RESUMEN
The study is aimed at assessing the impact that periodontal disease and chronic hepatitis C could have on gingival crevicular fluid levels of the NLRP3 inflammasome, caspase-1 (CASP-1), and interleukin-18 (IL-18) and at evaluating whether the increased local inflammatory reaction with clinical periodontal consequences is correlated to their upregulation. Patients were divided into four groups, according to their periodontal status and previously diagnosed hepatitis C, as follows: (i) CHC group, chronic hepatitis C patients; (ii) P group, periodontal disease patients, systemically healthy; (iii) CHC + P group, patients suffering from both conditions; and (iv) H group, systemically and periodontally healthy controls. Gingival crevicular samples were collected for quantitative analysis of the NLRP3 inflammasome, CASP-1, and IL-18. CHC + P patients expressed the worse periodontal status and the highest NLRP3, CASP-1, and IL-18 levels, the difference being statistically significant (p < 0.05). The P group patients also expressed significantly more elevated NLRP3, CASP-1, and IL-18 levels, as compared to nonperiodontal patients (CHC and H groups). Chronic hepatitis C and periodontal disease could have a significant influence on the upregulation of NLRP3 inflammasome and its components, possibly contributing to an increased local inflammatory reaction and clinical periodontal consequences.
Asunto(s)
Periodontitis Crónica/inmunología , Líquido del Surco Gingival/inmunología , Hepatitis C Crónica/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Caspasa 1/análisis , Femenino , Humanos , Mediadores de Inflamación/análisis , Interleucina-18/análisis , Masculino , Persona de Mediana EdadRESUMEN
We aimed to investigate the effects of ethanol and its metabolites (ß-hydroxybutyrate and sodium acetate) in the effector functions of macrophages in response to Paracoccidioides brasiliensis yeast cells and to determine their influence in the development of the adaptive response. Purified peripheral blood monocytes were differentiated into macrophages and were treated with ethanol, ß-hydroxybutyrate, and sodium acetate, and stimulated with P. brasiliensis yeast cells and evaluated for their phenotypic characteristics, functional activity, and capability to induce T cells activation/differentiation. We found that the ethanol treatment diminished the expression of HLA-AB, HLA-DR, CD80, and CD86, modulating the expression of dectin-1, as well as Syk phosphorylation. The ethanol treatment increased the phagocytic activity, expression of CD206, and IL-10 production; however, reduced ROS production, fungicidal activity, caspase-1 cleavage, and IL-1ß and IL-6 production. Our data also showed that the presence of ethanol reduced the differentiation of Th1 and Th17 cells and increased the frequency of Th2 cells. Our results indicated that ethanol exposure could suppress effector function of macrophages, possibly leading to the polarization of M2 macrophages. The ethanol modulates the expression of costimulatory and antigen-presentation molecules and interferes with the NLRP3 inflammasome. Altogether, these alterations affect the development of the adaptive response, decreasing the frequency of IL-17, IL-22, and IFN- γ producing cells, and increasing the frequency of IL-4 producing cells. Therefore, exposure to ethanol can impair the capability of macrophages to exert their effector functions and activate the acquired response related to resistance to P. brasiliensis infection.
