RESUMEN
Dengue virus (DENV) is the most widespread arbovirus worldwide and is responsible for major outbreaks. The host's immune response plays a crucial role in controlling this infection but might also contribute to the promotion of viral spread and immunopathology. In response to DENV infection, NK cells preferentially produce cytokines and are cytotoxic in the presence of specific antibodies. Here, we identified that DENV-2 inhibits glycogen synthase kinase 3 (GSK-3) activity to subsequently induce MHC class-1-related chain (MIC) A and MIC-B expression and IL-12 production in monocyte-derived DCs, independently of the STAT-3 pathway. The inhibition of GSK-3 by DENV-2 or small molecules induced MIC-A/B expression on monocyte-derived DCs, resulting in autologous NK cells of a specific increase in IFN-γ and TNF-α production, in the absence of direct cytotoxicity. Together, these findings identified GSK-3 as a regulator of MIC-A/B expression and suggested its role in DENV-2 infection to specifically induce cytokine production by NK cells.
Asunto(s)
Dengue/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Células Cultivadas , Citocinas/biosíntesis , HumanosRESUMEN
The past few years have witnessed exciting progress in the application of immune check-point blockade (ICB) for the treatment of various human cancers. ICB was first used against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to demonstrate durable anti-tumor responses followed by ICB against programmed cell death-1 (PD-1) or its ligand, PD-L1. Present approaches involve the use of combinations of blocking antibodies against CTLA-4, PD-1 and other inhibitory receptors (IRs) such as TIM3, TIGIT and LAG3. Despite this success, most patients are not cured by ICB therapy and there are limitations to the use of antibodies including cost, tumor penetration, the accessibility of receptors, and clearance from the cell surface as well as inflammatory and autoimmune complications. Recently, we demonstrated that the down-regulation or inhibition of glycogen synthase kinase 3 (GSK-3) down-regulates PD-1 expression in infectious diseases and cancer (Taylor et al., 2016 Immunity 44, 274-86; 2018 Cancer Research 78, 706-717; Krueger and Rudd 2018 Immunity 46, 529-531). In this Review, we outline the use of small molecule inhibitors (SMIs) that target intracellular pathways for co-receptor blockade in cancer immunotherapy.
Asunto(s)
Glucógeno Sintasa Quinasa 3/inmunología , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidoresRESUMEN
Regenerative dentistry is attracting growing interest in the scientific community, mainly because of its translational and promising therapeutic approach. The latest research carried out by the scientific community are aimed at triggering the local cellular response, in order to induce a physiological self-repairing of damaged oral tissues. Such physiological processes mainly involve the activation of local stem cell populations: mesenchymal stem cells, in fact, retain the ability to proliferate and to differentiate towards functional mature elements, thus leading towards healing of damaged tissues. Glycogen Synthase Kinase-3 (GSK-3) is a key-regulator of the Wnt/ß-catenin pathway; it phosphorylates ß-catenin, that then is degraded in the cytosol. The activation of such signalling, mediated by Wnt ligand/receptor association, inhibits GSK-3, leading to translocation of ß-catenin to the nucleus and to gene transcription. Selective inhibitors of GSK-3 have been linked to the activity of Wnt signalling and to the regeneration of injured tissues, including complex dental and oral structures. Small Molecule GSK-3 Antagonists are the most interesting class of molecules acting with a "Bystander effect": reducing local inflammation and local bone resorption and triggering the activity and differentiation of resident "sleeping" MSCs. The aim of this narrative topical review is to describe the current knowledge on the role of small molecule GSK-3 antagonists in regenerative dentistry, with strategic insights towards the translational applications in nanomaterials in dentistry and in dental repairing.
