RESUMEN
The dynamics that develop between cells and molecules in the host against infection by Mycobacterium bovis, leads to the formation of granulomas mainly present in the lungs and regional lymph nodes in cattle. Cell death is one of the main features in granuloma organization, however, it has not been characterized in granulomatous lesions caused by M. bovis. In this study we aimed to identify the profiles of cell death in the granuloma stages and its relationship with the accumulation of bacteria. We identified necrosis, activated caspase-3, LC3B/p62 using immunohistochemistry and digital pathology analysis on 484 granulomatous lesions in mediastinal lymph nodes from 23 naturally infected cattle. Conclusions: greater amounts of mycobacterial antigens were identified in granulomas from calves compared with adult cattle. The highest percentage of necrosis and quantity of mycobacterial antigens were identified in granuloma stages (III/IV) from adults. The LC3B/p62 profile was heterogeneous in granulomas between adults and calves. Our data suggest that necrosis is associated with a higher amount of mycobacterial antigens in the late stages of granuloma and the development of autophagy appears to play an heterogeneous effector response against infection in adults and calves. These results represent one of the first approaches in the identification of cell death in the four stages of granulomas in bovine tuberculosis.
Asunto(s)
Antígenos Bacterianos , Granuloma , Mycobacterium bovis , Necrosis , Tuberculosis Bovina , Animales , Bovinos , Granuloma/veterinaria , Granuloma/inmunología , Granuloma/microbiología , Granuloma/patología , Mycobacterium bovis/inmunología , Mycobacterium bovis/patogenicidad , Necrosis/veterinaria , Necrosis/inmunología , Necrosis/microbiología , Tuberculosis Bovina/inmunología , Tuberculosis Bovina/microbiología , Tuberculosis Bovina/patología , Antígenos Bacterianos/inmunología , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Caspasa 3/inmunología , Inmunohistoquímica/veterinariaRESUMEN
BACKGROUND: Hydatid disease is caused by the larval stages of the canine tapeworm Echinococcus granulosus. It is one of the most critical helminthic diseases, representing worldwide public health and socio-economic concern. AIM: This study aimed to investigate the expression of apoptosis and immune response within hepatic tissues of humans and sheep infected with the Hydatid cyst. METHODS: Paraffin-embedded tissue was prepared from each tissue sample and used for histopathological examination by Haematoxylin- Eosin. Also, toluidine blue staining was used for mast cell detection, while an immunohistochemical study was performed to assess CD3 T lymphocytes, CD4 helper T lymphocytes, CD8 cytotoxic T lymphocytes, CD20 memory B lymphocytes, CD68 macrophage, and caspase-3 antibodies. RESULTS: The histological examination revealed significant changes, including the infiltration of inflammatory cells, predominantly lymphocytes with scattered giant cells, necrotic hepatic tissue, and fibrosis. Toluidine blue stain revealed a higher number of mast cells (5 cells/field) in humans compared to sheep (3.6 cells/field). The immunohistochemical analysis confirmed that the CD3 were the most predominant inflammatory cell in the hepatic tissue of humans (intensive 70%), and sheep (moderate 38.47%). Caspase-3 was observed in all samples in different grades and mostly in human liver tissue. CONCLUSION: This data could aid in recognizing immunological markers for differentiating disease progression, as well as enhance the understanding of local immune responses to cystic Echinococcosis (CE). The findings could provide preliminary data for future studies on immune responses associated with Hydatid cysts.
Asunto(s)
Equinococosis Hepática , Enfermedades de las Ovejas , Animales , Ovinos/inmunología , Equinococosis Hepática/inmunología , Equinococosis Hepática/veterinaria , Equinococosis Hepática/parasitología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/parasitología , Humanos , Hígado/parasitología , Hígado/inmunología , Hígado/patología , Masculino , Femenino , Equinococosis/inmunología , Equinococosis/veterinaria , Echinococcus granulosus/inmunología , Apoptosis/inmunología , Caspasa 3/inmunología , AdultoRESUMEN
BACKGROUND: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.
