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Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R–MyD88–IRAK2–PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity. Obesity is often accompanied by chronic inflammation. Li and colleagues show that, in mice fed high-fat diets, IL-1 signaling in adipocytes induces an unconventional IRAK2 translocation to mitochondria and suppresses respiratory super-complex formation to alter mitochondrial function, and exacerbates obesity.

Although Act1 (adaptor for IL-17 receptors) is necessary for IL-17-mediated inflammatory responses, Act1- (but not Il17ra-, Il17rc-, or Il17rb-) deficient mice develop spontaneous SLE- and Sjögren’s-like diseases. Here, we show that Act1 functions as a negative regulator in T and B cells via direct inhibition of STAT3. Mass spectrometry analysis detected an Act1–STAT3 complex, deficiency of Act1 (but not Il17ra-, Il17rc-, or Il17rb) results in hyper IL-23- and IL-21-induced STAT3 activation in T and B cells, respectively. IL-23R deletion or blockade of IL-21 ameliorates SLE- and Sjögren’s-like diseases in Act1−/− mice. Act1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T–B cell interaction for B cell expansion and antibody production. Moreover, anti-IL-21 ameliorates the SLE- and Sjögren’s-like diseases in Act1-deficient mice. Thus, IL-21 blocking antibody might be an effective therapy for treating SLE- and Sjögren’s-like syndrome in patients containing Act1 mutation., Adaptor for IL-17 receptors (Act1) is known to be crucial for IL-17-mediated immune responses. Here the authors show that Act1 also functions as a negative regulator of T and B cells by direct inhibition of STAT3.

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type–specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A–induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A–induced stress fiber formation in ASMCs and attenuated IL-17A–enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM-caspase-8-ASC complex formation, resulting in the activation of caspase-8 and IL-1β release in microglia. Noncanonical inflammasome-derived IL-1β produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1β via noncanonical caspase-8-dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation.

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation., eLife digest The innate immune system is the body’s first line of defense against invading microbes. Some immune cells carry specific receptor proteins called Toll-like receptors that can identify microbes and the signals they emit. As soon as the receptors have detected a threat – for example through sensing oily molecules that make up the cell membranes of microbes – they produce signaling proteins called cytokines and chemokines to alert other immune cells. The DNA in the cell’s nucleus carries the instructions needed to make proteins. To produce proteins, including cytokines and chemokines, the information first has to be transferred into mRNA templates, which carry the instructions to the sites in the cell where the proteins are made. Cytokine and chemokine mRNAs are generally short-lived, but previous studies in 2009 and 2011 have shown that an enzyme called IRAK2 can stabilize them to make them last longer. IRAK enzymes are activated by the Toll-like receptors after a threat has been detected. However, until now it was not known whether IRAK2 also helps to transport the mRNAs of cytokines and chemokines out of the cell nucleus. Using immune cells of mice, Zhou et al. – including some of the researchers involved in the previous studies – discovered that IRAK2 helped to export the mRNAs of cytokines and chemokines from the immune cell nucleus into the surrounding cell fluid. The Toll-like receptors recognized the oily molecules of the microbes and consequently activated IRAK2, which lead to IRAK2 being moved into the cell nucleus. Once activated, IRAK2 helped to assemble the export machinery that moved selected mRNAs out of the nucleus to build the proteins. To do so, IRAK2 stopped a destabilizing protein from binding to the mRNA, so that instead the export machinery could transport the mRNA of the cytokines and chemokines out of the cell nucleus. A next step will be to test whether IRAK2 is required to guide exported mRNA tothe sites in the cell where the proteins are made. This new insight could help to develop new treatments for various diseases. For example, diseases in which the immune system attacks the cells of the body, rather than invaders, can be caused by too many cytokines and chemokines. Since IRAK2 helps to control the availability of cytokines and chemokines it may in future be used as a new drug target.

