RESUMEN
Allergic diseases are pathological immune responses with significant morbidity, which are closely associated with allergic mediators as released by allergen-stimulated mast cells (MCs). Prophylactic stabilization of MCs is regarded as a practical approach to prevent allergic diseases. However, most of the existing small molecular MC stabilizers exhibit a narrow therapeutic time window, failing to provide long-term prevention of allergic diseases. Herein, ceria nanoparticle (CeNP-) based phosphatase-mimetic nano-stabilizers (PMNSs) with a long-term therapeutic time window are developed for allergic disease prevention. By virtue of the regenerable catalytic hotspots of oxygen vacancies on the surface of CeNPs, PMNSs exhibit sustainable phosphatase-mimetic activity to dephosphorylate phosphoproteins in allergen-stimulated MCs. Consequently, PMNSs constantly modulate intracellular phospho-signaling cascades of MCs to inhibit the degranulation of allergic mediators, which prevents the initiation of allergic mediator-associated pathological responses, eventually providing protection against allergic diseases with a long-term therapeutic time window.
Asunto(s)
Alérgenos/inmunología , Degranulación de la Célula/inmunología , Hipersensibilidad/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Animales , Biomimética , Modelos Animales de Enfermedad , Mastocitos , RatonesRESUMEN
Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ΔptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection.
Asunto(s)
Arginina/análogos & derivados , Interacciones Huésped-Patógeno/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Staphylococcus aureus/patogenicidad , Factores de Virulencia/inmunología , Animales , Arginina/inmunología , Ratones , Compuestos Organofosforados/inmunologíaRESUMEN
Suppression and modulation of the host immune response to parasitic nematodes have been extensively studied. In the present study, we cloned and produced recombinant phosphotyrosyl phosphatase activator protein from Haemonchus contortus (rHCPTPA), a parasitic nematode of small ruminants, and studied the effect of this protein on modulating the immune response of goat peripheral blood mononuclear cells. Enzymatic assays revealed that rHCPTPA enhanced the p-nitrophenylphosphate phosphatase activity of bovine PP2A1. Immunohistochemical tests verified that the HCPTPA protein was localised mainly in the bowel wall and on the body surface of worms. It was also shown that serum produced by goats artificially infected with H. contortus successfully recognised rHCPTPA, which conjugated with goat peripheral blood mononuclear cells. The rHCPTPA was then co-incubated with goat peripheral blood mononuclear cells to assess its immunomodulatory effects on proliferation, apoptosis, cytokine secretion, migration and nitric oxide production. Our results showed that rHCPTPA suppressed the proliferation of goat peripheral blood mononuclear cells stimulated by concanavalin A and induced apoptosis in goat peripheral blood mononuclear cells. After rHCPTPA exposure, IFN-γ and IL-2 expression was markedly reduced, whereas secretion of IL-10 and IL-4 was significantly elevated, in goat peripheral blood mononuclear cells. Moreover, rHCPTPA down-regulated nitric oxide production and migration of goat peripheral blood mononuclear cells in a dose-dependent manner. These results illuminate the interaction between parasites and hosts at the molecular level, suggest a possible immunomodulatory target and contribute to the search for innovative proteins that might be candidate targets for drugs and vaccines.
Asunto(s)
Haemonchus , Proteínas del Helminto/inmunología , Leucocitos Mononucleares , Monoéster Fosfórico Hidrolasas/inmunología , Animales , Apoptosis , Bovinos , Citocinas/inmunología , Enfermedades de las Cabras , Cabras , Hemoncosis , Haemonchus/enzimología , Inmunidad , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/parasitologíaRESUMEN
Antibodies are widely used both in clinical practice and in research. However, the development of methods to increase the ratio of antibodies to recognize phosphorylated proteins remains challenging. In this study, we report a novel and useful method for the efficient production of antibodies for phosphorylated proteins. Based on our previously developed vaccine adjuvant Nap-GDFDFDY, we prepared hydrogels by the Ca2+-induced self-assembly of a phosphorylated peptide gelator Nap-GDFDFpDY. The hydrogel could protect phosphorylated antigens from being dephosphorylated by endogenous phosphatase, thus selectively increasing the ratio of the antibodies for phosphorylated proteins. Our study provides a useful strategy for the production of antibodies to recognize proteins with specific posttranslational modifications.