Asunto(s)
Odontología , Glucógeno Sintasa Quinasa 3 , Inflamación/metabolismo , Regeneración/fisiología , Vía de Señalización Wnt/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Resorción Ósea , Diferenciación Celular , Odontología/métodos , Odontología/tendencias , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre/citología , Células Madre/metabolismo , beta Catenina/metabolismoRESUMEN
Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664-75. ©2017 AACR.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células A549 , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Femenino , Glucógeno Sintasa Quinasa 3/inmunología , Humanos , Interleucina-15/farmacología , Células K562 , Células Asesinas Naturales/enzimología , Ratones , Ratones Endogámicos NOD , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Piridinas/farmacología , Pirimidinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic rhinosinusitis without nasal polyps (CRSsNP) is one of the most common otorhinolaryngologic diseases worldwide. However, the underlying mechanism remains unclear. In this study, the expression of glycogen synthase kinase 3 (GSK-3) was quantitatively evaluated in patients with CRSsNP (n = 20) and healthy controls (n = 20). The mRNA levels of GSK-3α and GSK-3ß were examined by qPCR, the immunoreactivities of GSK-3ß and nuclear factor-κB (NF-κB) were examined by immunohistochemistry (IHC) staining, and the protein levels of GSK-3ß, phospho-GSK-3ß (p-GSK-3ß, s9) and NF-κB were examined using Western blot analysis. We found that GSK-3 was highly expressed in both CRSsNP and control groups without significant difference in both GSK-3ß mRNA and protein levels. However, when compared with healthy control group, the GSK-3ß activation index, defined as the ratio of GSK-3ß over p-GSK-3ß, was significantly decreased, whereas the NF-κB protein abundance was significantly increased in CRSsNP group (P < 0.05). Strikingly, the GSK-3ß activation index, was highly correlated with NF-κB protein level, as well as CT scores in CRSsNP group (P < 0.05). It was also highly correlated with the mRNA expressions of inflammation-related genes, including T-bet, IFN-γ and IL-4 in CRSsNP group (P < 0.05). Our findings suggest that GSK-3ß activation index, reflecting the inhibitory levels of GSK-3ß through phosphorylation, may be a potential indicator for recurrent inflammation of CRSsNP, and that the insufficient inhibitory phosphorylation of GSK-3ß may play a pivotal role in the pathogenesis of CRSsNP.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/genética , FN-kappa B/genética , ARN Mensajero/genética , Rinitis/diagnóstico , Sinusitis/diagnóstico , Adolescente , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3 beta/inmunología , Humanos , Inflamación , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Masculino , Persona de Mediana Edad , FN-kappa B/inmunología , Pólipos Nasales , Fosforilación , ARN Mensajero/inmunología , Recurrencia , Rinitis/genética , Rinitis/metabolismo , Rinitis/fisiopatología , Transducción de Señal , Sinusitis/genética , Sinusitis/metabolismo , Sinusitis/fisiopatología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunologíaRESUMEN
Glycogen synthase kinase-3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-ß from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF-α production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis.
Asunto(s)
Braquiuros/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Hemocitos/inmunología , Inmunidad Innata/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Braquiuros/enzimología , Hemocitos/metabolismo , Fagocitosis/inmunologíaRESUMEN
The Wnt signaling pathway plays a key role in many biological aspects, such as cellular proliferation, tissue regeneration, embryonic development, and other systemic effects. Under a physiological condition, it is tightly controlled at different layers and arrays, and a dysregulated activation of this signaling has been implicated into the pathogenesis of various human disorders, including autoimmune diseases. Despite the fact that therapeutic interventions are available for ameliorating disease manifestations, there is no curative therapy currently available for autoimmune disorders. Increasing lines of evidence have suggested a crucial role of Wnt signaling during the pathogenesis of many autoimmune diseases; in addition, some of microRNAs (miRNAs), a class of small, noncoding RNA molecules capable of transcriptionally regulating gene expression, have also recently been demonstrated to possess both physiological and pathological roles in autoimmune diseases by regulating the Wnt signaling pathway. This review summarizes currently our understanding of the pathogenic roles of Wnt signaling in several major autoimmune disorders and miRNAs, those targeting Wnt signaling in autoimmune diseases, with a focus on the implication of the Wnt signaling as potential biomarkers and therapeutic targets in immune diseases, as well as miRNA-mediated regulation of Wnt signaling activation in the development of autoimmune diseases.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Lupus Eritematoso Sistémico/inmunología , Esclerodermia Sistémica/inmunología , Espondilitis Anquilosante/inmunología , Proteínas Wnt/inmunología , Vía de Señalización Wnt/inmunología , Animales , Proteína Axina/genética , Proteína Axina/inmunología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3 beta , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , MicroARNs/genética , MicroARNs/inmunología , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Espondilitis Anquilosante/genética , Espondilitis Anquilosante/patología , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/inmunologíaRESUMEN