Asunto(s)
Antígenos CD4 , Antígenos CD8 , Caspasa 3 , Criptosporidiosis , FN-kappa B , Vacunas Antiprotozoos , Animales , Ratones , Caspasa 3/biosíntesis , Caspasa 3/inmunología , Antígenos CD4/biosíntesis , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/biosíntesis , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Criptosporidiosis/prevención & control , Criptosporidiosis/parasitología , Cryptosporidium , Cryptosporidium parvum/inmunología , Inmunohistoquímica , FN-kappa B/biosíntesis , FN-kappa B/inmunología , Vacunas Antiprotozoos/uso terapéutico , VacunasRESUMEN
BACKGROUND: One hallmark of sepsis is the reduced number of lymphocytes, termed lymphopenia, that occurs from decreased lymphocyte proliferation or increased cell death contributing to immune suppression. Histone modification enzymes regulate immunity by their epigenetic and non-epigenetic functions; however, the role of these enzymes in lymphopenia remains elusive. METHODS: We used molecular biological approaches to investigate the high expression and function of a chromatin modulator protein arginine N-methyltransferase 4 (PRMT4)/coactivator-associated arginine methyltransferase 1 in human samples from septic patients and cellular and animal septic models. RESULTS: We identified that PRMT4 is elevated systemically in septic patients and experimental sepsis. Gram-negative bacteria and their derived endotoxin lipopolysaccharide (LPS) increased PRMT4 in B and T lymphocytes and THP-1 monocytes. Single-cell RNA sequencing results indicate an increase of PRMT4 gene expression in activated T lymphocytes. Augmented PRMT4 is crucial for inducing lymphocyte apoptosis but not monocyte THP-1 cells. Ectopic expression of PRMT4 protein caused substantial lymphocyte death via caspase 3-mediated cell death signalling, and knockout of PRMT4 abolished LPS-mediated lymphocyte death. PRMT4 inhibition with a small molecule compound attenuated lymphocyte death in complementary models of sepsis. CONCLUSIONS: These findings demonstrate a previously uncharacterised role of a key chromatin modulator in lymphocyte survival that may shed light on devising therapeutic modalities to lessen the severity of septic immunosuppression.
Asunto(s)
Linfopenia , Proteína-Arginina N-Metiltransferasas , Sepsis , Animales , Humanos , Arginina/genética , Caspasa 3/genética , Caspasa 3/inmunología , Cromatina , Lipopolisacáridos/farmacología , Linfopenia/etiología , Linfopenia/genética , Linfopenia/inmunología , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Sepsis/complicaciones , Sepsis/genética , Sepsis/inmunologíaRESUMEN
Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8 DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.
Asunto(s)
Caspasa 3/inmunología , Caspasa 8/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Infecciones por Orthomyxoviridae/inmunología , Proteínas de Unión a Fosfato/inmunología , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Muerte Celular , Citocinas , Virus de la Influenza A/inmunología , Virus de la Influenza A/metabolismo , Ratones , Infecciones por Orthomyxoviridae/metabolismoRESUMEN
Two sets of innate immune proteins detect pathogens. Pattern recognition receptors (PRRs) bind microbial products, whereas guard proteins detect virulence factor activities by the surveillance of homeostatic processes within cells. While PRRs are well known for their roles in many types of infections, the role of guard proteins in most infectious contexts remains less understood. Here, we demonstrated that inhibition of protein synthesis during viral infection is sensed as a virulence strategy and initiates pyroptosis in human keratinocytes. We identified the BCL-2 family members MCL-1 and BCL-xL as sensors of translation shutdown. Virus- or chemical-induced translation inhibition resulted in MCL-1 depletion and inactivation of BCL-xL, leading to mitochondrial damage, caspase-3-dependent cleavage of gasdermin E, and release of interleukin-1α (IL-1α). Blocking this pathway enhanced virus replication in an organoid model of human skin. Thus, MCL-1 and BCL-xL can act as guard proteins within barrier epithelia and contribute to antiviral defense.