Asunto(s)
Anticuerpos/química , Anticuerpos/inmunología , Formación de Anticuerpos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Monoéster Fosfórico Hidrolasas/análisis , Monoéster Fosfórico Hidrolasas/inmunología , Hidrogel de Polietilenoglicol-Dimetacrilato/síntesis química , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Monoéster Fosfórico Hidrolasas/metabolismo , FosforilaciónRESUMEN
BACKGROUND: Latent autoimmune diabetes in adults (LADA) is determined by both a noninsulin-dependent clinical presentation and an autoimmune pathogenic process. Glutamic acid decarboxylase antibody (GADA) constitutes the most important marker, although IA-2A and ZnT8A also define LADA presentation. Type 2 diabetes mellitus (T2DM) is the most prevalent type particularly over 65 years old. Studies about autoimmunity in this age group are scarce. OBJECTIVE: The aim of this work was to determine whether three autoantibodies for diabetes autoimmunity were present in elderly T2DM patients, and to assess the distinctive clinical features of autoantibody-positive patients. RESEARCH DESIGN AND METHODS: We recruited 153 patients with diabetes with onset of diabetes after 65 years of age and a BMI under 30 kg/m2 . RESULTS: The prevalence of at least one of the autoantibodies was 15.68% (24/153). The most prevalent autoantibody was GADA with 8.49% (13/153), followed by ZnT8A with 6.50% (10/153) and IA2A with 1.96% (3/153). The autoimmunity-positive group presented higher HbA1c (7.01 ± 1.98 vs 6.35 ± 1.01; P = 0.007) and more prevalent insulin therapy (25% vs 10.85%; P = 0.047). GADA-positive patients with diabetes presented higher FPG (7.79 ± 3.79 mmol/L vs 6.43 ± 1.6 mmol/L; P = 0.014) and insulin therapy more frequently (46% vs 10.71%; p = 0.015). GADA titre levels in the individuals with BMI under 27 kg/m2 were higher (35.00 ± 4.20) than those in the group with BMI over 27 kg/m2 (8.83 ± 3.041; P = 0.0005). CONCLUSION: Autoantibodies GADA and Znt8A may be useful markers in identifying a subgroup of older patients with a clinical presentation of diabetes which could be characterized as latent autoimmune diabetes in the elderly.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Autoinmune Latente del Adulto/inmunología , Diabetes Autoinmune Latente del Adulto/patología , Edad de Inicio , Anciano , Anciano de 80 o más Años , Autoanticuerpos/análisis , Autoinmunidad/fisiología , Biomarcadores , Estudios Transversales , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Diabetes Autoinmune Latente del Adulto/diagnóstico , Diabetes Autoinmune Latente del Adulto/epidemiología , Masculino , Monoéster Fosfórico Hidrolasas/inmunología , Pronóstico , Transportador 8 de Zinc/inmunologíaRESUMEN
Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumourtargeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an NiNTA column and a G50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by highperformance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec1, respectively. The antihMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Enzimas Reparadoras del ADN/inmunología , Enzimas Reparadoras del ADN/metabolismo , Desarrollo de Medicamentos/métodos , Monoéster Fosfórico Hidrolasas/inmunología , Monoéster Fosfórico Hidrolasas/metabolismo , Anticuerpos Monoclonales/aislamiento & purificación , Línea Celular Tumoral , Clonación Molecular , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/aislamiento & purificación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Pruebas de Enzimas/métodos , Humanos , Oxidación-Reducción , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Enfermedades de los Peces/prevención & control , Perciformes/inmunología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/inmunología , Infecciones Estreptocócicas/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/genética , Citocinas/genética , Citocinas/inmunología , Enfermedades de los Peces/inmunología , Riñón/inmunología , Muramidasa/sangre , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , ARN Mensajero/metabolismo , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus/inmunología , VacunaciónRESUMEN
B cell antigen receptor (BCR) signaling is a tightly regulated process governed by both positive and negative mediators/regulators to ensure appropriate responses to exogenous and autologous antigens. Upon naïve B cell recognition of antigen CD79 [the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling subunit of the BCR] is phosphorylated and recruits Src and Syk family kinases that then phosphorylate proximal intermediaries linked to downstream activating signaling circuitry. This plasma membrane localized signalosome activates PI3K leading to generation of PIP3 critical for membrane localization and activation of plecktrin homology domain-containing effectors. Conversely, in anergic B cells, chronic antigen stimulation drives biased monophosphorylation of CD79 ITAMs leading to recruitment of Lyn, but not Syk, which docks only to bi-phosphorylated ITAMS. In this context, Lyn appears to function primarily as a driver of inhibitory signaling pathways promoting the inhibition of the PI3K pathway by inositol phosphatases, SHIP-1 and PTEN, which hydrolyze PIP3 to PIP2. Lyn may also exert negative regulation of signaling through recruitment of SHP-1, a tyrosine phosphatase that dephosphorylates activating signaling molecules. Alleles of genes that encode or regulate expression of components of this axis, including SHIP-1, SHP-1, Csk/PTPn22, and Lyn, have been shown to confer risk of autoimmunity. This review will discuss functional interplay of components of this pathway and the impact of risk alleles on its function.
