RESUMEN
Multiple bacterial genera take advantage of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin to invade host cells. Secretion of the MARTX toxin by Vibrio vulnificus, a deadly opportunistic pathogen that causes primary septicemia, the precursor of sepsis, is a major driver of infection; however, the molecular mechanism via which the toxin contributes to septicemia remains unclear. Here, we report the crystal and cryo-electron microscopy (EM) structures of a toxin effector duet comprising the domain of unknown function in the first position (DUF1)/Rho inactivation domain (RID) complexed with human targets. These structures reveal how the duet is used by bacteria as a potent weapon. The data show that DUF1 acts as a RID-dependent transforming NADase domain (RDTND) that disrupts NAD+ homeostasis by hijacking calmodulin. The cryo-EM structure of the RDTND-RID duet complexed with calmodulin and Rac1, together with immunological analyses in vitro and in mice, provide mechanistic insight into how V. vulnificus uses the duet to suppress ROS generation by depleting NAD(P)+ and modifying Rac1 in a mutually-reinforcing manner that ultimately paralyzes first line immune responses, promotes dissemination of invaders, and induces sepsis. These data may allow development of tools or strategies to combat MARTX toxin-related human diseases.
Asunto(s)
Toxinas Bacterianas , Microscopía por Crioelectrón , Vibrio vulnificus , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidad , Animales , Humanos , Ratones , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Femenino , NAD/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sepsis/microbiología , Dominios Proteicos , Vibriosis/microbiología , NAD+ Nucleosidasa/metabolismo , NAD+ Nucleosidasa/química , Cristalografía por Rayos XRESUMEN
Commensal bacteria are critically involved in the establishment of tolerance against inflammatory challenges, the molecular mechanisms of which are just being uncovered. All kingdoms of life produce aminoacyl-tRNA synthetases (ARSs). Thus far, the non-translational roles of ARSs have largely been reported in eukaryotes. Here, we report that the threonyl-tRNA synthetase (AmTARS) of the gut-associated bacterium Akkermansia muciniphila is secreted and functions to monitor and modulate immune homeostasis. Secreted AmTARS triggers M2 macrophage polarization and orchestrates the production of anti-inflammatory IL-10 via its unique, evolutionary-acquired regions, which mediates specific interactions with TLR2. This interaction activates the MAPK and PI3K/AKT signaling pathways, which converge on CREB, leading to an efficient production of IL-10 and suppression of the central inflammatory mediator NF-κB. AmTARS restores IL-10-positive macrophages, increases IL-10 levels in the serum, and attenuates the pathological effects in colitis mice. Thus, commensal tRNA synthetases can act as intrinsic mediators that maintain homeostasis.
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Treonina-ARNt Ligasa , Animales , Ratones , Treonina-ARNt Ligasa/metabolismo , Interleucina-10/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Verrucomicrobia/metabolismo , Homeostasis , ARN de Transferencia/metabolismoRESUMEN
The AKT signaling pathway plays critical roles in the resolution of inflammation. However, the underlying mechanisms of anti-inflammatory regulation and signal coordination remain unclear. Here, we report that anti-inflammatory AKT signaling is coordinated by glutamyl-prolyl-tRNA synthetase 1 (EPRS1). Upon inflammatory activation, AKT specifically phosphorylates Ser999 of EPRS1 in the cytoplasmic multi-tRNA synthetase complex, inducing release of EPRS1. EPRS1 compartmentalizes AKT to early endosomes via selective binding to the endosomal membrane lipid phosphatidylinositol 3-phosphate and assembles an AKT signaling complex specific for anti-inflammatory activity. These events promote AKT activation-mediated GSK3ß phosphorylation, which increase anti-inflammatory cytokine production. EPRS1-deficient macrophages do not assemble the early endosomal complex and consequently exacerbate inflammation, decreasing the survival of EPRS1-deficient mice undergoing septic shock and ulcerative colitis. Collectively, our findings show that the housekeeping protein EPRS1 acts as a mediator of inflammatory homeostasis by coordinating compartment-specific AKT signaling.
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Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antiinflamatorios/farmacología , InflamaciónRESUMEN
Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.
