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1.
Methods Mol Biol ; 2276: 203-213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060043

RESUMO

To evaluate how a cell responds to the external stimuli, treatment, or alteration of the microenvironment, the quantity and quality of mitochondria are commonly used as readouts. However, it is challenging to apply mitochondrial analysis to the samples that are composed of mixed cell populations originating from tissues or when multiple cell populations are of interest, using methods such as Western blot, electron microscopy, or extracellular flux analysis.Flow cytometry is a technique allowing the detection of individual cell status and its identity simultaneously when used in combination with surface markers. Here we describe how to combine mitochondria-specific dyes or the dyes targeting the superoxide produced by mitochondria with surface marker staining to measure the mitochondrial content and activity in live cells by flow cytometry. This method can be applied to all types of cells in suspension and is particularly useful for analysis of samples composed of heterogeneous cell populations.


Assuntos
Células Sanguíneas/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Baço/metabolismo , Superóxidos/metabolismo , Animais , Células Sanguíneas/citologia , Células Sanguíneas/ultraestrutura , Humanos , Baço/citologia , Baço/ultraestrutura
2.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070449

RESUMO

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-ß, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.


Assuntos
Neoplasias da Mama/metabolismo , Mucina-1/metabolismo , Células Supressoras Mieloides/imunologia , Neoplasias Pancreáticas/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-1/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/metabolismo , Fator de Crescimento Transformador beta/sangue , Microambiente Tumoral/genética
3.
Ecotoxicol Environ Saf ; 220: 112386, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34090108

RESUMO

Atrazine (ATR) is a herbicide used widely worldwide. Because of its prolonged persistence in the environment and accumulation in the body, ATR exposure is a potential threat to human health. Our previous study showed that subacute exposure to ATR suppresses cellular immune function in mice. In this study, the effects of long-term exposure to ATR on rat immunological system function were measured. Four-week-old female Sprague-Dawley (SD) rats were treated with 0.4 µmol/L, 2 µmol/L and 10 µmol/L ATR for 24 weeks. The results showed that the spleen index increased, white blood cells decreased, and monocytes and eosinophils increased. No obvious changes were detected in the numbers of neutrophils and lymphocytes. Th1, Th2, and Th17 cells decreased significantly, while Treg cells increased after long-term ATR exposure. Moreover, serum levels of cytokines, including TNF-α, INF-γ, IL-6, and IL-12, decreased, while IL-1, IL-4, and IL-5 increased. Degenerative changes and cell apoptosis were found in the spleen; Caspase-3 and Caspase-9 were upregulated, and Bcl-2 was downregulated. These results suggested that long-term ATR exposure may inhibit immune system function.


Assuntos
Apoptose/efeitos dos fármacos , Atrazina/toxicidade , Herbicidas/toxicidade , Baço/efeitos dos fármacos , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Linfócitos T Auxiliares-Indutores
4.
Nat Commun ; 12(1): 2856, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001893

RESUMO

Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.


Assuntos
Neutrófilos/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Ontologia Genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Peritonite/genética , Peritonite/patologia , Pneumonia/genética , Pneumonia/patologia , Baço/citologia , Baço/metabolismo
5.
Front Immunol ; 12: 581799, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953705

RESUMO

Forkhead box O 3 (FOXO3) is a transcription factor involved in cell metabolism, inflammation and longevity. Here, we investigated if metformin can activate FOXO3 in human immune cells and affects the subsequent level of reactive oxygen/nitrogen species (ROS/RNS) in immune cells. AMP-activated protein kinase (AMPK) and FOXO3 activation were investigated by immunoblot or flow cytometry (FC) analysis, respectively. FOXO3 target gene expression was quantified by real-time PCR. ROS/RNS measurement using dichlorodihydrofluorescein diacetate (DCFH-DA) dye was investigated by FC. The role of the FOXO3 single nucleotide polymorphisms (SNPs) rs12212067, rs2802292 and rs12206094 on ROS/RNS production was studied using allelic discrimination PCR. Metformin induced activation of AMPK (pT172) and FOXO3 (pS413). ROS/RNS level was reduced in immune cells after metformin stimulation accompanied by induction of the FOXO3 targets mitochondrial superoxide dismutase and cytochrome c. Studies in Foxo3 deficient (Foxo3-/- ) mouse splenocytes confirmed that metformin mediates its effects via Foxo3 as it attenuates ROS/RNS in myeloid cells of wildtype (WT) but not of Foxo3-/- mice. Our results suggest that FOXO3 can be activated by metformin leading to reduced ROS/RNS level in immune cells. This may add to the beneficial clinical effects of metformin observed in large cohort studies on longevity, cardiovascular and cancer risk.