Most psychiatric and neurological diseases are exacerbated by stress. Because this may partially result from stress-induced inflammation, we examined factors involved in this stress response. After a paradigm of inescapable foot shock stress that causes learned helplessness depression-like behavior, eighteen cytokines and chemokines increased in mouse hippocampus, peaking 6-12h after stress. A 24h prior pre-conditioning stress accelerated the rate of stress-induced hippocampal cytokine and chemokine increases, with most reaching peak levels after 1-3h, often without altering the maximal levels. Toll-like receptor 4 (TLR4) was involved in this response because most stress-induced hippocampal cytokines and chemokines were attenuated in TLR4 knockout mice. Stress activated glycogen synthase kinase-3 (GSK3) in wild-type mouse hippocampus, but not in TLR4 knockout mice. Administration of the antidepressant fluoxetine or the GSK3 inhibitor TDZD-8 reduced the stress-induced increases of most hippocampal cytokines and chemokines. Stress increased hippocampal levels of the danger-associated molecular pattern (DAMP) protein high mobility group box 1 (HMGB1), activated the inflammatory transcription factor NF-κB, and the NLRP3 inflammasome. Knockdown of HMGB1 blocked the acceleration of cytokine and chemokine increases in the hippocampus caused by two successive stresses. Fluoxetine treatment blocked stress-induced up-regulation of HMGB1 and subsequent NF-κB activation, whereas TDZD-8 administration attenuated NF-κB activation downstream of HMGB1. To test if stress-induced cytokines and chemokines contribute to depression-like behavior, the learned helplessness model was assessed. Antagonism of TNFα modestly reduced susceptibility to learned helplessness induction, whereas TLR4 knockout mice were resistant to learned helplessness. Thus, stress-induces a broad inflammatory response in mouse hippocampus that involves TLR4, GSK3, and downstream inflammatory signaling, and these stress responses contribute to susceptibility to depression-like behavior in mice.
Asunto(s)
Depresión/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Estrés Fisiológico/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Citocinas/inmunología , Citocinas/metabolismo , Depresión/genética , Depresión/inmunología , Fluoxetina/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/inmunología , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Neuroinmunomodulación , Transducción de Señal , Estrés Fisiológico/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting a critical transcription factor, C/EBPß. Specifically, IL-17 causes GSK-3ß-dependent phosphorylation of C/EBPß, which is associated with diminished C/EBPß binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3ß level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.
Asunto(s)
Pérdida de Hueso Alveolar/inmunología , Proteína beta Potenciadora de Unión a CCAAT/inmunología , Proteínas Portadoras/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Interleucina-17/inmunología , Periodontitis/inmunología , Pérdida de Hueso Alveolar/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Unión al Calcio , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Ácidos Docosahexaenoicos/farmacología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Encía/metabolismo , Glucógeno Sintasa Quinasa 3/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta , Células Endoteliales de la Vena Umbilical Humana , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Noqueados , Periodontitis/genética , Peroxidasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease that is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), whereas selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Overexpression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of glycogen synthase kinase-3ß (GSK3ß). Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis.
Asunto(s)
Apoptosis/inmunología , Enterocolitis Necrotizante/prevención & control , Enterocitos/inmunología , Leche/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Animales , Apoptosis/genética , Línea Celular , Enterocolitis Necrotizante/genética , Enterocolitis Necrotizante/inmunología , Enterocolitis Necrotizante/patología , Enterocitos/patología , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/inmunología , Receptores ErbB/genética , Receptores ErbB/inmunología , Femenino , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3 beta , Ratones , Transducción de Señal/genética , Receptor Toll-Like 4/genéticaRESUMEN
TRAF6 is critical for the production of inflammatory cytokines in various TLR-mediated signalling pathways. However, it is poorly understood how TRAF6 regulates TLR3 responses. Here we demonstrate that GSK3ß interacts with TRAF6 and positively regulates the TLR3-mediated signalling. Suppression of GSK3ß expression or its kinase activity drastically reduces the production of inflammatory cytokines and the induction of c-Fos by decreasing ERK and p38 phosphorylation. GSK3ß physically associates with TRAF6 in a TLR3 ligand poly I:C-dependent manner. TRAF6 is determined to be a direct E3 ligase for GSK3ß, and TRAF6-mediated GSK3ß ubiquitination is essential for poly I:C-dependent cytokine production by promoting the TLR3 adaptor protein TRIF-assembled signalling complex.
