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Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca 2+ ) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP 3 R), a homo- or heterotetramer of the IP 3 R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca 2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP 3 R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP 3 R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca 2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca 2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP 3 R3 in IP 3 R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca 2+ channels and immunodeficiency and identify IP 3 Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca 2+ -associated immunodeficiency from store-operated entry to impaired Ca 2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca 2+ signaling., (© 2022. The Author(s).)

Antigen recognition by the T-cell receptor induces a cytosolic Ca 2+ signal that is crucial for T-cell function. The Ca 2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca 2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca 2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro , Trpm2 -/- and WT CD4 + and CD8 + T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2 -/- CD8 + T cells and unimpaired differentiation of CD4 + T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo , Trpm2 -/- and WT CD8 + T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2 -/- mice and after transfer of WT and Trpm2 -/- CD8 + T cells into infected recipients. CD4 + T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2 -/- CD4 + T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lory, Nawrocki, Corazza, Schmid, Schumacher, Bedke, Menzel, Koch-Nolte, Guse, Huber and Mittrücker.)

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca 2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kim, Yi, Kwon, Park, Kwon and Kim.)

Intestinal inflammation is a major threat to the health and growth of young animals such as piglets. As a next-generation probiotics, limited studies have shown that Akkermansia muciniphila could alleviate inflammation of intestinal epithelial cells (IECs). In this study, a TNF-α-induced inflammatory model of IPEC-J2 cells, the intestinal porcine enterocytes, was built to evaluate the effects of active or inactive A. muciniphila on the inflammation of IECs. The viability of IPEC-J2 cells was the highest when treated with active (10 8 copies/mL) or inactive (10 9 copies/mL) A. muciniphila for 7.5 h ( P < 0.01). Treated with 20 ng/mL of TNF-α and followed by a treatment of A. muciniphila , the mRNA level of proinflammatory cytokines ( IL-8, IL-1β , IL-6 and TNF-α ) was remarkably reduced ( P < 0.05) along with the increased mRNA level of tight junction proteins ( ZO-1 and Occludin , P < 0.05). Flow cytometry analysis showed that active or inactive A. muciniphila significantly suppressed the rate of the early and total apoptotic of the inflammatory IPEC-J2 cells ( P < 0.05). According to results of transcriptome sequencing, active and inactive A. muciniphila may decline cell apoptosis by down-regulating the expression of key genes in calcium signaling pathway, or up-regulating the expression of key genes in cell cycle signaling pathway. And the bacterium may alleviate the inflammation of IECs by down-regulating the expression of PI3K upstream receptor genes. Our results indicate that A. muciniphila may be a promising NGP targeting intestinal inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Luo, Lan, Xie, Li, Devillard, He, Liu, Cai, Tian, Wu, Ren, Chen, Yu, Huang, Zheng, Mao, Yu, Luo, Yan, Wang, Wang and Tang.)

CRAC, which plays important role in Ca 2+ -dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca 2+ -imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca 2+ -response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca 2+ -oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Most agonists stimulate platelet Ca 2+ rises via G-protein coupled receptors (GPCRs) or ITAM-linked receptors (ILRs). Well studied are the GPCRs stimulated by the soluble agonists thrombin (PAR1, PAR4), ADP (P2Y 1 , P2Y 12 ), and thromboxane A 2 (TP), signaling via phospholipase (PLC)β isoforms. The platelet ILRs glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2), and FcγRIIa are stimulated by adhesive ligands or antibody complexes and signal via tyrosine protein kinases and PLCγ isoforms. Marked differences exist between the GPCR- and ILR-induced Ca 2+ signaling in: (i) dependency of tyrosine phosphorylation; (ii) oscillatory versus continued Ca 2+ rises by mobilization from the endoplasmic reticulum; and (iii) smaller or larger role of extracellular Ca 2+ entry via STIM1/ORAI1. Co-stimulation of both types of receptors, especially by thrombin (PAR1/4) and collagen (GPVI), leads to a highly enforced Ca 2+ rise, involving mitochondrial Ca 2+ release, which activates the ion and phospholipid channel, anoctamin-6. This highly Ca 2+ -dependent process causes swelling, ballooning, and phosphatidylserine expression, establishing a unique platelet population swinging between vital and necrotic (procoagulant 'zombie' platelets). Additionally, the high Ca 2+ status of procoagulant platelets induces a set of additional events: (i) Ca 2+ dependent cleavage of signaling proteins and receptors via calpain and ADAM isoforms; (ii) microvesiculation; (iii) enhanced coagulation factor binding; and (iv) fibrin-coat formation involving transglutaminases. Given the additive roles of GPCR and ILR in Ca 2+ signal generation, high-throughput screening of biomolecules or small molecules based on Ca 2+ flux measurements provides a promising way to find new inhibitors interfering with prolonged high Ca 2+ , phosphatidylserine expression, and hence platelet procoagulant activity.

Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca 2+ from intracellular stores in a wide variety of cell types, tissues and organisms. Current evidence suggests that NAADP may function as a trigger to initiate a Ca 2+ signal that is then amplified by other Ca 2+ release mechanisms. A fundamental question that remains unanswered is the identity of the NAADP receptor. Our recent studies have identified HN1L/JPT2 as a high affinity NAADP binding protein that is essential for the modulation of Ca 2+ channels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Walseth and Guse.)