Assuntos
Proteína Forkhead Box O3/metabolismo , Sistema Imunitário/efeitos dos fármacos , Metformina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Proteína Forkhead Box O3/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo
6.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33827945

RESUMO

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Memória Imunológica , Vírus da Influenza A Subtipo H1N1/imunologia , Células Matadoras Naturais/imunologia , Infecções por Orthomyxoviridae/imunologia , Transferência Adotiva , Animais , Antígenos Ly/análise , Antígenos Ly/metabolismo , Benzotiazóis/farmacologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cocultura , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Receptor 1 Desencadeador da Citotoxicidade Natural/análise , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Baço/citologia , Baço/imunologia , Tolueno/análogos & derivados , Tolueno/farmacologia
7.
Nutrients ; 13(4)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801785

RESUMO

Combination feeding (human milk and formula) is common and influences immune development compared to exclusive breastfeeding. Infant formulas contain prebiotics, which influence immune development. Herein, immune development of combination-fed (CF), sow-reared (SR) and formula-fed (FF) piglets, and the effect of prebiotics was tested. Piglets (n = 47) were randomized to: SR, FF, CF, FF+prebiotic (FP), and CF+prebiotic (CP). FP and CP received formula with galactooligosaccharides and inulin (4 g/L in a 4:1 ratio). CF and CP piglets were sow-reared for until d5 and then rotated between a sow and formula every 12 h. On day 21, piglets received an intraperitoneal injection of lipopolysaccharide 2 h prior to necropsy. Immune cells from blood, mesenteric lymph nodes (MLN), and spleen were phenotyped. Classical (nitric oxide synthase) and alternative (arginase activity) activation pathways were measured in isolated macrophages. Serum IL-6 and TNF-α were measured by ELISA. SR piglets had lower (p < 0.0001) CD4+ T-helper cells and higher (p < 0.0001) B-cells in PBMC than all other groups. CP piglets had higher (p < 0.0001) arginase activity compared to all other groups. FF piglets had higher (p < 0.05) IL-6 compared to both CF and SR, but were similar to FP and CP. Thus, CF, with or without prebiotics, differentially affected immunity compared to exclusively fed groups.


Assuntos
Ração Animal , Dieta/veterinária , Alimentos Formulados , Prebióticos , Suínos/crescimento & desenvolvimento , Fenômenos Fisiológicos da Nutrição Animal , Animais , Animais Lactentes , Citocinas/genética , Citocinas/metabolismo , Feminino , Linfonodos/citologia , Baço/citologia
8.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801683

RESUMO

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.


Assuntos
Fatores de Transcrição Forkhead/genética , Inativação Gênica , Imunogenicidade da Vacina , Oligonucleotídeos Antissenso/química , Sporothrix/imunologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Farmacêuticos , Animais , Sistema Imunitário , Interferon gama/metabolismo , Subunidade p35 da Interleucina-12/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Linfócitos T Reguladores/metabolismo
9.
Toxins (Basel) ; 13(5)2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926136

RESUMO

The objective of this study was to determine the impact of Ageratina adenophora (A. adenophora) on splenic immune function in a rat model. Rats were fed with 10 g/100 g normal feed and an experimental feed, which was composed of 3:7 A. adenophora powder and normal feed for 60 days. On days 14, 28, and 60, subsets of rats (n = 8 rats/group/time point) were selected for blood and spleen tissue sample collection. The results showed that the proportion of CD3+ T cells in the spleen was decreased at day 60 (vs. control). Also, mRNA and protein expression of chemokines CCL21 and CCL19 and functional protein gp38 in spleen decreased significantly versus the control at day 60. In addition, ER-TR7 antigen protein expression was also decreased at day 60. Levels of T-helper (Th)1 cells significantly increased, whereas those of Th2 cells decreased significantly versus the control at day 60 in spleen. The finding revealed that A. adenophora could affect splenic immune function in rats by altering the fibroblast reticulocyte (FRC) network, as well as by causing an imbalance in Th1/Th2 cell ratios. This research provides new insights into potential mechanisms of spleen immunotoxicity due to exposures to A. Adenophora.