Asunto(s)
Etanol/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Paracoccidioides/fisiología , Paracoccidioidomicosis/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Antifúngicos/farmacología , Complejo CD3/análisis , Caspasa 1/análisis , Citocinas/análisis , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Receptores de Lipopolisacáridos/análisis , Macrófagos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Peróxidos/metabolismo , Fagocitosis/efectos de los fármacosRESUMEN
OBJECTIVE: The orf8b protein of the coronavirus SARS-CoV, analogous to SARS-CoV-2, triggers the NLRP3 inflammasome in macrophages in vitro. Deregulated inflammasome-mediated release of interleukin-1 family cytokines is important in hyper-inflammatory syndromes, like happens in SARS-CoV-2-mediated cytokine release syndrome. We propose that an intense inflammasome formation characterizes the lungs of patients with fatal COVID-19 disease due to pneumonia and acute respiratory distress syndrome (ARDS). METHODS: Samples from four patients with confirmed COVID-19 pneumonia who had been hospitalized at the Hospital of the University of Trieste (Italy) and died of ARDS and four lung samples from a historical repository from subjects who had died of cardiopulmonary arrest and had not been placed on mechanical ventilation and without evidence of pulmonary infection at postmortem examination were collected. Pathology samples had been fixed in formalin 10% at time of collection and subsequently embedded in paraffin. We conducted staining for ASC (Apoptosis-associated Speck-like protein containing a Caspase recruitment domain), NLRP3 (NACHT, LRR, and PYD domains-containing protein 3), and cleaved caspase-1. RESULTS: Intense expression of the inflammasome was detected, mainly in leukocytes, within the lungs of all patients with fatal COVID-19 in the areas of lung injury. The number of ASC inflammasome specks per high power fields was significantly higher in the lungs of patients with fatal COVID-19 as compared with the lungs of control subjects (52 ± 22 vs 6 ± 3, P = 0.0064). CONCLUSIONS: These findings identify the presence of NLRP3 inflammasome aggregates in the lungs of fatal COVID-19 pneumonia thus providing the potential molecular link between viral infection and cytokine release syndrome.
Asunto(s)
COVID-19/patología , Inflamasomas , Pulmón/patología , Adulto , Anciano , Autopsia , Proteínas Adaptadoras de Señalización CARD/análisis , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/análisis , Caspasa 1/metabolismo , Síndrome de Liberación de Citoquinas/metabolismo , Síndrome de Liberación de Citoquinas/patología , Femenino , Paro Cardíaco/etiología , Humanos , Leucocitos/patología , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neumonía Viral/etiología , Neumonía Viral/patología , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
Neuroinflammation plays an important role in brain damage after acute carbon monoxide poisoning (ACOP). The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing (NLRP) 3 inflammasome triggers the activation of inflammatory caspases and maturation of interleukin (IL)-1ß and -18, and has been linked to various human autoinflammatory and autoimmune diseases. In this study we investigated the effects of hyperbaric oxygen (HBO2) on NLRP3 inflammasome activation after ACOP. Mice were randomly divided into four groups: sham group (exposure to normobaric air - i.e., 21% O2 at 1 atmosphere absolute); HBO2-only group; CO + normobaric air group; and CO + HBO2 group. Cognitive function was evaluated with the Morris water maze; myelin injury was assessed by FluoroMyelin GreenTM fluorescent myelin staining and myelin basic protein (MBP) immunostaining; and mRNA and protein levels of NLRP3 inflammasome complex proteins were measured by quantitative real-time PCR and Western blot, respectively. Additionally, serum and brain levels of IL-1ßß and -18 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined by enzyme-linked immunosorbent assay. It was found that HBO2 improved learning and memory, and alleviated myelin injury in mice subjected to acute CO exposure. Furthermore, HBO2 decreased NLRP3, absent in melanoma 2 (AIM2), caspase-1, and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain mRNA and protein levels, and reduced brain and serum concentrations of IL-1ß and -18 and NADPH oxidase. These results indicate that HBO2 suppresses the inflammatory response after ACOP by blocking NLRP3 inflammasome activation, thereby alleviating cognitive deficits.