Asunto(s)
Linfocitos B/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Proteínas Quinasas/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Anergia Clonal , HumanosRESUMEN
T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (â¼50%) than the smaller R0 isoform (â¼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.
Asunto(s)
Antígenos Comunes de Leucocito/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígeno B7-H1/inmunología , Línea Celular , Membrana Celular/inmunología , Humanos , Cinética , Ligandos , Activación de Linfocitos/inmunología , Fosforilación/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Unión Proteica/inmunología , Células Sf9 , Transducción de Señal/inmunologíaRESUMEN
Leishmania donovani resides within the host macrophages by dampening host defence mechanisms and thereby it modulates the host cell functions for its survival. Multiple host cell factors compete during the interplay between the host and the parasite. Roles for dual-specificity phosphatases (DUSPs) are implicated in various pathological conditions. However, the reciprocity of these DUSPs was unknown in L. donovani infection in a susceptible model. Here, we show that Mycobacterium indicus pranii (Mw), an immunomodulator, reciprocally regulates DUSP1 and DUSP6 through the TLR4 pathway. Association of PKC-ß with DUSP6 increases after Mw treatment resulting in decreased IL-10, phosphorylation of ERK1/2 and Arginase-1, whereas Mw treatment decreases the association between PKC-ε and DUSP1 resulting in increased IL-12, phosphorylation of p38 and inducible nitric oxide synthase expression. Silencing of DUSP1 or over-expression of DUSP6 in L. donovani-infected BALB/c mice decreases the parasite burden by inducing IL-12 and reducing IL-10 production. Therefore, we identify DUSP1 and DUSP6 as therapeutic targets, functions of which could be favourably modulated by Mw during L. donovani infection.
Asunto(s)
Factores Inmunológicos/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Mycobacterium/inmunología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Ratones , Ratones Endogámicos BALB C , Monoéster Fosfórico Hidrolasas/inmunología , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.
Asunto(s)
Apoptosis/fisiología , Estrés del Retículo Endoplásmico/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Estrés Fisiológico/fisiología , Células 3T3 , Animales , Anticuerpos/inmunología , Apoptosis/genética , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/inmunología , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Borrelia burgdorferi possesses a sophisticated chemotaxis signaling system; however, the roles of the majority of the chemotaxis proteins in the infectious life cycle have not yet been demonstrated. Specifically, the role of CheD during host colonization has not been demonstrated in any bacterium. Here, we systematically characterized the B. burgdorferi CheD homolog using genetics and biochemical and mouse-tick-mouse infection cycle studies. Bacillus subtilis CheD plays an important role in chemotaxis by deamidation of methyl-accepting chemotaxis protein receptors (MCPs) and by increasing the receptor kinase activity or enhancing CheC phosphatase activity, thereby regulating the levels of the CheY response regulator. Our biochemical analysis indicates that B. burgdorferi CheD significantly enhances CheX phosphatase activity by specifically interacting with the phosphatase. Moreover, CheD specifically binds two of the six MCPs, indicating that CheD may also modulate the receptor proteins. Although the motility of the cheD mutant cells was indistinguishable from that of the wild-type cells, the mutant did exhibit reduced chemotaxis. Importantly, the mutant showed significantly reduced infectivity in C3H/HeN mice via needle inoculation. Mouse-tick-mouse infection assays indicated that CheD is dispensable for acquisition or transmission of spirochetes; however, the viability of cheD mutants in ticks is marginally reduced compared to that of the wild-type or complemented cheD spirochetes. These data suggest that CheD plays an important role in the chemotaxis and pathogenesis of B. burgdorferi We propose potential connections between CheD, CheX, and MCPs and discuss how these interactions play critical roles during the infectious life cycle of the spirochete.