Asunto(s)
Citocinas/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Inmunidad Innata/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Receptor Toll-Like 3/inmunología , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Animales , Citocinas/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Inmunidad Innata/genética , Inflamación , Quinasas Quinasa Quinasa PAM/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Noqueados , Fosforilación/inmunología , Poli I-C/inmunología , Proteínas Proto-Oncogénicas c-fos/inmunología , Factor 6 Asociado a Receptor de TNF/genética , Ubiquitinación/genética , Ubiquitinación/inmunologíaRESUMEN
In neuronal cultures, glycogen synthase kinase 3(GSK3) is truncated at the N-terminal end by calpain downstream of activated glutamate receptors. However, the in vivo biological significance of that truncation has not been explored. In an attempt to elucidate if GSK3 truncation has a pathophysiological relevance, we have used intraperitoneal injections of kainic acid (KA) in rats and intra-amygdala KA microinjections in mice as in vivo models of excitotoxicity. Spectrin cleavage analyzed by immunohistochemistry was observed in the CA1 hippocampal field in KA-intraperitoneal treated rats while the CA3 region was the hippocampal area affected after intra-amygdala KA microinjections. GSK3ß immunofluorescence did not colocalize with truncated spectrin in both treatments using an antibody that recognize the N-terminal end of GSK3ß. Thus, those neurons which are spectrin-positive do not show GSK3ß immunolabelling. To study GSK3ß truncation in vitro, we exposed organotypic hippocampal slices and cultured cortical neurons to KA leading to the truncation of GSK3 and we found that truncation was blocked by the calpain inhibitor calpeptin. These data suggest a relationship between N-terminal GSK3ß truncation and excitotoxicity. Overall, our data reinforces the important relationship between glutamate receptors and GSK3 and their role in neurodegenerative processes in which excitotoxicity is involved.
Asunto(s)
Agonistas de Aminoácidos Excitadores/toxicidad , Glucógeno Sintasa Quinasa 3/metabolismo , Hipocampo/enzimología , Ácido Kaínico/toxicidad , Neuronas/enzimología , Amígdala del Cerebelo/efectos de los fármacos , Animales , Anticuerpos , Células Cultivadas , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Espectrina/metabolismoRESUMEN
IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3ß. GSK3ß was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3ß binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3ß activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3ß might serve as a unique strategy to lessen pulmonary inflammation.
Asunto(s)
Quinasa 1 de Adhesión Focal/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Interleucinas/inmunología , Receptores de Interleucina-1/inmunología , Transducción de Señal/inmunología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Activación Enzimática/genética , Activación Enzimática/inmunología , Quinasa 1 de Adhesión Focal/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Interleucina-33 , Interleucinas/genética , Ratones , Mutación Missense , Unión Proteica , Receptores de Interleucina-1/genética , Transducción de Señal/genéticaRESUMEN
Previous investigations demonstrated that protein tyrosine phosphatase, receptor type, O (PTPRO) acts as a tumor suppressor in liver cancer; however, little is known about its role in liver inflammation. Thus, we investigated the role of PTPRO in fulminant hepatitis (FH) using a Con A-induced mouse model. Significantly more severe liver damage, but attenuated inflammation, was detected in PTPRO-knockout (KO) mice, and PTPRO deficiency could confer this phenotype to wild-type mice in bone marrow transplantation. Moreover, hepatocytes with PTPRO depletion were more sensitive to TNF-α-induced apoptosis, and secretion of cytokines was significantly decreased in both T and NK/NKT cells and led to marked impairment of NF-κB activation. Intriguingly, wild-type and PTPRO-KO cells responded equally to TNF-α in activation of IKK, but NF-κB activation was clearly decreased in PTPRO-KO cells. PTPRO associated with ErbB2, and loss of PTPRO potentiated activation of the ErbB2/Akt/GSK-3ß/ß-catenin cascade. Increased ß-catenin formed a complex with NF-κB and attenuated its nuclear translocation and activation. Importantly, in humans, PTPRO was much decreased in FH, and this was associated with enhanced ß-catenin accumulation but reduced IFN-γ secretion. Taken together, our study identified a novel PTPRO/ErbB2/Akt/GSK-3ß/ß-catenin/NF-κB axis in FH, which suggests that PTPRO may have therapeutic potential in this liver disease.
