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A method for high-content functional imaging of intracellular calcium responses in gelatin-immobilized non-adherent cells.
Ritter, P, Bye, LJ, Finol-Urdaneta, RK, Lesko, C, Adams, DJ, Friedrich, O, Gilbert, DF
Experimental cell research. 2020;(2):112210
Abstract
Functional imaging of the intracellular calcium concentration [Ca2+]i using fluorescent indicators is a powerful and frequently applied method for assessing various biological questions in vitro, including ion channel function and intracellular signaling in homeostasis and disease. In functional [Ca2+]i imaging experiments, the fluorescence intensity of single cells is typically recorded during application of a chemical stimulus, i.e. by exchange of modified extracellular media, exposure to drugs and/or ligands. The concomitant mechanical perturbation caused by the perfusion of different solution during experimentation severely hinders calcium imaging in non-adherent cells, including peripheral immune cells, as cells in suspension are dislocated by turbulent flow during chemical stimulation. The quantitative analysis, involving time-courses of intracellular fluorescence signal changes, necessitates cells to remain at the same position throughout the experiment. To prevent dislocation of cells during solution exchange, and to enable imaging as well as analysis of Ca2+ responses in immune cells, a gelatin-based method for immobilization of non-adherent cells was developed. Gelatin has been a long-serving material for cell immobilization, e.g. in 3D bio-printing of cells and has thus, also been employed in the context of this study. To demonstrate the applicability of the established method for functional Ca2+ imaging in gelatin-immobilized suspension cells, a proof-of-concept study was conducted using human peripheral blood model cell lines (Jurkat/T-lymphocytes and THP-1/monocytes), Ca2+ indicators (Fluo-4 and Fura-2) and two different fluorescence microscopy rigs. The data presented that the established methodology is applicable for studying Ca2+ signaling by in vitro high-content functional imaging of [Ca2+]i in suspension cells, including but not restricted to human immune cells.
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Microglial Store-operated Calcium Signaling in Health and in Alzheimer's Disease.
McLarnon, JG
Current Alzheimer research. 2020;(12):1057-1064
Abstract
The dysregulation of calcium signaling mechanisms in neurons has been considered a contributing factor to the pathogenesis evident in early-onset Alzheimer's Disease (AD). However, considerably less is known concerning the possible impairment of Ca2+ mobilization in resident immune cell microglia. This review considers findings which suggest that a prominent pathway for non-excitable microglial cells, store-operated calcium entry (SOCE), is altered in the sporadic form of AD. The patterns of Ca2+ mobilization are first discussed with platelet-activating factor (PAF) stimulation of SOCE in adult, fetal and immortalized cell-line, human microglia in the healthy brain. In all cases, PAF was found to induce a rapid transient depletion of Ca2+ from endoplasmic reticulum (ER) stores, followed by a sustained entry of Ca2+ (SOCE). A considerably attenuated duration of SOCE is observed with ATP stimulation of human microglia, suggested as due to agonist actions on differential subtype purinergic receptors. Microglia obtained from AD brain tissue, or microglia treated with full-length amyloid-β peptide (Aβ42), show significant reductions in the amplitude of SOCE relative to controls. In addition, AD brain and Aβ42-treated microglia exhibit decreased levels of Ca2+ release from ER stores compared to controls. Changes in properties of SOCE in microglia could lead to altered immune cell response and neurovascular unit dysfunction in the inflamed AD brain.
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3.
Combustible Cigarette and Smokeless Tobacco Product Preparations Differentially Regulate Intracellular Calcium Mobilization in HL60 Cells.
Arimilli, S, Makena, P, Prasad, GL
Inflammation. 2019;(5):1641-1651
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Abstract
Changes in the level of intracellular calcium ([Ca2+]i) are central to leukocyte signaling and immune response. Although evidence suggests that cigarette smoking affects inflammatory response via an increase in intracellular calcium, it remains unclear if the use of smokeless tobacco (e.g., moist snuff) elicits a similar response. In this study, we evaluated the effects of tobacco product preparations (TPPs), including total particulate matter (TPM) from 3R4F reference cigarettes, smokeless tobacco extract (STE) from 2S3 reference moist snuff, and nicotine alone on Ca2+ mobilization in HL60 cells. Treatment with TPM, but not STE or nicotine alone, significantly increased [Ca2+]i in a concentration-dependent manner in HL60 cells. Moreover, TPM-induced [Ca2+]i increase was not related to extracellular Ca2+ and did not require the activation of the IP3 pathway nor involved the transient receptor potential (TRP) channels. Our findings indicate that, in cells having either intact or depleted endoplasmic reticulum (ER) Ca2+ stores, TPM-mediated [Ca2+]i increase involves cytosolic Ca2+ pools other than thapsigargin-sensitive ER Ca2+ stores. These results, for the first time, demonstrate that TPM triggers [Ca2+]i increases, while significantly higher nicotine equivalent doses of STE or nicotine alone, did not affect [Ca2+]i under the experimental conditions. In summary, our study suggests that in contrast with STE or nicotine preparations, TPM activates Ca2+ signaling pathways in HL60 cells. The differential effect of combustible and non-combustible TPPs on Ca2+ mobilization could be a useful in vitro endpoint for tobacco product evaluation.
