Publications

To overcome oxidative, inflammatory, and metabolic stress, cells have evolved networks of cytoprotective proteins controlled by nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator the Kelch-like ECH associated protein 1 (Keap1). Here, we used high-resolution mass-spectrometry to characterize the proteomes of macrophages with genetically altered Nrf2 status. Our analysis revealed significant differences among the genotypes in cellular metabolism and redox homeostasis, which we validated with respirometry and metabolomics, as well as in anti-viral immune pathways and the cell cycle. Nrf2 status significantly affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity observed. Of note, Nrf2 activation was found to promote mitochondrial fusion in inflammatory macrophages. The Keap1 inhibitor, 4-octyl itaconate (4-OI), a derivative of the mitochondrial immunometabolite itaconate, remodeled the inflammatory macrophage proteome, increasing redox and suppressing anti-viral immune effectors in a Nrf2-dependent manner. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages. Graphical abstract Highlights First high-resolution proteome of macrophages with genetically altered Nrf2 status Nrf2 is key regulator of macrophage redox and intermediary metabolism Nrf2 finetunes the inflammatory response suppressing anti-viral immune and cytokine effectors, whilst promoting T cell activation factors Nrf2 regulates mitochondrial adaptation in inflammatory macrophages promoting the formation of a fused network 4-octyl itaconate (4-OI) suppresses anti-viral immune effectors in inflammatory macrophages in a Nrf2-dependent manner

No abstract is provided for this article.

The IκB kinases (IKKs) IKK-α and IKK-β, and the IKK-related kinases TBK1 and IKK-ɛ, have essential roles in innate immunity through signal-induced activation of NF-κB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-κB essential modulator coordinates some IKK complexes, whereas TANK, NF-κB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-ɛ complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-ɛ subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity. * Authors contributed equally to this work.

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1β and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1-/- macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1β. The excessive prooxidant and proinflammatory phenotype of Bmal1-/- macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1β are known to play a role.

No abstract is provided for this article.

An inducible program of inflammatory gene expression is central to anti-microbial defenses. Signal-dependent activation of transcription factors, transcriptional co-regulators and chromatin modifying factors collaborate to control this response. Large intervening non-coding RNAs (LincRNAs) are a relatively new class of genes identified as key regulators of distinct cellular functions. LincRNAs are typically 200 to thousands of nucleotides in length. It has been known for many years that a small number of lincRNAs play roles in regulating epigenetic processes such as X chromosome inactivation and imprinting, however their role in innate immunity has not been examined. Toll like receptors sense microbial infection and activate an inducible inflammatory gene expression program, important in host defense. Here we identify lincRNA-Cox2 as a highly inducible non coding RNA that acts as a key regulator of this inflammatory response. LincRNA-Cox2 is transcribed in the opposite orientation to its proximal gene Ptgs2 (Cox2). It is 1.8kb in length and we have identified three splice variants of the transcript. Expression of lincRNA-Cox2 can be induced by ligands such as LPS, Pam3CSK4 and R848 in a MyD88 and NFkB dependent manner. To define the functional role of lincRNA-Cox2 in inflammatory gene expression, we generated macrophage cell lines in which lincRNA-Cox2 expression was silenced by shRNA. We conducted whole transcriptome analysis on macrophages deficient in lincRNA-Cox2 and results show that this lincRNA mediates both the activation and repression of distinct classes of innate immune genes. Knockdown of lincRNA-Cox2 directly resulted in increased basal levels of a number of interferon stimulated genes such as Ccl5 (Rantes) and IP10. 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 TLR-induced inflammatory response.

<b>Introduction:</b> Severe, steroid-resistant (SSR) asthma is the major unmet need in asthma management and obese asthmatics are more likely to have therapy-resistant disease. Recent studies strongly implicate a role for NLRP3 inflammasome responses in driving obesity-associated AHR, however, these studies did not assess its role in steroid resistance or therapeutic modulation in obesity-associated, SSR asthma. <b>Aims and Objectives:</b> To elucidate the role of NLRP3 inflammasome responses in obesity-associated, SSR asthma. <b>Methods:</b> We developed and characterised a novel murine model of high-fat diet (HFD)-induced, obesity-associated, SSR allergic airways disease (SSR AAD) and assessed the effects of steroid treatment. We also assessed the roles and potential for targeting the NLRP3 inflammasome, and IL-5/IL-13 responses, in our model of HFD-induced, obesity-associated, SSR AAD. <b>Results:</b> HFD administration results in increased weight gain and adiposity; and the development of steroid-resistant AHR. Interestingly, HFD-induced obesity results in increased levels of NLRP3 in the airways and caspase-1 activation in the lung, that are suppressed by treatment with the NLRP3 inflammasome-specific inhibitor, MCC950. Furthermore, combinatorial treatment with anti-IL-5 and anti-IL-13 neutralising antibodies suppresses airway NLRP3 inflammasome responses and steroid-resistant AHR in our model. <b>Conclusions:</b> Our study highlights an unrecognised role for HFD-induced, obesity-associated, NLRP3 inflammasome responses in steroid-resistant AHR. Furthermore, we identify a novel role for IL-5 and IL-13 responses upstream of the NLRP3 inflammasome in the development of obesity-associated, SSR asthma.

The cytokine interleukin-10 (IL-10) limits the immune response and promotes resolution of acute inflammation. Because of its immunosuppressive effects, IL-10 up-regulation is a common feature of tumor progression and metastasis. Recently, IL-10 regulation has been shown to depend on mitochondria and redox-sensitive signals. We have found that Suppressor of site III Qo Electron Leak 1.2 (S3QEL 1.2), a specific inhibitor of reactive oxygen species (ROS) production from mitochondrial complex III, and myxothiazol, a complex III inhibitor, decrease IL-10 in lipopolysaccharide (LPS)–activated macrophages. IL-10 down-regulation is likely to be mediated by suppression of c-Fos, which is a subunit of activator protein 1 (AP1), a transcription factor required for IL-10 gene expression. S3QEL 1.2 impairs IL-10 production in vivo after LPS challenge and promotes the survival of mice bearing B16F10 melanoma by lowering tumor growth. Our data identify a link between complex III–dependent ROS generation and IL-10 production in macrophages, the targeting of which could have potential in boosting antitumor immunity.

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A wide variety of electrophilic derivatives of itaconate, the Kreb's cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1β in response to these innate activators. In contrast, the production of interferon (IFN)β, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.