Asunto(s)
Hepatitis Animal/inmunología , Hepatocitos/inmunología , Hígado/inmunología , FN-kappa B/inmunología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/inmunología , beta Catenina/inmunología , Enfermedad Aguda , Animales , Concanavalina A , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3 beta , Hepatitis Animal/inducido químicamente , Hepatitis Animal/mortalidad , Hepatitis Animal/patología , Hepatocitos/patología , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/mortalidad , Inflamación/patología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Hígado/patología , Masculino , Ratones , Ratones Noqueados , FN-kappa B/agonistas , FN-kappa B/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Índice de Severidad de la Enfermedad , Transducción de Señal , Análisis de Supervivencia , Linfocitos T/inmunología , Linfocitos T/patología , beta Catenina/genéticaRESUMEN
The classical activation of macrophages, one of major innate effector cells, requires IFN-γ pretreatment (priming) and subsequent TLR stimuli (triggering). The priming effect of IFN-γ can promote macrophages to secrete higher level of proinflammatory cytokines but lower level of the anti-inflammatory cytokines, enhancing microbicidal and tumoricidal activity of macrophages. However, the underlying molecular mechanisms for IFN-γ-priming effect on macrophage activation remain to be fully understood. microRNAs (miRNAs) are now emerging as important regulators in immune response, including signaling transduction in immune cell function. In this study, we explored the effect of IFN-γ on miRNA expression profiling in macrophages and tried to identify the definite miRNA involved in the priming effect of IFN-γ. We discovered that miR-3473b, which was significantly downregulated after IFN-γ priming, could attenuate the priming effect of IFN-γ. miR-3473b promoted Akt/glycogen synthase kinase 3 signaling and IL-10 production through directly targeting phosphatase and tensin homolog (PTEN) to suppress activation of macrophages and inflammatory response. Our data indicate that IFN-γ beefs up macrophage innate response and cytotoxicity by downregulating miR-3473b to release PTEN from suppression, and then the increase of PTEN contributes to the full activation of IFN-γ-primed macrophages. Our results provide mechanistic insight to priming effect of IFN-γ on macrophage classical activation by identifying an IFN-γ/miR-3473b/PTEN regulatory loop in the regulation of macrophage function.
Asunto(s)
Interferón gamma/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , MicroARNs/biosíntesis , Fosfohidrolasa PTEN/inmunología , Animales , Citocinas/biosíntesis , Citocinas/metabolismo , Regulación hacia Abajo , Femenino , Glucógeno Sintasa Quinasa 3/inmunología , Células HEK293 , Humanos , Interleucina-10/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , MicroARNs/genética , Neoplasias/inmunología , Fosfohidrolasa PTEN/biosíntesis , Proteínas Proto-Oncogénicas c-akt/inmunología , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Autophagy, an evolutionarily conserved homeostasis process regulating biomass quantity and quality, plays a critical role in the host response to sepsis. Recent studies show its calcium dependence, but the calcium-sensitive regulatory cascades have not been defined. In this study, we describe a novel mechanism in which calcium/calmodulin-dependent protein kinase IV (CaMKIV), through inhibitory serine phosphorylation of GSK-3ß and inhibition of FBXW7 recruitment, prevents ubiquitin proteosomal degradation of mammalian target of rapamycin (mTOR) and thereby augments autophagy in both the macrophage and the kidney. Under the conditions of sepsis studied, mTOR expression and activity were requisite for autophagy, a paradigm countering the current perspective that prototypically, mTOR inhibition induces autophagy. CaMKIV-mTOR-dependent autophagy was fundamentally important for IL-6 production in vitro and in vivo. Similar mechanisms were operant in the kidney during endotoxemia and served a cytoprotective role in mitigating acute kidney injury. Thus, CaMKIV-mTOR-dependent autophagy is conserved in both immune and nonimmune/parenchymal cells and is fundamental for the respective functional and adaptive responses to septic insult.