Asunto(s)
Apoptosis/inmunología , Células Epiteliales/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Piroptosis/inmunología , Receptores de Estrógenos/inmunología , Virus/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/inmunología , Caspasa 3/inmunología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Interleucina-1alfa/inmunología , Ratones , Mitocondrias/inmunología , Células 3T3 NIH , Células Vero , Replicación Viral/inmunología , Proteína bcl-X/inmunologíaRESUMEN
Cellular inflammasome activation causes caspase-1 cleavage of the pore-forming protein gasdermin D (GSDMD) with subsequent pyroptotic cell death and cytokine release. Here, we clarify the ambiguous role of the related family member gasdermin E (GSDME) in this process. Inflammasome stimulation in GSDMD-deficient cells led to apoptotic caspase cleavage of GSDME. Endogenous GSDME activation permitted sublytic, continuous interleukin-1ß (IL-1ß) release and membrane leakage, even in GSDMD-sufficient cells, whereas ectopic expression led to pyroptosis with GSDME oligomerization and complete liberation of IL-1ß akin to GSDMD pyroptosis. We find that NLRP3 and NLRP1 inflammasomes ultimately rely concurrently on both gasdermins for IL-1ß processing and release separately from their ability to induce cell lysis. Our study thus identifies GSDME as a conduit for IL-1ß release independent of its ability to cause cell death.
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Inflamasomas/genética , Interleucina-1beta/genética , Macrófagos/inmunología , Proteínas de Unión a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Piroptosis/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 3/genética , Caspasa 3/inmunología , Línea Celular Transformada , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas de Unión a Fosfato/deficiencia , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/inmunología , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Salmonella typhimurium/química , Salmonella typhimurium/patogenicidad , Transducción de Señal , Células THP-1RESUMEN
Natural polysaccharides have attracted considerable interests due to diverse biological activities. Succinoglycan is an extracellular polysaccharide produced by most Agrobacterium strains. Here, we confirmed riclin was a typical succinoglycan by NMR and methylation analysis, and investigated the antitumor effects of riclin in sarcoma 180 tumor-bearing mice. The results showed that riclin inhibited the tumor growth significantly as well as cyclophosphamide (CTX). While CTX caused serious damage to spleen structure, riclin increased the spleen index and promoted lymphocytes proliferation in peripheral blood, spleen and lymph nodes. Riclin decreased splenocytes apoptosis as evidenced by alterations of B-cell lymphoma-2 family proteins and Cleaved Caspase-3 protein. Moreover, 1H nuclear magnetic resonance (NMR)-based metabolomics analysis revealed that riclin partially altered the metabolic profiles of splenocytes. In conclusion, riclin is a succinoglycan that performed strong immunogenicity and suppressed sarcoma growth in mice. Succinoglycan riclin could be a potential antitumor agent for functional food and pharmaceutical purpose.
Asunto(s)
Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Factores Inmunológicos/farmacología , Linfocitos/efectos de los fármacos , Polisacáridos Bacterianos/farmacología , Sarcoma 180/tratamiento farmacológico , Agrobacterium/química , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Secuencia de Carbohidratos , Caspasa 3/genética , Caspasa 3/inmunología , Proliferación Celular/efectos de los fármacos , Ciclofosfamida/farmacología , Factores Inmunológicos/química , Factores Inmunológicos/aislamiento & purificación , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfocitos/inmunología , Linfocitos/patología , Masculino , Metaboloma/inmunología , Metilación , Ratones , Ratones Endogámicos C57BL , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Sarcoma 180/genética , Sarcoma 180/inmunología , Sarcoma 180/patología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: Salmonella enterica serovar Typhimurium, a non-typhoidal food-borne pathogen, causes acute enterocolitis, bacteremia, extraintestinal focal infections in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) contribute to invading into host cellular cytosol, residing in Salmonella-containing vacuoles for intracellular survival, and inducing cellular apoptosis. This study aimed to better understand the mechanism underlying apoptosis in Salmonella-infected macrophages. METHODS: S. Typhimurium SL1344 was used to evaluate extrinsic and intrinsic apoptosis pathways in THP-1 monocyte-derived macrophages in response to Salmonella infection. RESULTS: Activated caspase-3-induced apoptosis pathways, including extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, in Salmonella-infected macrophages were verified. THP-1 cells with dysfunction of TLR-4 and TLR-5 and Salmonella SPI-1 and SPI-2 mutants were constructed to identify the roles of the genes associated with programmed cell death in the macrophages. Caspase-3 activation in THP-1 macrophages was induced by Salmonella through TLR-4 and TLR-5 signaling pathways. We also identified that SPI-1 structure protein PrgH and effectors SipB and SipD, but not SPI-2 structure protein SsaV, could induce apoptosis via caspase-3 activation and reduce the secretion of inflammation marker TNF-α in the Salmonella-infected cells. The two effectors also reduced the translocation of the p65 subunit of NF-κB into the nucleus and the expression of TNF-α, and then inflammation was diminished. CONCLUSION: Non-typhoid Salmonella induced apoptosis of macrophages and thereby reduced inflammatory cytokine production through the expression of SPI-1. This mechanism in host-pathogen interaction may explain why Salmonella usually manifests as occult bacteremia with less systemic inflammatory response syndrome in the bloodstream infection of children.
