ABSTRACT
The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.
Subject(s)
Mechanistic Target of Rapamycin Complex 1 , Membrane Proteins , Proto-Oncogene Proteins c-akt , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Humans , Cell Proliferation , Guanosine Triphosphate/metabolism , Immunoprecipitation , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Ras Homolog Enriched in Brain Protein/metabolism , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis Complex 1 Protein/metabolism , Tuberous Sclerosis Complex 2 Protein/metabolismABSTRACT
Transcription factor NF-κB and its activating kinase IKKß are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKß-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKß (Ikkß(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkß(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkß(Δ)Il1r1(-/-) mice, unlike Ikkß(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKß-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.
Subject(s)
Bacterial Infections/immunology , Granulocyte-Macrophage Progenitor Cells/metabolism , I-kappa B Kinase/metabolism , Interleukin-1beta/metabolism , Neutrophils/metabolism , Animals , Cell Count , Cell Growth Processes/genetics , Cell Survival/genetics , Cells, Cultured , Disease Susceptibility , Granulocyte-Macrophage Progenitor Cells/immunology , Granulocyte-Macrophage Progenitor Cells/pathology , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Immunity, Innate/genetics , Interleukin-1beta/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microarray Analysis , Neutrophils/immunology , Neutrophils/pathology , Receptors, Interleukin-1 Type I/genetics , Up-Regulation/geneticsABSTRACT
BACKGROUND: A major feature of the microenvironment in pancreatic ductal adenocarcinoma (PDAC) is the significant amount of extracellular matrix produced by pancreatic stellate cells (PSCs), which have been reported to enhance the invasiveness of pancreatic cancer cells and negatively impact the prognosis. METHODS: We analyzed the data from two publicly available microarray datasets deposited in the Gene Expression Omnibus and found candidate genes that were differentially expressed in PDAC cells with metastatic potential and PDAC cells cocultured with PSCs. We studied the interaction between PDAC cells and PSCs in vitro and verified our finding with the survival data of patients with PDAC from the website of The Human Protein Atlas. RESULTS: We found that PSCs stimulated PDAC cells to secrete S100A9, which attracted circulatory monocytes into cancer tissue and enhanced the expression of programmed death-ligand 1 (PD-L1) on macrophages. When analyzing the correlation of S100A9 and PD-L1 expression with the clinical outcomes of patients with PDAC, we ascertained that high expression of S100A9 and PD-L1 was associated with poor survival in patients with PDAC. CONCLUSIONS: PSCs stimulated PDAC cells to secrete S100A9, which acts as a chemoattractant to attract circulatory monocytes into cancer microenvironment and induces expression of PD-L1 on macrophages. High expression of S100A9 and PD-L1 was associated with worse overall survival in a cohort of patients with PDAC.
Subject(s)
Calgranulin B/genetics , Carcinoma, Pancreatic Ductal/etiology , Carcinoma, Pancreatic Ductal/metabolism , Cell Communication , Pancreatic Neoplasms/etiology , Pancreatic Neoplasms/metabolism , Stromal Cells/metabolism , Biomarkers , Calgranulin B/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Carcinoma, Pancreatic Ductal/mortality , Carcinoma, Pancreatic Ductal/pathology , Cell Communication/genetics , Cell Communication/immunology , Cell Line, Tumor , Cell Movement/drug effects , Coculture Techniques , Culture Media, Conditioned/pharmacology , Disease Susceptibility , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Neoplasm Grading , Neoplasm Staging , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Pancreatic Stellate Cells/metabolism , Pancreatic Stellate Cells/pathology , Prognosis , RNA Interference , Stromal Cells/pathology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Inflammasome is an intracellular protein complex that serves as cytosolic pattern recognition receptor (PRR) to engage with pathogens and to process cytokines of the interleukin-1 (IL-1) family into bioactive molecules. It has been established that interleukin-1ß (IL-1ß) is important to host defense against Histoplasma capsulatum infection. However, the detailed mechanism of how H. capsulatum induces inflammasome activation leading to IL-1ß production has not been studied. Here, we showed in dendritic cells (DCs) that H. capsulatum triggers caspase-1 activation and IL-1ß production through NLRP3 inflammasome. By reciprocal blocking of Dectin-1 or Dectin-2 in single receptor-deficient DCs and cells from Clec4n-/-, Clec7a-/-, and Clec7a-/-Clec4n-/- mice, we discovered that while Dectin-2 operates as a primary receptor, Dectin-1 serves as a secondary one for NLRP3 inflammasome. In addition, both receptors trigger Syk-JNK signal pathway to activate signal 1 (pro-IL-1ß synthesis) and signal 2 (activation of caspase-1). Results of pulmonary infection with H. capsulatum showed that CD103+ DCs are one of the major producers of IL-1ß and Dectin-2 and Dectin-1 double deficiency abolishes their IL-1ß response to the fungus. While K+ efflux and cathepsin B (but not ROS) function as signal 2, viable but not heat-killed H. capsulatum triggers profound lysosomal rupture leading to cathepsin B release. Interestingly, cathepsin B release is regulated by ERK/JNK downstream of Dectin-2 and Dectin-1. Our study demonstrates for the first time the unique roles of Dectin-2 and Dectin-1 in triggering Syk-JNK to activate signal 1 and 2 for H. capsulatum-induced NLRP3 inflammasome activation.