Asunto(s)
Encéfalo/metabolismo , Intoxicación por Monóxido de Carbono/metabolismo , Oxigenoterapia Hiperbárica , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad Aguda , Animales , Presión Atmosférica , Química Encefálica , Proteínas Adaptadoras de Señalización CARD/análisis , Caspasa 1/análisis , Proteínas de Unión al ADN/análisis , Interleucina-18/análisis , Interleucina-1beta/análisis , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina , NADP/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/genética , ARN Mensajero/metabolismo , Distribución AleatoriaRESUMEN
Single cell analysis has been used to reveal cellular heterogeneity and to gain deeper insight into some of life's mysteries. In this work, we developed a high throughput profiling platform for single polar cell analysis with controllable cell polarity on anisotropic microwell arrays. Due to the spatial confinement effect, the cells were individually trapped in the microwells and polarized to an assigned polarity (aspect ratio). Given the significance of polarity in biology, quantitative evaluation of the drug response on single polarized cells may provide guidance for chemotherapy. With this aim, we studied the interaction of the single polarized cells with drugs (doxorubicin and paclitaxel) to reveal the possible apoptosis mechanisms involved. Cell death features including cellular morphologies, nucleus pycnosis and the formation of inflammasome provided the indicators of programmed cell death and inflammatory death. To trace the apoptosis pathway, cell death enzyme cascades were quantitatively evaluated, especially the regulated cell death executor (caspase 3) and inflammatory death (caspase 1). The results showed that DOX killed the tumour cells not only through a regular cell death program, but also an inflammatory death pathway, while paclitaxel only led to a regular cell death program. Furthermore, the cells with different polarities respond to the drugs at different rates, namely the round cells (in their unpolarized phase) being the most sensitive to the drug molecules. These results provided significant information about guiding the treatment of tumours with chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Polaridad Celular/fisiología , Doxorrubicina/farmacología , Paclitaxel/farmacología , Análisis de la Célula Individual/métodos , Anisotropía , Caspasa 1/análisis , Caspasa 1/metabolismo , Caspasa 3/análisis , Caspasa 3/metabolismo , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Humanos , Prueba de Estudio Conceptual , Piroptosis/efectos de los fármacos , Análisis de la Célula Individual/instrumentaciónRESUMEN
Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIXpromotor )-controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIXpromotor -AIM2 adenoviruses (Ad-CAIXpromotor -AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad-CAIXpromotor , Ad-CAIXpromotor -AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786-O cells or OSRC-2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC-2-xenograft mice. The tumour growth was remarkably inhibited in Ad-CAIXpromotor -AIM2-treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD-CMK resulted in the reduction of anti-tumour activity by Ad-CAIXpromotor -AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad-CAIXpromotor -AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour-specific Ad-CAIXpromotor -AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2-based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.
Asunto(s)
Adenovirus Humanos/fisiología , Anhidrasa Carbónica IX/genética , Carcinoma de Células Renales/terapia , Proteínas de Unión al ADN/fisiología , Neoplasias Renales/terapia , Viroterapia Oncolítica , Regiones Promotoras Genéticas/genética , Proteínas E1A de Adenovirus/biosíntesis , Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Clorometilcetonas de Aminoácidos/farmacología , Animales , Carcinoma de Células Renales/secundario , Caspasa 1/análisis , Línea Celular , Línea Celular Tumoral , Citocinas/análisis , Proteínas de Unión al ADN/genética , Genes Sintéticos , Humanos , Inflamasomas/efectos de los fármacos , Neoplasias Renales/patología , Túbulos Renales/citología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Ensayo de Tumor de Célula Madre , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bile cast nephropathy (BCN) is an underdiagnosed cause of acute kidney injury (AKI). The precise pathogenesis of bilirubin tubular toxicity remains unknown. The aim of this study is to explore the cellular and molecular pathophysiology of human BCN. Paraffin-embedded sections of renal biopsy tissue from a BCN patient were stained by immunohistochemistry (IHC) for oxidative stress (4-hydroxynonenal), immune cell subpopulations, including dendritic cells (CD1c), macrophages (CD68) and T cells (CD3), and inflammasome activation by staining for active-caspase-1 and the inflammasome adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain). Quantitative analyses of IHC staining were compared to healthy renal cortical tissue. We identified yellow to brown granular casts within the BCN case, consistent with the presence of bile pigment. The presence of bile pigment was associated with strong tubular 4-hydroxynonenal staining intensity, a marker of oxidative stress. Diffuse tubulointerstitial inflammatory cell infiltrate was detected, with elevated CD1c, CD68 and CD3 staining. Foci of inflammasome activity were co-localized with this intense immune cell infiltration, with increased active-caspase-1 and ASC staining. Our findings are the first to suggest that bile casts may lead to oxidative stress and trigger the inflammasome signalling cascade, leading to interstitial inflammation and driving AKI pathobiology. SUMMARY AT A GLANCE The report suggests that bile casts may lead to oxidative stress and trigger the inflammasome signalling cascade, leading to interstitial inflammation and driving bile cast nephropathy pathobiology.
