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1.
FASEB J ; 33(11): 12500-12514, 2019 11.
Article in English | MEDLINE | ID: mdl-31408613

ABSTRACT

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.


Subject(s)
Cell Nucleus/immunology , Kangai-1 Protein/immunology , Macrophages/immunology , Oligodeoxyribonucleotides/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 9/immunology , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/genetics , Active Transport, Cell Nucleus/immunology , Animals , Cell Nucleus/genetics , Cytokines/genetics , Cytokines/immunology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/pathology , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Kangai-1 Protein/genetics , Macrophages/pathology , Mice , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/immunology , RAW 264.7 Cells , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 9/genetics
2.
Virulence ; 9(1): 1150-1162, 2018.
Article in English | MEDLINE | ID: mdl-29962263

ABSTRACT

Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC50 of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC50 in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections.


Subject(s)
Antifungal Agents/pharmacology , Biguanides/pharmacology , Candida glabrata/drug effects , Candida/drug effects , Amphotericin B/pharmacology , Candida glabrata/growth & development , Drug Combinations , Drug Resistance, Fungal , Echinocandins/pharmacology , Fluconazole/pharmacology , Humans , Lipopeptides/pharmacology , Metformin/pharmacology , Micafungin , Microbial Sensitivity Tests , Mycoses/drug therapy , Mycoses/microbiology , TOR Serine-Threonine Kinases/drug effects , Voriconazole/pharmacology
3.
Infect Immun ; 85(3)2017 03.
Article in English | MEDLINE | ID: mdl-28031265

ABSTRACT

Dematiaceous molds are found ubiquitously in the environment and cause a wide spectrum of human disease, including infections associated with high rates of mortality. Despite this, the mechanism of the innate immune response has been less well studied, although it is key in the clearance of fungal pathogens. Here, we focus on Exserohilum rostratum, a dematiaceous mold that caused 753 infections during a multistate outbreak due to injection of contaminated methylprednisolone. We show that macrophages are incapable of phagocytosing Exserohilum Despite a lack of phagocytosis, macrophage production of tumor necrosis factor alpha is triggered by hyphae but not spores and depends upon Dectin-1, a C-type lectin receptor. Dectin-1 is specifically recruited to the macrophage-hyphal interface but not the macrophage-spore interface due to differences in carbohydrate antigen expression between these two fungal forms. Corticosteroid and antifungal therapy perturb this response, resulting in decreased cytokine production. In vivo soft tissue infection in wild-type mice demonstrated that Exserohilum provokes robust neutrophilic and granulomatous inflammation capable of thwarting fungal growth. However, coadministration of methylprednisolone acetate results in robust hyphal tissue invasion and a significant reduction in immune cell recruitment. Our results suggest that Dectin-1 is crucial for macrophage recognition and the macrophage response to Exserohilum and that corticosteroids potently attenuate the immune response to this pathogen.


Subject(s)
Ascomycota/immunology , Host-Pathogen Interactions/immunology , Lectins, C-Type/metabolism , Mycoses/immunology , Mycoses/metabolism , Adrenal Cortex Hormones/pharmacology , Antifungal Agents/pharmacology , Ascomycota/drug effects , Carbohydrates/immunology , Cell Wall/immunology , Cytokines/biosynthesis , Humans , Hyphae , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Mycoses/microbiology , Phagocytosis , Spores, Fungal , Tumor Necrosis Factor-alpha/metabolism
4.
Nat Commun ; 7: 10917, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26965188

ABSTRACT

Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or ß3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity.


Subject(s)
B-Lymphocytes/immunology , Integrin alphaV/immunology , Microtubule-Associated Proteins/immunology , Protein Transport/immunology , Toll-Like Receptors/immunology , Animals , Autophagy , Autophagy-Related Protein 5 , Blotting, Western , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , In Vitro Techniques , Integrin alphaV/genetics , Integrin beta3/genetics , Mice , Mice, Knockout , Microscopy, Confocal , Signal Transduction/immunology
5.
J Immunol ; 196(5): 2249-61, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26829985

ABSTRACT

Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate ß-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that ß-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing ß-1,3 glucan beads. Dectin-1-mediated spleen tyrosine kinase activation is required for TLR9 trafficking to ß-1,3 glucan-, A. fumigatus-, and C. albicans-containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of ß-1,3 glucan by Dectin-1 triggers TLR9 trafficking to ß-1,3 glucan-containing phagosomes, which may be critical in coordinating innate antifungal defense.


