RESUMEN
During peritoneal dialysis (PD), the peritoneum is exposed to a bioincompatible dialysate, deteriorating the tissue and limiting the long-term effectiveness of PD. Peritoneal fibrosis is triggered by chronic inflammation induced by a variety of stimuli, including peritonitis. Exposure to PD fluid alters peritoneal macrophages phenotype. Inflammasome activation triggers chronic inflammation. First, it was determined whether inflammasome activation causes peritoneal deterioration. In the in vivo experiments, the increased expression of the inflammasome components, caspase-1 activity, and concomitant overproduction of IL-1ß and IL-18 were observed in a mouse model of peritoneal fibrosis. ASC-positive and F4/80-positive cells colocalized in the subperitoneal mesothelial cell layer. These macrophages expressed high CD44 levels indicating that the CD44-positive macrophages contribute to developing peritoneal deterioration. Furthermore, intravital imaging of the peritoneal microvasculature demonstrated that the circulating CD44-positive leukocytes may contribute to peritoneal fibrosis. Bone marrow transplantation in ASC-deficient mice suppressed inflammasome activation, thereby attenuating peritoneal fibrosis in a high glucose-based PD solution-injected mouse model. Our results suggest inflammasome activation in CD44-positive macrophages may be involved in developing peritoneal fibrosis. The inflammasome-derived pro-inflammatory cytokines might therefore serve as new biomarkers for developing encapsulating peritoneal sclerosis.
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Fibrosis Peritoneal , Peritonitis , Animales , Ratones , Peritoneo , Inflamasomas , Modelos Animales de Enfermedad , InflamaciónRESUMEN
Chronic kidney disease is a common problem in the elderly and is associated with increased mortality. We have reported on the role of nitric oxide, which is generated from endothelial nitric oxide synthase (eNOS), in the progression of aged kidneys. To elucidate the role of endothelial dysfunction and the lack of an eNOS-NO pathway in ageing kidneys, we conducted experiments using eNOS and ASC-deficient mice. C57B/6 J mice (wild type (WT)), eNOS knockout (eNOS KO), and ASC knockout (ASC KO) mice were used in the present study. Then, eNOS/ASC double-knockout (eNOS/ASC DKO) mice were generated by crossing eNOS KO and ASC KO mice. These mice were sacrificed at 17-19 months old. The Masson positive area and the KIM-1 positive area tended to increase in eNOS KO mice, compared with WT mice, but not eNOS/ASC DKO mice. The COX-positive area was significantly reduced in eNOS KO mice, compared with WT and eNOS/ASC DKO mice. To determine whether inflammasomes were activated in infiltrating macrophages, the double staining of IL-18 and F4/80 was performed. IL-18 and F4/80 were found to be co-localised in the tubulointerstitial areas. Inflammasomes play a pivotal role in inflammaging in ageing kidneys. Furthermore, inflammasome activation may accelerate cellular senescence via mitochondrial dysfunction. The importance of endothelial function as a regulatory mechanism suggests that protection of endothelial function may be a potential therapeutic target.
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Envejecimiento , Endotelio/fisiopatología , Inflamasomas , Riñón/fisiopatología , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Endotelio/enzimología , Endotelio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/fisiología , Óxido Nítrico/metabolismo , Enfermedades Vasculares/fisiopatologíaRESUMEN
To selectively and continuously produce anti-cancer molecules specifically in malignant tumors, we have established an in situ delivery and production system (iDPS) with Bifidobacterium as a micro-factory of various anti-cancer agents. By focusing on the characteristic hypoxia in cancer tissue for a tumor-specific target, we employed a gene-engineered obligate anaerobic and non-pathogenic bacterium, Bifidobacterium, as a tool for systemic drug administration. This review presents and discusses the anti-tumor effects and safety of the iDPS production of numerous anti-cancer molecules and addresses the problems to be improved by directing attention mainly to the hallmark vasculature and so-called enhanced permeability and retention effect of tumors.