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4.
Calcium signalling in T cells.
Trebak, M, Kinet, JP
Nature reviews. Immunology. 2019;(3):154-169
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Abstract
Calcium (Ca2+) signalling is of paramount importance to immunity. Regulated increases in cytosolic and organellar Ca2+ concentrations in lymphocytes control complex and crucial effector functions such as metabolism, proliferation, differentiation, antibody and cytokine secretion and cytotoxicity. Altered Ca2+ regulation in lymphocytes leads to various autoimmune, inflammatory and immunodeficiency syndromes. Several types of plasma membrane and organellar Ca2+-permeable channels are functional in T cells. They contribute highly localized spatial and temporal Ca2+ microdomains that are required for achieving functional specificity. While the mechanistic details of these Ca2+ microdomains are only beginning to emerge, it is evident that through crosstalk, synergy and feedback mechanisms, they fine-tune T cell signalling to match complex immune responses. In this article, we review the expression and function of various Ca2+-permeable channels in the plasma membrane, endoplasmic reticulum, mitochondria and endolysosomes of T cells and their role in shaping immunity and the pathogenesis of immune-mediated diseases.
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Ion channels and anti-cancer immunity.
Panyi, G, Beeton, C, Felipe, A
Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 2014;(1638):20130106
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Abstract
The outcome of a malignant disease depends on the efficacy of the immune system to destroy cancer cells. Key steps in this process, for example the generation of a proper Ca(2+) signal induced by recognition of a specific antigen, are regulated by various ion channel including voltage-gated Kv1.3 and Ca(2+)-activated KCa3.1 K(+) channels, and the interplay between Orai and STIM to produce the Ca(2+)-release-activated Ca(2+) (CRAC) current required for T-cell proliferation and function. Understanding the immune cell subset-specific expression of ion channels along with their particular function in a given cell type, and the role of cancer tissue-dependent factors in the regulation of operation of these ion channels are emerging questions to be addressed in the fight against cancer disease. Answering these questions might lead to a better understanding of the immunosuppression phenomenon in cancer tissue and the development of drugs aimed at skewing the distribution of immune cell types towards killing of the tumour cells.
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Calcium signalling in diabetes.
Guerrero-Hernandez, A, Verkhratsky, A
Cell calcium. 2014;(5):297-301
Abstract
Molecular cascades responsible for Ca(2+) homeostasis and Ca(2+) signalling could be assembled in highly plastic toolkits that define physiological adaptation of cells to the environment and which are intimately involved in all types of cellular pathology. Control over Ca(2+) concentration in different cellular compartments is intimately linked to cell metabolism, because (i) ATP production requires low Ca(2+), (ii) Ca(2+) homeostatic systems consume ATP and (iii) Ca(2+) signals in mitochondria stimulate ATP synthesis being an essential part of excitation-metabolic coupling. The communication between the ER and mitochondria plays an important role in this metabolic fine tuning. In the insulin resistance state and diabetes this communication has been impaired leading to different disorders, for instance, diminished insulin production by pancreatic β cells, reduced heart and skeletal muscle contractility, reduced NO production by endothelial cells, increased glucose production by liver, increased lipolysis by adipose cells, reduced immune responses, reduced cognitive functions, among others. All these processes eventually trigger degenerative events resulting in overt diabetes due to reduction of pancreatic β cell mass, and different complications of diabetes, such as retinopathy, nephropathy, neuropathy, and different cardiovascular diseases.
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Ca(2+) -related signaling events influence TLR9-induced IL-10 secretion in human B cells.
Ziegler, S, Gartner, K, Scheuermann, U, Zoeller, T, Hantzschmann, J, Over, B, Foermer, S, Heeg, K, Bekeredjian-Ding, I
European journal of immunology. 2014;(5):1285-98
Abstract
Suppressory B-cell function controls immune responses and is mainly dependent on IL-10 secretion. Pharmacological manipulation of B-cell-specific IL-10 synthesis could, thus, be therapeutically useful in B-cell chronic lymphocytic leukemia, transplantation, autoimmunity and sepsis. TLR are thought to play a protagonistic role in the formation of IL-10-secreting B cells. The aim of the study was to identify the molecular events selectively driving IL-10 production in TLR9-stimulated human B cells. Our data highlight the selectivity of calcineurin inhibitors in blocking TLR9-induced B-cell-derived IL-10 transcription and secretion, while IL-6 transcription and release, B-cell proliferation, and differentiation remain unaffected. Nevertheless, TLR9-induced IL-10 production was found to be independent of calcineurin phosphatase activity and was even negatively regulated by NFAT. In contrast to TLR9-induced IL-6, IL-10 secretion was highly sensitive to targeting of spleen tyrosine kinase (syk) and Bruton's tyrosine kinase. Further analyses demonstrated increased phosphorylation of Ca(2+) /calmodulin kinase II (CaMKII) in TLR9-stimulated B cells and selective reduction of TLR9-induced secretion of IL-10 upon treatment with CaMKII inhibitors, with negligible impact on IL-6 levels. Altogether, our results identify calcineurin antagonists as selective inhibitors of IL-10 transcription and syk/Bruton´s tyrosine kinase-induced Ca(2+) /calmodulin- and CaMKII-dependent signaling as a pathway regulating the release of TLR9-induced B-cell-derived IL-10.
