“…Wild-type AB zebrafish were bred and maintained in a circulating water bath at 28°C under standard conditions (11). All fish used in the experiments were offspring of a single AB strain parent pair after five generations of partial inbreeding (12).…”
Section: Methodsmentioning
confidence: 99%
“…Fluorescence signals were determined by using a FACScan flow cytometer (BD Bio-sciences) at 488 nm. FCM analysis was based on forward/side scatter (FSC/SSC) characteristics and PE/FITC-conjugated fluorescence with CellQuest program as previously described (11, 25). At least 10,000 events were collected from the lymphocyte gate.…”
Section: Methodsmentioning
confidence: 99%
“…As a downstream event of CD4 + T cell activation, the B cell activation was further examined on the basis of the percentage of mIgM + CD40 + B cells in leukocytes from the peripheral blood, spleen, and head kidney, as determined using mouse anti- Dr IgM and rabbit anti- Dr CD40 primary Abs followed by FITC- and PE-conjugated anti-rabbit IgG and anti-mouse IgG secondary Abs. The expression of Lck, CD154, mIgM, and CD40 was determined to evaluate the activation of CD4 + T cells and B cells through real-time PCR (11). …”
γδ T cells represent an evolutionarily primitive T cell subset characterized by distinct T cell receptors (TCRs) and innate and adaptive immune functions. However, the presence of this T cell subset in ancient vertebrates remains unclear. In this study, γδ T cells from a zebrafish (Danio rerio) model were subjected to molecular and cellular characterizations. The constant regions of zebrafish TCR-γ (DrTRGC) and δ (DrTRDC) were initially identified. Zebrafish γδ T cells accounted for 7.7–20.5% of the total lymphocytes in spleen, head kidney, peripheral blood, skin, gill, and intestine tissues. They possess typical morphological features of lymphocytes with a surface phenotype of γ+δ+CD4−CD8+. Zebrafish γδ T cells functionally showed a potent phagocytic ability to both soluble and particulate antigens. They can also act as an antigen-presenting cell to initiate antigen (KLH)-specific CD4+ TKLH cell activation and to induce B cell proliferation and IgM production. Particularly, zebrafish γδ T cells also play a critical role in antigen-specific IgZ production in intestinal mucus. These findings demonstrated that γδ T cells had been originated as early as teleost fish, which providing valuable insights into the evolutionary history of T cell subset. It is anticipated that this study would be used as a guide to develop a zebrafish model for the cross-species investigation of γδ T cell biology.
“…Wild-type AB zebrafish were bred and maintained in a circulating water bath at 28°C under standard conditions (11). All fish used in the experiments were offspring of a single AB strain parent pair after five generations of partial inbreeding (12).…”
Section: Methodsmentioning
confidence: 99%
“…Fluorescence signals were determined by using a FACScan flow cytometer (BD Bio-sciences) at 488 nm. FCM analysis was based on forward/side scatter (FSC/SSC) characteristics and PE/FITC-conjugated fluorescence with CellQuest program as previously described (11, 25). At least 10,000 events were collected from the lymphocyte gate.…”
Section: Methodsmentioning
confidence: 99%
“…As a downstream event of CD4 + T cell activation, the B cell activation was further examined on the basis of the percentage of mIgM + CD40 + B cells in leukocytes from the peripheral blood, spleen, and head kidney, as determined using mouse anti- Dr IgM and rabbit anti- Dr CD40 primary Abs followed by FITC- and PE-conjugated anti-rabbit IgG and anti-mouse IgG secondary Abs. The expression of Lck, CD154, mIgM, and CD40 was determined to evaluate the activation of CD4 + T cells and B cells through real-time PCR (11). …”
γδ T cells represent an evolutionarily primitive T cell subset characterized by distinct T cell receptors (TCRs) and innate and adaptive immune functions. However, the presence of this T cell subset in ancient vertebrates remains unclear. In this study, γδ T cells from a zebrafish (Danio rerio) model were subjected to molecular and cellular characterizations. The constant regions of zebrafish TCR-γ (DrTRGC) and δ (DrTRDC) were initially identified. Zebrafish γδ T cells accounted for 7.7–20.5% of the total lymphocytes in spleen, head kidney, peripheral blood, skin, gill, and intestine tissues. They possess typical morphological features of lymphocytes with a surface phenotype of γ+δ+CD4−CD8+. Zebrafish γδ T cells functionally showed a potent phagocytic ability to both soluble and particulate antigens. They can also act as an antigen-presenting cell to initiate antigen (KLH)-specific CD4+ TKLH cell activation and to induce B cell proliferation and IgM production. Particularly, zebrafish γδ T cells also play a critical role in antigen-specific IgZ production in intestinal mucus. These findings demonstrated that γδ T cells had been originated as early as teleost fish, which providing valuable insights into the evolutionary history of T cell subset. It is anticipated that this study would be used as a guide to develop a zebrafish model for the cross-species investigation of γδ T cell biology.
“…Plenty of data suggest that several TIM molecules play important roles in the immune responses of Th1 and Th2 cells [17]. There are reports showing that DC-derived TIM4 was able to promote CD4+ T cells to differentiate into Th2 cells [18]. On a specific microenvironment, DCs are a group of cells that possess the ability to promote differentiation of CD4+ T cells into either Th1 or Th2 cells [19].…”
Smoking is considered to be the main source of indoor pollution, and it has been identified as an important environmental factor contributing to asthma onset. We know that T helper 2 (Th2) response plays a crucial role in the process of asthma disease. We have investigated the reaction of cigarette smoke extract (CSE) on Th polarization which is controlled by dendritic cells (DCs). Stimulated by CSE, immature DCs from murine bone marrow showed upregulated levels of TIM4. Cocultured with CD4+ T cells, stimulated DCs increased the ratio of IL-4+ versus IFN-γ+ of CD4+ T cells. This suggests a differentiation towards Th2 response. Moreover, antibodies against TIM4 reversed the upexpression of the IL-4+/IFN-γ+ ratio provoked by CSE, indicating that the Th2 polarization which was induced by CSE is via TIM4 mechanisms. CSE could activate mitogen-activated protein kinase pathways like ERK and p38. Upregulation of TIM4 expression by CSE stimulation was found to be inhibited by an ERK inhibitor but not p38 and JNK. In conclusion, DC-induced Th2 polarization is a hallmark of CSE allergy, and this aspect can be explained by CSE-induced TIM4 expression.
“…The Genome and Expressed Sequence Tags databases maintained by the National Center for Biotechnology Information (NCBI), the University of California Santa Cruz, and Ensembl were used to predict NLRP1 homolog in zebrafish as previously described (32). Total RNA was isolated from zebrafish embryos and tissues by using an RNAiso Plus kit (Takara Bio).…”
NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, NLRP1 (NLRP1) from a zebrafish () model. This NLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation of zebrafish inflammatory caspases ( Caspase [Caspase]-A and Caspase-B) and maturation of IL-1β in a ASC (ASC)-dependent manner. In this process, NLRP1 promotes the aggregation ofASC into a filament with ASC core and ASC cluster. The assembly of NLRP1 inflammasome depends on the CARD-CARD homotypic interaction betweenNLRP1 and ASC core, and PYD-PYD interaction between Caspase-A/B andASC cluster. The FIIND domain in NLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the twoCaspases are coordinated in NLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the recruitment and activation ofCaspase-A/B in the inflammasome is shown in an alternate manner, with a preference for Caspase-A followed by a subsequent selection forCaspase-B. By using morpholino oligonucleotide-based knockdown assays, the NLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome-mediated immunology and diseases.
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