RESUMEN
APS001F is a strain of Bifidobacterium longum genetically engineered to express cytosine deaminase that converts 5-fluorocytosine (5-FC) to 5-fluorouracil. In the present study, antitumor effects of APS001F plus 5-FC (APS001F/5-FC) in combination with anti-PD-1 monoclonal antibody were investigated using a CT26 syngeneic mouse model. Both of dosing of APS001F/5-FC before and after anti-PD-1 mAb in the combination dosing exhibited antitumor effects as well as prolonged survival over the nontreated control. The survival rate in the combination therapy significantly increased over the monotherapy with APS001F/5-FC and that with anti-PD-1 mAb. Regulatory T cells among CD4+ T cells in tumor decreased in the combination therapy, while the ratio of CD8+ T cells was maintained in all groups. Taken these results together, APS001F/5-FC not only demonstrates a direct antitumor activity, but also immunomodulatory effects once localized in the hypoxic region of the tumor, which allows anti-PD-1 mAb to exert potentiated antitumor effects.
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Anticuerpos Monoclonales/inmunología , Antineoplásicos/farmacología , Bifidobacterium longum/fisiología , Flucitosina/farmacología , Ingeniería Genética , Receptor de Muerte Celular Programada 1/inmunología , Animales , Bifidobacterium longum/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , RatonesRESUMEN
Short-chain fatty acids (SCFAs) produced by gastrointestinal microbiota regulate immune responses, but host molecular mechanisms remain unknown. Unbiased screening using SCFA-conjugated affinity nanobeads identified apoptosis-associated speck-like protein (ASC), an adaptor protein of inflammasome complex, as a noncanonical SCFA receptor besides GPRs. SCFAs promoted inflammasome activation in macrophages by binding to its ASC PYRIN domain. Activated inflammasome suppressed survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages by pyroptosis and facilitated neutrophil recruitment to promote bacterial elimination and thus inhibit systemic dissemination in the host. Administration of SCFAs or dietary fibers, which are fermented to SCFAs by gut bacteria, significantly prolonged the survival of S. Typhimurium-infected mice through ASC-mediated inflammasome activation. SCFAs penetrated into the inflammatory region of the infected gut mucosa to protect against infection. This study provided evidence that SCFAs suppress Salmonella infection via inflammasome activation, shedding new light on the therapeutic activity of dietary fiber.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Ácidos Grasos Volátiles/metabolismo , Inflamasomas/inmunología , Inflamasomas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Infecciones por Salmonella/prevención & control , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Femenino , Microbioma Gastrointestinal/inmunología , Células HEK293 , Humanos , Inmunidad Innata/fisiología , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Infecciones por Salmonella/genética , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/inmunología , Células U937RESUMEN
The aryl hydrocarbon receptor (AhR) is a transcription factor known as a dioxin receptor. Recently, Ahr-/- mice were revealed to develop cecal tumors with inflammation and Wnt/ß-catenin pathway activation. However, whether ß-catenin degradation is AhR dependent remains unclear. To determine whether other signaling pathways function in Ahr-/- cecal tumorigenesis, we investigated histologic characteristics of the tumors and cytokine/chemokine production in tumors and Ahr-/- peritoneal macrophages. AhR expression was also assessed in human colorectal carcinomas. Of the 28 Ahr-/- mice, 10 developed cecal lesions by 50 weeks of age, an incidence significantly lower than previously reported. Cecal lesions of Ahr-/- mice developed from serrated hyperplasia to adenoma/dysplasia-like neoplasia with enhanced proliferation. Macrophage and neutrophil infiltration into the lesions was also observed early in serrated hyperplasia, although adjacent mucosa was devoid of inflammation. Il1b, Il6, Ccl2, and Cxcl5 were up-regulated at lesion sites, whereas only IL-6 production increased in Ahr-/- peritoneal macrophages after lipopolysaccharide + ATP stimulation. Neither Myc (alias c-myc) up-regulation nor ß-catenin nuclear translocation was observed, unlike previously reported. Interestingly, enhanced phosphorylation of extracellular signal-regulated kinase, Src, and epidermal growth factor receptor and Amphiregulin up-regulation