Subject(s)
Dendritic Cells/immunology , Histoplasma/physiology , Histoplasmosis/immunology , Inflammasomes/immunology , Lectins, C-Type/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Animals , Caspase 1/genetics , Caspase 1/immunology , Dendritic Cells/microbiology , Histoplasma/genetics , Histoplasmosis/genetics , Histoplasmosis/microbiology , Humans , Inflammasomes/genetics , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Lectins, C-Type/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/geneticsABSTRACT
Highly pathogenic avian influenza A H5N1 virus causes pneumonia and acute respiratory distress syndrome in humans. Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a ß-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here, we describe galectin-3 up-regulation in mouse lung tissue after challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable with wild-type (WT) mice after infections. Compared with infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced IL-1ß levels in bronchoalveolar lavage fluid. In addition, the bone marrow-derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains and secreted less IL-1ß compared with BMMs from WT mice. However, similar levels of the inflammasome component of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) were observed in two genotypes of BMMs. Co-immunoprecipitation data indicated galectin-3 and NLRP3 interaction in BMMs infected with H5N1. An association was also observed between galectin-3 and NLRP3/apoptosis-associated speck-like proteins containing caspase-associated recruitment domain complex. Combined, our results suggest that endogenous galectin-3 enhances the effects of H5N1 infection by promoting host inflammatory responses and regulating IL-1ß production by macrophages via interaction with NLRP3.
Subject(s)
Birds/virology , Galectin 3/metabolism , Influenza A Virus, H5N1 Subtype/physiology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pneumonia/metabolism , Pneumonia/virology , Animals , CARD Signaling Adaptor Proteins/metabolism , Dogs , HEK293 Cells , Humans , Interleukin-1beta/metabolism , Lung/pathology , Lung/virology , Macrophages/metabolism , Madin Darby Canine Kidney Cells , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , Pneumonia/pathology , Pyroptosis , Survival Analysis , Up-RegulationABSTRACT
Psoriasis is a chronic inflammatory skin disorder that affects ~2%-3% of the worldwide population. Inappropriate and excessive activation of endosomal Toll-like receptors 7, 8, and 9 (TLRs 7-9) at the psoriatic site has been shown to play a pathogenic role in the onset of psoriasis. Macrophage is a major inflammatory cell type that can be differentiated into phenotypes M1 and M2. M1 macrophages produce proinflammatory cytokines, and M2 macrophages produce anti-inflammatory cytokines. The balance between these two types of macrophages determines the progression of various inflammatory diseases; however, whether macrophage polarization plays a role in psoriatic inflammation activated by endosomal TLRs has not been investigated. In this study, we investigated the function and mechanism of macrophages related to the pathogenic role of TLRs 7-9 in the progression of psoriasis. Analysis of clinical data in database revealed significantly increased expression of macrophage markers and inflammatory cytokines in psoriatic tissues over those in normal tissues. In animal studies, depletion of macrophages in mice ameliorated imiquimod, a TLR 7 agonist-induced psoriatic response. Imiquimod induced expression of genes and cytokines that are signature of M1 macrophage in the psoriatic lesions. In addition, treatment with this TLR 7 agonist shifted macrophages in the psoriatic lesions to a higher M1/M2 ratio. Both of the exogenous and endogenous TLR 7-9 ligands activated M1 macrophage polarization. M1 macrophages expressed higher levels of proinflammatory cytokines and TLRs 7-9 than M2 macrophages. These results suggest that by rendering macrophages into a more inflammatory status and capable of response to their ligands in the psoriatic sites, TLR 7-9 activation drives them to participate in endosomal TLR-activated psoriatic inflammation, resulting in an amplified inflammatory response. Our results also suggest that blocking M1 macrophage polarization could be a strategy which enables inhibition of psoriatic inflammation activated by these TLRs.
