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1.
J Exp Med ; 221(8)2024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-38869500

RESUMEN

UNC93B1 is a transmembrane domain protein mediating the signaling of endosomal Toll-like receptors (TLRs). We report five families harboring rare missense substitutions (I317M, G325C, L330R, R466S, and R525P) in UNC93B1 causing systemic lupus erythematosus (SLE) or chilblain lupus (CBL) as either autosomal dominant or autosomal recessive traits. As for a D34A mutation causing murine lupus, we recorded a gain of TLR7 and, to a lesser extent, TLR8 activity with the I317M (in vitro) and G325C (in vitro and ex vivo) variants in the context of SLE. Contrastingly, in three families segregating CBL, the L330R, R466S, and R525P variants were isomorphic with respect to TLR7 activity in vitro and, for R525P, ex vivo. Rather, these variants demonstrated a gain of TLR8 activity. We observed enhanced interaction of the G325C, L330R, and R466S variants with TLR8, but not the R525P substitution, indicating different disease mechanisms. Overall, these observations suggest that UNC93B1 mutations cause monogenic SLE or CBL due to differentially enhanced TLR7 and TLR8 signaling.


Asunto(s)
Eritema Pernio , Lupus Eritematoso Sistémico , Receptor Toll-Like 7 , Femenino , Humanos , Masculino , Eritema Pernio/genética , Mutación con Ganancia de Función , Células HEK293 , Lupus Eritematoso Cutáneo/genética , Lupus Eritematoso Cutáneo/patología , Lupus Eritematoso Sistémico/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación Missense , Linaje , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/metabolismo , Preescolar , Niño , Adulto Joven , Adulto
2.
Immunology ; 171(2): 181-197, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37885279

RESUMEN

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.


Asunto(s)
Transducción de Señal , Receptor Toll-Like 4 , Humanos , Receptor Toll-Like 4/metabolismo , Sitios de Unión , Citocinas/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Lipopolisacáridos
3.
Cell Rep Med ; 4(12): 101256, 2023 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-38118422

RESUMEN

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector T cells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.


Asunto(s)
Neoplasias , Linfocitos T Reguladores , Humanos , Ratones , Animales , Células Asesinas Naturales , Células Dendríticas , Homeostasis
4.
Curr Opin Immunol ; 84: 102375, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37562076

RESUMEN

Intracellular Toll-like receptors (TLRs) are key components of the innate immune system. Their expression in antigen-presenting cells (APCs), and in particular dendritic cells (DCs), makes them critical in the induction of the adaptive immune response. In DCs, they interact with the chaperone UNC93B1 that mediates their trafficking from the endoplasmic reticulum (ER) to endosomes where they are cleaved by proteases and activated. All these different steps are also shared by major histocompatibility complex class-II (MHCII) molecules. Here, we will discuss the tight relationship intracellular TLRs have with the antigen processing machinery in APCs for their trafficking and activation.


Asunto(s)
Presentación de Antígeno , Transducción de Señal , Humanos , Receptores Toll-Like , Antígenos de Histocompatibilidad Clase II , Endosomas , Células Dendríticas
5.
Cell Mol Life Sci ; 80(5): 138, 2023 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-37145189

RESUMEN

Numerous evidences support that microglia contributes to the progression of Alzheimer's disease. P2X4 receptors are ATP-gated channels with high calcium permeability, which are de novo expressed in a subset of reactive microglia associated with various pathological contexts, contributing to microglial functions. P2X4 receptors are mainly localized in lysosomes and trafficking to the plasma membrane is tightly regulated. Here, we investigated the role of P2X4 in the context of Alzheimer's disease (AD). Using proteomics, we identified Apolipoprotein E (ApoE) as a specific P2X4 interacting protein. We found that P2X4 regulates lysosomal cathepsin B (CatB) activity promoting ApoE degradation; P2rX4 deletion results in higher amounts of intracellular and secreted ApoE in both bone-marrow-derived macrophage (BMDM) and microglia from APPswe/PSEN1dE9 brain. In both human AD brain and APP/PS1 mice, P2X4 and ApoE are almost exclusively expressed in plaque-associated microglia. In 12-month-old APP/PS1 mice, genetic deletion of P2rX4 reverses topographical and spatial memory impairment and reduces amount of soluble small aggregates of Aß1-42 peptide, while no obvious alteration of plaque-associated microglia characteristics is observed. Our results support that microglial P2X4 promotes lysosomal ApoE degradation, indirectly altering Aß peptide clearance, which in turn might promotes synaptic dysfunctions and cognitive deficits. Our findings uncover a specific interplay between purinergic signaling, microglial ApoE, soluble Aß (sAß) species and cognitive deficits associated with AD.


