayed increased susceptibility to oral but not intraperitoneal infection with Salmonella Typhimurium. Bone marrow chimeras demonstrated that mice with Mal-deficient non-hematopoietic cells were more susceptible to infection, indicating a role for Mal in non-myeloid cells. We observed perturbed barrier function in Mal(-/-) mice, as indicated by reduced electrical resistance and increased mucosa blood permeability following infection. Altered expression of occludin, Zonula occludens-1, and claudin-3 in intestinal epithelia from Mal(-/-) mice suggest that Mal regulates tight junction formation, which may in part contribute to intestinal integrity. Mal interacted with several protein kinase C (PKC) isoforms in a Caco-2 model of intestinal epithelia and inhibition of Mal or PKC increased permeability and bacterial invasion via a paracellular route, while a pan-PKC inhibitor increased susceptibility to oral infection in mice. Mal signaling is therefore beneficial to the integrity of the intestinal barrier during infection.Mucosal Immunology advance online publication 24 April 2013. doi:10.1038/mi.2013.24.
[show abstract][hide abstract] ABSTRACT: Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the 'GABA (γ-aminobutyric acid) shunt' pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.
[show abstract][hide abstract] ABSTRACT: Metabolic changes in cells that participate in inflammation, such as activated macrophages and T-helper 17 cells, include a shift towards enhanced glucose uptake, glycolysis and increased activity of the pentose phosphate pathway. Opposing roles in these changes for hypoxia-inducible factor 1α and AMP-activated protein kinase have been proposed. By contrast, anti-inflammatory cells, such as M2 macrophages, regulatory T cells and quiescent memory T cells, have lower glycolytic rates and higher levels of oxidative metabolism. Some anti-inflammatory agents might act by inducing, through activation of AMP-activated protein kinase, a state akin to pseudo-starvation. Altered metabolism may thus participate in the signal-directed programs that promote or inhibit inflammation.
[show abstract][hide abstract] ABSTRACT: The pro-inflammatory danger signal, IL-33 which is released from damaged or dying cells, achieves its effects via the IL-1R family member ST2L. The detection of IL-33 by ST2L initiates downstream signalling pathways which result in the activation of MAP kinases and NF-κB. Here we show that transmembrane emp24 domain-containing protein 1 (TMED1) associates with ST2L. Using a series of mutation and deletion constructs we have demonstrated that this interaction is mediated by the GOLD domain of TMED1 and the TIR domain of ST2L. Our findings have also demonstrated that TMED1 is required for optimal IL-33 induced IL-8 and IL 6 production. This discovery provides additional support to the concept that the TMED family are important players in innate immune signalling.
Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.
PLoS ONE 01/2013; 8(8):e70622. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: B cells signal through both the B cell receptor (BCR) which binds antigens and Toll-like receptors (TLRs) including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK) is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.
PLoS ONE 01/2013; 8(8):e74103. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background: Obesity is characterized by chronic inflammation, immune dysregulation and alteration of gene expression, associated with Type 2 Diabetes Mellitus (T2DM) and cardiovascular disease. The degree to which these changes occur in childhood obesity is not fully defined. Aims and Methods: The aim was to investigate the effect of childhood obesity on immune cell frequency, macrophage activation, cytokine production and specific regulators of metabolic gene expression. Profiling was performed on peripheral blood from 29 obese and 20 non-obese children using real-time PCR, ELISA and flow cytometry. Results: Fasting glucose was similar in both groups but there was a higher degree of insulin resistance in obese subjects (HOMA IR 4.8vs0.84,p<0.001). Soluble CD163, a marker of macrophage polarization to a pro-inflammatory profile, was elevated in the obese compared to non-obese children (135vs105 ng/ml,p=0.03). Invariant natural killer T cells were reduced in the obese children (%CD3 TCells 0.31%vs0.53%,p=0.001). Cytokine profiling revealed significantly elevated TNF-α (6.7vs5.1pg/ml,p=0.01) and leptin (1186vs432pg/ml,p<0.001) and reduced adiponectin (884vs1321pg/ml,p=0.001) in obese compared to non-obese children. Stimulation of peripheral blood mononuclear cells from obese children resulted in higher levels of IL-1β (2100vs1500pg/ml,p=0.018). There was a four-fold increase in expression of microRNA33a(p=0.001) and a three-fold increase in microRNA33b(p=0.017) in obese children. Conclusion: Childhood obesity is associated with changes in immune cell frequency, inflammatory environment and regulation of metabolic gene expression. These changes have been causally linked to the onset of metabolic disease in adulthood and suggest the future trajectory of obese children to the development of T2DM and premature cardiovascular disease.
The Journal of clinical endocrinology and metabolism 01/2013; · 6.50 Impact Factor