at Ahr-/- lesion sites were detected. In human serrated lesions, however, AhR expression in epithelial cells was up-regulated despite morphologic similarity to Ahr-/- cecal lesions. Our results suggest novel mechanisms underlying Ahr-/- cecal tumorigenesis, depending primarily on cecum-specific mitogen-activated protein kinase pathway activation and inflammation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Carcinogénesis/patología , Neoplasias del Ciego/patología , Neoplasias Colorrectales/patología , Inflamación/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Hidrocarburo de Aril/fisiología , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinogénesis/inmunología , Carcinogénesis/metabolismo , Neoplasias del Ciego/inmunología , Neoplasias del Ciego/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Femenino , Hiperplasia/inmunología , Hiperplasia/metabolismo , Hiperplasia/patología , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Chronic activation of the IL-1ß system in adipose tissue on metabolic disorders is well demonstrated. However, a mechanism for its expression and activation in the tissue has remained unexplored. Here, we demonstrate that IL-1ß transcript was enriched in neutrophils of white adipose tissue (WAT) from lean mice. Mechanistically, the interaction of neutrophils with adipocytes induced IL-1ß expression via NF-κB pathway. Lipolysis of adipocytes accumulated neutrophils prior to macrophages in WAT and produced high levels of IL-1ß via an inflammasome pathway. Leukotriene B4 (LTB4) production in WAT also contributed to neutrophil accumulation. Furthermore, an LTB4-inflammasome axis contributed to the expression of chemotactic molecules involved in high-fat diet-induced macrophage infiltration into WAT. We have identified previously unappreciated roles for neutrophils in the development of adipose tissue inflammation: robust IL-1ß production and infiltration of macrophages to initiate chronic inflammation.-Watanabe, Y., Nagai, Y., Honda, H., Okamoto, N., Yanagibashi, T., Ogasawara, M., Yamamoto, S., Imamura, R., Takasaki, I., Hara, H., Sasahara, M., Arita, M., Hida, S., Taniguchi, S., Suda, T., Takatsu, K. Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation.
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Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Inflamación/metabolismo , Neutrófilos/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Inflamasomas/metabolismo , Lipólisis/fisiología , Macrófagos/metabolismo , Ratones Transgénicos , Obesidad/metabolismoRESUMEN
Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1ß (IL-1ß-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1ß, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1ß-/- and sham-operated IL-1ß-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1ß-/- mouse atria compared with sham-operated WT and IL-1ß-/- mouse atria. In contrast, the IL-1ß gene was not detected in either TAC-operated or sham-operated IL-1ß-/- mouse atria. These results suggest that an IL-1ß activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.
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Fibrilación Atrial/metabolismo , Hipertensión/metabolismo , Interleucina-1beta/metabolismo , Animales , Fibrilación Atrial/genética , Presión Sanguínea , Proteínas Adaptadoras de Señalización CARD/genética , Quimiocina CCL2/genética , Atrios Cardíacos/metabolismo , Hipertensión/genética , Interleucina-1beta/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
The NLRP3 inflammasome has important roles in the pathogenesis of various inflammatory diseases. However, the regulatory mechanisms of the NLRP3 inflammasome are not fully understood. In this study, we attempted to identify molecules that interact with NLRP3 upon its activation. We identified G protein subunit ß 1 (GNB1), a downstream molecule of G protein-coupled receptors (GPCRs), which regulates the NLRP3 inflammasome activation. GNB1 was physically associated with NLRP3 via the pyrin domain of NLRP3. Activation of the NLRP3 inflammasome was enhanced in GNB1-knockdown or GNB1-deficient murine macrophages, although a lack of GNB1 did not affect activation of the AIM2 inflammasome. ASC oligomerization induced by NLRP3 was enhanced by GNB1 deficiency. Conversely, NLRP3-dependent ASC oligomerization was inhibited by the overexpression of GNB1. This study indicates that GNB1 negatively regulates NLRP3 inflammasome activation by suppressing NLRP3-dependent ASC oligomerization, and it provides a regulatory mechanism of the NLRP3 inflammasome.