Background: Bronchial remodeling is a key feature of asthma that is already present in preschoolers with wheezing. Moreover, bronchial smooth muscle (BSM) remodeling at preschool age is predictive of asthma at school age. However, the mechanism responsible for BSM remodeling in preschoolers with wheezing remains totally unknown. In contrast, in adult asthma, BSM remodeling has been associated with an increase in BSM cell proliferation related to increased mitochondrial mass and biogenesis triggered by an altered calcium homeostasis. Indeed, BSM cell proliferation was decreased in vitro by the calcium channel blocker gallopamil., Objective: Our aim was to investigate the mechanisms involved in BSM cell proliferation in preschoolers with severe wheezing, with special attention to the role of mitochondria and calcium signaling., Methods: Bronchial tissue samples obtained from 12 preschool controls without wheezing and 10 preschoolers with severe wheezing were used to measure BSM mass and establish primary BSM cell cultures. BSM cell proliferation was assessed by manual counting and flow cytometry, ATP content was assessed by bioluminescence, mitochondrial respiration was assessed by using either the Seahorse or Oroboros technique, mitochondrial mass and biogenesis were assessed by immunoblotting, and calcium response to carbachol was assessed by confocal microscopy. The effect of gallopamil was also evaluated., Results: BSM mass, cell proliferation, ATP content, mitochondrial respiration, mass and biogenesis, and calcium response were all increased in preschoolers with severe wheezing compared with in the controls. Gallopamil significantly decreased BSM mitochondrial biogenesis and mass, as well as cell proliferation., Conclusion: Mitochondria are key players in BSM cell proliferation in preschoolers with severe wheezing and could represent a potential target to treat BSM remodeling at an early stage of the disease., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca 2+ entry (SOCE) is now recognized as the main mechanism of the majority of nonexcitable cell calcium influx. Calcium overload is a primary mechanism of endothelial cell injury during systemic inflammatory response and sepsis. Whether STIM1-mediated SOCE plays a role in calcium overload in vascular endothelial cell injury remains unclear., Materials and Methods: To explore the role of STIM1-gated SOCE in vascular endothelial cell calcium overload and inflammation, we established a human septic serum or lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell (HUVEC) experimental system and derived ribonucleic acid interference (RNAi)-mediated STIM1, ORAI1 (orai gene [HGNC: 25896 Entrez Gene: 84876] coding protein, ORAI Calcium Release-Activated Calcium Modulator 1), and transient receptor potential channel 1 (TRPC1) (core components of store-operated Ca 2+ [SOC]) downregulated HUVECs, as well as STIM1 overinduced HUVECs., Results: Our results show that sepsis serum or LPS stimulation increased STIM1 in HUVECs and increased all cytokines except for VEGF and the inflammatory mediators tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1 in a time-dependent manner. RNAi-mediated knockdown of STIM1 significantly inhibited serum or LPS-induced inflammatory cytokine expression, and STIM1 overexpression in HUVECs promoted LPS-mediated induction of these cytokines. Meanwhile, similar to the blocking effect of the specific SOC inhibitors Gd 3+ and La 3+ on LPS-induced calcium influx, RNAi-mediated depletion of STIM1 or the SOC proteins TRPC1 and ORAI1 could significantly inhibit serum or LPS-induced extracellular calcium influx, as well as the expression of the inflammatory cytokines tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1. Simultaneous downregulation of the SOCE core units TRPC1 and ORAI1 inhibited LPS-induced calcium influx and cytokine expression, which could not be restored by inducing STIM1. Forced expression of nuclear factor-κB (NF-κB) in HUVECs significantly induced STIM1 expression, whereas RNAi-mediated depletion of NF-κB significantly inhibited STIM1 mRNA levels and significantly reduced the thapsigargin-mediated SOCE calcium influx, which was similar to results with the NF-κB inhibitor wogonin., Conclusions: Septic serum stimulates the expression of STIM1, cytokines, and inflammatory mediators in HUVECs. STIM1-mediated SOCE is required for Ca 2+ influx induced by LPS or septic serum and contributes cytokines and inflammatory mediators in septic serum-stimulated HUVECs. In addition, STIM1-mediated SOCE on Ca 2+ influx by septic serum or LPS involves NF-κB signaling., (Copyright © 2020. Published by Elsevier Inc.)

Alveolar macrophages (AM) maintain airway immune balance; however, the regulation of heterogeneity of AMs is incompletely understood. We demonstrate that RGS1 coregulates the immunophenotype of AM subpopulations, including pro- and anti-inflammatory, injury- and repair-associated, and pro- and antifibrotic phenotypes, through the PLC-IP3R signal-dependent intracellular Ca 2+ response. Flt3 + AMs and Tie2 + AMs had different immune properties, and RGS1 expression in the cells was targeted by exosomes (EXOs) containing miR-223 and miR-27b-3p that were derived from vascular endothelial cells (EnCs) and type II alveolar epithelial cells (EpCs-II), respectively. Imbalance of AMs was correlated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis (PF) caused a lack of secretion of CD31 + and CD74 + EXOs derived from EnCs and EpCs-II. Timely treatment with EXOs significantly improved endotoxin-induced ALI/ARDS and bleomycin-induced PF in mice. Thus, EnC- and EpC-II-derived EXOs regulate the immune balance of AMs and can be used as potential therapeutic drugs.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8 + and CD4 + T cell locomotion in tissues. Expression of β-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Glycerol monolaurate (GML), a naturally occurring monoglyceride, is widely used commercially for its antimicrobial properties. Interestingly, several studies have shown that GML not only has antimicrobial properties but is also an anti-inflammatory agent. GML inhibits peripheral blood mononuclear cell proliferation and inhibits T cell receptor (TCR)-induced signaling events. In this study, we perform an extensive structure activity relationship analysis to investigate the structural components of GML necessary for its suppression of human T cell activation. Human T cells were treated with analogs of GML, differing in acyl chain length, head group, linkage of acyl chain, and number of laurate groups. Treated cells were then tested for changes in membrane dynamics, LAT clustering, calcium signaling, and cytokine production. We found that an acyl chain with 12-14 carbons, a polar head group, an ester linkage, and a single laurate group at any position are all necessary for GML to inhibit protein clustering, calcium signaling, and cytokine production. Removing the glycerol head group or replacing the ester linkage with a nitrogen prevented derivative-mediated inhibition of protein cluster formation and calcium signaling, while still inhibiting TCR-induced cytokine production. These findings expand our current understanding of the mechanisms of action of GML and the of GML needed to function as a novel immunosuppressant.

Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators., Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel., Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2., Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abji, Rasti, Gómez-Aristizábal, Muytjens, Saifeddine, Mihara, Motahhari, Gandhi, Viswanathan, Hollenberg, Oikonomopoulou and Chandran.)

Exhausted immune responses to chronic diseases represent a major challenge to global health. We study CD4 + T cells in a mouse model with regulatable antigen presentation. When the cells are driven through the effector phase and are then exposed to different levels of persistent antigen, they lose their T helper 1 (Th1) functions, upregulate exhaustion markers, resemble naturally anergic cells, and modulate their MAPK, mTORC1, and Ca 2+ /calcineurin signaling pathways with increasing dose and time. They also become unable to help B cells and, at the highest dose, undergo apoptosis. Transcriptomic analyses show the dynamic adjustment of gene expression and the accumulation of T cell receptor (TCR) signals over a period of weeks. Upon antigen removal, the cells recover their functionality while losing exhaustion and anergy markers. Our data suggest an adjustable response of CD4 + T cells to different levels of persisting antigen and contribute to a better understanding of chronic disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca 2+ signaling. Especially immune cells highly rely on Ca 2+ -dependent processes and a strictly regulated Ca 2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca 2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca 2+ signaling due to channels and receptors in T cells and beyond in the context of aging.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. In the past years, new therapeutic approaches (e.g., ibrutinib or venetoclax) have been established and greatly improved treatment of CLL. However, complete control or cure of the disease have not been reached so far. Thus, reliable prognostic markers are an imperative for treatment decisions. Recent studies have revealed an essential role for B cell receptor (BCR) signaling in the pathogenesis, prognosis, and therapy of CLL. A heterogeneous response to receptor stimulation with anti-IgM treatment culminating in different calcium flux capabilities has been demonstrated by several authors. However, the methods employed have not reached clinical application. Here, we report on a flow cytometry-based assay to evaluate calcium flux capabilities in CLL and demonstrate that compromised BCR signaling with diminished calcium flux is associated with a significantly better clinical outcome and progression free survival. In summary, our data strongly support the role of compromised BCR signaling as an important prognostic marker in CLL and establish a novel diagnostic tool for its assessment in clinical settings., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people worldwide. Unfortunately, none of the current treatments are effective at improving cognitive function in AD patients and, therefore, there is an urgent need for the development of new therapies that target the early cause(s) of AD. Intracellular calcium (Ca 2+ ) regulation is critical for proper cellular and neuronal function. It has been suggested that Ca 2+ dyshomeostasis is an upstream factor of many neurodegenerative diseases, including AD. For this reason, chemical agents or small molecules aimed at targeting or correcting this Ca 2+ dysregulation might serve as therapeutic strategies to prevent the development of AD. Moreover, neurons are not alone in exhibiting Ca 2+ dyshomeostasis, since Ca 2+ disruption is observed in other cell types in the brain in AD. In this review, we examine the distinct Ca 2+ channels and compartments involved in the disease mechanisms that could be potential targets in AD.

An expansion of CD21 low B cells has been described in a variety of diseases associated with persistent immune stimulation as in chronic infection, immunodeficiency, or autoimmunity. Different developmental stages of CD21 low B cells have been highlighted in specific diseases; however, a systematic comparison of distribution, phenotype, and signaling capacity of these populations has not yet been performed to delineate the pivotal character of this unusual B cell population. Screening of more than 200 patients with autoimmune disease demonstrated that the prevalence of patients with expanded CD21 low B cells varies between diseases. The expansion was frequent in patients with systemic lupus erythematosus, in which it correlated to relative B cell lymphopenia and duration of disease. Different proportions of distinct developmental stages of CD21 low B cells co-occur in nearly all patients with autoimmune disease. Although in most patients, naive-like and CD27 - switched memory B cells were the most prominent CD21 low subpopulations, there was no detectable association of the pattern with the underlying disease. Despite their distinct developmental stage, all CD21 low B cells share a common core phenotype including the increased expression of inhibitory receptors, associated with an elevated constitutive phosphorylation of proximal signaling molecules downstream of the BCR but impaired Ca 2+ mobilization and NF-κB activation after BCR stimulation. Further, this was accompanied by impaired upregulation of CD69, although CD86 upregulation was preserved. Beyond maturation-associated differences, the common core characteristics of all CD21 low B cell populations suggests either a common ancestry or a shared sustained imprint by the environment they originated in., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. β 2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α-mediated β 2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)-regulated calcium influx. Furthermore, we show that CD4 + CD28 null T cells represent a highly reactive subset showing 25-fold stronger β 2 integrin activation upon SDF-1α stimulation compared with CD28 + T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28 null subset compared with the CD28 + T cells, whereas the established mediators of the classical pathways for β 2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28 null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28 + and CD28 null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H 2 O 2 Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Calcium (Ca 2+ ) signals play fundamental roles in immune cell function. The main sources of Ca 2+ influx in mammalian lymphocytes following antigen receptor stimulation are Ca 2+ release-activated Ca 2+ (CRAC) channels. These are formed by ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the endoplasmic reticulum (ER). Human loss-of-function (LOF) mutations in ORAI1 and STIM1 that abolish Ca 2+ influx cause a unique disease syndrome called CRAC channelopathy that is characterized by immunodeficiency autoimmunity and non-immunological symptoms. Studies in mice lacking Stim and Orai genes have illuminated many cellular and molecular mechanisms by which these molecules control lymphocyte function. CRAC channels are required for the differentiation and function of several T lymphocyte subsets that provide immunity to infection, mediate inflammation and prevent autoimmunity. This review examines new insights into how CRAC channels control T cell-mediated immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