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Células Madre Pluripotentes , Fibrosis Pulmonar , Células Epiteliales Alveolares , Bleomicina/metabolismo , Humanos , Pulmón , Macrófagos Alveolares , Organoides , Células Madre Pluripotentes/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Microtomografía por Rayos XRESUMEN
BACKGROUND: Triggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer's disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells. RESULTS: Here, we demonstrated for the first time that CD3-CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice. CONCLUSIONS: Collectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.
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Células de la Médula Ósea/inmunología , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Trasplante de Médula Ósea , Complejo CD3/metabolismo , Diferenciación Celular , Femenino , Humanos , Inmunoterapia Adoptiva , Subunidad beta del Receptor de Interleucina-2/metabolismo , Melanoma/terapia , Melanoma Experimental , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales , Receptores Inmunológicos/genéticaRESUMEN
Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor expressed on macrophages, microglial cells, and pre-osteoclasts, and that participates in diverse cellular function, including inflammation, bone homeostasis, neurological development, and coagulation. In spite of the indispensable role of the TREM2 protein in the maintenance of immune homeostasis and osteoclast differentiation, the exact ligand for TREM2 has not yet been identified. Here, we report a putative TREM2 ligand which is secreted from MC38 cells and identified as a cyclophilin A (CypA). A specific interaction between CypA and TREM2 was shown at both protein and cellular levels. Exogenous CypA specifically interacted and co-localized with TREM2 in RAW264.7 cells, and the physical interactions were shown to regulate TREM2 signaling transduction. The Pro144 residue in the extracellular domain of TREM2 was found to be the specific binding site of CypA. When considered together, this provides evidence that CypA interacts specifically with TREM2 as a potent ligand.
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Ciclofilina A/metabolismo , Ligandos , Microglía/metabolismo , Células Mieloides/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Humanos , Macrófagos/metabolismo , Osteoclastos/metabolismoRESUMEN
Gut-homing γδ T cells are induced by chemokines and cell adhesion molecules and play a critical role in homeostasis and mucosal immunity; however, little is known regarding their upstream regulators. We investigated the role of Axl as a specific regulator of chemokines and cell adhesion molecule in the distribution of intestinal γδ T cells. The population of γδ T-cell receptor-positive cells including Vγ1 and Vγ7 subsets was remarkably increased in the intraepithelial lymphocytes of Axl-/- mice compared with those of wild-type (WT) mice. An increased number of migrated γδ T cells were observed in the coculture with intraepithelial cells from Axl-/- mice. The mRNA expression level of chemokine (C-C motif) ligand (CCL) 25 was specifically higher in the small intestine of Axl-/- mice than in WT mice. In adoptive transfer, the migration of both thymic and extrathymic γδ T cells was increased in Axl-/- mice. The activation of Axl signaling down-regulated CCL25 expression via ERK signaling pathway and reduced the population of γδ T cells. Systemic dissemination was suppressed in Axl-/- mice infected with Salmonella typhimurium. Thus, our findings suggest that Axl plays a critical role in regulating the migration of γδ T cells for the maintenance of homeostasis and bacterial resistance.-Kim, S.-M., Park, M., Yee, S.-M., Ji, K.-Y., Lee, E.-H., Nguyen, T.-V., Nguyen, T. H.-L., Jang, J., Kim, E.-M., Choi, H.-R., Yun, C.-H., Kang, H.-S. Axl is a key regulator of intestinal γδ T-cell homeostasis.
Asunto(s)
Células Epiteliales/inmunología , Homeostasis , Intestino Delgado/inmunología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Fiebre Tifoidea/inmunología , Animales , Movimiento Celular , Células Cultivadas , Quimiocinas CC/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestino Delgado/metabolismo , Intestino Delgado/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Salmonella typhimurium/fisiología , Fiebre Tifoidea/metabolismo , Fiebre Tifoidea/microbiología , Tirosina Quinasa del Receptor AxlRESUMEN
TREM2 (triggering receptor expressed on myeloid cells) is involved in the development of malignancies. However, the function of TREM2 in colorectal cancer has not been clearly elucidated. Here, we investigated TREM2 function for the first time in colorectal epithelial cancer cells and demonstrated that TREM2 is a novel tumor suppressor in colorectal carcinoma. Blockade of TREM2 significantly promoted the proliferation of HT29 colorectal carcinoma cells by regulating cell cycle-related factors, such as p53 phosphorylation and p21 and cyclin D1 protein levels. HT29 cell migration was also increased by TREM2 inhibition via MMP9 (matrix metalloproteinase 9) expression upregulation. Furthermore, we found that the tumor suppressor effects of TREM2 were associated with Wnt/ß-catenin and extracellular signal-regulated kinase (ERK) signaling. Importantly, the effect of TREM2 in the suppression of tumor development was demonstrated by in vivo and in vitro assays, as well as in human colon cancer patient tissue arrays. Overall, our results identify TREM2 as a potential prognostic biomarker and therapeutic target for colorectal cancer.