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Subunidades beta de la Proteína de Unión al GTP/inmunología , Inflamasomas/inmunología , Macrófagos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Animales , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Hypertension causes vascular complications, such as stroke, cardiovascular disease, and chronic kidney disease (CKD). The relationship between endothelial dysfunction and progression of kidney disease is well known. However, the relationship between the eNOS-NO pathway and chronic inflammation, which is a common pathway for the progression of kidney disease, remains unexplored. We performed in vivo experiments to determine the role of the eNOS-NO pathway by using eNOS-deficient mice in a hypertensive kidney disease model. All mice were unilateral nephrectomized (Nx). One week after Nx, the mice were randomly divided into two groups: the aldosterone infusion groups and the vehicle groups. All mice also received a 1% NaCl solution instead of drinking water. The aldosterone infusion groups were treated with hydralazine to correct blood pressure differences. After four weeks of drug administration, all mice were euthanized, and blood and kidney tissue samples were collected. In the results, NLRP3 inflammasome activation was elevated in the kidneys of the eNOS-deficient mice, and tubulointerstitial fibrosis was accelerated. Suppression of inflammasome activation by knocking out ASC prevented tubulointerstitial injury in the eNOS knockout mice, indicating that the eNOS-NO pathway is involved in the development of kidney dysfunction through acceleration of NLRP3 inflammasome in macrophages. We revealed that endothelial function, particularly the eNOS-NO pathway, attenuates the progression of renal tubulointerstitial injury via suppression of inflammasome activation. Clinically, patients who develop vascular endothelial dysfunction have lifestyle diseases, such as hypertension or diabetes, and are known to be at risk for CKD. Our study suggests that the eNOS-NO pathway could be a therapeutic target for the treatment of chronic kidney disease associated with endothelial dysfunction.
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Hipertensión Renal/metabolismo , Hipertensión/metabolismo , Inflamasomas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Insuficiencia Renal Crónica/metabolismo , Aldosterona/farmacología , Animales , Antihipertensivos/administración & dosificación , Modelos Animales de Enfermedad , Endotelio/patología , Endotelio/fisiopatología , Fibrosis , Humanos , Hidralazina/administración & dosificación , Hipertensión/complicaciones , Hipertensión Renal/tratamiento farmacológico , Hipertensión Renal/patología , Inflamasomas/efectos de los fármacos , Riñón/patología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Óxido Nítrico Sintasa de Tipo III/genética , Cultivo Primario de Células , Insuficiencia Renal Crónica/inducido químicamente , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/patología , Vasodilatadores/administración & dosificaciónRESUMEN
Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-ß expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-ß, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection.
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Proteínas Portadoras/metabolismo , Neoplasias del Colon/metabolismo , Proteína 58 DEAD Box/metabolismo , Quinasa I-kappa B/metabolismo , Interferón beta/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/virología , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Interferón beta/genética , Interferón beta/inmunología , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Receptores Inmunológicos , Virosis/genética , Virosis/inmunología , Virosis/metabolismoRESUMEN
Primary lung cancer is the most frequent cause of cancer-related deaths worldwide. Cisplatin has been used as a key drug in the treatment for patients with lung cancer; however, most of the patients failed to respond to cisplatin within several months, and the mechanisms underlying the cisplatin resistance have not been fully elucidated. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a key adaptor protein in the formation of inflammasomes. ASC is also involved in apoptotic signaling. Importantly, ASC expression is decreased in lung cancer and various cancers, but its precise function in tumor progression remains unknown. To explore the hitherto unknown role of ASC in lung cancer, we initially searched for lung cancer cell lines with higher expression levels of ASC using Cancer Cell Line Encyclopedia (CCLE) database, thereby identifying the A549 human non-small cell lung cancer cell line. Accordingly, with retroviral shRNA, the expression of ASC was forced to decrease in A549 cells. Stable ASC-knockdown cells, thus established, showed the increased activities of proliferation, motility, and invasion, compared with control cells. Importantly, ASC-knockdown cells also became resistant to cisplatin, but not to other anti-cancer agents, 5-fluorouracil and paclitaxel. Bcl-2 and phospho-Src levels were increased in ASC-knockdown cells. A Bcl-2 inhibitor, ABT-199, induced an apoptotic response in ASC-knockdown cells, and dasatinib, a Src inhibitor, blocked cell invasiveness. Thus, ASC may be involved in tumor suppression and cell death via Bcl-2 and pSrc. Targeting Bcl-2 and Src in ASC-downregulated populations of lung cancer may improve treatment outcome.