WNT proteins constitute a very conserved family of secreted glycoproteins that act as short-range ligands for signaling with critical roles in hematopoiesis, embryonic development, and tissue homeostasis. These proteins transduce signals via the canonical pathway, which is β-catenin-mediated and better-characterized, or via more diverse noncanonical pathways that are β-catenin independent and comprise the planar cell polarity (PCP) pathway and the WNT/Ca ++ pathways. Several proteins regulate Wnt signaling through a variety of sophisticated mechanisms. Disorders within the pathway can contribute to various human diseases, and the dysregulation of Wnt pathways by different molecular mechanisms is implicated in the pathogenesis of many types of cancer, including the hematological malignancies. The types of leukemia differ considerably and can be subdivided into chronic, myeloid or lymphocytic, and acute, myeloid or lymphocytic, leukemia, according to the differentiation stage of the predominant cells, the progenitor lineage, the diagnostic age strata, and the specific molecular drivers behind their development. Here, we review the role of Wnt signaling in normal hematopoiesis and discuss in detail the multiple ways canonical Wnt signaling can be dysregulated in acute leukemia, including alterations in gene expression and protein levels, epigenetic regulation, and mutations. Furthermore, we highlight the different impacts of these alterations, considering the distinct forms of the disease, and the therapeutic potential of targeting Wnt signaling., (©2020 Society for Leukocyte Biology.)

NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc-gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca 2+ signaling and NK cell-mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca 2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low-affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high-affinity (VV) and VF SNP variants was two- to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca 2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low-affinity FF variation. In conclusion, this is the first study correlating Ca 2+ signaling and NK cell-mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy., (©2020 Society for Leukocyte Biology.)

Systemic lupus erythematosus (SLE) is an autoimmune multisystem disease featured by an increased cardiovascular risk that may lead to premature patient's death. It has been demonstrated that SLE patients suffer from early onset endothelial dysfunction which is due to the impairment of endogenous vascular repair mechanisms. Vascular integrity and homeostasis are maintained by endothelial progenitor cells (EPCs), which are mobilized in response to endothelial injury to replace damaged endothelial cells. Two main EPCs subpopulations exist in peripheral blood: endothelial colony forming cells (ECFCs), which represent truly endothelial precursors and can physically engraft within neovessels, and myeloid angiogenic cells (MACs), which sustain angiogenesis in a paracrine manner. Emerging evidence indicates that ECFCs/MACs are down-regulated and display compromised angiogenic activity in SLE, thereby contributing to the pathogenesis of this disease. Intracellular calcium (Ca 2+ ) signaling plays a crucial role in maintaining vascular integrity by stimulating migration, proliferation and tube formation in both ECFCs and MACs. Herein, we illustrate the evidences that support the role played by EPCs dysfunction in SLE. Subsequently, we discuss about the hypothesis that the Ca 2+ handling machinery is compromised in SLE-derived ECFCs and MACs, thereby resulting in their reduced pro-angiogenic activity. Finally, we speculate about the proposal to exploit intracellular Ca 2+ signaling to improve ECFCs' reparative phenotype and suggest this strategy as a new approach to treat SLE patients., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 ( PLCG2 ), a B cell-specific enzyme that stimulates intracellular Ca 2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca + signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca + flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca + signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca + signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Rheumatoid arthritis (RA) is closely associated with shared epitope (SE)-coding HLA-DRB1 alleles and circulating anticitrullinated protein Abs (ACPA), but neither the respective pathogenic roles of SE and ACPA in RA nor the mechanisms underlying their coassociation are known. It was recently shown that the SE functions as a signal transduction ligand that activates a cell surface calreticulin-mediated, proarthritogenic, bone erosive pathway in an experimental model of RA. In this study, we demonstrate that stimulation of murine macrophages with LPS or DTT facilitated cell surface translocation of calreticulin, which in turn enabled increased SE-activated calcium signaling and activation of peptidylarginine deiminase with the resultant increased cellular abundance of citrullinated proteins. The i.p. administration of LPS to transgenic mice carrying a human SE-coding HLA-DRB1 allele lead to increased serum levels of TNF-α and anticitrullinated cyclic peptide Abs, along with terminal phalanx bone destruction. These data uncover a previously unknown signal transduction pathway by which the SE facilitates protein citrullination, ACPA production, and bone destruction., (Copyright © 2020 by The American Association of Immunologists, Inc.)

TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNβ in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNβ induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury.

The large family of C-type lectin (CLEC) receptors comprises carbohydrate-binding proteins that require Ca 2+ to bind a ligand. The prototypic receptor is the asialoglycoprotein receptor-1 (ASGR1, CLEC4H1) that is expressed primarily by hepatocytes. The early work on ASGR1, which is highly specific for N-acetylgalactosamine (GalNAc), established the foundation for understanding the overall function of CLEC receptors. Cells of the immune system generally express more than one CLEC receptor that serve diverse functions such as pathogen-recognition, initiation of cellular signaling, cellular adhesion, glycoprotein turnover, inflammation and immune responses. The receptor CLEC10A (C-type lectin domain family 10 member A, CD301; also called the macrophage galactose-type lectin, MGL) contains a carbohydrate-recognition domain (CRD) that is homologous to the CRD of ASGR1, and thus, is also specific for GalNAc. CLEC10A is most highly expressed on immature DCs, monocyte-derived DCs, and alternatively activated macrophages (subtype M2a) as well as oocytes and progenitor cells at several stages of embryonic development. This receptor is involved in initiation of T H 1, T H 2, and T H 17 immune responses and induction of tolerance in naïve T cells. Ligand-mediated endocytosis of CLEC receptors initiates a Ca 2+ signal that interestingly has different outcomes depending on ligand properties, concentration, and frequency of administration. This review summarizes studies that have been carried out on these receptors.

The cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyt ) regulates a vast number of cellular functions, including insulin secretion from beta cells. The major physiological insulin secretagogue, glucose, triggers [Ca 2+ ] cyt oscillations in beta cells. Synchronization of the oscillations among the beta cells within an islet underlies the generation of pulsatile insulin secretion. This review describes the mechanisms generating [Ca 2+ ] cyt oscillations, the interactions between [Ca 2+ ] cyt and cell metabolism, as well as the contribution of various organelles to the shaping of [Ca 2+ ] cyt signals and insulin secretion. It also discusses how Ca 2+ signals are coordinated and spread throughout the islets and data indicating that altered Ca 2+ signaling is associated with beta cell dysfunction and development of type 2 diabetes., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While B cells play a significant role in the onset of type-1 diabetes (T1D), little is know about their role in those early stages. Thus, to gain new insights into the role of B cells in T1D, we converted a physiological early pancreas-infiltrating B cell into a novel BCR mouse model using Somatic Cell Nuclear Transfer (SCNT). Strikingly, SCNT-derived B1411 model displayed neither developmental block nor anergy. Instead, B1411 underwent spontaneous germinal center reactions. Without T cell help, B1411-Rag1 -/- was capable of forming peri-/intra-pancreatic lymph nodes, and undergoing class-switching. RNA-Seq analysis identified 93 differentially expressed genes in B1411 compared to WT B cells, including Irf7, Usp18 , and Mda5 that had been linked to a potential viral etiology of T1D. We also found various members of the oligoadenylate synthase (OAS) family to be enriched in B1411, such as Oas1 , which had recently also been linked to T1D. Strikingly, when challenged with glucose B1411-Rag1 -/- mice displayed impaired glucose tolerance., (Copyright © 2020 Phillips, Ke, Nham, Seidl, Freeman, Abadejos, Xiao, Nemazee, Ku and Kirak.)

Ellagic acid, a naturally occurring phenol found in a variety of fruits and nuts has been shown to possess anti-inflammatory properties. However, the mechanism of action behind its anti-inflammatory action is unclear. Using human Jurkat T cells, our study examined the effects of ellagic acid (EA) on Ca 2+ handling, in particular, store-operated Ca 2+ entry (SOCE), a process critical to proper T cell function. We observed that the acute addition of EA-induced Ca 2+ release with an EC 50 of 63 μM. The Ca 2+ release was significantly attenuated by Xestospongin C, a known inhibitor of the Inositol 1,4,5-trisphosphate receptor (IP 3 R) channel and was unaffected by the phospholipase C (PLC) inhibitor, U73122. Furthermore, chronic incubation of Jurkat T cells with EA not only decreased the ATP-induced Ca 2+ release but also diminished the SOCE-mediated Ca 2+ influx in a dose-dependent manner. This inhibition was confirmed by reduced Mn 2+ entry rates in the EA-treated cells. The ATP-induced Ca 2+ entry was also attenuated in EA-treated HEK293 cells transiently transfected with SOCE channel Orai1-myc and ER-sensor stromal interaction molecule (STIM1) (HEK STIM/Orai ). Moreover, EA treatment interfered with the Orai1 and STIM1 coupling by disrupting STIM1 puncta formation in the HEK STIM/Orai cells. We observed that EA treatment reduced cytokine secretion and nuclear factor of activated T-cell transcriptional activity in stimulated T cells. Hence, by inhibiting SOCE mediated Ca 2+ influx, EA decreased downstream activation of pro-inflammatory mediators. These results suggest a novel target for EA-mediated effects and provide insight into the mechanisms underlying EA-mediated anti-inflammatory effects., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sterile inflammation contributes to many pathological states associated with mitochondrial injury. Mitochondrial injury disrupts calcium homeostasis and results in the release of CpG-rich mitochondrial DNA. The role of CpG-stimulated TLR9 innate immune signalling and sterile inflammation is well studied; however, how calcium dyshomeostasis affects this signalling is unknown. Therefore, we interrogated the relationship beτween intracellular calcium and CpG-induced TLR9 signalling in murine macrophages. We found that CpG-ODN-induced NFκB-dependent IL1α and IL1β expression was significantly attenuated by both calcium chelation and calcineurin inhibition, a finding mediated by inhibition of degradation of the NFκB inhibitory protein IκBβ. In contrast, calcium ionophore exposure increased CpG-induced IκBβ degradation and IL1α and IL1β expression. These results demonstrate that through its effect on IκBβ degradation, increased intracellular Ca 2+ drives a pro-inflammatory TLR9-mediated innate immune response. These results have implications for the study of innate immune signalling downstream of mitochondrial stress and injury., (© 2020 John Wiley & Sons Ltd.)

