RESUMO
TCRαß+CD4-CD8α+CD8ß- intestinal intraepithelial lymphocytes (CD8αα IELs) are an abundant population of thymus-derived T cells that protect the gut barrier surface. We sought to better define the thymic IEL precursor (IELp) through analysis of its maturation, localization and emigration. We defined two precursor populations among TCRß+CD4-CD8- thymocytes by dependence on the kinase TAK1 and rigorous lineage-exclusion criteria. Those IELp populations included a nascent PD-1+ population and a T-bet+ population that accumulated with age. Both gave rise to intestinal CD8αα IELs after adoptive transfer. The PD-1+ IELp population included more strongly self-reactive clones and was largely restricted by classical major histocompatibility complex (MHC) molecules. Those cells localized to the cortex and efficiently emigrated in a manner dependent on the receptor S1PR1. The T-bet+ IELp population localized to the medulla, included cells restricted by non-classical MHC molecules and expressed the receptor NK1.1, the integrin CD103 and the chemokine receptor CXCR3. The two IELp populations further differed in their use of the T cell antigen receptor (TCR) α-chain variable region (Vα) and ß-chain variable region (Vß). These data provide a foundation for understanding the biology of CD8αα IELs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Mucosa Intestinal/imunologia , Células Precursoras de Linfócitos T/imunologia , Timócitos/imunologia , Imunidade Adaptativa/imunologia , Transferência Adotiva , Animais , Antígenos CD , Antígenos Ly/imunologia , Antígenos CD8/imunologia , Linhagem da Célula , Movimento Celular/imunologia , Citometria de Fluxo , Imunofluorescência , Antígenos de Histocompatibilidade/imunologia , Imunidade nas Mucosas/imunologia , Cadeias alfa de Integrinas , Mucosa Intestinal/citologia , Linfócitos , Camundongos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Fenótipo , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores CXCR3 , Receptores de Lisoesfingolipídeo/imunologia , Receptores de Esfingosina-1-Fosfato , Proteínas com Domínio T/imunologia , Timócitos/citologia , Timo/citologiaRESUMO
Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Expressão Gênica , Tolerância Imunológica , Peptídeos/genética , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/imunologia , Autoimunidade , Deleção Clonal/genética , Deleção Clonal/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Genes Reporter , Camundongos , Camundongos Transgênicos , Peptídeos/química , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismoRESUMO
Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.
Assuntos
Organoides/patologia , Organoides/fisiologia , Regeneração , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/fisiopatologia , Bexiga Urinária/patologia , Bexiga Urinária/fisiologia , Adulto , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Ouriços/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organoides/fisiopatologia , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/patologia , Células-Tronco/fisiologia , Transcriptoma , Bexiga Urinária/citologia , Infecções Urinárias/metabolismo , Infecções Urinárias/patologiaRESUMO
Invariant natural killer T cells (iNKT cells) can produce copious amounts of interleukin 4 (IL-4) early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we have defined three subsets of iNKT cells (NKT1, NKT2 and NKT17) that produced distinct cytokines; these represented diverse lineages and not developmental stages, as previously thought. These subsets exhibited substantial interstrain variation in numbers. In several mouse strains, including BALB/c, NKT2 cells were abundant and were stimulated by self ligands to produce IL-4. In those strains, steady-state IL-4 conditioned CD8(+) T cells to become 'memory-like', increased serum concentrations of immunoglobulin E (IgE) and caused dendritic cells to produce chemokines. Thus, iNKT cell-derived IL-4 altered immunological properties under normal steady-state conditions.