Subject(s)
Inflammation/immunology , Inflammation/metabolism , Macrophages/cytology , Macrophages/metabolism , Psoriasis/immunology , Psoriasis/metabolism , Animals , Cell Cycle/drug effects , Cell Differentiation/drug effects , Computational Biology , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Imidazoles/pharmacology , Macrophages/drug effects , Mice , Mice, Inbred BALB C , THP-1 Cells , Toll-Like Receptor 7/metabolism , Toll-Like Receptors/metabolismABSTRACT
Innate immune responses are important for pathogen elimination and adaptive immune response activation. However, excess inflammation may contribute to immunopathology and disease progression (e.g. inflammation-associated hepatocellular carcinoma). Immune modulation resulting from pattern recognition receptor-induced responses is a potential strategy for controlling immunopathology and related diseases. This study demonstrates that the mycotoxin patulin suppresses Toll-like receptor- and RIG-I/MAVS-dependent cytokine production through GSH depletion, mitochondrial dysfunction, the activation of p62-associated mitophagy, and p62-TRAF6 interaction. Blockade of autophagy restored the immunosuppressive activity of patulin, and pharmacological activation of p62-dependent mitophagy directly reduced RIG-I-like receptor-dependent inflammatory cytokine production. These results demonstrated that p62-dependent mitophagy has an immunosuppressive role to innate immune response and might serve as a potential immunomodulatory target for inflammation-associated diseases.
Subject(s)
Immunity, Innate/drug effects , Mitophagy/drug effects , Mycotoxins/pharmacology , Patulin/pharmacology , Sequestosome-1 Protein/immunology , Animals , HEK293 Cells , Humans , Mice , Mitophagy/immunology , RAW 264.7 CellsABSTRACT
The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1ß and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)-dependent caspase-1 activation and IL-1ß secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro-IL-1ß processing. Likewise, overexpressing Beclin 1 in PAI-2-deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1ß-driven inflammation.
Subject(s)
Autophagy/immunology , Carrier Proteins/metabolism , Inflammasomes/metabolism , Plasminogen Activator Inhibitor 2/metabolism , Proteolysis , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Animals , Apoptosis Regulatory Proteins , CARD Signaling Adaptor Proteins , Caspase 1/metabolism , Cytokines/metabolism , Cytoskeletal Proteins/metabolism , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Silencing , Immunoblotting , Interleukin-1beta/metabolism , L-Lactate Dehydrogenase/metabolism , Macrophages/metabolism , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns and immune-compromised adults. The pore-forming toxin (PFT) ß hemolysin/cytolysin (ßh/c) is a major virulence factor for GBS, which is generally attributed to its cytolytic functions. Here we show ßh/c has immunomodulatory properties on macrophages at sub-lytic concentrations. ßh/c-mediated activation of p38 MAPK drives expression of the anti-inflammatory and immunosuppressive cytokine IL-10, and inhibits both IL-12 and NOS2 expression in GBS-infected macrophages, which are critical factors in host defense. Isogenic mutant bacteria lacking ßh/c fail to activate p38-mediated IL-10 production in macrophages and promote increased IL-12 and NOS2 expression. Furthermore, targeted deletion of p38 in macrophages increases resistance to invasive GBS infection in mice, associated with impaired IL-10 induction and increased IL-12 production in vivo. These data suggest p38 MAPK activation by ßh/c contributes to evasion of host defense through induction of IL-10 expression and inhibition of macrophage activation, a new mechanism of action for a PFT and a novel anti-inflammatory role for p38 in the pathogenesis of invasive bacterial infection. Our studies suggest p38 MAPK may represent a new therapeutic target to blunt virulence and improve clinical outcome of invasive GBS infection.
Subject(s)
Bacterial Proteins/metabolism , Hemolysin Proteins/metabolism , Interleukin-10/biosynthesis , Macrophages/immunology , Macrophages/microbiology , Streptococcal Infections/immunology , Streptococcus agalactiae/pathogenicity , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Immunity, Innate , Interleukin-12/biosynthesis , JNK Mitogen-Activated Protein Kinases/metabolism , Macrophage Activation , Macrophages/metabolism , Mice , Nitric Oxide Synthase/biosynthesis , Streptococcal Infections/microbiology , Streptococcus agalactiae/immunology , Streptococcus agalactiae/metabolism , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.