Cytotoxic lymphocytes, including natural killer (NK) cells and T cells are distinguished by their ability to eliminate target cells through release of secretory lysosomes. Conventional lysosomes and secretory lysosomes are part of the pleomorphic endolysosomal system and characterized by its highly dynamic nature. Several calcium-permeable TRP calcium channels play an essential role in endolysosomal calcium signaling to ensure proper function of these organelles. In NK cells, the expression of self MHC-specific inhibitory receptors dynamically tunes their secretory potential in a non-transcriptional, calcium-dependent manner. New insights suggest that TRPML1-mediated lysosomal calcium fluxes are tightly interconnected to NK cell functionality through modulation of granzyme B and perforin content of the secretory lysosome. Lysosomal TRP channels show a subset-specific expression pattern during NK differentiation, which is paralleled with gradually increased loading of effector molecules in secretory lysosomes. Methodological advances, including organellar patch-clamping, specific pharmacological modulators, and genetically-encoded calcium indicators open up new possibilities to investigate how TRP channels influence communication between intracellular organelles in immune cells. This review discusses our current understanding of lysosome biogenesis in NK cells with an emphasis on the TRP mucolipin family and the implications for NK cell functionality and cancer immunotherapy., (Copyright © 2020 Clement, Goodridge, Grimm, Patel and Malmberg.)

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca 2+ concentration ([Ca 2+ ] i ) in both BMMC and PMC, robust [Ca 2+ ] i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca 2+ ] i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca 2+ oscillations in BMMC and robust [Ca 2+ ] i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca 2+ ] i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca 2+ ] i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca 2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca 2+ ] i rise was reduced by ~20% in PMC from Trpc1/4/6 -/- mice compared to Trpc1/4 -/- littermatched control mice, whereas FcεRI-evoked [Ca 2+ ] i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca 2+ ] i rise in Trpc1/4/6 -/- PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca 2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified., (Copyright © 2020 Tsvilovskyy, Solis-Lopez, Almering, Richter, Birnbaumer, Dietrich and Freichel.)

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP 2 into the second messengers IP 3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP 2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP 3 generation), and eventually maintains intracellular Ca 2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca 2+ -activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation., (Copyright © 2020 by The American Association of Immunologists, Inc.)

During infection, phagocytic cells pursue homeostasis in the host via multiple mechanisms that control microbial invasion. Neutrophils respond to infection by exerting a variety of cellular processes, including chemotaxis, activation, phagocytosis, degranulation and the generation of reactive oxygen species (ROS). Calcium (Ca 2+ ) signaling and the activation of specific Ca 2+ channels are required for most antimicrobial effector functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating Ca 2+ mobilization and oxidative stress in neutrophils. In the present study, we use a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils' functions during infection. We show that the susceptibility of Trpm2 -/- mice to L. monocytogenes infection is characterized by increased migration rates of neutrophils and monocytes to the liver and spleen in the first 24 h. During the acute phase of L. monocytogenes infection, Trpm2 -/- mice developed septic shock, characterized by increased serum levels of TNF-α, IL-6, and IL-10. Furthermore, in vivo depletion of neutrophils demonstrated a critical role of these immune cells in regulating acute inflammation in Trpm2 -/- infected mice. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2 -/- neutrophils. Finally, the increased inflammatory properties of Trpm2 -/- neutrophils correlated with the dysregulated cytoplasmic concentration of Ca 2+ and potentiated membrane depolarization, in response to L. monocytogenes . In conclusion, our findings suggest that the TRPM2 channel plays critical functional roles in regulating the inflammatory properties of neutrophils and preventing tissue damage during Listeria infection., (Copyright © 2020 Robledo-Avila, Ruiz-Rosado, Brockman and Partida-Sánchez.)

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the β 2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process., (Copyright © 2019 Lőrincz, Szeifert, Bartos, Szombath, Mócsai and Ligeti.)

Many of the 25 members of the selenoprotein family function as enzymes that utilize their selenocysteine (Sec) residues to catalyze redox-based reactions. However, some selenoproteins likely do not exert enzymatic activity by themselves and selenoprotein K (SELENOK) is one such selenoprotein family member that uses its Sec residue in an alternative manner. SELENOK is an endoplasmic reticulum (ER) transmembrane protein that has been shown to be important for ER stress and for calcium-dependent signaling. Molecular mechanisms for the latter have recently been elucidated using knockout mice and genetically manipulated cell lines. These studies have shown that SELENOK interacts with an enzyme in the ER membrane, DHHC6 (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain), and the SELENOK/DHHC6 complex catalyzes the transfer of acyl groups such as palmitate to cysteine residues in target proteins, i.e., palmitoylation. One protein palmitoylated by SELENOK/DHHC6 is the calcium channel protein, the inositol 1,4,5-trisphosphate receptor (IP3R), which is acylated as a means for stabilizing the tetrameric calcium channel in the ER membrane. Factors that lower SELENOK levels or function impair IP3R-driven calcium flux. This role for SELENOK is important for the activation and proliferation of immune cells, and recently, a critical role for SELENOK in promoting calcium flux for the progression of melanoma has been demonstrated. This review provides a summary of these findings and their implications in terms of designing new therapeutic interventions that target SELENOK for treating cancers like melanoma.

Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca 2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca 2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca 2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H 2 O 2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca 2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays., (© 2019 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Amplitude and kinetics of intracellular Ca 2+ signals ([Ca 2+ ] int ) determine many immune cell functions. To mimic in vivo changes of [Ca 2+ ] int in human immune cells, two approaches may be best suited: 1) Analyze primary human immune cells taken from blood under conditions resembling best physiological or pathophysiological conditions. 2.) Analyze the immune system in vivo or ex vivo in explanted tissue from small vertebrate animals, such as mice. With the help of genetically encoded Ca 2+ indicators and intravital microscopy, [Ca 2+ ] int have been investigated in murine T lymphocytes (T cells) in vivo during the last five years and in explanted lymph node (LN) during the last 10 years. There are several important reasons to compare [Ca 2+ ] int measured in primary murine T lymphocytes in vivo and in vitro with [Ca 2+ ] int measured in primary human T lymphocytes in vitro. First, how do human and murine data compare? Second, how do in vivo and in vitro data compare? Third, can in vitro data predict in vivo data? The last point is particularly important considering the many technical challenges that limit in vivo measurements and to reduce the number of animals sacrificed. This review summarizes and compares the results of the available publications on in vivo and in vitro [Ca 2+ ] int measurements in T lymphocytes stimulated focally by antigen-presenting cells (APC) after forming an immunological synapse., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Calcium is a key regulator of the T cell immune response. Depending on the spatial properties (nucleus versus cytoplasm) of the calcium signals generated after CD3xCD28 stimulation, primary human T cells either mount a productive immune response or develop tolerance. Nuclear calcium acts as a genomic decision maker: during T cell activation, it drives expression of genes associated with a productive immune response while in its absence, stimulated T cells acquire an anergy-like gene profile. Selective inhibition of nuclear calcium signaling in stimulated T cells blocks the productive immune response and directs the cells towards an anergy-like state. Here we show that the two transcriptional programs that include, respectively, the 'activation gene', interleukin 2 (IL2) and 'anergy-related genes', EGR2, EGR3, and CREM have different requirements for transmembrane calcium flux. By either lowering extracellular calcium concentrations with EGTA or using low concentrations of the ORAI blockers, BTP2 or RO2959, we reduced transmembrane calcium flux in human primary T cells stimulated with CD3xCD28. These 'conditions of limited calcium influx' (CLCI) blocked CD3xCD28-induced IL2 expression but only moderately affected induction of the anergy-related genes EGR2, EGR3, and CREM. We observed no difference in NFAT2 nuclear translocation after CD3xCD28 stimulation between normal conditions and CLCI. These results indicate that CLCI favors expression of anergy-related genes in activated human T cells. CLCI may be used to develop novel means for pro-tolerance immunosuppressive treatments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The gut microbiota influences several biological functions including immune responses. Inflammatory bowel disease is favorably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts, and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolizes ellagic acid resulting in the formation of bioactive urolithins A, B, C, and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, whether gut metabolite UA affects the function of immune cells remains incompletely understood. T cell proliferation is stimulated by store operated Ca 2+ entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4 + T cells with UA (10 μM, 3 days) significantly blunted SOCE in CD4 + T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4 + T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4 + T cells. UA further decreased CD4 + T cell proliferation. Thus, the gut bacterial metabolite UA increases miR-10a-5p levels thereby downregulating Orai1/STIM1/STIM2 expression, store operated Ca 2+ entry, and proliferation of murine CD4 + T cells.

The pancreatic islet is a complex micro-organ containing numerous cell types, including endocrine, immune, and endothelial cells. The communication of these systems is lost upon isolation of the islets, and therefore the pathogenesis of diabetes can only be fully understood by studying this organized, multicellular environment in vivo. We have developed several adaptable tools to create a versatile platform to interrogate β-cell function in vivo. Specifically, we developed β-cell-selective virally-encoded fluorescent protein biosensors that can be rapidly and easily introduced into any mouse. We then coupled the use of these biosensors with intravital microscopy, a powerful tool that can be used to collect cellular and subcellular data from living tissues. Together, these approaches allowed the observation of in vivo β-cell-specific ROS dynamics using the Grx1-roGFP2 biosensor and calcium signaling using the GcAMP6s biosensor. Next, we utilized abdominal imaging windows (AIW) to extend our in vivo observations beyond single-point terminal measurements to collect longitudinal physiological and biosensor data through repeated imaging of the same mice over time. This platform represents a significant advancement in our ability to study β-cell structure and signaling in vivo, and its portability for use in virtually any mouse model will enable meaningful studies of β-cell physiology in the endogenous islet niche.