Assuntos
Linhagem da Célula/imunologia , Imunidade Inata , Interleucina-4/biossíntese , Células T Matadoras Naturais/imunologia , Fatores Etários , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica , Variação Genética/imunologia , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Memória Imunológica , Imunofenotipagem , Interleucina-4/imunologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Camundongos , Células T Matadoras Naturais/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Especificidade da Espécie , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologiaRESUMO
Three subsets of invariant natural killer T (iNKT) cells have been identified, NKT1, NKT2, and NKT17, which produce distinct cytokines when stimulated, but little is known about their localization. Here, we have defined the anatomic localization and systemic distribution of these subsets and measured their cytokine production. Thymic NKT2 cells that produced interleukin-4 (IL-4) at steady state were located in the medulla and conditioned medullary thymocytes. NKT2 cells were abundant in the mesenteric lymph node (LN) of BALB/c mice and produced IL-4 in the T cell zone that conditioned other lymphocytes. Intravenous injection of α-galactosylceramide activated NKT1 cells with vascular access, but not LN or thymic NKT cells, resulting in systemic interferon-γ and IL-4 production, while oral α-galactosylceramide activated NKT2 cells in the mesenteric LN, resulting in local IL-4 release. These findings indicate that the localization of iNKT cells governs their cytokine response both at steady state and upon activation.
Assuntos
Citocinas/imunologia , Células T Matadoras Naturais/imunologia , Especificidade de Órgãos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Galactosilceramidas/imunologia , Galactosilceramidas/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Fígado/citologia , Fígado/imunologia , Fígado/metabolismo , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/classificação , Células T Matadoras Naturais/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Baço/citologia , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Timócitos/imunologia , Timócitos/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.
Assuntos
Diferenciação Celular/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Células Th1/citologia , Células Th1/imunologia , Transferência Adotiva , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linfócitos B/imunologia , Proteínas de Ligação a DNA/biossíntese , Regulação para Baixo , Fator de Transcrição GATA3/biossíntese , Técnicas de Inativação de Genes , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Lectinas Tipo C/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6 , Receptores de Lisoesfingolipídeo/biossíntese , Receptores de Lisoesfingolipídeo/metabolismo , Receptores de Esfingosina-1-Fosfato , Proteínas com Domínio T/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismoRESUMO
Interleukin-4 (IL-4) is produced by a unique subset of invariant natural killer T (iNKT) cells (NKT2) in the thymus in the steady state, where it conditions CD8+ T cells to become "memory-like" among other effects. However, the signals that cause NKT2 cells to constitutively produce IL-4 remain poorly defined. Using histocytometry, we observed IL-4-producing NKT2 cells localized to the thymic medulla, suggesting that medullary signals might instruct NKT2 cells to produce IL-4. Moreover, NKT2 cells receive and require T cell receptor (TCR) stimulation for continuous IL-4 production in the steady state, since NKT2 cells lost IL-4 production when intrathymically transferred into CD1d-deficient recipients. In bone marrow chimeric recipients, only hematopoietic, not stromal, antigen-presenting cells (APCs), provided such stimulation. Furthermore, using different Cre-recombinase transgenic mouse strains to specifically target CD1d deficiency to various APCs, together with the use of diphtheria toxin receptor (DTR) transgenic mouse strains to deplete various APCs, we found that macrophages were the predominant cell to stimulate NKT2 IL-4 production. Thus, NKT2 cells appear to encounter and require different activating ligands for selection in the cortex and activation in the medulla.