Asunto(s)
Lesión Renal Aguda/etiología , Bilis/metabolismo , Inflamasomas/fisiología , Inflamación/complicaciones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Antígenos CD1/análisis , Bilirrubina/metabolismo , Caspasa 1/análisis , Glicoproteínas/análisis , Humanos , Riñón/patología , Masculino , Persona de Mediana Edad , Estrés OxidativoRESUMEN
Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. OBJECTIVE: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. METHODOLOGY: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. RESULTS: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1ß (IL-1ß) and IL-6 protein expression. CONCLUSIONS: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Calcitriol/farmacología , FN-kappa B/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Periodontitis/tratamiento farmacológico , Periodontitis/metabolismo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Pérdida de Hueso Alveolar , Animales , Western Blotting , Conservadores de la Densidad Ósea/análisis , Calcitriol/análisis , Caspasa 1/análisis , Encía/efectos de los fármacos , Encía/metabolismo , Encía/patología , Inmunohistoquímica , Interleucina-1beta/análisis , Interleucina-6/análisis , Masculino , Ratones Endogámicos C57BL , FN-kappa B/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Periodontitis/patología , Porphyromonas gingivalis , Receptores de Hidrocarburo de Aril/análisis , Valores de Referencia , Reproducibilidad de los Resultados , Resultado del TratamientoRESUMEN
Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro-IL-1ß and pro-IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow-derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.
Asunto(s)
Técnicas Biosensibles/métodos , Caspasa 1/análisis , Inflamación/etiología , Animales , Apoptosis , Colitis/enzimología , Progresión de la Enfermedad , Enfermedad Injerto contra Huésped/enzimología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Infecciones Estafilocócicas/enzimología , Células THP-1RESUMEN
The type of cell death triggered by a particular environmental stimulus influences the outcome of infection or inflammatory disease processes. The ability to identify the cell death pathway that is activated in response to infection is essential for understanding the pathogenesis and host response to infection. Activation of the cysteine protease caspase-1 in various inflammasome complexes indicates that cells are undergoing pyroptosis, a regulated, proinflammatory cell death. Inflammasome assembly and caspase activation can be measured by various methods ranging from detection of inflammasome-dependent cell death, cytokine secretion, cleavage of caspase-1, or the formation of "puncta" within the cell that contain inflammasome components, such as caspase-1 or the adapter protein ASC. Here we describe a method for detecting caspase-1 activation on a single cell level in the context of infection by the Gram-negative pathogen Yersinia using immunofluorescence microscopy. We previously used this approach to quantify caspase-1 puncta formation in cells containing Yersinia translocon components (Zwack et al., MBio 6:e02095-14, 2015). This is a modification of methods used previously by Broz et al. (Cell Host Microbe 8:471-483, 2010) and Case and Roy (MBio 2:e00117-11, 2011). By taking a microscopy-based approach that allows us to quantify puncta as well as other cell-biological features of infection (i.e., number of bacteria associated with a particular cell; levels of bacterial effector or translocon proteins in caspase-1 puncta-containing cells; or levels or localization of host cellular proteins), we can better quantify the heterogeneity between cells undergoing pyroptosis and cells that are not under the same infection conditions. These approaches have the potential to generate hypotheses that can enable further mechanistic insight into activation of pyroptosis in response to bacterial infection.
