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BMC Veterinary Research May 2022Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune...
BACKGROUND
Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 and CD8 T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days.
RESULTS
The proportions of CD4 T lymphocytes (18.70 ± 4.21%) and CD8 T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 T lymphocytes was significantly (p < 0.001) higher than that in CD4 T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 T lymphocytes was significantly (p < 0.001) higher than that in CD8 T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 (2.83 ± 0.98%) and CD8 T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period.
CONCLUSIONS
Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.
Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Flow Cytometry; Interleukin-17; Interleukin-4; Ionomycin; Leukocytes, Mononuclear; Lymphocyte Activation; Sheep; Tetradecanoylphorbol Acetate
PubMed: 35513847
DOI: 10.1186/s12917-022-03268-7 -
Tissue Engineering. Part A Jul 2022Ion signaling through Ca and Na plays a key role in mechanotransduction and encourages a chondrogenic phenotype and tissue maturation. In this study, we propose that the...
Ion signaling through Ca and Na plays a key role in mechanotransduction and encourages a chondrogenic phenotype and tissue maturation. In this study, we propose that the pleiotropic effects of Ca and Na modulation can be used to induce maturation and improvement of neocartilage derived from redifferentiated expanded chondrocytes from minipig rib cartilage. Three ion modulators were employed: (1) 4α-phorbol-12,13-didecanoate (4-αPDD), an agonist of the Ca-permeable transient receptor potential vanilloid 4 (TRPV4), (2) ouabain, an inhibitor of the Na/K pump, and (3) ionomycin, a Ca ionophore. These ion modulators were used individually or in combination. While no beneficial effects were observed when using combinations of the ion modulators, single treatment of constructs with the three ion modulators resulted in multiple effects in structure-function relationships. The most significant findings were related to ionomycin. Treatment of neocartilage with ionomycin produced 61% and 115% increases in glycosaminoglycan and pyridinoline crosslink content, respectively, compared with the control. Moreover, treatment with this Ca ionophore resulted in a 45% increase of the aggregate modulus, and a 63% increase in the tensile Young's modulus, resulting in aggregate and Young's moduli of 567 kPa and 8.43 MPa, respectively. These results support the use of ion modulation to develop biomimetic neocartilage using expanded redifferentiated costal chondrocytes. Impact Statement New cost-effective, replicable, and highly controllable strategies are required to develop neocartilage with biomimetic properties akin to native tissue. Ion signaling plays a key role in mechanotransduction, promoting chondrogenic phenotype. Using rib cartilage, we proposed that Ca and Na modulation could be used to induce maturation of neotissue derived from redifferentiated, expanded costal chondrocytes, improving its mechanical properties. Our results indicate that Ca modulation with ionomycin, which stimulated extracellular matrix deposition and collagen crosslinking, improved morphological and mechanical features of neocartilage constructs, and holds potential as a powerful tool to engineer hyaline-like tissues.
Topics: Animals; Calcium; Cartilage, Articular; Chondrocytes; Ionomycin; Ionophores; Mechanotransduction, Cellular; Ribs; Sodium; Swine; Swine, Miniature; Tissue Engineering
PubMed: 34877888
DOI: 10.1089/ten.TEA.2021.0169 -
Redox Biology Aug 2023Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process...
Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.
Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.
Topics: Citrullination; Actins; Ionomycin; Nuclear Proteins; Ligands; Proteomics; Neutrophils; Extracellular Traps; Chromatin; DNA; Cytoskeleton
PubMed: 37356135
DOI: 10.1016/j.redox.2023.102784 -
Frontiers in Immunology 2022The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between...
The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8 CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8β T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8βCD27CD11a), intermediate differentiated (CD8βCD27CD11a), and terminally differentiated cells (CD8βCD27CD11a). Cells were analyzed in condition as well as upon stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8 T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (, , , , , , and ) and late (, , , , , , and ) stages of CD8 T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8 T-cell subsets, respectively. Intermediate differentiated CD8 T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (, , , , and ) were highly expressed in terminally and intermediate differentiated CD8 T-cell subsets, while naïve CD8 T cells lacked expression even after stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8 T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail.