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T-cell artificial focal triggering tools: linking surface interactions with cell response.
Carpentier, B, Pierobon, P, Hivroz, C, Henry, N
PloS one. 2009;(3):e4784
Abstract
T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.
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CD38/cyclic ADP-ribose regulates astrocyte calcium signaling: implications for neuroinflammation and HIV-1-associated dementia.
Banerjee, S, Walseth, TF, Borgmann, K, Wu, L, Bidasee, KR, Kannan, MS, Ghorpade, A
Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology. 2008;(3):154-64
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Abstract
CD38 is a 45-kD ectoenzyme involved in the synthesis of potent calcium (Ca(2+))-mobilizing agents, cyclic adenosine diphosphate-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP+). In HIV-1-infected patients, increased CD38 expression on CD8+ T cells is linked to immune system activation and progression of HIV-1 infection. However, the role of CD38 upregulation in astrocyte function and HIV-1-associated dementia (HAD-now called HAND HIV-1-associated neurocognitive disorder) neuropathogenesis is unclear. To these ends, we used interleukin (IL)-1beta and HIV-1gp120 to activate primary human astrocytes and measured CD38 expression using real-time polymerase chain reaction and CD38 function by ADP-ribosyl cyclase activity. We also determined cADPR-mediated changes in single-cell intracellular Ca(2+) transients in activated astrocytes in presence or absence of ethylene glycol tetraacetic acid. CD38 levels were downregulated using CD38 small-interfering RNA (siRNA) and intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured. We previously reported a approximately 20-fold rise in CD38 messenger RNA levels in IL-1beta-activated astrocytes. We extend this observation and report that HIV-1gp120 potentiated CD38 expression in a dose-dependent manner and also increased CD38 enzyme activity in control and IL-1beta-activated astrocytes. We demonstrate higher cADPR levels in IL-1beta-activated astrocytes with a corresponding rise in [Ca(2+)](i) upon cADPR application and its non-hydrolysable analog, 3-deaza-cADPR. In activated astrocytes, pre-treatment with the cADPR-specific antagonist 8-Br-cADPR and CD38 siRNA transfection returned elevated [Ca(2+)](i) to baseline, thus confirming a CD38-cADPR specific response. These data are important for unraveling the mechanisms underlying the role of astrocyte-CD38 in HAD and have broader implications in other inflammatory diseases involving astrocyte activation and CD38 dysregulation.
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HTLV-I infection of WE17/10 CD4+ cell line leads to progressive alteration of Ca2+ influx that eventually results in loss of CD7 expression and activation of an antiapoptotic pathway involving AKT and BAD which paves the way for malignant transformation.
Akl, H, Badran, BM, Zein, NE, Bex, F, Sotiriou, C, Willard-Gallo, KE, Burny, A, Martiat, P
Leukemia. 2007;(4):788-96
Abstract
Adult T-cell leukemia/lymphoma (ATLL) is a malignancy slowly emerging from human T-cell leukemia virus type 1 (HTLV-I)-infected mature CD4(+) T-cells. To characterize the molecular modifications induced by HTLV-I infection, we compared HTLV-I-infected WE17/10 cells with control cells, using micro-arrays. Many calcium-related genes were progressively downmodulated over a period of 2 years. Infected cells acquired a profound decrease of intracellular calcium levels in response to ionomycin, timely correlated with decreased CD7 expression. Focusing on apoptosis-related genes and their relationship with CD7, we observed an underexpression of most antiapoptotic genes. Western blotting revealed increasing Akt and Bad phosphorylation, timely correlated with CD7 loss. This was shown to be phosphatidylinositol 3-kinase (PI3K)-dependent. Activation of PI3K/Akt induced resistance to the apoptotic effect of interleukin-2 deprivation. We thus propose the following model: HTLV-I infection induces a progressive decrease in CD3 genes expression, which eventually abrogates CD3 expression; loss of CD3 is known to perturb calcium transport. This perturbation correlates with loss of CD7 expression and induction of Akt and Bad phosphorylation via activation of PI3K. The activation of the Akt/Bad pathway generates a progressive resistance to apoptosis, at a time HTLV-I genes expression is silenced, thus avoiding immune surveillance. This could be a major event in the process of the malignant transformation into ATLL.