Asunto(s)
Caspasa 1/inmunología , Inflamasomas/inmunología , Microscopía Fluorescente/métodos , Yersiniosis/inmunología , Yersinia/inmunología , Animales , Caspasa 1/análisis , Células Cultivadas , Humanos , Inflamasomas/análisis , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Coloración y Etiquetado/métodos , Yersinia/aislamiento & purificaciónRESUMEN
Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStreamX instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/análisis , Caspasa 1/análisis , Citometría de Flujo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Inflamasomas/metabolismo , Análisis de la Célula Individual/métodos , Fluorescencia , Células HEK293 , Humanos , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR/agonistas , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células THP-1RESUMEN
Abstract Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. Objective: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. Methodology: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. Results: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1β (IL-1β) and IL-6 protein expression. Conclusions: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.
Asunto(s)
Animales , Masculino , Periodontitis/metabolismo , Periodontitis/tratamiento farmacológico , Calcitriol/farmacología , FN-kappa B/efectos de los fármacos , Conservadores de la Densidad Ósea/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Periodontitis/patología , Valores de Referencia , Calcitriol/análisis , Inmunohistoquímica , Western Blotting , Reproducibilidad de los Resultados , Pérdida de Hueso Alveolar , FN-kappa B/análisis , Interleucina-6/análisis , Resultado del Tratamiento , Receptores de Hidrocarburo de Aril/análisis , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Porphyromonas gingivalis , Caspasa 1/análisis , Conservadores de la Densidad Ósea/análisis , Interleucina-1beta/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Encía/efectos de los fármacos , Encía/metabolismo , Encía/patología , Ratones Endogámicos C57BLRESUMEN
Activatable 19F MRI nanoprobes for sensing caspase-1 activity were developed. Tandem repetition of substrate peptide sequences improved the turn-on response of nanoprobes, allowing detection of caspase-1 activity by 19F MRI. In vivo immune response was successfully imaged using the new nanoprobe.
Asunto(s)
Caspasa 1/análisis , Medios de Contraste/química , Imagen por Resonancia Magnética con Fluor-19/métodos , Flúor/química , Nanopartículas/química , Dióxido de Silicio/química , Animales , Caspasa 1/metabolismo , Pruebas de Enzimas/métodos , RatonesRESUMEN
OBJECTIVES: The purpose of this study was to evaluate the effects of transcutaneous electrical nerve stimulation (TENS)-like stimulation on the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 in PC-12 cells, which are commonly used as neuronal cell models. METHODS: Nerve growth factor-differentiated PC-12 cells were exposed to electrical stimulation for 15 minutes at 1 mA, 200 µs, and 100 Hz. Cell lysate from stimulated and control cells was assayed for TNF-α, IL-1ß, and IL-6. In 6 trials, cells were preincubated with the L-type ion channel blocker nicardipine. Cultured cells were also incubated with Alexa Fluor 488 and visualized by fluorescence microscopy to determine the nuclear vs cytoplasmic distribution of the p65 sub-unit of NF-κB RESULTS: Compared with control (unstimulated) cells, the stimulated cells had a downregulation of the assayed cytokines. However, preincubation with the L-type ion channel blocker nicardipine blocked this effect of stimulation. Additionally, it was noted that TENS-like stimulation promoted a relative sequestration of the p65 subunit of NF-κB in the cytoplasm vs the nucleus. CONCLUSIONS: It appears that in this cell line and with these stimulation parameters, TENS-like stimulation attenuated the expression of the assayed proinflammatory cytokines, in part by promoting the relative sequestration of the p65 subunit of NF-κB in the cytoplasm, and that voltage-dependent calcium channels have a role in the cascade of events initiated by the TENS-like stimulation.