Assuntos
Ageratina/efeitos adversos , Reticulócitos/efeitos dos fármacos , Baço/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Animais , Fibroblastos/efeitos dos fármacos , Contagem de Linfócitos , Masculino , Folhas de Planta , Ratos , Ratos Sprague-Dawley , Baço/citologia , Baço/imunologia
10.
Food Funct ; 12(9): 4176-4198, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33861291

RESUMO

Herein, polyphenols were extracted from Pinus koraiensis bark and characterized. Besides, the in vitro antioxidant activity, inhibition effect on cancer cells and the activity of the immune system were investigated. The results showed that the main component of Pinus koraiensis bark was 3,5,7,3',5'-pentahydroxydihydroflavone. PKB polyphenols demonstrated a high antioxidant activity during in vitro investigation. In vivo immunological function studies on oxidatively injured mice revealed that Pinus koraiensis bark polyphenols effectively improved the survival status of irradiated mice. PKBP also increased the spleen and thymus index of mouse immunoregulatory organs. The results indicated that the phagocytic ability of mononuclear macrophages was increased. Comparing the cell distribution of the PKBP administered group and the model group, the PKBP-administered group reduced the cells arrested in the G1 phase, while the number of cells increased in the S and G2 phases. PKBP effectively protected the mouse immune system and reduced the immune suppression caused by radiation. These findings also confirmed that oxidative damaged cells induced by radiation could be repaired. PKBP had the highest inhibitory activity on colon cancer cells HT29, breast cancer cells MFC-7, gastric cancer cells BGC-823 and cervical cancer HeLa and HT29 cancer cells. PKB polyphenols could effectively induce the production of DNA-Ladder fragments and cause DNA damage in cancer cells. PKBP also blocked the cycle of cancer cells in the G2 phase, stopped cell division and induced cancer cell apoptosis. Analysis of cell apoptosis by Annexin V-FTIC/PI double staining indicated that PKBP inhibited HT29 cancer cell proliferation.


Assuntos
Antioxidantes , Neoplasias/patologia , Pinus/química , Polifenóis/química , Polifenóis/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Contagem de Células Sanguíneas , Peso Corporal/efeitos dos fármacos , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular , Sobrevivência Celular , Feminino , Sequestradores de Radicais Livres/farmacologia , Radicais Livres , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Estrutura Molecular , Oxirredução , Estresse Oxidativo/imunologia , Fagocitose , Casca de Planta/química , Polifenóis/isolamento & purificação , Baço/citologia
11.
Int J Mol Sci ; 22(8)2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33920631

RESUMO

BACKGROUND: The adaptor protein Src homology 3 domain-binding protein 2 (SH3BP2) is widely expressed in immune cells. It controls intracellular signaling pathways. The present study was undertaken to investigate the role of SH3BP2 in a murine systemic lupus erythematosus model. METHODS: For the lupus model, we used Faslpr/lpr mice. Clinical and immunological phenotypes were compared between Faslpr/lpr and SH3BP2-deficient Faslpr/lpr mice. Splenomegaly and renal involvement were assessed. Lymphocyte subsets in the spleen were analyzed by flow cytometry. To examine the role of SH3BP2 in specific cells, B cell-specific SH3BP2-deficient lupus mice were analyzed; T cells and bone marrow-derived dendritic cells and macrophages were analyzed in vitro. RESULTS: SH3BP2 deficiency significantly reduced lupus-like phenotypes, presented as splenomegaly, renal involvement, elevated serum anti-dsDNA antibody, and increased splenic B220+CD4-CD8- T cells. Notably, SH3BP2 deficiency in B cells did not rescue the lupus-like phenotypes. Furthermore, SH3BP2 deficiency did not substantially affect the characteristics of T cells and macrophages in vitro. Interestingly, SH3BP2 deficiency suppressed the differentiation of dendritic cells in vitro and reduced the number of dendritic cells in the spleen of the lupus-prone mice. CONCLUSIONS: SH3BP2 deficiency ameliorated lupus-like manifestations. Modulating SH3BP2 expression could thus provide a novel therapeutic approach to autoimmune diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Lúpus Eritematoso Sistêmico/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Rim/citologia , Rim/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia
12.
Methods Mol Biol ; 2285: 35-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928541

RESUMO

The development of T helper (Th) cell subsets requires activated T cells that respond to a polarizing cytokine environment, resulting in the activation and expression of specific transcription factors. The subset-specific transcription factors are located either in the cytoplasm or in the nucleus, which determine the functional profile of Th populations, inducing the production of specific effector cytokines and functions. Flow cytometry analysis of transcription factors has become very common not only in research but also in immunologic follow-up protocols of patients recruited in clinical trials (as evaluation of CD4+CD25+ FOXP3+ T regulatory cells). Here, we propose and describe one-step protocols to evaluate the expression of transcription factors in mouse and human CD4+ lymphocytes, focusing the critical points of this cytometric approach.