Asunto(s)
Vectores Arácnidos/microbiología , Borrelia burgdorferi/genética , Quimiotaxis/inmunología , Regulación Bacteriana de la Expresión Génica , Enfermedad de Lyme/inmunología , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Garrapatas/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Borrelia burgdorferi/crecimiento & desarrollo , Borrelia burgdorferi/patogenicidad , Quimiotaxis/genética , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/patología , Proteínas Quimiotácticas Aceptoras de Metilo/inmunología , Ratones , Ratones Endogámicos C3H , Mutación , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , VirulenciaRESUMEN
In mouse models of infection with the gastrointestinal parasite Trichuris muris, appropriate dendritic-cell (DC) Ag sampling, migration, and presentation to T cells are necessary to mount a protective Th2-polarized adaptive immune response, which is needed to clear infection. SH2-containing inositol 5'-phosphatase 1 (SHIP-1) has been shown to be an important regulator of DC function in vitro through the negative regulation of the phosphoinositide 3-kinase (PI3K) pathway, but its role in vivo is relatively unexplored. In the current work, mice with a specific deletion of SHIP-1 in DCs (Ship1(ΔDC) ) were infected with the parasite T. muris. Ship1(ΔDC) mice were susceptible to infection due to ineffective priming of Th2-polarized responses. This is likely due to an increased production of interleukin (IL) 12p40 by SHIP-1-deficient DCs, as in vivo antibody blockade of IL-12p40 was able to facilitate the clearing of infection in Ship1(ΔDC) mice. Our results describe a critical role for SHIP-1 in regulating the ability of DCs to efficiently prime Th2-type responses.
Asunto(s)
Células Dendríticas/inmunología , Activación de Linfocitos/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Células Th2/inmunología , Tricuriasis/inmunología , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Mutantes , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trichuris/inmunologíaRESUMEN
The receptor-interacting protein kinase 3 (RIPK3) plays crucial roles in programmed necrosis and innate inflammatory responses. However, a little is known about the involvement of RIPK3 in NKT cell-mediated immune responses. Here, we demonstrate that RIPK3 plays an essential role in NKT cell function via activation of the mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5). RIPK3-mediated activation of PGAM5 promotes the expression of cytokines by facilitating nuclear translocation of NFAT and dephosphorylation of dynamin-related protein 1 (Drp1), a GTPase is essential for mitochondrial homoeostasis. Ripk3(-/-) mice show reduced NKT cell responses to metastatic tumour cells, and both deletion of RIPK3 and pharmacological inhibition of Drp1 protects mice from NKT cell-mediated induction of acute liver damage. Collectively, the results identify a crucial role for RIPK3-PGAM5-Drp1/NFAT signalling in NKT cell activation, and further suggest that RIPK3-PGAM5 signalling may mediate crosstalk between mitochondrial function and immune signalling.
Asunto(s)
Dinaminas/inmunología , Inmunidad Celular/inmunología , Hígado/inmunología , Células T Asesinas Naturales/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transporte Activo de Núcleo Celular , Animales , Western Blotting , Citocinas/inmunología , Dinaminas/metabolismo , Células HEK293 , Hepatocitos , Humanos , Inflamación , Interferón gamma/inmunología , Interleucina-4/inmunología , Células Jurkat , Melanoma Experimental , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Factores de Transcripción NFATC/metabolismo , Trasplante de Neoplasias , Fosfoproteínas Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Dectin-1 (Clec7a) is a paradigmatic C-type lectin receptor that binds Syk through a hemITAM motif and couples sensing of pathogens such as fungi to induction of innate responses. Dectin-1 engagement triggers a plethora of activating events, but little is known about the modulation of such pathways. Trying to define a more precise picture of early Dectin-1 signaling, we explored the interactome of the intracellular tail of the receptor in mouse dendritic cells. We found unexpected binding of SHIP-1 phosphatase to the phosphorylated hemITAM. SHIP-1 colocalized with Dectin-1 during phagocytosis of zymosan in a hemITAM-dependent fashion. Moreover, endogenous SHIP-1 relocated to live or heat-killed Candida albicans-containing phagosomes in a Dectin-1-dependent manner in GM-CSF-derived bone marrow cells (GM-BM). However, SHIP-1 absence in GM-BM did not affect activation of MAPK or production of cytokines and readouts dependent on NF-κB and NFAT. Notably, ROS production was enhanced in SHIP-1-deficient GM-BM treated with heat-killed C. albicans, live C. albicans, or the specific Dectin-1 agonists curdlan or whole glucan particles. This increased oxidative burst was dependent on Dectin-1, Syk, PI3K, phosphoinositide-dependent protein kinase 1, and NADPH oxidase. GM-BM from CD11c∆SHIP-1 mice also showed increased killing activity against live C. albicans that was dependent on Dectin-1, Syk, and NADPH oxidase. These results illustrate the complexity of myeloid C-type lectin receptor signaling, and how an activating hemITAM can also couple to intracellular inositol phosphatases to modulate selected functional responses and tightly regulate processes such as ROS production that could be deleterious to the host.