Asunto(s)
Citocinas/metabolismo , Regulación hacia Abajo , Estimulación Eléctrica Transcutánea del Nervio/métodos , Proteína ADAM17/análisis , Caspasa 1/análisis , Células Cultivadas , Humanos , Interleucina-6/análisis , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
Both Caspase 1-induced cell death and Caspase 3-induced cell death were reported to be the causes of CD4+ T cell depletion in HIV infection. We measured by flow cytometry the expression of key proteins associated with pyroptosis (Caspase 1), apoptosis (Caspase 3, Caspase 8, Caspase 9), and immune activation in peripheral T cells. The percentages of CD4+ T cells that expressed Caspase 1 and Caspase 3 were significantly higher in untreated human immunodeficiency virus 1 (HIV-1) patients compared with healthy control (Caspase 1: 19.40% vs. 4.65%, p = .006; Caspase 3: 12.75% vs. 4.18%, p < .001). However, the percentages of Caspase 3 in CD8+ T cells increased significantly, while the percentages of Caspase 1 in CD8+ T cells did not change significantly (Caspase 1: 3.33% vs. 1.99%, p = .821; Caspase 3: 20.35% vs 4.74%, p < .001). The percentages of HLA-DR+ CD38+ CD8+ T cells were positively correlated with those of Caspase 1+ CD4+ T cells, but not with those of Caspase 3+ CD4+ T cells. After highly active antiretroviral therapy, the percentages of Caspase 1, but not of Caspase 3, -expressing CD4+ T cells decreased to a level comparable with those of healthy controls (Caspase 1: 6.05% vs. 4.65%, p = .514; Caspase 3: 9.67% vs. 4.18%, p < .001). Our study indicated that CD4+ T cells experience both pyroptosis and apoptosis, while CD8+ T cells undergo only apoptosis in HIV-1 infection. Pyroptosis, but not apoptosis, in CD4+ T cells may be inhibited by effective antiretroviral therapy.
Asunto(s)
Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos/química , Caspasa 1/análisis , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Adulto , Antígenos CD/análisis , Linfocitos T CD8-positivos/química , Caspasa 3/análisis , Femenino , Citometría de Flujo , Antígenos HLA-DR/análisis , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: To explore the effect of taxifolin on H2O2-induced pyroptosis in H9C2 cells and the possible mechanisms.â© Methods: The H9C2 cells was divided into 3 groups: a control group, a hydrogen peroxide (H2O2)group and a taxifolin group. The morphology of H9C2 cells was observed by inverted phase contrast microscope. The mitochondrial membrane potential was measured by JC-1 staining and flow cytometry. The alteration of the level of reactive oxygen species (ROS) was detected by specific mitochondrial probe. The protein levels of cysteinyl aspartate specific proteinase-1 (caspase-1)was determined by Western blot. The mRNA levels of interleukin-18 (IL-18), interleukin-1a (IL-1a), interleukin-1b (IL-1b), absent in melanoma 2 (AIM2), apoptosis-associated apeck-like protein (ASC), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)and nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain-containing protein 4 (NLRC4) were determined by reverse transcription-polymerase chain reaction (RT-PCR).â© Results: Compared with the control group, the morphology of H9C2 cells obviously changed in the H2O2-treated group, which was guadually improved in the presence of taxifolin. Compared with the control group, the mitochondrial membrane potential was markedly decreased in the H2O2-treated cells, accompanied by the increase ofROS (both P<0.05). Compared with the H2O2 group, the mitochondrial membrane potential changes in the taxifolin group was increased while the ROS was decreased, with significant difference (both P<0.05). Compared with the control group, the protein level of caspase-1 and the mRNA levels of IL-18, IL-1a, IL-1b, AIM2, ASC, NLRP3 and NLRC4 in the H2O2-treated group were significantly increased (all P<0.05), which were attenuated in the presence of taxifolin (all P<0.05).â© Conclusion: Taxifolin can protect H9C2 cells from oxidative injury, and it is able to suppress the H2O2-induced H9C2 cell pyroptosis through inhibition of AIM2, NLRP3 and NLRC4 in flammasome.