Assuntos
Separação Celular , Citometria de Fluxo , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Humanos , Linfonodos/citologia , Camundongos , Fenótipo , Projetos de Pesquisa , Baço/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Fluxo de Trabalho
13.
Methods Mol Biol ; 2285: 131-139, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928549

RESUMO

A critical property of T cells when activated by their cognate antigen-MHC complex is the initiation of cell cycle activity and clonal expansion. In this chapter, we describe how the proliferation of T cells can be assessed on the single cell level by flow cytometry and how this can be used to identify and potentially isolate antigen-reactive T cells.


Assuntos
Antígenos , Proliferação de Células , Citometria de Fluxo , Ativação Linfocitária , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Linfócitos T/imunologia , Animais , Separação Celular , Células Cultivadas , Corantes Fluorescentes/metabolismo , Humanos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Projetos de Pesquisa , Baço/citologia , Linfócitos T/metabolismo , Fluxo de Trabalho
14.
Front Immunol ; 12: 613597, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746957

RESUMO

Increased concentrations of circulating chromatin, especially oligo-nucleosomes, are observed in sepsis, cancer and some inflammatory autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, circulating nucleosomes mainly result from increased apoptosis and decreased clearance of apoptotic cells. Once released, nucleosomes behave both as an autoantigen and as a damage-associated molecular pattern (DAMP) by activating several immune cells, especially pro-inflammatory cells. Deoxyribonuclease 1 (DNase1) is a major serum nuclease whose activity is decreased in mouse and human lupus. Likewise, the mitochondrial chaperone tumor necrosis factor (TNF) receptor-associated protein-1 (Trap1) protects against oxidative stress, which is increased in SLE. Here, using wild type, DNase1-deficient and DNase1/Trap1-deficient mice, we demonstrate that DNase1 is a major serum nuclease involved in chromatin degradation, especially when the plasminogen system is activated. In vitro degradation assays show that chromatin digestion is strongly impaired in serum from DNase1/Trap1-deficient mice as compared to wild type mice. In vivo, after injection of purified chromatin, clearance of circulating chromatin is delayed in DNase1/Trap1-deficient mice in comparison to wild type mice. Since defective chromatin clearance may lead to chromatin deposition in tissues and subsequent immune cell activation, spleen cells were stimulated in vitro with chromatin. Splenocytes were activated by chromatin, as shown by interleukin (IL)-12 secretion and CD69 up-regulation. Moreover, cell activation was exacerbated when Trap1 is deficient. Importantly, we also show that cytokines involved in lupus pathogenesis down-regulate Trap1 expression in splenocytes. Therefore, combined low activities of both DNase1 and Trap1 lead to an impaired degradation of chromatin in vitro, delayed chromatin clearance in vivo and enhanced activation of immune cells. This situation may be encountered especially, but not exclusively, in SLE by the negative action of cytokines on Trap1 expression.


Assuntos
Cromatina/metabolismo , Citocinas/biossíntese , Desoxirribonuclease I/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mediadores da Inflamação/metabolismo , Animais , Desoxirribonuclease I/sangue , Espaço Extracelular , Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Leucócitos/metabolismo , Leucócitos/patologia , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Knockout , Baço/citologia , Baço/metabolismo
15.
J Cell Mol Med ; 25(10): 4870-4876, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33733611

RESUMO

Under steady-state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8+ T cells compared with WT counterparts. Finally, Med1-deficient CD8+ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.


Assuntos
Linfócitos T CD8-Positivos , Subunidade 1 do Complexo Mediador/imunologia , Receptores de Interleucina-7/imunologia , Baço/imunologia , Animais , Apoptose , Células da Medula Óssea , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Subunidade 1 do Complexo Mediador/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Baço/citologia
16.
Methods Mol Biol ; 2265: 111-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704709

RESUMO

Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.


Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Técnicas de Cultura de Células/métodos , Morte Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Feminino , Corantes Fluorescentes , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Baço/citologia , Baço/imunologia
17.
J Vis Exp ; (168)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33645564

RESUMO

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.


Assuntos
Separação Celular/métodos , Células T Matadoras Naturais/imunologia , Animais , Antígenos CD4/metabolismo , Proliferação de Células , Ativação Linfocitária/imunologia , Camundongos , Células T Matadoras Naturais/citologia , Baço/citologia
18.
Yonsei Med J ; 62(2): 137-148, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33527793

RESUMO

PURPOSE: In organ transplantation, the need for immune modulation rather than immune suppression has been emphasized. In this study, we investigated whether combinatorial treatments of with thalidomide (TM) and dexamethasone (DX) might be new approaches to induce systemic immunomodulation on T cells and other immune cells that regulate the expression of co-inhibitory molecules. MATERIALS AND METHODS: Naïve splenic T cells from C57BL/6 mice were sort-purified and cultured in vitro for CD4+ T cell proliferation and regulatory T cell (Treg) conversion in the presence of TM or/and DX. Expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) in proliferated and converted T cells was quantified by flow cytometry. We also quantified in vivo expression of CTLA-4 and PD-1 on splenic CD4+ T cells and other immune cells isolated from TM- or/and DX-treated mice. Mixed lymphocytes reactions (MLR) were performed to evaluate the capacity of immune cells in carrying out immune responses. RESULTS: CTLA-4 expressions in effector T cells in vivo and in Tregs in vivo/vitro significantly increased upon TM/DX combinatorial treatment. Corresponding to increased CTLA-4 expression in T cells, the expression of ligand molecules for CTLA-4 significantly increased in splenic dendritic cells in TM/DX-treated groups. In addition, MLR results demonstrated that splenocytes isolated from TM/DX-treated mice significantly suppressed the proliferation of T cells isolated from other strains. CONCLUSION: Based on these results, we suggest that TM/DX combinatorial treatments might be efficient immunomodulatory methods for regulating T cell immunity.


Assuntos
Dexametasona/farmacologia , Imunomodulação/efeitos dos fármacos , Linfócitos T/imunologia , Talidomida/farmacologia , Animais , Antígeno CTLA-4/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Citometria de Fluxo , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/metabolismo , Baço/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/imunologia
19.
Mol Immunol ; 133: 86-94, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33636433

RESUMO

Regulatory B cells (Bregs), a subset of B lymphocytes discovered in the past few decades, have the capacity to suppress the immune response and dampen inflammation by secreting cytokines (IL-10 and TGF-ß). Whether Bregs are involved in Trichinella spiralis infection and the phenotypic characteristics of these cells after infection are still unknown. We investigated the phenotype of and dynamic changes in IL-10-producing Bregs in Trichinella spiralis infection in BALB/c mice. We used multicolour fluorescence immunostaining of microwave-treated paraffin sections to investigate the number of Bregs in T. spiralis infection. Flow cytometry (FCM) was used to determine the frequency of Bregs and related subgroups and cytokines in the spleen and mesenteric lymph nodes (MLNs). High levels of IL-10 were detected in the spleen and MLNs of mice after infection with T. spiralis. Furthermore, the frequencies of IL-10-producing CD19+CD1dhighCD5+ regulatory B cells and CD19+ cells were increased during T. spiralis infection. We also showed that the induced phenotype was similar to that of transitional type 2 marginal zone precursor B cells (T-MZP) cells after T. spiralis infection in mice. This study is the first demonstration of the expansion of Bregs following T. spiralis infection.


Assuntos
Linfócitos B Reguladores/imunologia , Interleucina-10/metabolismo , Baço/metabolismo , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Proliferação de Células , Feminino , Interleucina-10/biossíntese , Interleucina-10/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Fator de Crescimento Transformador beta1/imunologia , Triquinelose/patologia
20.
Biomed Res Int ; 2021: 6626851, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33623783

RESUMO

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice's immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.


Assuntos
Antígenos de Neoplasias , Neoplasias do Colo/imunologia , Imunoterapia , Linfoma/imunologia , Células Tumorais Cultivadas/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Autofagia , Técnicas de Cultura de Células , Citocinas/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia
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