Asunto(s)
Secuencias de Aminoácidos/inmunología , Candida albicans/inmunología , Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Especies Reactivas de Oxígeno/inmunología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/microbiología , Candida albicans/fisiología , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interacciones Huésped-Patógeno/inmunología , Inositol Polifosfato 5-Fosfatasas , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones Noqueados , Microscopía Confocal , Datos de Secuencia Molecular , FN-kappa B/inmunología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/inmunología , Factores de Transcripción NFATC/metabolismo , Fagocitosis/inmunología , Fagosomas/inmunología , Fagosomas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Unión Proteica/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunologíaRESUMEN
SHIP1 is a 5'-inositol phosphatase known to negatively regulate the signaling product of the PI3K pathway, phosphatidylinositol (3-5)-trisphosphate. SHIP1 is recruited to a large number of inhibitory receptors expressed on invariant NK (iNKT) cells. We hypothesized that SHIP1 deletion would have major effects on iNKT cell development by altering the thresholds for positive and negative selection. Germline SHIP1 deletion has been shown to affect T cells as well as other immune cell populations. However, the role of SHIP1 on T cell function has been controversial, and its participation on iNKT cell development and function has not been examined. We evaluated the consequences of SHIP1 deletion on iNKT cells using germline-deficient mice, chimeric mice, and conditionally deficient mice. We found that T cell and iNKT cell development are impaired in germline-deficient animals. However, this phenotype can be rescued by extrinsic expression of SHIP1. In contrast, SHIP1 is required cell autonomously for optimal iNKT cell cytokine secretion. This suggests that SHIP1 calibrates the threshold of iNKT cell reactivity. These data further our understanding of how iNKT cell activation is regulated and provide insights into the biology of this unique cell lineage.
Asunto(s)
Diferenciación Celular/inmunología , Proliferación Celular , Células T Asesinas Naturales/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Animales , Western Blotting , Trasplante de Médula Ósea/métodos , Diferenciación Celular/genética , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Inositol Polifosfato 5-Fosfatasas , Hígado/inmunología , Hígado/metabolismo , Recuento de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/inmunología , Timo/metabolismoRESUMEN
Mutations in the adaptor protein PSTPIP2 are the cause of the autoinflammatory disease chronic multifocal osteomyelitis in mice. This disease closely resembles the human disorder chronic recurrent multifocal osteomyelitis, characterized by sterile inflammation of the bones and often associated with inflammation in other organs, such as the skin. The most critical process in the disease's development is the enhanced production of IL-1ß. This excessive IL-1ß is likely produced by neutrophils. In addition, the increased activity of macrophages, osteoclasts, and megakaryocytes has also been described. However, the molecular mechanism of how PSTPIP2 deficiency results in this phenotype is poorly understood. Part of the PSTPIP2 inhibitory function is mediated by protein tyrosine phosphatases from the proline-, glutamic acid-, serine- and threonine-rich (PEST) family, which are known to interact with the central part of this protein, but other regions of PSTPIP2 not required for PEST-family phosphatase binding were also shown to be indispensable for PSTPIP2 function. In this article, we show that PSTPIP2 binds the inhibitory enzymes Csk and SHIP1. The interaction with SHIP1 is of particular importance because it binds to the critical tyrosine residues at the C terminus of PSTPIP2, which is known to be crucial for its PEST-phosphatase-independent inhibitory effects in different cellular systems. We demonstrate that in neutrophils this region is important for the PSTPIP2-mediated suppression of IL-1ß processing and that SHIP1 inhibition results in the enhancement of this processing. We also describe deregulated neutrophil response to multiple activators, including silica, Ab aggregates, and LPS, which is suggestive of a rather generalized hypersensitivity of these cells to various external stimulants.