RESUMEN
BACKGROUND: The roots of Korean red ginseng (Panax ginseng C.A.Mey.; KGC) have been used as an herbal supplement to enhance vital energy and immune capacity. Salvia plebeia R.Br. has been used to treat inflammatory diseases. PURPOSE: The aim of this study was to examine the anti-asthmatic effects of a mixture of Korean red ginseng and Salvia plebeia R.Br. (KGC3P), its component nepetin, and their modes of action in alleviating ovalbumin (OVA)-induced asthma in mice. METHOD: BALB/c mice were sensitized with OVA then subjected to intratracheal, intraperitoneal, and aerosol challenges. KGC3P and nepetin were administered orally for four weeks. Airway hyperresponsiveness (AHR), OVA-specific IgE levels, and Th2 cytokine- and gene expression levels in bronchoalveolar lavage fluid (BALF) and splenocytes were measured. Histological and immune cell subtype analyses were performed. PTEN and Akt phosphorylation levels were also evaluated. RESULTS: KGC3P reduced OVA-induced AHR, serum IgE levels, histological changes, and eosinophils infiltration but also the absolute number of immune cell subtypes including CD3+/CD4+, CD3+/CD8+, CD4+/CD69+, and Gr-1+/CD11b+ in the lungs, BALF, and mesenteric lymph nodes (MLN). KGC3P also lowered the Th2 cytokines IL-4, IL-5, and IL-13 in the BALF and splenocytes and downregulated the IL-4, IL-13, IL-17, TNF-α, and MUC5AC genes in the lung. KGC3P upregulated the peroxisome proliferator-activated receptor (PPAR)γ gene but downregulated the p-Akt and p-PTEN phosphorylation. Similar results were obtained with nepetin treatment. CONCLUSION: KGC3P and nepetin are anti-asthmatic because they reduce various immune cells such as eosinophils and Th2 cell as well as Th2 cytokines. These mechanisms may be accompanied by the regulation of PPARγ expression and the PTEN pathway. Taken together, our results indicate that KGC3P and nepetin may potentially prevent and treat asthma.
Asunto(s)
Antiasmáticos/farmacología , Fosfohidrolasa PTEN/metabolismo , Panax/química , Salvia/química , Animales , Antiasmáticos/química , Asma/tratamiento farmacológico , Asma/metabolismo , Líquido del Lavado Bronquioalveolar , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Quimioterapia Combinada , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Eosinófilos/patología , Flavonas/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratones Endogámicos BALB C , Ovalbúmina/efectos adversos , Células Th2/efectos de los fármacos , Células Th2/patologíaRESUMEN
The effects of daucosterol have been identified in cancer therapy and neuronal diseases. However, the regulatory function of daucosterol in DSS-induced colitis has not yet been investigated. In this study, we evaluated the immunological and therapeutic effects of daucosterol in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Unlike vehicle mice, mice pre- or post-treated with daucosterol showed inhibition of body weight loss and the decrease in the disease activity index (DAI). In addition, daucosterol treatment rescued the DSS-induced decrease in colon length and disruption of the epithelial lining. Furthermore, it reduced DSS-induced production of reactive oxygen species (ROS), infiltration of macrophages, and expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß. Mice with colitis showed a decreased population of Foxp3+ cells, which was upregulated by daucosterol treatment. Furthermore, daucosterol increased natural killer (NK) cell activity and inhibited excessive IgA levels in mice with DSS-induced colitis. Collectively, our findings demonstrated that daucosterol significantly alleviated DSS-induced colitis, indicating the possibility of daucosterol as a therapeutic option for colitis.