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Apoptosis , Proteínas Adaptadoras de Señalización CARD/metabolismo , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Células A549 , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Fenotipo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
A monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2), trastuzumab has become a standard treatment for HER2-positive breast cancer. Recent advancements in antibody engineering have enabled the efficient generation of the trastuzumab single-chain variable fragment (scFv). In this study, we genetically engineered Bifidobacterium, a bacterial strain shown to accumulate safely and selectively in hypoxic tumor sites by intravenous (iv) injection, to express and secrete the trastuzumab scFv. The recombinant scFv bound to cell surface HER2 and inhibited in vitro growth of HER2-positive human cancer cells. Moreover, iv-injected recombinant bacteria specifically localized and secreted trastuzumab scFv in xenografted human HER2-positive tumors and consequently inhibited tumor growth. The development and results of this novel in situ delivery and production system for trastuzumab scFv with Bifidobacterium represents a promising avenue for future application in cancer treatment.
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Bifidobacterium/fisiología , Neoplasias de la Mama/microbiología , Neoplasias de la Mama/terapia , Receptor ErbB-2/metabolismo , Trastuzumab/administración & dosificación , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Región Variable de Inmunoglobulina/administración & dosificación , Anticuerpos de Cadena Única/administración & dosificación , Resultado del TratamientoRESUMEN
Chronic inflammation can be a major driver of the failure of a variety of organs, including chronic kidney disease (CKD). The NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been shown to play a pivotal role in inflammation in a mouse kidney disease model. Nuclear factor erythroid 2-related factor 2 (Nrf2), the master transcription factor for anti-oxidant responses, has also been implicated in inflammasome activation under physiological conditions. However, the mechanism underlying inflammasome activation in CKD remains elusive. Here, we show that the loss of Nrf2 suppresses fibrosis and inflammation in a unilateral ureter obstruction (UUO) model of CKD in mice. We consistently observed decreased expression of inflammation-related genes NLRP3 and IL-1ß in Nrf2-deficient kidneys after UUO. Increased infiltration of M1, but not M2, macrophages appears to mediate the suppression of UUO-induced CKD symptoms. Furthermore, we found that activation of the NLRP3 inflammasome is attenuated in Nrf2-deficient bone marrow-derived macrophages. These results demonstrate that Nrf2-related inflammasome activation can promote CKD symptoms via infiltration of M1 macrophages. Thus, we have identified the Nrf2 pathway as a promising therapeutic target for CKD.
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Macrófagos/inmunología , Macrófagos/metabolismo , Factor 2 Relacionado con NF-E2/deficiencia , Obstrucción Ureteral/etiología , Obstrucción Ureteral/metabolismo , Animales , Modelos Animales de Enfermedad , Fibrosis , Perfilación de la Expresión Génica , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Riñón/metabolismo , Riñón/patología , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Ratones , Ratones Noqueados , Obstrucción Ureteral/patología , Obstrucción Ureteral/terapiaRESUMEN
The n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in fish oil, exert a number of beneficial effects, and they are used in the treatment of hyperlipidemia. In recent years, EPA and DHA have been found to affect cancer cell proliferation. In the present study, PC3 cells, which are androgen-independent prostate cancer cells that resemble castration-resistant prostate cancer cells, were used to investigate a possible novel treatment for castration-resistant prostate cancer. The PC3 cells were cultured and incubated with various concentrations of EPA or DHA. Cancer proliferation was confirmed by trypan blue microscopy. Invasion and migration assays were used in the upper chamber in PC3 cells, and serum-free medium and various concentrations of EPA or DHA were placed in the lower chamber in serum-containing medium. EPA and DHA decreased PC3 cell proliferation, invasion and migration. The effect of EPA on PC3 cells was dose-dependent and significant differences were observed at concentrations of 100 and 200 µg/ml. The effect of DHA on PC3 cells was similar to that of EPA. In the migration assay, EPA exerted almost no effects at 25 µg/ml, but migration was reduced at 50 µg/ml. Similar to EPA, DHA exerted almost no effects at 25 µg/ml, but further reduction was observed at the 50 µg/ml concentration. In the invasion assay, EPA at 25 µg/ml was not significantly different from the control, but suppressed invasion at 50 µg/ml. DHA decreased invasion compared with the control at 25 µg/ml, whereas invasion was significantly reduced at a DHA concentration of 50 µg/ml. In conclusion, it was demonstrated that EPA and DHA were effective in decreasing the proliferation, invasion and migration of prostate PC3 cancer cells. However, the detailed underlying mechanisms have not yet been fully elucidated.