Asunto(s)
Apoptosis , Islas Genómicas/genética , Interacciones Huésped-Patógeno , Macrófagos/microbiología , Macrófagos/patología , Salmonella typhimurium/genética , Caspasa 3/genética , Caspasa 3/inmunología , Línea Celular , Citocinas/genética , Citocinas/inmunología , Humanos , Inflamación , Macrófagos/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Células THP-1 , VirulenciaRESUMEN
BACKGROUND: There are two distinctive acral manifestations of COVID-19 embodying disparate clinical phenotypes. One is perniosis occurring in mildly symptomatic patients, typically children and young adults; the second is the thrombotic retiform purpura of critically ill adults with COVID-19. OBJECTIVES: To compare the clinical and pathological profiles of these two different cutaneous manifestations of COVID-19. METHODS: We compared the light microscopic, phenotypic, cytokine and SARS-CoV-2 protein and RNA profiles of COVID-19-associated perniosis with that of thrombotic retiform purpura in critical patients with COVID-19. RESULTS: Biopsies of COVID-19-associated perniosis exhibited vasocentric and eccrinotropic T-cell- and monocyte-derived CD11c+ , CD14+ and CD123+ dendritic cell infiltrates. Both COVID-associated and idiopathic perniosis showed striking expression of the type I interferon-inducible myxovirus resistance protein A (MXA), an established marker for type I interferon signalling in tissue. SARS-CoV-2 RNA, interleukin-6 and caspase 3 were minimally expressed and confined to mononuclear inflammatory cells. The biopsies from livedo/retiform purpura showed pauci-inflammatory vascular thrombosis without any MXA decoration. Blood vessels exhibited extensive complement deposition with endothelial cell localization of SARS-CoV-2 protein, interleukin-6 and caspase 3; SARS-CoV-2 RNA was not seen. CONCLUSIONS: COVID-19-associated perniosis represents a virally triggered exaggerated immune reaction with significant type I interferon signaling. This is important to SARS-CoV-2 eradication and has implications in regards to a more generalized highly inflammatory response. We hypothesize that in the thrombotic retiform purpura of critically ill patients with COVID-19, the vascular thrombosis in the skin and other organ systems is associated with a minimal interferon response. This allows excessive viral replication with release of viral proteins that localize to extrapulmonary endothelium and trigger extensive complement activation.