Assuntos
Interleucina-4/metabolismo , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Timo/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD1/genética , Antígenos CD1/metabolismo , Células Cultivadas , Interleucina-4/genética , Ativação Linfocitária , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Timo/citologiaRESUMO
We previously demonstrated that Bordetella bronchiseptica (B. bronchiseptica) antigen (Ag) enhances the Mycoplasma hyopneumoniae Ag-specific immune response. The focus of this study was whether acellular bacterin of B. bronchiseptica could be used as an adjuvant to increase antigen-presenting capability of dendritic cells (DCs) by increasing the level of activation. The metabolic activity of DCs was increased by B. bronchiseptica, similar to lipopolysaccharide (LPS). Flow cytometry analysis revealed that B. bronchiseptica increases the expression of major histocompatibility complex class-2, cluster of differentiation (CD)40, CD54, and CD86 which are closely related to DC-mediated immune responses. B. bronchiseptica enhanced the production of cytokines related to adaptive immune responses. Furthermore, the survival rate of B. bronchiseptica-injected groups was 100% at 15 and 20 mg/kg doses, whereas that of LPS-injected groups was only 20%, 0% at 15 and 20 mg/kg doses respectively, and so B. bronchiseptica is likely to be safer than LPS. Taken together, these results indicate that B. bronchiseptica can be used as an adjuvant to enhance the antigen-presenting capability of DCs. B. bronchiseptica is a candidate for producing vaccines, especially in case of DC-mediating efficacy and safety demands. This study provides researchers and clinicians with valuable information regarding the usage of B. bronchiseptica as a safe bacteria-derived immunostimulating agent for developing efficient vaccines.
RESUMO
AIMS: Although peroxisome proliferator-activated receptors (PPARs)α/γ dual agonists can be beneficial for treatment of dyslipidemia in patients with type 2 diabetes, their use is limited owing to various side effects, including body weight gain, edema, and heart failure. We aimed to demonstrate that amodiaquine, an antimalarial agent, has potential as a PPARα/γ dual agonist with low risk of adverse effects. METHODS: We screened a Prestwick library (Prestwick Chemical; Illkirch, France) to identify novel PPARα/γ dual agonists and selected amodiaquine (4-[(7-chloroquinolin-4-yl)amino]-2-[(diethylamino)methyl]phenol), which activated both PPAR-α & -γ, for further investigation. We performed both in vitro, including glucose uptake assay and fatty acid oxidation assay, and in vivo studies to elucidate the anti-diabetic and anti-obesity effects of amodiaquine. RESULTS: Amodiaquine selectively activated the transcriptional activities of PPARα/γ and enhanced both fatty acid oxidation and glucose uptake without altering insulin secretion in vitro. In high-fat diet-induced obese and genetically modified obese/diabetic mice, amodiaquine not only remarkably ameliorated insulin resistance, hyperlipidemia, and fatty liver but also decreased body weight gain. CONCLUSION: Our findings suggest that amodiaquine exerts beneficial effects on glucose and lipid metabolism by concurrent activation of PPARα/γ. Furthermore, amodiaquine acts as an alternative insulin-sensitizing agent with a positive influence on lipid metabolism and has potential to prevent and treat type 2 diabetes while reducing the risk of lipid abnormalities.
Assuntos
Amodiaquina/farmacologia , Antimaláricos/farmacologia , Glicemia/efeitos dos fármacos , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , PPAR alfa/agonistas , PPAR gama/agonistas , Células 3T3-L1 , Animais , Glicemia/metabolismo , Peso Corporal , Proliferação de Células , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fígado Gorduroso , Hiperlipidemias , Técnicas In Vitro , Fígado/metabolismo , Camundongos , Camundongos Obesos , Oxirredução , Triglicerídeos/metabolismoRESUMO
Invariant NKT cells differentiate into three predominant effector lineages in the steady state. To understand these lineages, we sorted undifferentiated invariant NK T progenitor cells and each effector population and analyzed their transcriptional profiles by RNAseq. Bioinformatic comparisons were made to effector subsets among other lymphocytes, specifically Th cells, innate lymphoid cells (ILC), and γδ T cells. Myc-associated signature genes were enriched in NKT progenitors, like in other hematopoietic progenitors. Only NKT1 cells, but not NKT2 and NKT17 cells, had transcriptome similarity to NK cells and were also similar to other IFN-γ-producing lineages such as Th1, ILC1, and intraepithelial γδ T cells. NKT2 and NKT17 cells were similar to their analogous subsets of γδ T cells and ILCs, but surprisingly, not to Th2 and Th17 cells. We identified a set of genes common to each effector lineage regardless of Ag receptor specificity, suggesting the use of conserved regulatory cores for effector function.