Topics: Animals; CD8-Positive T-Lymphocytes; Gene Expression Profiling; Ionomycin; Lymphocyte Activation; Mice; Swine; T-Lymphocyte Subsets
PubMed: 35265090
DOI: 10.3389/fimmu.2022.849922 -
Cell Death & Disease Oct 2022Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in...
Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in CLL, the development of resistance diminishes their therapeutic activity. This is also true for the Bcl-2 antagonist venetoclax. We investigated the molecular mechanisms that drive venetoclax resistance in CLL, with a clear focus to provide new strategies to successfully combat it. Activation of CLL cells with IFNγ, PMA/ionomycin, and sCD40L diminished the cytotoxicity of venetoclax. We demonstrated that the metabolic activity of cells treated with 1 nM venetoclax alone was 48% of untreated cells, and was higher for cells co-treated with IFNγ (110%), PMA/ionomycin (78%), and sCD40L (62%). As of molecular mechanism, we showed that PMA/ionomycin and sCD40L triggered translocation of NFκB in primary CLL cells, while IFNγ activated p38 MAPK, suppressed spontaneous and venetoclax-induced apoptosis and induced formation of the immunoproteasome. Inhibition of immunoproteasome with ONX-0914 suppressed activity of immunoproteasome and synergized with venetoclax against primary CLL cells. On the other hand, inhibition of p38 MAPK abolished cytoprotective effects of IFNγ. We demonstrated that venetoclax-resistant (MEC-1 VER) cells overexpressed p38 MAPK and p-Bcl-2 (Ser70), and underexpressed Mcl-1, Bax, and Bak. Inhibition of p38 MAPK or immunoproteasome triggered apoptosis in CLL cells and overcame the resistance to venetoclax of MEC-1 VER cells and venetoclax-insensitive primary CLL cells. In conclusion, the p38 MAPK pathway and immunoproteasome represent novel targets to combat venetoclax resistance in CLL.
Topics: Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Drug Resistance, Neoplasm; Humans; Ionomycin; Leukemia, Lymphocytic, Chronic, B-Cell; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; bcl-2-Associated X Protein; p38 Mitogen-Activated Protein Kinases
PubMed: 36209148
DOI: 10.1038/s41419-022-05287-6 -
PloS One 2012Ionomycin is a Ca(2+)-selective ionophore that is widely used to increase intracellular Ca(2+) levels in cell biology laboratories. It is also occasionally used to...
Ionomycin is a Ca(2+)-selective ionophore that is widely used to increase intracellular Ca(2+) levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca(2+) levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca(2+) increase. The ionomycin-induced Ca(2+) rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca(2+) response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation.
Topics: Actins; Animals; Calcium; Calcium Signaling; Embryonic Development; Exocytosis; Female; Fertilization; Intracellular Space; Ionomycin; Microinjections; Microvilli; Oocytes; Ovum; Starfish
PubMed: 22723970
DOI: 10.1371/journal.pone.0039231 -
PloS One 2016Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a...
Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes.
Topics: Cell Degranulation; Cell Line; Flow Cytometry; Humans; Interferon-gamma; Interleukin-2; Ionomycin; Killer Cells, Natural; Transcription, Genetic
PubMed: 27007115
DOI: 10.1371/journal.pone.0150998 -
Biochimica Et Biophysica Acta.... Nov 2018ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates...
ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates including TGF-alpha, Amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). We recently presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. Anoctamin-6 (ANO6) has Ca-dependent phospholipid scramblase activity and it followed that the functions of ANO6 and ADAM17 might be linked. We report that overexpression of ANO6 in HEK293T cells led to increased Ca-mediated PS-exposure that was indeed accompanied by enhanced release of AREG and TGF-alpha. The effect was not observed when cells were treated with the PKC-dependent ADAM17 activator PMA. Transformation of cells with a constitutively active ANO6 mutant led to spontaneous PS-exposure and to the release of ADAM17-substrates in the absence of any stimuli. Inhibitor experiments indicated that ANO6-mediated enhancement of substrate cleavage simultaneously broadened the spectrum of participating metalloproteinases. In complementary experiments, siRNA-mediated downregulation of ANO6 was shown to decrease ionophore-mediated release of TNFR1 in human umbilical vein endothelial cells (HUVECs). We conclude that ANO6, by virtue of its scramblase activity, may play a role as an important regulator of the ADAM-network in the plasma membrane.