Asunto(s)
COVID-19/complicaciones , Eritema Pernio/diagnóstico , Livedo Reticularis/diagnóstico , Púrpura/diagnóstico , SARS-CoV-2/inmunología , Adolescente , Factores de Edad , Anciano , Biopsia , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/virología , Caspasa 3/inmunología , Caspasa 3/metabolismo , Eritema Pernio/inmunología , Eritema Pernio/patología , Diagnóstico Diferencial , Femenino , Pie , Mano , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Livedo Reticularis/inmunología , Livedo Reticularis/patología , Livedo Reticularis/virología , Masculino , Persona de Mediana Edad , Proteínas de Resistencia a Mixovirus/análisis , Proteínas de Resistencia a Mixovirus/metabolismo , Púrpura/inmunología , Púrpura/patología , Púrpura/virología , ARN Viral/aislamiento & purificación , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la Enfermedad , Piel/inmunología , Piel/patología , Piel/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificaciónRESUMEN
Enterovirus 71 (EV71)-induced T lymphocyte apoptosis plays an important role in hand, foot, and mouth disease (HFMD), and granzyme B (GZMB) has been shown to be critical for this process. However, the mechanisms underlying GZMB-mediated apoptosis of T lymphocytes remain unknown. In this study, we investigated whether transcription factors and microRNAs (miRNAs) are involved in GZMB-mediated apoptosis of T lymphocytes in response to EV71 infection. Our findings indicated that EV71 infection significantly induced apoptosis in Jurkat cells, a human T lymphocytes cell line, as revealed in flow cytometric analysis. Furthermore, EV71 increased the expression of pro-apoptosis Bcl-2-associated X (Bax) and cleaved caspase 3 but decreased the expression of anti-apoptosis B-cell lymphoma protein 2 (Bcl2). GZMB knockdown decreased cell apoptosis and prevented EV71-induced changes in the expression of Bax, cleaved caspase 3, and Bcl2 in Jurkat cells, highlighting the role of GZMB as a key factor in EV71-induced apoptosis. Our study also indicated that overexpression of the transcription factors GATA binding factor 1 (GATA1) and specificity protein 1 (SP1) significantly increased luciferase activity when this gene was inserted in the GZMB 3' untranslated region (3'UTR). GATA1/SP1 overexpression induced cell apoptosis, increased the expression of Bax and cleaved caspase 3, and decreased the expression of Bcl2. Finally, our results suggested that miR-874 plays an essential role in GZMB-mediated cell apoptosis, since an miR-874 mimic decreases the expression of GZMB by targeting its 3'UTR. Collectively, these data indicated that GATA1/SP1 and miR-874 mediate EV71-induced apoptosis in a granzyme B-dependent manner. This signaling pathway may provide a new pharmacological target for the prevention and treatment of HFMD.
Asunto(s)
Enterovirus Humano A/genética , Infecciones por Enterovirus/virología , Factor de Transcripción GATA1/metabolismo , Granzimas/metabolismo , MicroARNs/metabolismo , Factor de Transcripción Sp1/metabolismo , Apoptosis , Caspasa 3/inmunología , Enterovirus Humano A/fisiología , Humanos , Células Jurkat , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its metabolites (N-desmethyltamoxifen and endoxifen) on the mitochondrial membrane potential assay by flow cytometry, and the activation of effector caspase-3 through immunoblotting, in peripheral blood neutrophils obtained from healthy horses (n = 5). Results show that tamoxifen, N-desmethyltamoxifen and endoxifen depolarize the mitochondrial membrane and activate caspase-3 in healthy equine neutrophils in vitro. These findings suggest that tamoxifen and its metabolites may activate the intrinsic apoptotic pathway in equine neutrophils. However, more studies are necessary to further explore the signalling pathways of these drugs in the induction of apoptosis.
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Antiasmáticos/farmacología , Caspasa 3/inmunología , Caballos/inmunología , Inmunidad Innata/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Animales , Femenino , Citometría de Flujo/veterinaria , Immunoblotting/veterinaria , Masculino , Membranas Mitocondriales/fisiología , Neutrófilos/inmunologíaRESUMEN
To evaluate the effect of GuttaFlow bioseal (GFB) and MTA Fillapex (MTAF) in comparison with Endofill (EF) in the subcutaneous tissue. Polyethylene tubes with GFB, MTAF, EF or empty tubes (control group; CG) were implanted into subcutaneous of rats. After 7, 15, 30 and 60 days, the capsule thickness, inflammatory reaction, interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), caspase-3, TUNEL-positive cells, von Kossa and ultrastructural features were evaluated. The data were statistically analyzed (p ≤ 0.05). At all periods, the number of IL-6- and VEGF-immunolabelled cells, and capsule thickness were lower in GFB than MTAF, which was lower than EF (p < 0.0001). At 60 days, the number of inflammatory cells was similar in GFB and MTAF (p = 0.58). Significant differences in the number of TUNEL- and caspase-3-positive cells were not observed among GFB, MTAF and CG whereas the highest values were found in EF specimens. The EF specimens exhibited several cells with condensed chromatin, typical of apoptosis. von Kossa-positive and birefringent structures were only observed in GFB and MTAF, suggesting the presence of calcite crystals. Taken together, these results show that cellular and structural damage induced by GFB and MTAF sealers were recovery over time. Moreover, these sealers express bioactive potential in subcutaneous tissue.