Assuntos
Linhagem da Célula/imunologia , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Transcriptoma , Animais , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Citometria de Fluxo , Imunidade Inata/genética , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Anisotropic snowman-like silver nanoparticles (AgNPs) were synthesized using the extract of Caesalpinia sappan heartwood as a reducing agent in the presence of cetyltrimethylammonium bromide. Two surface plasmon resonance bands of the orange solution were observed at 446 nm and 539 nm in UV-visible spectra. High-resolution X-ray diffraction analysis confirmed the face-centered cubic structure of the AgNPs. High-resolution transmission electron microscopy images clearly revealed snowman-like AgNPs with an average size of 34.36 ± 11.44 nm. The C-O functional group was most likely involved in the synthesis of the AgNPs, which was demonstrated by Fourier transform infrared spectra. Most interestingly, the snowman-like AgNPs exhibited higher antibacterial activity than the spherical AgNPs and the extract alone. Among the tested strains, the snowman-like AgNPs showed the highest activity against Staphylococcus aureus, with minimum inhibitory concentrations of 4.69 µg/mL for the extract and 0.443 µg/mL for the silver. The antibacterial activity of the snowman-like AgNPs increased 24-fold against S. aureus. These results strongly suggested that the snowman-like AgNPs synthesized from C. sappan extract have potential for treating infected disease caused by S. aureus when the antibacterial activity was combined from plant extract and AgNPs. To our knowledge, the present report is the first in which the snowman-like AgNPs synthesized using a plant extract as a reducing agent showed excellent In Vitro antibacterial activity.
Assuntos
Antibacterianos/farmacologia , Caesalpinia , Nanopartículas Metálicas , Extratos Vegetais , Prata , Staphylococcus aureusRESUMO
A prominent population of innate CD8+ T cells develops in the thymus of several gene-deficient mouse strains, including Itk, KLF2, CBP and Id3. These cells have the phenotype and function of memory CD8+ T cells, without previous exposure to antigen. Surprisingly, the cytokine IL-4 plays a key role in their development. As this developmental mechanism was discovered, it came to light that innate CD8+ T cells exist also in normal mice and in humans. In this review, we discuss how these cells develop, compare and contrast them to other CD8 memory cells, and discuss their potential physiological relevance.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula , Memória Imunológica , Animais , Humanos , Imunidade Inata , Interleucina-4/imunologia , Células T Matadoras Naturais/imunologiaRESUMO
We have recently shown that MHC class II-dependent thymocyte-thymocyte (T-T) interaction successfully generates CD4(+) T cells (T-T CD4(+) T cells), and that T-T CD4(+) T cells expressing promyelocytic leukemia zinc finger protein (PLZF) show an innate property both in mice and humans. In this article, we report that the thymic T-T interaction is essential for the conversion of CD8(+) T cells into innate phenotype in the physiological condition. CD8(+) T cells developed in the presence of PLZF(+) CD4(+) T cells showed marked upregulation of eomesodermin (Eomes), activation/memory phenotype, and rapid production of IFN-γ on ex vivo stimulation. Their development was highly dependent on the PLZF expression in T-T CD4(+) T cells and the IL-4 secreted by PLZF(+) T-T CD4(+) T cells. The same events may take place in humans, as a substantial number of Eomes expressing innate CD8(+) T cells were found in human fetal thymi and spleens. It suggests that PLZF(+) T-T CD4(+) T cells in combination with Eomes(+) CD8(+) T cells might actively participate in the innate immune response against various pathogens, particularly in human perinatal period.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Inata , Fatores de Transcrição Kruppel-Like/genética , Timo/citologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Citometria de Fluxo , Humanos , Interferon gama/biossíntese , Interleucina-4/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Fenótipo , Reação em Cadeia da Polimerase , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas com Domínio T/metabolismo , Timo/embriologia , Timo/imunologia , Timo/metabolismoRESUMO