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CD4+ Th cells play crucial roles in orchestrating immune responses against pathogenic microbes, after differentiating into effector subsets. Recent research has revealed the importance of IFN-γ and IL-17 double-producing CD4+ Th cells, termed Th17/Th1 cells, in the induction of autoimmune and inflammatory diseases. In addition, Th17/Th1 cells are involved in the regulation of infection caused by the intracellular bacterium Mycobacterium tuberculosis in humans. However, the precise mechanism of Th17/Th1 induction during pathogen infection is unclear. In this study, we showed that the inflammasome and Fas-dependent IL-1ß induces Th17/Th1 cells in mice, in response to infection with the pathogenic intracellular bacterium Listeria monocytogenes In the spleens of infected wild-type mice, Th17/Th1 cells were induced, and expressed T-bet and Rorγt. In Pycard-/- mice, which lack the adaptor molecule of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain), Th17/Th1 induction was abolished. In addition, the Fas-mediated IL-1ß production was required for Th17/Th1 induction during bacterial infection: Th17/Th1 induction was abolished in Fas-/- mice, whereas supplementation with recombinant IL-1ß restored Th17/Th1 induction via IL-1 receptor 1 (IL-1R1), and rescued the mortality of Fas-/- mice infected with Listeria IL-1R1, but not apoptosis-associated speck-like protein containing a caspase recruitment domain or Fas on T cells, was required for Th17/Th1 induction, indicating that IL-1ß stimulates IL-1R1 on T cells for Th17/Th1 induction. These results indicate that IL-1ß, produced by the inflammasome and Fas-dependent mechanisms, contributes cooperatively to the Th17/Th1 induction during bacterial infection. This study provides a deeper understanding of the molecular mechanisms underlying Th17/Th1 induction during pathogenic microbial infections in vivo.
Asunto(s)
Inflamasomas/inmunología , Interleucina-1beta/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Células TH1/inmunología , Células Th17/inmunología , Receptor fas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD , Diferenciación Celular , Interleucina-1beta/administración & dosificación , Listeria monocytogenes/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/inmunología , Bazo/inmunología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Receptor fas/deficiencia , Receptor fas/genéticaRESUMEN
BACKGROUND: Muscle atrophy with aging is closely associated with chronic systemic inflammation and lifestyle-related diseases. In the present study, we assessed whether post-exercise milk product intake during 5-month interval walking training (IWT) enhanced the increase in thigh muscle strength and ameliorated susceptibility to inflammation in older women. METHODS: Subjects [n = 37, 66±5 (standard deviation) yrs] who had been performing IWT for >6 months participated in this study. They were randomly divided into the following 3 groups: IWT alone (CNT, n = 12), IWT + low-dose post-exercise milk product intake (LD, n = 12; 4 g protein and 3 g carbohydrate) or IWT + a 3-times higher dose of milk product intake than the LD group (HD, n = 13). They were instructed to repeat ≥5 sets of fast and slow walking for 3 min each at ≥70% and 40% peak aerobic capacity for walking, respectively, per day for ≥4 days/week. RESULTS: After IWT, thigh muscle strength increased in the HD group (8±2%) more than in the CNT group (-2±3%, P = 0.022), despite similar IWT achievements between the groups (P>0.15). Pyrosequencing analysis using whole blood showed that methylation of NFKB1 and NFKB2, master genes of inflammation, was enhanced in the HD group (29±7% and 44±11%, respectively) more than in the CNT group (-20±6% and -10±6%, respectively; P<0.001). Moreover, the genome-wide DNA methylation analysis showed that several inflammation-related genes were hyper-methylated in the HD group compared with that in the CNT group, suggesting greater pro-inflammatory cytokine gene suppression in the HD group. CONCLUSION: HD milk product intake after exercise produced a greater percent increase in thigh muscle strength and NFKB1 and NFKB2 gene methylation during IWT in physically active older women. TRIAL REGISTRATION: UMIN-CTR No. UMIN000024544 and No. UMIN000024912.