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Apoptosis/efectos de los fármacos , Dimetilpolisiloxanos/farmacología , Gutapercha/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Tejido Subcutáneo/inmunología , Animales , Apoptosis/inmunología , Caspasa 3/inmunología , Combinación de Medicamentos , Inflamación/inmunología , Inflamación/patología , Interleucina-6/inmunología , Masculino , Ratas , Ratas Sprague-Dawley , Tejido Subcutáneo/patología , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
The use of terrestrial raw materials to replace fish meal (FM) and fish oil (FO) in marine fish diets may affect fish growth performance and health. In the last years functional additives have been profiled as good candidates to reduce the effects on health and disease resistance derived from this replacement, via reinforcement of the fish immune system. In the present study, three isoenergetic and isonitrogenous diets with low FM and FO (10% and 6% respectively) were tested based on supplementation either with 0.5% galactomannanoligosaccharides (GMOS diet) or 0.02% of a mixture of essential oils (PHYTO diet), a non-supplemented diet was defined as a control diet. Fish were fed the experimental diets in triplicate for 9 weeks and then they were subjected to a stress by confinement as a single challenge (C treatment) or combined with an experimental intestinal infection with Vibrio anguillarum (CI treatment). Along the challenge test, selected stress and immunological parameters were evaluated at 2, 24 and 168h after C or CI challenges. As stress indicators, circulating plasma cortisol and glucose concentrations were analyzed as well as the relative gene expression of cyp11b hydroxylase, hypoxia inducible factor, steroidogenic acute regulatory protein, heat shock protein 70 and heat shock protein 90 (cyp11b, hif-1α, StAR, hsp70 and hsp90). As immune markers, serum and skin mucus lysozyme, bactericidal and peroxidase activities were measured, as well as gene expression of Caspase-3 (casp-3) and interleukin 1ß (il-1ß). The use of functional additives induced a significant (p < 0.05) reduction of circulating plasma cortisol concentration when confinement was the unique challenge test applied. Supplementation of PHYTO induced a down-regulation of cyp11b, hif-1α, casp-3 and il-1ß gene expression 2h after stress test, whereas StAR expression was significantly (p < 0.05) up-regulated. However, when combination of confinement stress and infection was applied (CI treatment), the use of PHYTO significantly (p < 0.05) down-regulated StAR and casp-3 gene expression 2h after challenge test, denoting that PHYTO diet reinforced fish capacity of stress response via protection of head kidney leucocytes from stress-related apoptotic processes, with lower caspase-3 gene expression and a higher il-1ß gene expression when an infection occurs. Additionally, dietary supplementation with GMOS and PHYTO compounds increased fish serum lysozyme after infection. Both functional additives entailed a better capability of the animals to cope with infection in European sea bass when fed low FM and FO diets.
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Alimentación Animal/análisis , Lubina/inmunología , Suplementos Dietéticos/análisis , Aceites de Pescado/administración & dosificación , Prebióticos/administración & dosificación , Estrés Fisiológico , Animales , Acuicultura , Lubina/genética , Caspasa 3/genética , Caspasa 3/inmunología , Resistencia a la Enfermedad , Hidrocortisona/sangre , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Aceites Volátiles/administración & dosificación , Oligosacáridos/administración & dosificaciónRESUMEN
Varicella-zoster virus (VZV) is a pathogenic human herpesvirus that causes varicella (chickenpox) as a primary infection following which it becomes latent in ganglionic neurons. Following viral reactivation many years later VZV causes herpes zoster (shingles) as well as a variety of other neurological syndromes. The molecular mechanisms of the conversion of the virus from a lytic to a latent state in ganglia are not well understood. In order to gain insights into the neuron-virus interaction, we studied virus-induced apoptosis in cultures of both highly pure terminally differentiated human neurons and human fetal lung fibroblasts (HFL). It was found that (a) VZV DNA did not accumulate in infected human neurons; (b) VZV transcripts were present at lower levels at all days studied post-infection in neurons; (c) Western blot analysis showed less VZV IE 63 and very little detectable VZV gE proteins in infected neurons compared with HFL; (d) lower levels of the apoptotic marker cleaved Caspase-3 protein were detected in VZV-infected neurons compared with HFL, and higher levels of the known anti-apoptotic proteins Bcl2, Bcl-XL and also the mitochondrial MT-CO2 protein were found in VZV-infected neurons compared with uninfected cells; and (e) both the MT-CO2 protein and VZV IE 63-encoded protein were detected in infected neurons by dual immunofluorescence. These findings showed that neurons are resistant to VZV-induced apoptosis, which may have relevance to the switching of VZV from a lytic to latent ganglionic neuronal infection.