BACKGROUND: Recently, various hemostatic devices, such as metal or plastic clips, ultrasonic coagulating shears, and electrothermal bipolar vessel sealer, have been widely applied during an operation. The purpose of this study was to analyze the mechanical, histological, and biochemical differences of vessels sealed with various hemostatic devices. METHODS: Thirty New Zealand White rabbits were randomly allocated into five groups, and short gastric vessels were ligated with metal clip, plastic clip, Harmonic Ace, LigaSure, or tie ligation. These vessels were harvested 3 days after operation and histologically analyzed at the site of ligation (proximal) and 5 mm from it (distal). Perivascular fibrosis was assessed on a score of mild to severe according to the severity. Inducible and endothelial nitric oxide synthase (iNOS and eNOS) mRNA expressions were measured quantitatively by real-time PCR at the sites of ligation. Abdominal aorta and inferior vena cava also were harvested and divided with each tool, and bursting pressures were measured. RESULTS: Bursting pressures were measured in 164 arteries and 141 veins. LigaSure showed significantly lower bursting pressures in arteries (p < 0.001), and plastic clip showed significantly lower bursting pressures in veins (p < 0.001). There was a negative relationship between outer diameters and bursting pressures in both the arteries (rho = -0.505, p < 0.001) and the veins (rho = -0.106, p = 0.24). The degree of perivascular fibrosis was not statistically different in either the proximal (p = 0.447) or distal (p = 0.381) sites. The expressions of iNOS and eNOS were significantly lower in the LS group (p < 0.001, and p < 0.001, respectively). CONCLUSIONS: There might be no clinical limitations when applying various hemostatic devices to small vessels under physiologic blood pressures. There were no acute histological differences between the hemostatic devices. However, LS showed the lowest iNOS and eNOS expressions, which might be due to thermal injuries of the whole vessel wall.
Assuntos
Ligadura/métodos , Animais , Western Blotting , Falha de Equipamento , Ligadura/instrumentação , Masculino , Óxido Nítrico Sintase Tipo II/análise , Complicações Pós-Operatórias , Coelhos , Distribuição Aleatória , Fatores de RiscoRESUMO
Adjuvants are used to elicit strong immune responses for vaccines that show poor immunogenicity. Previously, we demonstrated that a sonicated bacterin of Bordetella bronchiseptica can be used as a safe adjuvant that enhances the antigen-presenting capability of dendritic cells (DCs). In this study, we purified the lipopolysaccharide (LPS) of B. bronchiseptica (Bb-LPS) and investigated its immunogenic effects on DCs compared to those of Escherichia coli O26:B6 (O26)-derived LPS (O26-LPS), a positive control. Bb-LPS was purified using an LPS extraction kit. Limulus amebocyte lysate assay was performed to determine the optimal concentration of Bb-LPS and O26-LPS for treatment. Bb-LPS increased the metabolic activity of DCs at a concentration of 0 to 250 EU/mL, similar to that of O26-LPS. Bb-LPS significantly increased the expression level of CD40 and CD54, related to the immune responses of DCs. Bb-LPS enhanced the antigen-presenting capability of DCs and significantly increased the interferon-gamma/interleukin-4 ratio of CD4+ T cells co-cultured with DCs to 0.95 (p < 0.05). Moreover, Bb-LPS increased the production of pro-inflammatory cytokines in a safer manner than that obtained by O26-LPS. In vivo safety tests revealed that Bb-LPS was less toxic than O26-LPS in mice. This study demonstrated that Bb-LPS showed unique immune characteristics and immunogenic effects on the antigen-presenting capability of DCs, which differed from those of O26-LPS. This study provides valuable information for basic and clinical research for developing safe vaccine adjuvants.