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Antiinflamatorios no Esteroideos/farmacología , Peróxido de Hidrógeno , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Piroptosis/efectos de los fármacos , Quercetina/análogos & derivados , Animales , Proteínas Adaptadoras de Señalización CARD/análisis , Caspasa 1/análisis , Línea Celular , Proteínas de Unión al ADN/análisis , Peróxido de Hidrógeno/antagonistas & inhibidores , Interleucinas/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Quercetina/farmacología , ARN Mensajero/análisis , Ratas , Especies Reactivas de Oxígeno/análisis , Receptores de Superficie Celular/análisisRESUMEN
In situ construction of self-assemblies with unique property in living systems is a promising direction in the biomedical field. The noninvasive methods for significant enzyme activity in living cells or living subjects are imperative and meantime challenge tasks. The dynamic process of self-assembly of chlorophyll-based molecules in complex biological systems can be monitored by photoacoustic signals, which supports a noninvasive way to understand and quantitatively measure the activity of caspase-1. Furthermore, the activity of caspase-1 enables reflection of the bacterial infection in the early stage. Here, we present a biocompatible self-assembly from chlorophyll-peptide derivatives and first correlate the dynamic equilibrium with ratiometric photoacoustic signals. The intracellular equilibrium was managed by a bacterial infection precaution protein, i.e., caspase-1. This system offers a trial of noninvasive method to quantitative detection and real-time monitoring of bacterial infection in the early stage.
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Caspasa 1/metabolismo , Clorofila/química , Macrófagos/enzimología , Nanoestructuras/química , Técnicas Fotoacústicas/métodos , Animales , Caspasa 1/análisis , Ratones , Células RAW 264.7 , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Infecciones Estafilocócicas/enzimología , Staphylococcus aureusRESUMEN
BACKGROUND: Modic changes are the MRI signal changes of degenerative lumbar vertebral endplate and which lead to or accelerate intervertebral disc degeneration. NLRP3, caspase-1, and interleukin-1ß (IL-1ß) play a pivotal role in the pathogenesis of many inflammatory diseases, such as osteoarthritis. However, the roles of IL-1ß and its activators caspase-1 and NLRP3 are unclear in the degenerative endplate. QUESTIONS/PURPOSES: We asked: (1) What are the degenerative changes of the histologic features and chondrogenic markers' gene expressions between the cartilaginous endplates of patients with Modic changes and trauma (control)? (2) How does the NLRP3/caspase-1/IL-1ß axis in the cartilaginous endplates of patients with Modic changes compare with control (trauma) specimens? METHODS: Surgical specimens of cartilaginous endplates were divided into Modic changes (n = 56) and the trauma control (n = 16) groups. Hematoxylin and eosin and safranin O staining of cartilaginous endplate tissues were done to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed on these tissues to investigate mRNA expression of type II collagen (Col II), SOX-9, matrix metalloproteinase-3, and a disintegrin like and metalloproteinase thrombospondin type I motifs-5. NLRP3, caspase-1, and IL-1ß were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: Hematoxylin and eosin and safranin O staining showed the extracellular matrix degraded in the cartilaginous endplates of patients with Modic changes but not in the control cartilaginous endplates. Chondrogenic Col II (p = 0.024) and SOX9 (p = 0.053) were downregulated in the Modic changes group compared with the control group. In contrast to the control group, the transcriptional levels of NLRP3 (p < 0.001), caspase-1 (p = 0.054), and IL-1ß (p = 0.001) were all upregulated in the Modic changes group. CONCLUSIONS: The expression of NLRP3, caspase-1, and IL-1ß was upregulated in the patients with low back pain and Modic changes on MRI compared with patients with vertebral burst fracture without degenerative changes on MRI. The data suggest the NLRP3/caspase-1/IL-1ß axis may be implicated in lumbar cartilaginous endplate degeneration. CLINICAL RELEVANCE: The NLRP3/caspase-1/IL-1ß axis is active in cartilaginous endplates of patients with Modic changes and inflammatory cascades can exacerbate the cartilaginous endplate degeneration which may act as a trigger for intervertebral disc degeneration and low back pain. If these findings can be confirmed by others, we hope that new and effective therapy could be developed directed against this target.