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Apoptosis/genética , ADN Viral/genética , Herpesvirus Humano 3/genética , Interacciones Huésped-Patógeno/genética , Neuronas/virología , Latencia del Virus/genética , Apoptosis/inmunología , Caspasa 3/genética , Caspasa 3/inmunología , Línea Celular , ADN Viral/inmunología , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/inmunología , Feto , Fibroblastos/inmunología , Fibroblastos/virología , Regulación de la Expresión Génica , Herpesvirus Humano 3/crecimiento & desarrollo , Herpesvirus Humano 3/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Neuronas/inmunología , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Transducción de Señal , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Latencia del Virus/inmunología , Proteína bcl-X/genética , Proteína bcl-X/inmunologíaRESUMEN
The prognostic value of Toll-like receptor 3 (TLR3) is debated in cancer, differing between tumor types, methods, and cell types. We recently showed for the first time that TLR3 expression on early stage non-small-cell lung cancer (NSCLC) results associated with a good prognosis. Here, we provide experimental evidences explaining the molecular reason behind TLR3's favorable prognostic role. We demonstrated that TLR3 activation in vitro induces apoptosis in lung cancer cell lines and, accordingly, that TLR3 expression is associated with caspase-3 activation in adenocarcinoma NSCLC specimens, both evaluated by immunohistochemistry. Moreover, we showed that TLR3 expression on cancer cells contributes to activate the CD103+ lung dendritic cell subset, that is specifically associated with processing of antigens derived from apoptotic cells and their presentation to CD8+ T lymphocytes. These findings point to the relevant role of TLR3 expression on lung cancer cells and support the use of TLR3 agonists in NSCLC patients to re-activate local innate immune response.
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Apoptosis/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Neoplasias Pulmonares/inmunología , Proteínas de Neoplasias/inmunología , Receptor Toll-Like 3/inmunología , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Caspasa 3/inmunología , Línea Celular Tumoral , Humanos , Inmunidad Innata , Inmunoterapia , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Ratones , Receptor Toll-Like 3/agonistasRESUMEN
Caspase 3 plays an important role in apoptotic pathways and contributes to maintaining the homeostasis of the immune system in organisms. The structure, functions, and characteristics of caspase 3 have been extensively investigated in many species, but the research is scarce when it comes to bivalves, particularly oysters. In this study, we identified and cloned a previously unknown caspase 3 gene, named ChCas 3, in C. hongkongensis. The full-length cDNA of ChCas 3 was 1562 bp and included a 175 bp 5'-untranslated region (UTR), a 141 bp 3'-UTR and a 1245 bp open reading frame (ORF) that encoded a polypeptide of 415 amino acids. Similar to caspase 3 in other species, ChCas 3 has a pro-domain, a conserved cysteine active site, a large p20 subunit and a small p10 subunit. Our findings demonstrated the expression of ChCas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. ChCas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1-ChCas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of ChCas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of ChCas 3 led to upregulation of the transcriptional activity of NF-κB and p53 reporter genes in HEK293T cells, which indicated that it was involved in innate immune responses. Finally, the apoptosis rate of the haemocytes declined considerably compared with that of the control group after the expression of ChCas 3 was successfully silenced by dsRNA, corroborating its sentinel role in apoptosis. This study provides comprehensive underpinning evidences, affirming caspase 3 crucial role against bacterial infection and apoptosis in C. hongkongensis.
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Apoptosis/genética , Caspasa 3/genética , Caspasa 3/inmunología , Crassostrea/genética , Crassostrea/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Animales , Células HEK293 , Hemocitos/metabolismo , HumanosRESUMEN
Effector CD8+ T cells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ T cells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ T cell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector T cell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ T cell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ T cells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ T cells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ T cell immunity.