Assuntos
Bordetella bronchiseptica , Lipopolissacarídeos , Camundongos , Animais , Lipopolissacarídeos/metabolismo , Adjuvantes de Vacinas , Adjuvantes Imunológicos/metabolismo , Vacinas Bacterianas , Células DendríticasRESUMO
Green synthesis strategies have been widely applied for the preparation of versatile nanomaterials. Gold nanospheres with an average size of 6.95 ± 2.25 nm were green synthesized by using a 70% ethanol extract of Korean red ginseng (Panax ginseng Meyer) root as a reducing agent. A seed-mediated synthesis was conducted to prepare Au-Ag bimetallic nanoparticles using gold nanospheres as seeds. Remarkably, Au-Ag bimetallic nanoparticles with an average size of 80.4 ± 11.9 nm were synthesized. Scanning transmission electron microscopy, energy dispersive X-ray spectroscopy and elemental mappings revealed bimetallic nanoparticles with Au-Ag alloy core and Au-rich shells. A face-centered cubic structure of Au-Ag bimetallic nanoparticles was confirmed by X-ray diffraction analysis. For Au-Ag bimetallic nanoparticles, the ratio of Ag/Au was 0.20 which was detected and analyzed by inductively coupled plasma-mass spectrometry. Gold nanospheres and Au-Ag bimetallic nanoparticles were functionalized by PEGylation, folic acid conjugation and grafting onto graphene oxide. Finally, docetaxel was loaded for evaluating the in vitro cell viability on cancer cells. Successful functionalization was confirmed by Fourier-transform infrared spectra. The anticancer activity of the docetaxel-loaded nanoparticles was higher than that of their non-docetaxel-loaded counterparts. The highest anticancer activity on human gastric adenocarcinoma cells (AGS) was observed in the docetaxel-loaded gold nanospheres that were functionalized by PEGylation, folic acid conjugation and grafting onto graphene oxide. Additionally, grafting onto graphene oxide and docetaxel loading induced high intracellular reactive oxygen species generation. For chemo-photothermal (PTT) anticancer therapy, cell viability was investigated using near-infrared laser irradiation at 808 nm. The highest chemo-PTT anticancer activity on AGS cells was observed in the docetaxel-loaded Au-Ag bimetallic nanoparticles. Therefore, the newly prepared docetaxel-loaded Au-Ag bimetallic nanoparticles in the current report have potential applications in chemo-PTT anticancer therapy.
RESUMO
Memory-phenotype (MP) CD4+ T cells are a substantial population of conventional T cells that exist in steady-state mice, yet their immunological roles in autoimmune disease remain unclear. In this work, we unveil a unique phenotype of MP CD4+ T cells determined by analyzing single-cell transcriptomic data and T cell receptor (TCR) repertoires. We found that steady-state MP CD4+ T cells in the spleen were composed of heterogeneous effector subpopulations and existed regardless of germ and food antigen exposure. Distinct subpopulations of MP CD4+ T cells were specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells exerted TCR-independent bystander effector functions similar to innate lymphoid cells. In particular, CCR6high subpopulation of MP CD4+ T cells were major responders to IL-23 and IL-1ß without MOG35-55 antigen reactivity, which gave them pathogenic Th17 characteristics and allowed them to contribute to autoimmune encephalomyelitis. We identified that Bhlhe40 in CCR6high MP CD4+ T cells as a key regulator of GM-CSF expression through IL-23 and IL-1ß signaling, contributing to central nervous system (CNS) pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal the clearly distinct effector-like heterogeneity of MP CD4+ T cells in the steady state and indicate that CCR6high MP CD4+ T cells exacerbate autoimmune neuroinflammation via the Bhlhe40/GM-CSF axis in a bystander manner.