Asunto(s)
Enfermedad de Alzheimer , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E/metabolismo , Modelos Animales de Enfermedad , Trastornos de la Memoria , Ratones Transgénicos , Microglía/metabolismo , Placa Amiloide/metabolismo , Receptores Purinérgicos P2X4/metabolismo
6.
Methods Mol Biol ; 2618: 279-288, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36905524

RESUMEN

Phagocytosis is a process by which specific immune cells such as macrophages or dendritic cells engulf large particles. It is an important innate immune defense mechanism for removing a wide variety of pathogens and apoptotic cells. Following phagocytosis, nascent phagosomes are formed which, when fused to lysosome to become phagolysosome containing acidic proteases, will allow the degradation of ingested material. This chapter describes in vitro and in vivo assays to measure phagocytosis by murine dendritic cells using amine beads coupled with streptavidin Alexa 488. This protocol can also be applied to monitor phagocytosis in human dendritic cells.


Asunto(s)
Fagocitos , Fagocitosis , Humanos , Ratones , Animales , Fagocitos/metabolismo , Macrófagos/metabolismo , Fagosomas/metabolismo , Células Dendríticas
7.
Proc Natl Acad Sci U S A ; 120(7): e2208509120, 2023 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-36745791

RESUMEN

Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the ß-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The ß-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.


Asunto(s)
Antígenos de Histocompatibilidad Clase I , Precursores del ARN , Animales , Ratones , Antígenos de Histocompatibilidad Clase I/genética , Precursores del ARN/genética , Linfocitos T CD8-positivos , Biosíntesis de Proteínas , Presentación de Antígeno , Ratones Endogámicos C57BL , Péptidos/metabolismo , Tolerancia Inmunológica/genética , Epítopos , Antígenos de Histocompatibilidad Clase II/genética
9.
EMBO Rep ; 23(12): e55470, 2022 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-36215666

RESUMEN

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway, and we propose one potential mechanism of action.


Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase II , Antígenos de Histocompatibilidad Clase II/genética , Autofagia , Péptidos
10.
Cell Immunol ; 374: 104484, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35247713

RESUMEN

The accumulation of protein aggregates is toxic and linked to different diseases such as neurodegenerative disorders, but the role of the immune system to target and destroy aggregate-carrying cells is still relatively unknown. Here we show a substrate-specific presentation of antigenic peptides to the direct MHC class I pathway via autophagy. We observed no difference in presentation of peptides derived from the viral EBNA1 protein following suppression of autophagy by knocking down Atg5 and Atg12. However, the same knock down treatment suppressed the presentation from ovalbumin. Fusing the aggregate-prone poly-glutamine (PolyQ) to the ovalbumin had no effect on antigen presentation via autophagy. Interestingly, fusing the EBNA1-derived gly-ala repeat (GAr) sequence to ovalbumin rendered the presentation Atg5/12 independent. We also demonstrate that the relative levels of protein expression did not affect autophagy-mediated antigen presentation. These data suggest a substrate-dependent presentation of antigenic peptides for the MHC class I pathway via autophagy and indicate that the GAr of the EBNA1 illustrates a novel virus-mediated mechanism for immune evasion of autophagy-dependent antigen presentation.


Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Antígenos , Autofagia , Antígenos de Histocompatibilidad Clase II/metabolismo , Evasión Inmune , Ovalbúmina
11.
Mol Immunol ; 145: 1-2, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35247872

RESUMEN

The Special Issue is dedicated to the 10th Antigen Processing and Presentation Workshop, which took place at Institut Cochin in Paris from May 28th to June 2nd, 2019. It contains several reviews or original articles from contributors to this workshop. It is also a vibrant Tribute to Nilabh Shastri, founder of the APP Workshops, who untimely passed away in 2021 and is deeply missed by his colleagues and friends.


Asunto(s)
Presentación de Antígeno , Historia del Siglo XX , Paris
12.
Mol Immunol ; 144: 44-48, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35184022

RESUMEN

Dendritic cells (DCs) have the unique capacity to link innate to adaptive immunity. While most cells that express major histocompatibility (MHC) molecules are able to present antigens to activated T cells, DCs possess the means for presenting antigens to naïve T cells, and, as such, are able to instruct T cells to initiate immune response. There are two cascades of events necessary for DCs to start their instructive function. First, DCs enzymatically process proteins to make T cells recognize an antigen as unique peptide-MHC complexes. Second, DCs provide secretory cytokines and co-stimulatory functions for T cells to respond to this antigen. Thus, the compartments for protein degradation and for protein synthesis are central to DC function. The endoplasmic reticulum (ER), a vast network of membranes and vesicles, connects these compartments and helps modulate DC-specific performance, such as antigen capture and presentation. However, while the health of ER appears relevant for DC function, the intersection between ER stress and antigen presentation remains to be explored.