Topics: ADAM Proteins; ADAM17 Protein; Anoctamins; Calcium; Cell Membrane; Gene Expression Regulation; HEK293 Cells; Humans; Ionomycin; Models, Biological; Mutation; Phosphatidylserines; Phospholipid Transfer Proteins; Phospholipids; Transforming Growth Factor alpha
PubMed: 30327201
DOI: 10.1016/j.bbamcr.2018.08.011 -
Molecular Systems Biology Mar 2017To monitor transcriptional regulation in human cells, rapid changes in enhancer and promoter activity must be captured with high sensitivity and temporal resolution....
To monitor transcriptional regulation in human cells, rapid changes in enhancer and promoter activity must be captured with high sensitivity and temporal resolution. Here, we show that the recently established protocol TT-seq ("transient transcriptome sequencing") can monitor rapid changes in transcription from enhancers and promoters during the immediate response of T cells to ionomycin and phorbol 12-myristate 13-acetate (PMA). TT-seq maps eRNAs and mRNAs every 5 min after T-cell stimulation with high sensitivity and identifies many new primary response genes. TT-seq reveals that the synthesis of 1,601 eRNAs and 650 mRNAs changes significantly within only 15 min after stimulation, when standard RNA-seq does not detect differentially expressed genes. Transcription of enhancers that are primed for activation by nucleosome depletion can occur immediately and simultaneously with transcription of target gene promoters. Our results indicate that enhancer transcription is a good proxy for enhancer regulatory activity in target gene activation, and establish TT-seq as a tool for monitoring the dynamics of enhancer landscapes and transcription programs during cellular responses and differentiation.
Topics: Base Pairing; Enhancer Elements, Genetic; Gene Expression Profiling; Gene Expression Regulation; Humans; Ionomycin; Jurkat Cells; RNA; Sequence Analysis, RNA; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transcriptional Activation
PubMed: 28270558
DOI: 10.15252/msb.20167507 -
Biomolecules Jun 2022HSPA1A is a molecular chaperone that regulates the survival of stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes and...
HSPA1A is a molecular chaperone that regulates the survival of stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes and embeds in the plasma membrane (PM) of stressed and tumor cells. Membrane-associated HSPA1A exerts immunomodulatory functions and renders tumors resistant to standard therapies. Therefore, understanding and manipulating HSPA1A's surface presentation is a promising therapeutic. However, HSPA1A's pathway to the cell surface remains enigmatic because this protein lacks known membrane localization signals. Considering that HSPA1A binds to lipids, like phosphatidylserine (PS) and monophosphorylated phosphoinositides (PIPs), we hypothesized that this interaction regulates HSPA1A's PM localization and anchorage. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A's PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that co-transfection of HSPA1A with lipid-biosensors masking PI(4)P and PI(3)P significantly reduced HSPA1A's heat-induced surface presentation. Next, we manipulated the cellular lipid content using ionomycin, phenyl arsine oxide (PAO), GSK-A1, and wortmannin. These experiments revealed that HSPA1A's PM localization was unaffected by ionomycin but was significantly reduced by PAO, GSK-A1, and wortmannin, corroborating the findings obtained by the co-transfection experiments. We verified these results by selectively depleting PI(4)P and PI(4,5)P using a rapamycin-induced phosphatase system. Our findings strongly support the notion that HSPA1A's surface presentation is a multifaceted lipid-driven phenomenon controlled by the binding of the chaperone to specific endosomal and PM lipids.
Topics: Cell Membrane; HSP70 Heat-Shock Proteins; Humans; Ionomycin; Phosphatidylinositol Phosphates; Wortmannin
PubMed: 35740982
DOI: 10.3390/biom12060856