Asunto(s)
Lesión Renal Aguda/inmunología , Autofagia/efectos de los fármacos , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/toxicidad , Macrófagos/inmunología , Serina-Treonina Quinasas TOR/inmunología , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Autofagia/genética , Autofagia/inmunología , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/genética , Línea Celular , Proteínas F-Box/genética , Proteínas F-Box/inmunología , Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación de la Expresión Génica/inmunología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3 beta , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Macrófagos/patología , Ratones , Ratones Noqueados , Serina-Treonina Quinasas TOR/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
Accumulative evidence shows a role of the immune system in susceptibility to depression. Proinflammatory cytokines have been shown to be involved in the induction of depressive behavior both in humans and mice, opening a new avenue of therapeutic strategy. Because glycogen synthase kinase-3 (GSK3) was recently identified to be controlling the production of proinflammatory cytokines, and GSK3 has been shown to be implicated in mood disorders for many years, it has been proposed that the proinflammatory action of GSK3 could be responsible for the increased susceptibility to depressive behavior. Moreover, besides regulating cytokines, GSK3 also promotes differentiation of proinflammatory subtypes of Th cells, which are sufficient to induce depressive behavior in mice. Although the clear involvement of the immune system during depressive behavior still needs to be firmly demonstrated, there is growing evidence for the involvement of inflammation in the induction of depressive behavior.
Asunto(s)
Glucógeno Sintasa Quinasa 3/inmunología , Inflamación/enzimología , Trastornos del Humor/enzimología , Linfocitos T/enzimología , Animales , Humanos , Inflamación/inmunología , Trastornos del Humor/inmunología , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: Our previous studies have demonstrated that glycogen synthase kinase 3ß (GSK3ß) activity is increased in the progression of acute liver failure (ALF), which aggravates liver injury, while its regulatory mechanism remains elusive. This study is designated to address whether oxidative stress activates GSK3ß to promote ALF. METHODS: In a murine model induced by D-galactosamine (D-GalN) (700 mg/kg) and LPS (10 µg/kg), N-acetylcysteine (300 mg/kg) or SB216763 (25 mg/kg) was used to inhibit oxidative stress or GSK3ß activity, respectively. Serum alanine aminotransferase and aspartate aminotransferase levels were assessed. The parameters of oxidative stress were evaluated in liver tissue. Whether GSK3ß inhibition protects hepatocytes from oxidative stress-induced cell apoptosis was investigated in vitro. Moreover, the activity of GSK3ß was measured in the liver of chronic hepatitis B (CHB) patients and ALF patients. RESULTS: In vivo, N-acetylcysteine ameliorated the D-GalN/LPS-induced hepatotoxicity and reduced GSK3ß activity; GSK3ß inhibition increased hepatic superoxide dismutase activity and the glutathione content, decreased malondialdehyde production in the liver tissues; while GSK3ß inhibition suppressed the JNK activation in the liver and decreased cytochrome c release from mitochondria. In vitro, GSK3ß inhibition lessened hepatocytes apoptosis induced by H2O2 or Antimycin A, as demonstrated by decreased LDH activity, and reduced cleavage of caspase-3 expression. Furthermore, GSK3ß activity in the CHB patients was increased in the phase of ALF. CONCLUSIONS: Results indicate that GSK3ß activation contributes to liver injury by participating in oxidative stress response in ALF and is, therefore, a potential therapeutic target for ALF.
Asunto(s)
Glucógeno Sintasa Quinasa 3/inmunología , Hepatitis B Crónica/inmunología , Fallo Hepático Agudo/inmunología , Adulto , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Línea Celular , Femenino , Galactosamina , Glucógeno Sintasa Quinasa 3 beta , Humanos , Lipopolisacáridos , Fallo Hepático Agudo/sangre , Fallo Hepático Agudo/inducido químicamente , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Estrés OxidativoRESUMEN
The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.
Asunto(s)
Monocitos/inmunología , FN-kappa B/inmunología , Proteína Fosfatasa 1/inmunología , Transducción de Señal/inmunología , Factor de Transcripción AP-1/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos/inmunología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3/metabolismo , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIA/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3ß as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.