Asunto(s)
Metilación de ADN , Productos Lácteos , Ejercicio Físico , Leche , Fuerza Muscular , FN-kappa B/genética , Muslo , Factores de Edad , Anciano , Animales , Células Sanguíneas/metabolismo , Islas de CpG , Femenino , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Persona de Mediana Edad , Cooperación del Paciente , Aptitud Física , Proyectos Piloto , Regiones Promotoras Genéticas , Transducción de Señal , Factores de Tiempo , CaminataRESUMEN
ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.
Asunto(s)
Caspasa 9/metabolismo , Proteínas del Citoesqueleto/metabolismo , Uniones Comunicantes/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Proteínas Adaptadoras de Señalización CARD , Caspasa 9/genética , Comunicación Celular/genética , Comunicación Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular , Conexina 43/genética , Proteínas del Citoesqueleto/genética , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Disorders of cytoskeletal remodeling and signal transduction are frequently involved in cancer progression. In particular, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) has been reported a proapoptotic molecule that is epigenetically silenced in several human cancers. ASC is a well-characterized adaptor protein involved in the formation of multiprotein oligomers, called inflammasomes, and plays a crucial role in the activation and secretion of interleukin-1ß and interleukin-18 in innate immune cells. However, the function of ASC in the regulation of tumor progression remains elusive. The present investigation examined the involvement of ASC in cancer progression and the acquisition of metastatic ability. To determine the effect of ASC depletion in in vitro and in vivo model systems, ASC was stably knocked down in B16 murine melanoma cell lines using retroviral transduction of shRNA. ASC suppression increased the motility of B16BL6 cells in scratch assays and augmented invasiveness as assessed by a Matrigel-coated transwell system. Invadopodia formation and Src phosphorylation level were markedly enhanced in ASC-knockdown cells as well. Since caspase-8 has been reported to enhance cellular migration by Tyr380 phosphorylation via Src, we examined Tyr380 phosphorylation of caspase-8 in ASC-knockdown cells and found it to be elevated in ASC-knockdown cells but attenuated by z-VAD-fmk or z-IETD-fmk. Moreover, ASC ablation increased pulmonary metastasis in mice after intravenous injection of B16BL6 cells. Our cumulative findings indicate that ASC suppresses cancer metastasis and progression via the modulation of cytoskeletal remodeling and the Src-caspase-8 signaling pathway.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/metabolismo , Fenotipo , Transducción de Señal , Animales , Caspasa 8/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Neoplasias Pulmonares/secundario , Melanoma Experimental , Ratones , Metástasis de la Neoplasia , Neoplasias/patología , Fosforilación , Familia-src Quinasas/metabolismoRESUMEN
AIMS: Controlling vascular integrity is expected to be a novel therapeutic target of cancers as well as cardiovascular diseases. Adrenomedullin (AM) and its receptor-modulating protein, RAMP2, have been identified as essential mediators of cardiovascular homeostasis. In this study, we used inducible vascular endothelial cell-specific RAMP2 knockout (DI-E-RAMP2(-/-)) mice to clarify the contribution made by the endogenous AM-RAMP2 system to angiogenesis and metastasis. METHODS AND RESULTS: Subcutaneously transplanted sarcoma or melanoma cells showed less growth and angiogenesis in DI-E-RAMP2(-/-) than in control mice. On the other hand, after the transplantation of B16BL6 melanoma cells into hindlimb footpads, spontaneous metastasis to the lung was enhanced in DI-E-RAMP2(-/-) mice. Early after RAMP2 gene deletion, DI-E-RAMP2(-/-) mice showed enhanced vascular permeability, endothelial-mesenchymal transition (EndMT)-like change, and systemic oedema. Within the lungs of DI-E-RAMP2(-/-) mice, pulmonary endothelial cells were deformed, and inflammatory cells infiltrated the vessel walls and expressed the chemotactic factors S100A8/9 and SAA3, which attract tumour cells and mediate the formation of a pre-metastatic niche. Conversely, the overexpression of RAMP2 suppressed tumour cell adhesion to endothelial cells, tumour metastasis, and improved survival. CONCLUSION: These findings indicate that the AM-RAMP2 system regulates vascular integrity, whereas RAMP2 deletion promotes vascular permeability and EndMT-like change within primary lesions and formation of pre-metastatic niches in distant organs by destabilizing the vascular structure and inducing inflammation. Vascular integrity regulated by the AM-RAMP2 system could thus be a hopeful therapeutic target for suppressing tumour metastasis.