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas del Citoesqueleto/inmunología , Osteomielitis/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Familia-src Quinasas/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Tirosina Quinasa CSK , Línea Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Inflamación/inmunología , Inositol Polifosfato 5-Fosfatasas , Interleucina-1beta/biosíntesis , Macrófagos/inmunología , Megacariocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neutrófilos/inmunología , Osteoclastos/inmunología , Osteomielitis/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Inpp5d (Src homology 2 domain-containing inositol-5-phosphatase [Ship1])-deficient mice experience spontaneous airway inflammation and have enhanced sensitivity to allergen-induced airway inflammation. OBJECTIVE: We hypothesized that lineage-specific deletion of Ship1 expression in cells known to be crucial for adaptive TH2 responses would uncover distinct roles that could either positively or negatively regulate susceptibility to allergic airway inflammation (AAI). METHODS: Ship1 expression was deleted in B cells, T cells, or dendritic cells (DCs), and the resulting Ship1(ΔB cell), Ship1(ΔT cell), Ship1(ΔDC), or Ship1(F/F) (wild-type) control mice were evaluated in a model of house dust mite (HDM)-induced AAI. RESULTS: Unlike germline panhematopoietic Ship1 deletion, deletion of Ship1 selectively in either the B-cell, T-cell, or DC lineages did not result in spontaneous airway inflammation. Strikingly, although loss of Ship1 in the B-cell lineage did not affect HDM-induced AAI, loss of Ship1 in either of the T-cell or DC lineages protected mice from AAI by skewing the typical TH2 immune response toward a TH1 response. CONCLUSIONS: Although panhematopoietic deletion of Ship1 leads to spontaneous lung inflammation, selective deletion of Ship1 in T cells or DCs impairs the formation of an adaptive TH2 response and protects animals from HDM-induced AAI.
Asunto(s)
Hiperreactividad Bronquial/inmunología , Linaje de la Célula/inmunología , Células Dendríticas/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Neumonía/inmunología , Linfocitos T/inmunología , Inmunidad Adaptativa , Alérgenos/administración & dosificación , Alérgenos/inmunología , Animales , Antígenos Dermatofagoides/administración & dosificación , Antígenos Dermatofagoides/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/patología , Linaje de la Célula/genética , Células Dendríticas/patología , Expresión Génica , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Noqueados , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Neumonía/inducido químicamente , Neumonía/genética , Neumonía/patología , Pyroglyphidae/química , Linfocitos T/patología , Balance Th1 - Th2RESUMEN
NK cells are an important component of host immune defense against malignancy and infection. NK cells are educated by MHC class I ligands to ensure self-tolerance while also promoting lytic competency against altered self and damaged self targets. However, the intracellular molecular events that culminate in tolerance and functional competency of educated NK cells remain undefined. Mice with germline deficiency in SHIP1 were shown to have a defective NK cell compartment. However, SHIP1 is expressed in all hematopoietic lineages, and consequently several hematolymphoid phenotypes have already been identified in certain cell types that are the result of SHIP1 deficiency in cells in separate and distinct lineages, that is, cell-extrinsic phenotypes. Thus, it was previously impossible to determine the NK cell-intrinsic role of SHIP1. In the present study, through the creation of an NK cell-specific deletion mouse model of SHIP1, we show that SHIP1 plays a profound NK lineage-intrinsic role in NK cell homeostasis, development, education, and cytokine production. Moreover, we show SHIP1 expression by NK cells is required for in vivo-mismatched bone marrow allograft rejection as well as for NK memory responses to hapten.
Asunto(s)
Trasplante de Médula Ósea/métodos , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Autotolerancia/inmunología , Animales , Citocinas/inmunología , Citocinas/metabolismo , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Citometría de Flujo , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Haptenos/inmunología , Histocompatibilidad/inmunología , Homeostasis/genética , Homeostasis/inmunología , Inositol Polifosfato 5-Fosfatasas , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Autotolerancia/genéticaRESUMEN
IgE-mediated, antigen-triggered activation of mast cells and basophils often results in bell-shaped dose-response curves for the release of various pro-inflammatory mediators. The degree of suppression of mediator release observed following supra-optimal stimulation varies widely for different allergens as well as for different experimental agents that cause crosslinking of high-affinity IgE receptors (FcÉRI) on these cells. While the reasons for these differences have not yet been resolved it has become increasingly apparent that supra-optimal stimulation in many cases causes a shift in the balance of stimulatory and inhibitory signal transduction mechanisms arising from FcÉRI triggering. In particular, the lipid phosphatase SHIP1 has been shown to be centrally involved in explaining the bell-shaped phenomena in both mast cells and basophils in different species and appears to play a fundamental role in limiting the IgE responsiveness of these allergic effector cells. Elucidating the nature of this inhibitory signaling pathway may provide crucial knowledge in order to optimize desensitization strategies in the treatment of allergic diseases.