NOTCH1 signalling contributes to defective remyelination by impairing differentiation of oligodendrocyte progenitor cells (OPCs). Here we report that IL-17 stimulation induces NOTCH1 activation in OPCs, contributing to Th17-mediated demyelinating disease. Mechanistically, IL-17R interacts with NOTCH1 via the extracellular domain, which facilitates the cleavage of NOTHC1 intracellular domain (NICD1). IL-17-induced NOTCH1 activation results in the interaction of IL-17R adaptor Act1 with NICD1, followed by the translocation of the Act1–NICD1 complex into the nucleus. Act1–NICD1 are recruited to the promoters of several NOTCH1 target genes (including STEAP4, a metalloreductase important for inflammation and cell proliferation) that are specifically induced in the spinal cord by Th17 cells. A decoy peptide disrupting the IL-17RA–NOTCH1 interaction inhibits IL-17-induced NOTCH1 activation and attenuates Th17-mediated experimental autoimmune encephalitis (EAE). Taken together, these findings demonstrate critical crosstalk between the IL-17 and NOTCH1 pathway, regulating Th17-induced inflammatory and proliferative genes to promote demyelinating disease., NOTCH signalling stimulates oligodendrocyte progenitor cell proliferation but how this regulates demyelinating disease is unclear. Here, the authors show that an IL-17 adaptor protein, Act1, interacts with the C-terminal fragment of NOTCH1 (NICD) to activate cell proliferation and an inflammatory response.

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.

Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.

SummaryInterleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3α were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3α and GSK3β, we found that GSK3α but not GSK3β formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3α phosphorylation at S21, thereby inactivating GSK3α to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.

Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development.

A hypothesis was formed that it would be possible to isolate an adequate amount of protein from a patient, having normal renal function, to identify biological markers of a particular disease state using a variety of proteomics techniques. To support this hypothesis, three samples of urine were collected from a volunteer: first when healthy, later when experiencing acute inflammation due to a pilonidal abcess, and again later still after successful recovery from the condition. The urine from these samples was processed by solid-phase extraction to concentrate and desalt the endogenous proteins and peptides. The proteins and peptides from these urine samples were analyzed in three different experiments: (1) traditional two-dimensional gel electrophoresis followed by proteolysis and mass spectrometric identification of various protein spots, (2) whole mixture proteolysis followed by one-dimensional packed capillary liquid chromatography and tandem mass spectrometry, (3) whole mixture proteolysis followed by two-dimensional capillary liquid chromatography and tandem mass spectrometry. In all three cases, a set of proteins was identified representing putative biomarkers. Each of these proteins was then found to have been previously linked in the scientific literature to inflammation. One acute phase reactant in particular, orosomucoid, was readily observed in all three experiments to dramatically increase in abundance, thereby supporting the hypothesis.

Efficient loading of major histocompatibility complex class II molecules with peptides requires the invariant chain (Ii) and the class II–like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii−/−M−/−). Antigen presenting cells (APCs) from Ii−/−M−/− mice, as compared with APCs from Ii−/− mice, exhibit a significant reduction in their ability to present self-peptides to a panel of class II I-Ab–restricted T cells. As a consequence of this defect in the loading of self peptides, CD4+ thymocyte development is profoundly impaired in Ii−/−M−/− mice, resulting in a peripheral CD4+ T cell population with low levels of T cell receptor expression. These findings are consistent with the idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.

Crop raiding by wild herbivores close to an area of protected wildlife is a serious problem that can potentially undermine conservation efforts. Since there is orders of magnitude difference between farmers' perception of damage and the compensation given by the government, an objective and realistic estimate of damage was found essential. We employed four different approaches to estimate the extent of and patterns in crop damage by wild herbivores along the western boundary of Tadoba-Andhari Tiger Reserve in the state of Maharashtra, central India. These approaches highlight different aspects of the problem but converge on an estimated damage of over 50% for the fields adjacent to the forest, gradually reducing in intensity with distance. We found that the visual damage assessment method currently employed by the government for paying compensation to farmers was uncorrelated to and grossly underestimated actual damage. The findings necessitate a radical rethinking of policies to assess, mitigate as well as compensate for crop damage caused by protected wildlife species.