Authors: Emil Lagumdzic, Clara Pernold, Marta Viano...

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

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.

Authors: Lorenna Rocha Reis, Douglas Ricardo Souza Junior, Rebeka Tomasin...

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

Authors: Jihui Yang, Yongxue Lv, Yazhou Zhu...

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

Authors: S Janna Bashar, Caitlyn L Holmes, Miriam A Shelef...

INTRODUCTION

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens as well as which peptidylarginine deiminases (PADs) are required for their production remain incompletely defined. Here, we investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins bound by APCAs, and if their formation requires PAD2 or PAD4.

METHODS

Thioglycolate-induced peritoneal macrophages from wild-type, PAD2, and PAD4 mice or human peripheral blood-derived M1 macrophages were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (citH4) antibody or sera from ACPA+ or ACPA- rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, and quantified.

RESULTS

We found that ionomycin and monosodium urate crystals reliably induced murine citH4+ METs, which were reduced in the absence of PAD2 and lost in the absence of PAD4. Also, IgG from ACPA+, but not ACPA-, rheumatoid arthritis sera bound to murine METs, and in the absence of PAD2 or PAD4, ACPA-bound METs were lost. Finally, ionomycin induced human METs that are citH4+ and ACPA-bound.

DISCUSSION

Thus, METs may contribute to the pool of citrullinated antigens bound by ACPAs in a PAD2- and PAD4-dependent manner, providing new insights into the targets of immune tolerance loss in rheumatoid arthritis.

Topics: Humans; Mice; Animals; Protein-Arginine Deiminases; Autoantibodies; Extracellular Traps; Protein-Arginine Deiminase Type 4; Ionomycin; Arthritis, Rheumatoid; Histones; Macrophages; Aminosalicylic Acids

PubMed: 38476240
DOI: 10.3389/fimmu.2024.1167362

Authors: Gaston A Otarola, Jerry C Hu, Kyriacos A Athanasiou...

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

Authors: Martin Veit, Katharina Isabelle Koyro, Björn Ahrens...

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

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca signaling and transcriptomic profiles.

Authors: Barbara Lisec, Tim Bozic, Iva Santek...

BACKGROUND

Disruption of Ca homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca kinetics and gene expression involved in the regulation of Casignaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1.

METHODS

CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca regulation ([Ca]) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl, and GdCl. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca signaling through RNA sequencing.

RESULTS

EA.hy926 cells, at increasing Ca concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca kinetics showed higher amplitudes in Ca responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca] changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca ions induced a significantly higher [Ca] rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca import and downregulated export genes in EA.hy926.

CONCLUSIONS

Our finding show that significant differences in CaEP response and [Ca] regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca] changes, which may be linked to overexpression of Ca-related genes and an inability to mitigate changes in [Ca]. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Topics: Calcium; Acetylcholine; Angiotensin II; Bradykinin; Ionomycin; Thapsigargin; Cell Line; Endothelial Cells; Endothelium, Vascular; Gene Expression Profiling; Electroporation; Adenosine Triphosphate

PubMed: 38347539
DOI: 10.1186/s12964-024-01503-2

Authors: Margaux Michel, Carina Demel, Benedikt Zacher...

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

Authors: Juliane Lokau, Christoph Garbers

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are two important pleiotropic cytokines, both of which signal through a homodimer of the β-receptor gp130. Specificity is gained through the unique, nonsignaling α-receptors IL-6R and IL-11R. Soluble variants of IL-6R and IL-11R also exist. Both membrane-bound receptors can be cleaved by the metalloprotease ADAM10. Here, we use ten different chimeric receptors consisting of different parts of IL-6R and IL-11R and analyze their susceptibility toward cleavage by ADAM10. As expected, all chimeras are substrates of ADAM10. However, we observed that cleavage of chimeric receptors containing the stalk region of the IL-11R could be blocked by the protease inhibitor GI (selective for ADAM10), but not by the protease inhibitor GW (selective for both ADAM10 and ADAM17), suggesting that another protease besides ADAM10 is involved in cleavage of these chimeras.

Topics: ADAM10 Protein; HEK293 Cells; Humans; Interleukin-6; Ionomycin; Protease Inhibitors; Proteolysis; Receptors, Interleukin-11; Recombinant Fusion Proteins

PubMed: 34778975
DOI: 10.1002/1873-3468.14230

Authors: Gema Romera-Cárdenas, L Michael Thomas, Sheila Lopez-Cobo...

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