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Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Fibrinógeno/inmunología , Receptores de IgG/inmunología , Adulto , Anciano , Animales , Caspasa 3/inmunología , Caspasa 7/inmunología , Línea Celular Tumoral , Femenino , Fibrinógeno/genética , Rechazo de Injerto/inmunología , Humanos , Inmunoglobulina G/inmunología , Terapia de Inmunosupresión , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores de IgG/genética , Adulto JovenRESUMEN
Ectopic lymphoid tissues (eLTs) characterized by B cell aggregation contribute to the local immunoglobulin production in nasal polyps (NPs). B cell-activating factor (BAFF) is vital for B cell survival, proliferation, and maturation. The purpose of this study is to investigate whether BAFF is involved in the B cell survival and eLT formation in NPs. The mRNA and protein levels of BAFF in NP tissues with and without eLTs were detected by PCR and ELISA assay, respectively. The cellular sources of BAFF and active caspase-3-positive B cells in NPs were studied by immunofluorescence staining. B cells purified from NP tissues were stimulated with BAFF and were analyzed by flow cytometry. Stromal cells purified from NP tissues were stimulated with lymphotoxin (LT) α1ß2, and BAFF levels in culture supernatants were analyzed by ELISA. Compared with those in control tissues and NPs without eLTs, the BAFF levels were elevated in NPs with eLTs. Abundant BAFF-positive cells and few active caspase-3-positive apoptotic B cells were found in NPs with eLTs, in contrast to those in NPs without eLTs. There was a negative correlation between the numbers of BAFF-positive cells and frequencies of apoptotic B cells in total B cells in NP tissues. BAFF protected nasal polyp B cells from apoptosis in vitro. Stromal cells were an important cellular source of BAFF in NPs with eLTs. LTα1ß2 induced BAFF production from nasal stromal cells in vitro. We propose that BAFF contribute to eLT formation in NPs by promoting B cell survival.
Asunto(s)
Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Pólipos Nasales/inmunología , Estructuras Linfoides Terciarias/inmunología , Apoptosis/inmunología , Linfocitos B/patología , Caspasa 3/inmunología , Supervivencia Celular , Femenino , Humanos , Heterotrímero de Linfotoxina alfa1 y beta2/inmunología , Masculino , Pólipos Nasales/patología , Estructuras Linfoides Terciarias/patologíaRESUMEN
Enterovirus A71 (EV-A71) is the major pathogen responsible for the severe hand, foot and mouth disease worldwide, for which few effective antiviral drugs are presently available. Interferon-α (IFN-α) has been used in antiviral therapy for decades; it has been reported that EV-A71 antagonizes the antiviral activity of IFN-α based on viral 2Apro-mediated reduction of the interferon-alpha receptor 1 (IFNAR1); however, the mechanism remains unknown. Here, we showed a significant increase in IFNAR1 protein induced by IFN-α in RD cells, whereas EV-A71 infection caused obvious down-regulation of the IFNAR1 protein and blockage of IFN-α signaling. Subsequently, we observed that EV-A71 2Apro inhibited IFNAR1 translation by cleavage of the eukaryotic initiation factor 4GI (eIF4GI), without affecting IFNAR1 mRNA levels induced by IFN-α. The inhibition of IFNAR1 translation also occurred in puromycin-induced apoptotic cells when caspase-3 cleaved eIF4GI. Importantly, we verified that 2Apro could activate cellular caspase-3, which was subsequently involved in eIF4GI cleavage mediated by 2Apro. Furthermore, inhibition of caspase-3 activation resulted in the partial restoration of IFNAR1 in cells transfected with 2A or infected with EV-A71, suggesting the pivotal role of both viral 2Apro and caspase-3 activation in the disturbance of IFN-α signaling. Collectively, we elucidate a novel mechanism by which cellular caspase-3 contributes to viral 2Apro-mediated down-regulation of IFNAR1 at the translation level during EV-A71 infection, indicating that caspase-3 inhibition could be a potential complementary strategy to improve clinical anti-EV-A71 therapy with IFN-α.