Asunto(s)
Presentación de Antígeno , Estrés del Retículo Endoplásmico , Antígenos , Células Dendríticas , Histocompatibilidad
15.
Free Radic Biol Med ; 173: 104-116, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34303829

RESUMEN

BACKGROUND: Chloroquine has been used successfully to treat Malaria, including by chloroquine-resistant Plasmodium sp., indicating that it has effects on disease itself. Since heme has inflammatory effects and contributes to the pathogenesis of hemolytic diseases, we hypothesize that the anti-inflammatory effect of chloroquine is partially due to its inhibitory effect on heme-induced macrophage activation and on inflammatory tissue damage. METHODS: Bone marrow derived macrophages (BMDMs) were incubated with chloroquine before stimulation with heme, in different conditions, to evaluate cytokines secretion, ROS production, mitogen activated protein kinases (MAPK) or spleen tyrosine kinase (Syk) activation, alone or combined with LPS. The effects of chloroquine upon heme inflammation were also evaluated in vivo, through simultaneous i.p. injection of LPS and heme, intratracheal instillation of Poly-IC followed by heme injection, and in a rhabdomyolysis model. RESULTS: Chloroquine inhibited TNF secretion, mitochondrial ROS production, MAPK, and Syk activation induced by heme. Inhibition of TNF production could be mimicked by zinc ionophore quercetin, but not by primaquine, a chloroquine analog with low affinity for heme. IL-6 and IL-1ß secretions induced by heme in the presence of PRRs agonists were inhibited by chloroquine, but not by calcium chelator BAPTA or inhibitor of endosomal acidification concamycin B. Chloroquine also protected mice from heme inflammatory effects in vivo, inhibiting lethal synergism with PRR agonists, lung pathology caused by heme injection after intratracheal instillation of Poly-IC, and delaying death after rhabdomyolisis. CONCLUSION: Our data indicate that chloroquine might be used as a supportive therapy to control heme-induced deleterious inflammation in different hemolytic diseases.


Asunto(s)
Cloroquina , Hemo , Animales , Citocinas , Lipopolisacáridos/toxicidad , Activación de Macrófagos , Macrófagos , Ratones
16.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-32699279

RESUMEN

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Asunto(s)
Linfocitos B/inmunología , Microambiente Celular/inmunología , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/inmunología , Inmunidad Adaptativa , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Línea Celular , Quimiocina CXCL13/inmunología , Simulación por Computador , Células Dendríticas Foliculares/citología , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Noqueados , Microscopía Fluorescente , Modelos Biológicos , Tonsila Palatina/citología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Células del Estroma/inmunología , Células del Estroma/metabolismo
17.
Bioconjug Chem ; 31(6): 1685-1692, 2020 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-32510940

RESUMEN

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.


Asunto(s)
Ciclooctanos/química , Receptor Toll-Like 2/metabolismo , Animales , Diseño de Fármacos , Humanos , Ligandos , Ratones , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Estereoisomerismo , Receptor Toll-Like 2/agonistas
18.
J Cell Sci ; 133(5)2020 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-32079661

RESUMEN

Toll-like receptor 7 (TLR7) is an endosomal receptor that recognizes single-stranded RNA from viruses. Its trafficking and activation is regulated by the endoplasmic reticulum (ER) chaperone UNC93B1 and lysosomal proteases. UNC93B1 also modulates major histocompatibility complex class II (MHCII) antigen presentation, and deficiency in MHCII protein diminishes TLR9 signaling. These results indicate a link between proteins that regulate both innate and adaptive responses. Here, we report that TLR7 resides in lysosomes and interacts with the MHCII-chaperone molecule, the invariant chain (Ii) or CD74, in B cells. In the absence of CD74, TLR7 displays both ER and lysosomal localization, leading to an increase in pro-inflammatory cytokine production. Furthermore, stimulation with TLR7 but not TLR9, is inefficient in boosting antigen presentation in Ii-deficient cells. In contrast, in B cells lacking TLR7 or mutated for UNC93B1, which are able to trigger TLR7 activation, antigen presentation is enhanced. This suggests that TLR7 signaling in B cells is controlled by the Ii chain.


Asunto(s)
Proteínas de Transporte de Membrana , Receptor Toll-Like 7 , Antígenos de Diferenciación de Linfocitos B/genética , Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo
19.
Methods Mol Biol ; 1988: 289-296, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31147948

RESUMEN

Proteases generate peptides that bind to MHC class II molecules to interact with a wide diversity of CD4+ T cells. They are expressed in dedicated organelles: endosomes and lysosomes of professional antigen-presenting cells (pAPCs) such as B cells, macrophages, and dendritic cells. The identification of endosomal proteases which produce antigenic peptides is important for example for better vaccination and to prevent autoimmune diseases. Here, we describe a panel of techniques (in vitro digestion assays of protein with recombinant proteases or purified endosomes/lysosomes, T cell stimulation) to monitor the production of MHC class II ligands.


Asunto(s)
Antígenos de Histocompatibilidad Clase II/metabolismo , Biología Molecular/métodos , Péptido Hidrolasas/metabolismo , Animales , Presentación de Antígeno/inmunología , Electroforesis en Gel de Poliacrilamida , Endosomas/metabolismo , Ligandos , Lisosomas/metabolismo , Ratones , Biosíntesis de Proteínas
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