Subject(s)
Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein , Caspase 8 , Fas-Associated Death Domain Protein , Humans , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/chemistry , Caspase 8/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Fas-Associated Death Domain Protein/metabolism , Fas-Associated Death Domain Protein/genetics , HEK293 Cells , Models, Molecular , Protein Binding , Protein Domains , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Signal TransductionABSTRACT
Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.
Subject(s)
COVID-19 , Vaccines , Animals , Mice , COVID-19 Vaccines , Toll-Like Receptor 9/agonists , SARS-CoV-2 , Oligodeoxyribonucleotides/pharmacology , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/chemistry , Cytokines , Immunity , Germinal CenterABSTRACT
Type I interferons are important antiviral cytokines, but prolonged interferon production is detrimental to the host. The TLR3-driven immune response is crucial for mammalian antiviral immunity, and its intracellular localization determines induction of type I interferons; however, the mechanism terminating TLR3 signaling remains obscure. Here, we show that the E3 ubiquitin ligase ZNRF1 controls TLR3 sorting into multivesicular bodies/lysosomes to terminate signaling and type I interferon production. Mechanistically, c-Src kinase activated by TLR3 engagement phosphorylates ZNRF1 at tyrosine 103, which mediates K63-linked ubiquitination of TLR3 at lysine 813 and promotes TLR3 lysosomal trafficking and degradation. ZNRF1-deficient mice and cells are resistant to infection by encephalomyocarditis virus and SARS-CoV-2 because of enhanced type I interferon production. However, Znrf1-/- mice have exacerbated lung barrier damage triggered by antiviral immunity, leading to enhanced susceptibility to respiratory bacterial superinfections. Our study highlights the c-Src-ZNRF1 axis as a negative feedback mechanism controlling TLR3 trafficking and the termination of TLR3 signaling.
Subject(s)
COVID-19 , Interferon Type I , Animals , Mice , Antiviral Agents , SARS-CoV-2 , Toll-Like Receptor 3 , Genes, srcABSTRACT
A design of microfluidic devices is presented to integrate single-mode, liquid-core waveguides with microfluidic channels that generate and deliver disklike emulsion microdroplet cavities doped with an organic dye. The microcavity modes can be directly coupled to the liquid waveguide. Cavity-enhanced spontaneous emission was observed at the waveguide with low pump pulse energy.
ABSTRACT
Autophagy is one of the downstream effector mechanisms for elimination of intracellular microbes following activation of the Toll-like receptors (TLRs). Although the detailed molecular mechanism for this cellular process is still unclear, Beclin 1, a key molecule for autophagy, has been suggested to play a role. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the stability of signaling proteins. Herein, we show that Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. In monocytic cells, geldanamycin (GA), an Hsp90 inhibitor, effectively promoted proteasomal degradation of Beclin 1 in a concentration-dependent (EC(50) 100 nM) and time-dependent (t(50) 2 h) manner. In contrast, KNK437/Hsp inhibitor I had no effect. Hsp90 specifically interacted with Beclin 1 but not with other adapter proteins in the TLR signalsome. Treatment of cells with GA inhibited TLR3- and TLR4-mediated autophagy. In addition, S. typhimurium infection-induced autophagy was blocked by GA treatment. This further suggested a role of the Hsp90/Beclin 1 in controlling autophagy in response to microbial infections. Taken together, our data revealed that by maintaining the homeostasis of Beclin 1, Hsp90 plays a novel role in TLR-mediated autophagy.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Autophagy/physiology , HSP90 Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Toll-Like Receptors/metabolism , Animals , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Autophagy/drug effects , Autophagy/immunology , Beclin-1 , Benzoquinones/pharmacology , Cell Line , Evolution, Molecular , HEK293 Cells , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Immunity, Innate , Interferon-beta/metabolism , Lactams, Macrocyclic/pharmacology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Models, Biological , Multiprotein Complexes , NF-kappa B/metabolism , Protein Interaction Domains and Motifs , Signal Transduction , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , UbiquitinationABSTRACT
We used an experimental murine cancer metastasis model in which a colon adenocarcinoma cell line generates lung metastases, whose growth is stimulated in response to injection of bacterial lipopolysaccharide (LPS), to investigate the role of NF-kappaB in inflammation-induced tumor growth. We found that LPS-induced metastatic growth response in this model depends on both TNFalpha production by host hematopoietic cells and NF-kappaB activation in tumor cells. Inhibition of NF-kappaB in both colon and mammary carcinoma cells converts the LPS-induced growth response to LPS-induced tumor regression. The latter response is TNFalpha-independent, but depends on another member of the TNF superfamily, TRAIL, whose receptor is induced in NF-kappaB-deficient cancer cells.