UVB-induced skin damage is attributable to reactive oxygen species, which are triggered by intracellular Ca 2+ signals. However, exactly how the reactive oxygen species are triggered by intracellular Ca 2+ upon UVB irradiation remains obscure. Here, we show that UVB induces Ca 2+ signals via sequential generation of the following Ca 2+ messengers: inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose. UVB induced H 2 O 2 production through NADPH oxidase 4 activation, which is downstream to inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate. H 2 O 2 derived from NADPH oxidase 4 activated CD38 to produce cyclic ADP-ribose. UVB first evoked the pannexin channel to release ATP, which acts on P2X7 receptor to generate inositol 1,4,5-trisphosphate. Inhibitors of these messengers, as well as antioxidants, blocked UVB-induced Ca 2+ signals and IL-1β secretion in keratinocytes. Furthermore, ablation of CD38 and NADPH oxidase 4 protected against UVB-induced inflammation and IL-1β secretion in the murine epidermis. These results show that UVB induces IL-1β secretion through cross-talk between Ca 2+ and reactive oxygen species, providing insight towards potential targets against UVB-induced inflammation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Appropriate T cell responses are controlled by strict balance between activatory and inhibitory pathways downstream of TCR. Although mice or humans with impaired TCR signaling develop autoimmunity, the precise molecular mechanisms linking reduced TCR signaling to autoimmunity are not fully understood. Engagement of TCR activates Ca 2+ signaling mainly through store-operated Ca 2+ entry activated by stromal interaction molecule (Stim) 1 and Stim2. Despite defective T cell activation, mice deficient in both Stim1 and Stim2 in T cells (conditional double knockout [cDKO]) developed lymphoproliferative disorders and skin inflammation with a concomitant increase in serum IgG1 and IgE levels. In cDKO mice, follicular helper T (Tfh) cells were dramatically increased in number, and they produced IL-4 spontaneously. These inflammatory symptoms were abolished by the deletion of IL-4 in cDKO mice. Tfh development and inflammatory symptoms in cDKO mice were abrogated by further deletion of NFAT2 in T cells. These findings suggest that Tfh cells spontaneously developed in the absence of Ca 2+ signaling and caused unregulated type 2 responses., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Calcium intake has been associated with a lower risk of colorectal cancer. Calcium signaling may enhance T-cell proliferation and differentiation, and contribute to T-cell-mediated antitumor immunity. In this prospective cohort study, we investigated the association between calcium intake and colorectal cancer risk according to tumor immunity status to provide additional insights into the role of calcium in colorectal carcinogenesis. The densities of tumor-infiltrating T-cell subsets [ CD3 + , CD8 + , CD45RO ( PTPRC ) + , or FOXP3 + cell] were assessed using IHC and computer-assisted image analysis in 736 cancer cases that developed among 136,249 individuals in two cohorts. HRs and 95% confidence intervals (CI) were calculated using Cox proportional hazards regression. Total calcium intake was associated with a multivariable HR of 0.55 (comparing ≥1,200 vs. <600 mg/day; 95% CI, 0.36-0.84; P trend = 0.002) for CD8 + T-cell-low but not for CD8 + T-cell-high tumors (HR = 1.02; 95% CI, 0.67-1.55; P trend = 0.47). Similarly, the corresponding HRs (95% CIs) for calcium for low versus high T-cell-infiltrated tumors were 0.63 (0.42-0.94; P trend = 0.01) and 0.89 (0.58-1.35; P trend = 0.20) for CD3 + ; 0.58 (0.39-0.87; P trend = 0.006) and 1.04 (0.69-1.58; P trend = 0.54) for CD45RO + ; and 0.56 (0.36-0.85; P trend = 0.006) and 1.10 (0.72-1.67; P trend = 0.47) for FOXP3 + , although the differences by subtypes defined by T-cell density were not statistically significant. These potential differential associations generally appeared consistent regardless of sex, source of calcium intake, tumor location, and tumor microsatellite instability status. Our findings suggest a possible role of calcium in cancer immunoprevention via modulation of T-cell function., (©2019 American Association for Cancer Research.)

Background: Dysregulation in calcium (Ca 2+ ) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca 2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca 2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca 2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study., Methods: An extensive analysis of the Ca 2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca 2+ entry, basal Ca 2+ levels, and store operated Ca 2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients., Results: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca 2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP 3 R (SOCE) Ca 2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1 PM ); and (vi) blocked when using a mAb targeting STIM1 PM . Next, we further established an association between an elevated expression of STIM1 PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1 PM CLL subgroup., Conclusions: These data establish the critical role of a newly discovered BCR independent Ca 2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

Basophils have nonredundant roles in various immune responses that require Ca 2+ influx. Here, we examined the role of two Ca 2+ sensors, stromal interaction molecule 1 and 2 (STIM1 and STIM2), in basophil activation. We found that loss of STIM1, but not STIM2, impaired basophil IL-4 production after stimulation with immunoglobulin E (IgE)-containing immune complexes. In contrast, when basophils were stimulated with IL-3, loss of STIM2, but not STIM1, reduced basophil IL-4 production. This difference in STIM proteins was associated with distinct time courses of Ca 2+ influx and transcription of the Il4 gene that were elicited by each stimulus. Similarly, basophil-specific STIM1 expression was required for IgE-driven chronic allergic inflammation in vivo, whereas STIM2 was required for IL-4 production after combined IL-3 and IL-33 treatment in mice. These data indicate that STIM1 and STIM2 have differential roles in the production of IL-4, which are stimulus dependent. Furthermore, these results illustrate the vital role of STIM2 in basophils, which is often considered to be less important than STIM1., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ion channels facilitate the movement of ions across the plasma and organellar membranes. A recent symposium brought together scientists who study ion channels and transporters in immune cells, which highlighted advances in this emerging field and served to chart new avenues for investigating the roles of ion channels in immunity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)