Asunto(s)
Antiinflamatorios/uso terapéutico , Colitis/tratamiento farmacológico , Sitoesteroles/uso terapéutico , Animales , Antiinflamatorios/farmacología , Colitis/inducido químicamente , Colitis/inmunología , Colitis/patología , Colon/efectos de los fármacos , Colon/patología , Citocinas/genética , Citocinas/inmunología , Sulfato de Dextran , Femenino , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ratones Endogámicos C57BL , Sitoesteroles/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: Hesperidin is a flavonoid with antioxidant, anti-inflammatory, and immune modulatory activities. Photoaging is a consequence of chronic exposure to the sun and ultraviolet (UV) radiation. This study was designed to evaluate the efficacy of hesperidin against photoaging of dorsal skin in hairless mice. METHODS: Hairless male mice (6-week-old) were divided into three groups (n = 7): control, UVB-treated vehicle, and UVB-treated hesperidin groups. UVB-irradiated mice from hesperidin group were orally administered 0.1 mL of water containing 100 mg/kg body weight per day hesperidin. RESULTS: The mean length and depth of wrinkles in the UVB-treated hesperidin group significantly improved after the oral administration of hesperidin, which significantly inhibited the increase in epidermal thickness and epidermal hypertrophy (P < 0.05). UVB irradiation of mice induced epidermal barrier dysfunction including an increase in the transepidermal water loss (TEWL); however, hesperidin decreased the TEWL. UVB irradiation increased the expression of MMP-9 and pro-inflammatory cytokines whereas UVB-treated hesperidin group showed reduced expression. These results indicate that hesperidin showed anti-photoaging activity in the UVB-irradiated hairless mice. In conclusion, hesperidin inhibited the UVB-induced increase in skin thickness, wrinkle formation, and collagen fiber loss in male hairless mice. CONCLUSIONS: These results suggest that hesperidin shows potent anti-photoaging activity by regulating MMP-9 expression through the suppression of MAPK-dependent signaling pathways.
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Antioxidantes/farmacología , Hesperidina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Envejecimiento de la Piel/efectos de los fármacos , Animales , Regulación hacia Abajo/efectos de los fármacos , Epidermis/efectos de los fármacos , Epidermis/efectos de la radiación , Inmunohistoquímica , Masculino , Ratones , Ratones Pelados , Envejecimiento de la Piel/efectos de la radiación , Rayos UltravioletaRESUMEN
TREM2 plays a critical role in the alleviation of Alzheimer's disease by promoting Aß phagocytosis by microglia, but the detailed molecular mechanism underlying TREM2-induced direct phagocytic activity of Aß remains to be revealed. We found that learning and memory functions were improved in aged TREM2 TG mice, with the opposite effects in KO mice. The amount of phagocytosed Aß was significantly reduced in the primary microglia of KO mice. CD36 expression in primary microglia was greater in TG than in WT mice but was substantially decreased in KO mice. The expression of C/EBPα, an upstream transcriptional activator of CD36, was also elevated in primary microglia of TG mice but decreased in KO mice. The transcription of CD36 was markedly increased by TREM2 overexpression, and this effect was suppressed by a mutation of the C/EBPα binding site on the CD36 promoter. The TREM2-induced expression of CD36 and C/EBPα was inhibited by treatment with PI3K/AKT signaling blockers, and phosphorylation of AKT was elevated in TREM2-overexpressing BV2 cells. The present study provides evidence that TREM2 is required for preventing loss of memory and learning in Alzheimer's disease by regulating C/EBPα-dependent CD36 expression and the consequent Aß phagocytosis.