Subject(s)
Lectins, C-Type/metabolism , Phagosomes/metabolism , Toll-Like Receptor 9/metabolism , beta-Glucans/metabolism , Animals , Aspergillus fumigatus/immunology , Candida albicans/immunology , Cell Line , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation , Gene Knockout Techniques , Humans , Hydrogen-Ion Concentration , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Mice , Models, Biological , Phagocytosis , Protein Transport , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Syk Kinase , Toll-Like Receptor 9/genetics
6.
J Autoimmun ; 37(1): 48-57, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21561736

ABSTRACT

Polymorphisms in the SLAM family of leukocyte cell surface regulatory molecules have been associated with lupus-like phenotypes in both humans and mice. The murine Slamf gene cluster lies within the lupus-associated Sle1b region of mouse chromosome 1. Non-autoreactive C57BL/6 (B6) mice that have had this region replaced by syntenic segments from other mouse strains (i.e. 129, NZB and NZW) are B6 congenic strains that spontaneously produce non-nephritogenic lupus-like autoantibodies. We have recently reported that genetic ablation of the SLAM family member CD48 (Slamf2) drives full-blown autoimmune disease with severe proliferative glomerulonephritis (CD48GN) in B6 mice carrying 129 sequences of the Sle1b region (B6.129CD48(-/-)). We also discovered that BALB/c mice with the same 129-derived CD48-null allele (BALB.129CD48(-/-)) have neither nephritis nor anti-DNA autoantibodies, indicating that strain specific background genes modulate the effects of CD48 deficiency. Here we further examine this novel model of lupus nephritis in which CD48 deficiency transforms benign autoreactivity into fatal nephritis. CD48GN is characterized by glomerular hypertrophy with mesangial expansion, proliferation and leukocytic infiltration. Immune complexes deposit in mesangium and in sub-endothelial, sub-epithelial and intramembranous sites along the glomerular basement membrane. Afflicted mice have low-grade proteinuria, intermittent hematuria and their progressive renal injury manifests with elevated urine NGAL levels and with uremia. In contrast to the lupus-like B6.129CD48(-/-) animals, neither BALB.129CD48(-/-) mice nor B6 × BALB/c F1.129CD48(-/-) progeny have autoimmune traits, indicating that B6-specific background genes modulate the effect of CD48 on lupus nephritis in a recessive manner.


Subject(s)
Antigens, CD/genetics , Antigens, CD/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lupus Nephritis/genetics , Animals , Antigen-Antibody Complex/genetics , Antigen-Antibody Complex/immunology , Autoimmunity/genetics , Autoimmunity/immunology , CD48 Antigen , Disease Models, Animal , Female , Genes, Recessive/genetics , Genes, Recessive/immunology , Glomerulonephritis/genetics , Glomerulonephritis/immunology , Glomerulonephritis/pathology , Kidney Glomerulus/immunology , Kidney Glomerulus/pathology , Kidney Glomerulus/ultrastructure , Lupus Erythematosus, Systemic/pathology , Lupus Nephritis/immunology , Lupus Nephritis/pathology , Mice , Mice, 129 Strain , Mice, Inbred BALB C , Mice, Inbred MRL lpr , Mice, Knockout
7.
Curr Biol ; 19(23): 2008-13, 2009 Dec 15.
Article in English | MEDLINE | ID: mdl-19932026

ABSTRACT

Mitochondria are pleomorphic organelles that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970 aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues, and antibodies to human Myo19 detect an approximately 109 kDa band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain of function where the majority of the mitochondria move continuously. Moving mitochondria travel for many micrometers with an obvious leading end and distorted shape. The motility and shape change are sensitive to latrunculin B, indicating that both are actin dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells.


Subject(s)
Mitochondria/metabolism , Myosins/genetics , Myosins/metabolism , Actins/metabolism , Cell Line , Gene Expression Regulation , Humans , Protein Structure, Tertiary
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