Subject(s)
Inflammation/pathology , Lung Neoplasms/enzymology , Membrane Glycoproteins/physiology , NF-kappa B/physiology , Tumor Necrosis Factor-alpha/physiology , Adenocarcinoma/secondary , Animals , Apoptosis Regulatory Proteins , Colonic Neoplasms/pathology , Enzyme Activation , Inflammation/chemically induced , Lipopolysaccharides , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, CulturedABSTRACT
Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.
Subject(s)
Signal Transduction , TNF Receptor-Associated Factor 3/metabolism , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptors/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Antigens, Differentiation/chemistry , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cell Line , Dimerization , Gene Expression Regulation , Immunity, Innate , Interferons/biosynthesis , Interleukin-10/biosynthesis , Mice , Myeloid Cells/metabolism , Myeloid Differentiation Factor 88 , Protein Serine-Threonine Kinases/metabolism , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Substrate Specificity , TNF Receptor-Associated Factor 6/deficiency , TNF Receptor-Associated Factor 6/geneticsABSTRACT
Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. Here, we show that TRAF6 E3 ubiquitin ligase and A20 deubiquitinase coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The ROS-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of VPS34-UVRAG complex, thereby stimulating autophagy. Notably, expression of the ATG9A ubiquitination mutants impairs ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreases IRF-3 phosphorylation in LPS-stimulated macrophages. Our findings provide important insights into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.
Subject(s)
TNF Receptor-Associated Factor 6 , Ubiquitin-Protein Ligases , Autophagy/physiology , Oxidative Stress , TNF Receptor-Associated Factor 6/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Transcription factor, nuclear factor kappaB (NF-kappaB), is required for osteoclast formation in vivo and mice lacking both of the NF-kappaB p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the IkappaB kinase (IKK) complex that mediate NF-kappaB activation, IKKalpha and IKKbeta, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKbeta subunit as a transducer of signals from receptor activator of NF-kappaB (RANK) to NF-kappaB. Although IKKalpha is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKbeta is required for osteoclastogenesis in vitro and in vivo. IKKbeta also protects osteoclasts and their progenitors from tumor necrosis factor alpha-induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.
Subject(s)
Bone Resorption/metabolism , NF-kappa B/metabolism , Osteoclasts/metabolism , Protein Precursors/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Apoptosis , Bone Resorption/genetics , Bone Resorption/pathology , Carrier Proteins/metabolism , Cell Differentiation , Cell Survival/genetics , Cell Survival/physiology , I-kappa B Kinase , Inflammation/genetics , Inflammation/metabolism , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Myeloid Progenitor Cells/metabolism , NF-kappa B/genetics , NF-kappa B p50 Subunit , NF-kappa B p52 Subunit , Protein Precursors/genetics , Protein Serine-Threonine Kinases/genetics , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
NOD2, a NOD-like receptor (NLR), is an intracellular sensor of bacterial muramyl dipeptide (MDP) that was suggested to promote secretion of the proinflammatory cytokine IL-1beta. Yet, the molecular mechanism by which NOD2 can stimulate IL-1beta secretion, and its biological significance were heretofore unknown. We found that NOD2 through its N-terminal caspase recruitment domain directly binds and activates caspase-1 to trigger IL-1beta processing and secretion in MDP-stimulated macrophages, whereas the C-terminal leucine-rich repeats of NOD2 prevent caspase-1 activation in nonstimulated cells. MDP challenge induces the association of NOD2 with another NLR protein, NALP1, and gel filtration analysis revealed the formation of a complex consisting of NOD2, NALP1, and caspase-1. Importantly, Bacillus anthracis infection induces IL-1beta secretion in a manner that depended on caspase-1 and NOD2. In vitro, Anthrax lethal toxin strongly potentiated IL-1beta secretion, and that response was NOD2 and caspase-1-dependent. Thus, NOD2 plays a key role in the B. anthracis-induced inflammatory response by being a critical mediator of IL-1beta secretion.