Assuntos
Encefalomielite Autoimune Experimental , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunidade Inata , Doenças Neuroinflamatórias , Encefalomielite Autoimune Experimental/metabolismo , Células Th17 , Interleucina-23 , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos , Camundongos Endogâmicos C57BL , Proteínas de Homeodomínio/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genéticaRESUMO
Despite a long-standing hypothesis that chronic graft-versus-host disease (cGVHD) is an autoimmune disorder, most mouse models of cGVHD have been developed on the assumption that donor T cells are essential for its development. Here we show that cGVHD may be caused by autoreactive host T cells in mice that have been lethally irradiated and grafted with T-cell-depleted allogeneic bone marrow cells. In this chimera, host T cells derived from radioresistant intrathymic T-cell precursors caused dermal fibrosis and periportal inflammation, without the requirement for donor T cells. The lack of host DCs within the thymus after high-dose irradiation allowed autoreactive host T cells to escape thymic negative selection. Moreover, the homeostatic expansion of these T cells may augment their autoreactivity. These findings indicate that host T-cell-mediated cGVHD is an autoimmune process that occurs following the grafting of T-cell-depleted BM cells into hosts with functioning thymuses. We propose, based on the present data, that host T-cell-dependent autoimmunity is a potential mechanism by which cGVHD is induced.
Assuntos
Transplante de Medula Óssea , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Esclerodermia Limitada/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade , Células Cultivadas , Doença Crônica , Seleção Clonal Mediada por Antígeno , Doença Enxerto-Hospedeiro/complicações , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Esclerodermia Limitada/etiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Timo/imunologia , Timo/patologia , Quimeras de TransplanteRESUMO
OBJECTIVE: To determine whether trophoblast-derived microparticles can induce different inflammatory responses of the peripheral blood mononuclear cells depending upon the state of trophoblast when the microparticles are generated. STUDY DESIGN: A trophoblast-derived cell line (ATCC no. CRL-1584) was cultured under normal or hypoxic conditions. Microparticles were isolated from the cell culture supernatants (microparticles from normal trophoblast; microparticles from hypoxic trophoblast). Peripheral blood mononuclear cells were cultured alone or cocultured with either microparticles from normal trophoblast or microparticles from hypoxic trophoblast. RESULTS: After 48 hours, the peripheral blood mononuclear cells cocultured with microparticles from normal trophoblast released higher concentrations of interleukin-6 than peripheral blood mononuclear cells cultured alone. The peripheral blood mononuclear cells cocultured with microparticles from hypoxic trophoblast showed higher concentration of interleukin-6 and tumor necrosis factor alpha than peripheral blood mononuclear cells cocultured with microparticles from normal trophoblast, after 24 hours and 48 hours. CONCLUSION: More intense and rapid inflammatory response of peripheral blood mononuclear cells was observed with microparticles from hypoxic trophoblast than with microparticles from normal trophoblast. This difference might explain the exaggerated systemic inflammatory response as a result of placental hypoxia in preeclampsia.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Micropartículas Derivadas de Células/patologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Hipóxia/patologia , Inflamação/patologia , Interleucina-6/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Alopecia areata (AA) is an autoimmune disease mediated by NKG2D-expressing cytotoxic T lymphocytes destroying hair follicles in the skin. It is one of the most common autoimmune diseases, but there is no effective treatment modality approved by the FDA. Regulatory T cells (Tregs) are crucial for suppressing autoreactive T cells, and in the skin, they promote hair growth by inducing anagen. Based on this, we tested the therapeutic potential of expanded Tregs in AA using the C3H/HeJ mouse model. In mice with AA, NKG2D-expressing CD8 T cells widely infiltrate both haired and hairless skin areas, which have tissue-resident memory T-cell phenotypes. Tregs in the skin express CD25, CTLA-4, GATA-3, and Jagged1 and efficiently proliferate with IL-2 cytokine antibody complex. However, expanding Tregs in the skin did not induce anagen in normal mice, indicating that they are necessary but not sufficient for anagen induction. Also, they fail to suppress autoreactive CD8 T cells in the skin to reverse established AA in C3H/HeJ mice. These results suggest that Treg expansion alone is not sufficient for AA treatment, and combined immunotherapy is required.