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The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1–generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.

The NLRP3 inflammasome has an important function in inflammation by promoting the processing of pro-IL-1β and pro-IL-18 to their mature bioactive forms, and by inducing cell death via pyroptosis. Here we show a critical function of the E3 ubiquitin ligase Pellino2 in facilitating activation of the NLRP3 inflammasome. Pellino2-deficient mice and myeloid cells have impaired activation of NLRP3 in response to toll-like receptor priming, NLRP3 stimuli and bacterial challenge. These functions of Pellino2 in the NLRP3 pathway are dependent on Pellino2 FHA and RING-like domains, with Pellino2 promoting the ubiquitination of NLRP3 during the priming phase of activation. We also identify a negative function of IRAK1 in the NLRP3 inflammasome, and describe a counter-regulatory relationship between IRAK1 and Pellino2. Our findings reveal a Pellino2-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome. The NLRP3 inflammasome is important for inducing IL-1β and IL-18 inflammatory responses. Here the authors show, by generating and characterizing Peli2 deficient mice and immune cells, that an E3 ubiquitin ligase Pellino2 promotes inflammasome priming by inducing NLRP3 ubiquitination and by targeting IRAK1.

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1 V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus. Rare genetic variants in UNC93B1 predispose to childhood-onset lupus via TLR7/-8–IRAK1/-4, validated with a corresponding mouse model.

It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the “master” protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1–dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1–induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.

Inflammation is a key factor in the pathogenesis of dry eye disease (DED). We aimed to investigate the role of microRNA-146a (miR-146a) in regulating corneal inflammation in a mouse model of benzalkonium chloride (BAC)-induced dry eye and the TNF-α-induced NF-κB signaling pathway in human corneal epithelial cells (HCECs). A mouse model of dry eye was established by administering with BAC to BALB/c mice, and the expression of TNF-α, IL-1β, IL-6, IL-8, cyclooxygenase 2 (COX2), interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) in the corneas of dry eye model mice was significantly increased; this was accompanied by the upregulation of miR-146a and activation of the NF-κB pathway. In vitro, TNF-α induced miR-146a expression in HCECs, while the NF-κB inhibitor SC-514 reduced the expression of miR-146a. Overexpression of miR-146a decreased the expression of IRAK1 and TRAF6, which have been identified as targets of miR-146a. Furthermore, overexpression of miR-146a suppressed NF-κB p65 translocation from the cytoplasm to the nucleus. Moreover, overexpression of miR-146a attenuated the TNF-α-induced expression of IL-6, IL-8, COX2 and intercellular adhesion molecule 1 (ICAM1), while inhibition of miR-146a exerted the opposite effect. Our results suggest that miR-146a mediates the inflammatory response in DED. MiR-146a negatively regulates inflammation in HCECs through the IRAK1/TRAF6/NF-κB pathway, and this may serve as a potential therapeutic approach for the treatment of DED.

Notch signaling is known to modulate macrophage polarization. Hu and colleagues show that the Notch–RBP-J axis controls the expression of M1 macrophage–specific genes by promoting translation of the transcription factor IRF8. Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes . Notch–RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage–associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2–dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch–RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical I κ B kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF- κ B essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF- κ B1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCF β TRCP , leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage.

Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel , while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-β. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts. Legionella pneumophila expresses a range of bacterial determinants that mimic eukaryotic functions. Here the authors show small RNAs of L.pneumophila mimic eukaryotic microRNA and modulate the host response to infection.

Thymoquinone (TQ) is a bioactive component of black seed ( Nigella sativa ) volatile oil and has been shown to have anti-oxidative, anti-inflammatory, and anti-cancer properties. In the present study, we explored the molecular mechanisms that underlie the anti-inflammatory effect of TQ and its target proteins using lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 and human monocyte-like U937 cells, together with LPS/D-galactosamine (GalN)-induced acute hepatitis and HCl/EtOH-induced gastritis mouse models. TQ strongly inhibited the production of nitric oxide (NO) and repressed NO synthase (iNOS), tumor necrosis factor (TNF)-α, cyclooxygenase (COX)−2, interleukin (IL)−6, and IL-1β expression in LPS-activated RAW264.7 cells. Treatment of LPS/D-GalN–induced hepatitis and EtOH/HCl–induced gastritis mouse models with TQ significantly ameliorated disease symptoms. Using luciferase reporter gene assays, we also showed that the nuclear levels of transcription factors and phosphorylation patterns of signaling proteins, activator protein (AP)−1, and nuclear factor (NF)-κB pathways were all affected by TQ treatment. Finally, we used additional kinase and luciferase validation assays with interleukin-1 receptor-associated kinase 1 (IRAK1) to show that IRAK1 is directly suppressed by TQ treatment. Together, these findings strongly suggest that the anti-inflammatory actions of TQ are caused by suppression of IRAK-linked AP-1/NF-κB pathways.

Macrophage-orchestrated, low-grade chronic inflammation plays a pivotal role in obesity and atherogenesis. However, the underlying regulatory mechanisms remain incompletely understood. Here, we identify major vault protein (MVP), the main component of unique cellular ribonucleoprotein particles, as a suppressor for NF-κB signaling in macrophages. Both global and myeloid-specific MVP gene knockout aggravates high-fat diet induced obesity, insulin resistance, hepatic steatosis and atherosclerosis in mice. The exacerbated metabolic disorders caused by MVP deficiency are accompanied with increased macrophage infiltration and heightened inflammatory responses in the microenvironments. In vitro studies reveal that MVP interacts with TRAF6 preventing its recruitment to IRAK1 and subsequent oligomerization and ubiquitination. Overexpression of MVP and its α-helical domain inhibits the activity of TRAF6 and suppresses macrophage inflammation. Our results demonstrate that macrophage MVP constitutes a key constraint of NF-κB signaling thereby suppressing metabolic diseases. Metabolic diseases are associated with chronic, low-grade inflammation. Here the authors show that major vault protein (MVP) suppresses NF-κB signalling in macrophages via an IRAK1–TRAF6 axis and that loss of MVP in myeloid cells exacerbates the inflammatory response in mice fed a high fat diet.