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Cartílago Articular/enzimología , Caspasa 1/análisis , Interleucina-1beta/análisis , Vértebras Lumbares/enzimología , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Enfermedades de la Columna Vertebral/enzimología , Adolescente , Adulto , Anciano , Cartílago Articular/patología , Estudios de Casos y Controles , Caspasa 1/genética , Matriz Extracelular/patología , Femenino , Humanos , Inmunohistoquímica , Interleucina-1beta/genética , Vértebras Lumbares/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades de la Columna Vertebral/genética , Enfermedades de la Columna Vertebral/patología , Transcripción Genética , Regulación hacia Arriba , Adulto JovenRESUMEN
BACKGROUND: Dermatomyositis (DM) and polymyositis (PM) are common inflammatory myopathies whose immunopathogenic mechanisms remain poorly understood. The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome is a type of cytoplasmic multiprotein inflammasome and is responsible for the activation of inflammatory reactivations. Responding to a wide range of exogenous and endogenous microbial or sterile stimuli, NLRP3 inflammasomes can cleave pro-caspase-1 into active caspase-1, which processes the pro-inflammatory cytokines pro-interleukin (IL)-1ß and pro-IL-18 into active and secreted IL-1ß and IL-18. The NLRP3 inflammasome is implicated in infectious and sterile inflammatory diseases. However, it remains unclear whether it is involved in the pathogenesis of DM/PM, which we aim to address in our research. METHODS: In this study, 22 DM/PM patients and 24 controls were recruited. The protein and RNA expression of IL-1ß, IL-18, NLRP3, and caspase-1 in serum and muscle samples were tested and compared between the two groups. RESULTS: The serum IL-1ß and IL-18 levels were significantly higher in DM/PM patients than those in the controls by enzyme linked immunosorbent assay (ELISA, DM vs. control, 25.02 ± 8.29 ng/ml vs. 16.49 ± 3.30 ng/ml,P < 0.001; PM vs. control, 26.49 ± 7.79 ng/ml vs. 16.49 ± 3.30 ng/ml,P < 0.001). Moreover, the real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showed that DM/PM patients exhibited higher RNA expression of IL-1ß, IL-18, and NLRP3 in the muscle (for IL-1ß, DM vs. control, P= 0.0012, PM vs. control, P= 0.0021; for IL-18, DM vs. control, P= 0.0045, PM vs. control, P= 0.0031; for NLRP3, DM vs. control, P= 0.0017, PM vs. control, P= 0.0006). Moreover, the protein expression of NLRP3 and caspase-1 in muscle samples of DM/PM patients were also significantly elevated compared to that in the muscles of the controls. CONCLUSIONS: Our findings demonstrate that the NLRP3 inflammasome is implicated in the pathogenesis of DM/PM. High NLRP3 expression led to elevated levels of IL-1ß and IL-18 and could be one of the factors promoting disease progress.
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Dermatomiositis/etiología , Inflamasomas/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Polimiositis/etiología , Adulto , Caspasa 1/análisis , Caspasa 1/genética , Femenino , Humanos , Interleucina-18/análisis , Interleucina-18/genética , Interleucina-1beta/análisis , Interleucina-1beta/genética , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/genéticaRESUMEN
Many pathogens trigger caspase-1-mediated innate immune responses. Avian leukosis virus subgroup J (ALV-J) causes serious immunosuppression and diverse tumors in chicks. The caspase-1 inflammasome mechanism of response to ALV-J invading remains unclear. Here we investigated the expression of caspase-1, the inflammasome adaptor NLRP3, IL-1ß and IL-18 in response to ALV-J infection in the liver of chick. We found caspase-1 mRNA expression was elevated at 5 dpi and peaked at 7 dpi in ALV-J infected animals. Corresponding to this, the expressions of NLRP3 and proinflammatory cytokines IL-1ß and IL-18 were significantly increased at 5 or 7 dpi. In addition, caspase-1 protein expression and inflammatory cell infiltration were induced after virus infection. These results indicated that ALV-J infection could trigger the caspase-1- mediated inflammatory response in chicks. Thus, an understanding of the inflammatory responses can provide a better insight into the pathogenicity of ALV-J and a possible anti-virus target for ALV-J infection.