ABSTRACT
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.
Subject(s)
Adipocytes/immunology , Inflammation/immunology , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-1/metabolism , Mitochondrial Membranes/metabolism , Obesity/immunology , Adipocytes/pathology , Animals , Cells, Cultured , Humans , Interleukin-1 Receptor-Associated Kinases/genetics , Male , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Oxidative Phosphorylation , Prohibitins , Protein Transport , Receptors, Interleukin-1/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Connexin 43/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Peptide Fragments/metabolism , Psoriasis/immunology , Th17 Cells/immunology , Animals , Cell Line , Connexin 43/genetics , Connexin 43/immunology , Disease Models, Animal , Genetic Predisposition to Disease , Humans , Interleukin-17/metabolism , Mice , Mice, Knockout , Molecular Chaperones/genetics , Mutation/genetics , Peptide Fragments/genetics , Peptide Fragments/immunology , Polymorphism, Genetic , Protein Binding/genetics , Protein Binding/immunology , Psoriasis/genetics , Signal TransductionABSTRACT
The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.
Subject(s)
Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Lupus Erythematosus, Systemic/drug therapy , Prednisolone/therapeutic use , Animals , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/physiology , Toll-Like Receptor 7/physiology , Toll-Like Receptor 9/physiologyABSTRACT
IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Chemokine CXCL1/genetics , Chemokine CXCL5/genetics , ELAV Proteins/metabolism , Interleukin-17/metabolism , RNA Stability , Animals , Cell Line , ELAV Proteins/genetics , HeLa Cells , Humans , Inflammation/immunology , Lung/metabolism , Mice , Mice, Knockout , Protein Binding , RNA, Messenger/metabolism , Respiratory Mucosa/metabolism , Signal Transduction , UbiquitinationABSTRACT
The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.
Subject(s)
Arthritis, Rheumatoid , Synovitis , Humans , CD8-Positive T-Lymphocytes/metabolism , Synovial Membrane/metabolism , Receptors, Antigen, T-Cell , Autoantigens , AutoantibodiesABSTRACT
IRAK4 is an attractive therapeutic target for the treatment of inflammatory conditions. Structure guided optimization of a nicotinamide series of inhibitors has been expanded to explore the IRAK4 front pocket. This has resulted in the identification of compounds such as 12 with improved potency and selectivity. Additionally 12 demonstrated activity in a pharmacokinetics/pharmacodynamics (PK/PD) model. Further optimization efforts led to the identification of the highly kinome selective 21, which demonstrated a robust PD effect and efficacy in a TLR7 driven model of murine psoriasis.
ABSTRACT
BACKGROUND: It has been proposed that ligation of CD80 and CD86 induces reverse signaling into antigen-presenting cells. In this study, we tested the ability of abatacept, a soluble human fusion protein comprising the extracellular domain of cytotoxic T lymphocyte antigen 4 and a fragment of the Fc domain of IgG(1), to activate antigen-presenting cells by measuring changes in global transcriptional responses. METHODS: Affymetrix chips were used to measure gene expression levels using mRNA isolated from immature and mature human dendritic cells and a B cell line following 6 h of treatment with abatacept. RESULTS: In contrast to robust transcriptional responses induced by the control treatment phorbol-12-myristate-13-acetate, abatacept induced minimal gene changes in three different populations of antigen-presenting cells. Furthermore, no gene changes were observed in response to belatacept, a modified version of abatacept that binds with higher avidity to CD80 and CD86. CONCLUSIONS: We conclude that reverse signaling in antigen-presenting cells is unlikely to occur in response to either abatacept or belatacept, thereby supporting the modulation of CD28 signaling on T cells as the main mechanism of action for these therapeutics.
Subject(s)
Antigen Presentation/drug effects , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Dendritic Cells/drug effects , Immunoconjugates/pharmacology , Immunosuppressive Agents/pharmacology , Abatacept , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B7-1 Antigen/immunology , B7-2 Antigen/immunology , Cell Line, Tumor , Cytokines/biosynthesis , Cytokines/immunology , Dendritic Cells/immunology , Gene Expression/drug effects , Humans , Phorbol Esters/pharmacologyABSTRACT
TYK2 is a nonreceptor tyrosine kinase involved in adaptive and innate immune responses. A deactivating coding variant has previously been shown to prevent receptor-stimulated activation of this kinase and provides high protection from several common autoimmune diseases but without immunodeficiency. An agent that recapitulates the phenotype of this deactivating coding variant may therefore represent an important advancement in the treatment of autoimmunity. BMS-986165 is a potent oral agent that similarly blocks receptor-stimulated activation of TYK2 allosterically and with high selectivity and potency afforded through optimized binding to a regulatory domain of the protein. Signaling and functional responses in human TH17, TH1, B cells, and myeloid cells integral to autoimmunity were blocked by BMS-986165, both in vitro and in vivo in a phase 1 clinical trial. BMS-986165 demonstrated robust efficacy, consistent with blockade of multiple autoimmune pathways, in murine models of lupus nephritis and inflammatory bowel disease, supporting its therapeutic potential for multiple immune-mediated diseases.