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Péptidos beta-Amiloides/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Antígenos CD36/genética , Glicoproteínas de Membrana/genética , Microglía/fisiología , Fagocitosis , Receptores Inmunológicos/genética , Animales , Biomarcadores , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , Fagocitos/inmunología , Fagocitos/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
Axl receptor tyrosine kinase is involved in the tumorigenesis and metastasis of many cancers. Axl expression was markedly higher in human papilloma virus type 16E6 (HPV16E6)-overexpressing HeLa (HE6F) cells and lower in HPV16E6-suppressing CaSki (CE6R) cells than in the controls. SiRNA-mediated knockdown of E6 expression led to increased phosphatase and tensin homolog (PTEN) phosphorylation at Ser380 and attenuated AKT phosphorylation. Expression of membrane-associated guanylate kinase inverted-2 (MAGI-2), an E6-induced degradation target, was induced in E6-siRNA-transfected cells. Moreover, myeloid zinc finger protein 1 (MZF1) binds directly to the Axl promoter in HE6F cells. Axl expression was regulated by HPV16E6-mediated PTEN/AKT signalling pathway, and Axl promoter activity was regulated through MZF1 activation in cervical cancer, which promoted malignancy. Axl silencing suppressed the metastasis of Caski cells and enhanced the susceptibility to NK cell-mediated killing of HE6F cells. In addition, the expression of Axl and MZF1 was highly correlated with clinical stage of cervical cancer and HPV16/18 infection. Taken together, Axl expression was induced by HPV16E6 in cervical cancer cells, suggesting that blockade of Axl signalling might be an effective way to reduce the progression of cervical cancer.
Asunto(s)
Carcinogénesis/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Represoras/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Represoras/genética , Transducción de Señal/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Tirosina Quinasa del Receptor AxlRESUMEN
Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma.
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Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor/fisiología , Linfoma de Células T/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Tirosina Quinasa del Receptor AxlRESUMEN
Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axl antibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rß (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment of Axl-Ig. Anti-Axl antibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axl antibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83 % by the administration of mNK cells treated with anti-Axl antibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.
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Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Melanoma/inmunología , Melanoma/terapia , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Tirosina Quinasas Receptoras/deficiencia , Tirosina Quinasa del Receptor AxlRESUMEN
Bacterial ß-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield ß-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial ß-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial ß-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial ß-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1ß, IL-6 and IL-17A/F, were markedly decreased in the colon of ß-(1,3)-glucan-pretreated mice. ß-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, ß-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial ß-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.
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Antiinflamatorios/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Linfocitos T Reguladores/inmunología , beta-Glucanos/uso terapéutico , Agrobacterium/metabolismo , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Proliferación Celular/efectos de los fármacos , Colon/citología , Colon/patología , Citocinas/genética , Sulfato de Dextran , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Heces/química , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Inmunoglobulina A/inmunología , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/citología , Masculino , Ratones Endogámicos C57BL , Proteoglicanos , Especies Reactivas de Oxígeno/inmunología , beta-Glucanos/metabolismo , beta-Glucanos/farmacologíaRESUMEN
We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Formaldehído/toxicidad , Células Asesinas Naturales/efectos de los fármacos , Melanoma Experimental/patología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Lavado Broncoalveolar , Carcinogénesis/inducido químicamente , Carcinogénesis/patología , Supervivencia Celular/efectos de los fármacos , Femenino , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The gamma-secretase complex is involved in the intramembranous proteolysis of a variety of substrates, including the amyloid precursor protein and the Notch receptor. Nicastrin (NCT) is an essential component of the gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we determined that serum- and glucocorticoid-induced protein kinase 1 (SGK1) markedly reduced the protein stability of NCT. The SGK1 kinase activity was decisive for NCT degradation and endogenous SGK1 inhibited gamma-secretase activity. SGK1 downregulates NCT protein levels via proteasomal and lysosomal pathways. Furthermore, SGK1 directly bound to and phosphorylated NCT on Ser437, thereby promoting protein degradation. Collectively, our findings indicate that SGK1 is a gamma-secretase regulator presumably effective through phosphorylation and degradation of NCT.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Células HEK293 , Humanos , Proteínas Inmediatas-Precoces/genética , Lisosomas/genética , Glicoproteínas de Membrana/genética , Ratones , Fosforilación/fisiología , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Serina-Treonina Quinasas/genética , Estabilidad ProteicaRESUMEN
Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.
Asunto(s)
Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Presenilina-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Cloruro de Amonio/farmacología , Línea Celular , Cloroquina/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/biosíntesis , Leupeptinas/farmacología , Lisosomas/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , Presenilina-1/genética , Presenilina-2/genética , Inhibidores de Proteasoma , Proteolisis , Receptor Notch1/metabolismo , Transcripción Genética , Activación Transcripcional , Ubiquitinación , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC-RBP-Jk complex after processing by γ-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.