Subject(s)
Autoimmunity/drug effects , Signal Transduction/drug effects , TYK2 Kinase/chemistry , Animals , Female , Healthy Volunteers , Heterocyclic Compounds/pharmacology , Humans , Interferon alpha-2/pharmacology , Mice , Mice, Inbred C57BL , Mice, SCID , Protein Kinase Inhibitors/pharmacology , TYK2 Kinase/antagonists & inhibitorsABSTRACT
OBJECTIVE: To develop an objective, readily measurable pharmacodynamic biomarker of glucocorticoid (GC) activity. METHODS: Genes modulated by prednisolone were identified from in vitro studies using peripheral blood mononuclear cells from normal healthy volunteers. Using the criteria of a >2-fold change relative to vehicle controls and an adjusted P value cutoff of less than 0.05, 64 up-regulated and 18 down-regulated genes were identified. A composite score of the up-regulated genes was generated using a single-sample gene set enrichment analysis algorithm. RESULTS: GC gene signature expression was significantly elevated in peripheral blood leukocytes from normal healthy volunteers following oral administration of prednisolone. Expression of the signature increased in a dose-dependent manner, peaked at 4 hours postadministration, and returned to baseline levels by 48 hours after dosing. Lower expression was detected in normal healthy volunteers who received a partial GC receptor agonist, which is consistent with the reduced transactivation potential of this compound. In cohorts of patients with systemic lupus erythematosus and patients with rheumatoid arthritis, expression of the GC signature was negatively correlated with the percentages of peripheral blood lymphocytes and positively correlated with peripheral blood neutrophil counts, which is consistent with the known biology of the GC receptor. Expression of the signature largely agreed with reported GC use in these populations, although there was significant interpatient variability within the dose cohorts. CONCLUSION: The GC gene signature identified in this study represents a pharmacodynamic marker of GC exposure.
Subject(s)
Gene Expression Regulation/drug effects , Glucocorticoids/administration & dosage , Leukocytes, Mononuclear/drug effects , Prednisolone/administration & dosage , Administration, Oral , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/genetics , Biomarkers/blood , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Female , Healthy Volunteers , Humans , Leukocyte Count , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/genetics , Male , Pharmacogenomic Testing , Up-Regulation/drug effectsABSTRACT
Agents targeting the PD1-PDL1 axis have transformed cancer therapy. Factors that influence clinical response to PD1-PDL1 inhibitors include tumor mutational burden, immune infiltration of the tumor, and local PDL1 expression. To identify peripheral correlates of the anti-tumor immune response in the absence of checkpoint blockade, we performed a retrospective study of circulating T cell subpopulations and matched tumor gene expression in melanoma and non-small cell lung cancer (NSCLC) patients. Notably, both melanoma and NSCLC patients whose tumors exhibited increased inflammatory gene transcripts presented high CD4+ and CD8+ central memory T cell (CM) to effector T cell (Eff) ratios in blood. Consequently, we evaluated CM/Eff T cell ratios in a second cohort of NSCLC. The data showed that high CM/Eff T cell ratios correlated with increased tumor PDL1 expression. Furthermore, of the 22 patients within this NSCLC cohort who received nivolumab, those with high CM/Eff T cell ratios, had longer progression-free survival (PFS) (median survival: 91 vs. 215 days). These findings show that by providing a window into the state of the immune system, peripheral T cell subpopulations inform about the state of the anti-tumor immune response and identify potential blood biomarkers of clinical response to checkpoint inhibitors in melanoma and NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/immunology , Lung Neoplasms/immunology , Melanoma/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , T-Lymphocyte Subsets/immunology , Aged , Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung/drug therapy , Humans , Lung Neoplasms/drug therapy , Male , Middle Aged , Nivolumab/therapeutic use , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , Progression-Free Survival , T-Lymphocyte Subsets/metabolismABSTRACT
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.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Interleukin-17/metabolism , Multiple Sclerosis/immunology , Oligodendrocyte Precursor Cells/physiology , Receptor, Notch1/immunology , Th17 Cells/immunology , Adaptor Proteins, Signal Transducing , Animals , Astrocytes , Cell Differentiation/immunology , Cell Proliferation/physiology , Coculture Techniques , Female , HEK293 Cells , HeLa Cells , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/immunology , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Interleukin-17/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Primary Cell Culture , Protein Binding/immunology , Protein Domains/physiology , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Receptors, Interleukin-17/metabolism , Remyelination/physiology , Signal Transduction/immunology , Th1 Cells/immunology , Th17 Cells/metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolismABSTRACT
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.
Subject(s)
Active Transport, Cell Nucleus , Interleukin-1 Receptor-Associated Kinases/metabolism , Macrophages/immunology , RNA, Messenger/metabolism , Animals , Lipopolysaccharides/metabolism , Macrophages/drug effects , Mice , Nucleocytoplasmic Transport Proteins/metabolism , Phosphorylation , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors/metabolism , SumoylationABSTRACT
Tristetraprolin (TTP) is a widely expressed, zinc finger-containing protein that has been implicated in the regulation of TNFalpha production in mice. Stimulus-dependent cytoplasmic translocation of TTP has been demonstrated in several cells. In this report we used the yeast two-hybrid screen to identify proteins able to interact with full length, human TTP. One of the isolated TTP-interacting clones encoded the FG repeat region of the nuclear pore protein Nup214. Full length Nup214 co-precipitated with TTP from resting and LPS-stimulated THP-1 cells, indicating that this interaction occurred in intact cells. The ability of TTP to associate with Nup214 was dependent on two intact zinc fingers within TTP. In contrast to wild type TTP that localized primarily in the cytosol, a mutant unable to associate with Nup214 localized throughout the cell, suggesting that the interaction with Nup214 regulates TTP localization.