Cavß1 regulates T cell expansion and apoptosis independently of voltage-gated Ca2+ channel function.

TCR stimulation triggers Ca2+ signals that are critical for T cell function and immunity. Several pore-forming α and auxiliary ß subunits of voltage-gated Ca2+ channels (VGCC) were reported in T cells, but their mechanism of activation remains elusive and their contribution to Ca2+ signaling in T cells is controversial. We here identify CaVß1, encoded by Cacnb1, as a regulator of T cell function. Cacnb1 deletion enhances apoptosis and impairs the clonal expansion of T cells after lymphocytic choriomeningitis virus (LCMV) infection. By contrast, Cacnb1 is dispensable for T cell proliferation, cytokine production and Ca2+ signaling. Using patch clamp electrophysiology and Ca2+ recordings, we are unable to detect voltage-gated Ca2+ currents or Ca2+ influx in human and mouse T cells upon depolarization with or without prior TCR stimulation. mRNAs of several VGCC α1 subunits are detectable in human (CaV3.3, CaV3.2) and mouse (CaV2.1) T cells, but they lack transcription of many 5' exons, likely resulting in N-terminally truncated and non-functional proteins. Our findings demonstrate that although CaVß1 regulates T cell function, these effects are independent of VGCC channel activity.

The volume-regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling.

The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2'3'cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c-/- mice have increased cell cycle progression, proliferation, survival, Ca2+ influx and cytokine production-a phenotype associated with downmodulation of p53 signaling. Mechanistically, LRRC8C mediates the transport of 2'3'cGAMP in T cells, resulting in STING and p53 activation. Inhibition of STING recapitulates the phenotype of LRRC8C-deficient T cells, whereas overexpression of p53 inhibits their enhanced T cell function. Lrrc8c-/- mice have exacerbated T cell-dependent immune responses, including immunity to influenza A virus infection and experimental autoimmune encephalomyelitis. Our results identify cGAMP uptake through LRRC8C and STING-p53 signaling as a new inhibitory signaling pathway in T cells and adaptive immunity.

STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of non-pathogenic Th17 cells.

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell-specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases.

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Eye on ion channels in immune cells.

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.

Targeting the anion exchanger 2 with specific peptides as a new therapeutic approach in B lymphoid neoplasms.

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.

Regulation of epithelial ion transport in exocrine glands by store-operated Ca2+ entry.

Store-operated Ca2+ entry (SOCE) is a conserved mechanism of Ca2+ influx that regulates Ca2+ signaling in many cell types. SOCE is activated by depletion of endoplasmic reticulum (ER) Ca2+ stores in response to physiological agonist stimulation. After it was first postulated by J.W. Putney Jr. in 1986, SOCE has been described in a large number of non-excitable cell types including secretory cells of different exocrine glands. Here we discuss the mechanisms by which SOCE controls salt and fluid secretion in exocrine glands, with a special focus on eccrine sweat glands. In sweat glands, SOCE plays an important, non-redundant role in regulating the function of Ca2+-activated Cl- channels (CaCC), Cl- secretion and sweat production. In the absence of key regulators of SOCE such as the CRAC channel pore subunit ORAI1 and its activator STIM1, the Ca2+-activated chloride channel TMEM16A is inactive and fails to secrete Cl-, resulting in anhidrosis in mice and human patients.

Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function.

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

Dental enamel cells express functional SOCE channels.

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

CD8+ T cells undergo activation and programmed death-1 repression in the liver of aged Ae2a,b-/- mice favoring autoimmune cholangitis.

UNLABELLED: Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl-/HCO3- anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b(-/-) mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b(-/-) mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥ 10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8(+) T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2'-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8(+) T cells from aged knockouts. CONCLUSION: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.

Ursodeoxycholic acid inhibits hepatic cystogenesis in experimental models of polycystic liver disease.

BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive biliary cystogenesis. Current therapies show short-term and/or modest beneficial effects. Cystic cholangiocytes hyperproliferate as a consequence of diminished intracellular calcium levels ([Ca(2+)]i). Here, the therapeutic value of ursodeoxycholic acid (UDCA) was investigated. METHODS: Effect of UDCA was examined in vitro and in polycystic (PCK) rats. Hepatic cystogenesis and fibrosis, and the bile acid (BA) content were evaluated from the liver, bile, serum, and kidneys by HPLC-MS/MS. RESULTS: Chronic treatment of PCK rats with UDCA inhibits hepatic cystogenesis and fibrosis, and improves their motor behaviour. As compared to wild-type animals, PCK rats show increased BA concentration ([BA]) in liver, similar hepatic Cyp7a1 mRNA levels, and diminished [BA] in bile. Likewise, [BA] is increased in cystic fluid of PLD patients compared to their matched serum levels. In PCK rats, UDCA decreases the intrahepatic accumulation of cytotoxic BA, normalizes their diminished [BA] in bile, increases the BA secretion in bile and diminishes the increased [BA] in kidneys. In vitro, UDCA inhibits the hyperproliferation of polycystic human cholangiocytes via a PI3K/AKT/MEK/ERK1/2-dependent mechanism without affecting apoptosis. Finally, the presence of glycodeoxycholic acid promotes the proliferation of polycystic human cholangiocytes, which is inhibited by both UDCA and tauro-UDCA. CONCLUSIONS: UDCA was able to halt the liver disease of a rat model of PLD through inhibiting cystic cholangiocyte hyperproliferation and decreasing the levels of cytotoxic BA species in the liver, which suggests the use of UDCA as a potential therapeutic tool for PLD patients.

Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells.

Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.

Mouse models of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic and progressive cholestatic liver disease of unknown etiopathogenesis that mainly affects middle-aged women. Patients show non-suppurative cholangitis with damage and destruction of small- and medium-sized intrahepatic bile ducts. Characteristically, the disease is strongly associated with autoimmune phenomena such as the appearance of serum antimitochondrial autoantibodies (AMA) and portal infiltrates with autoreactive T cells which recognize the inner lipoyl domain of the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Here we review the major characteristics of a series of inducible and genetically modified animal models of PBC and analyze their similarities and differences with PBC features in humans.

Anion exchanger 2 is critical for CD8(+) T cells to maintain pHi homeostasis and modulate immune responses.

Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses.

Identification of fibroblast growth factor 15 as a novel mediator of liver regeneration and its application in the prevention of post-resection liver failure in mice.

OBJECTIVE: Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. Fibroblast growth factor 15 (Fgf15, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf15 in the preservation of BA homeostasis after PH and its potential role in the regenerative process. DESIGN: Liver regeneration after PH was studied in Fgf15 (-/-) and Fgf15 (+/+) mice. The effects of the BA sequestrant cholestyramine and adenovirally delivered Fgf15 were examined in this model. The role of Fgf15 in BA-induced liver growth was tested in Fgf15 (-/-) mice upon cholic acid (CA) feeding. The direct mitogenic effect of Fgf15 was evaluated in cultured mouse hepatocytes and cholangiocytes. RESULTS: Fgf15 (-/-) mice showed marked liver injury and mortality after PH accompanied by persistently elevated intrahepatic BA levels. Cholestyramine feeding and adenovirally delivered Fgf15 reduced BA levels and significantly prevented this lethal outcome. Fgf15 also reduced mortality after extensive hepatectomy in Fgf15(+/+) animals. Liver growth elicited by CA feeding was significantly diminished in Fgf15 (-/-) mice. Proliferation of hepatocytes and cholangiocytes was also noticeably reduced in CA-fed Fgf15 (-/-) mice. Fgf15 induced intracellular signalling and proliferation of cultured hepatocytes and cholangiocytes. CONCLUSIONS: Fgf15 is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf15 is an essential mediator of the liver growth-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.

Role of AE2 for pHi regulation in biliary epithelial cells.

The Cl(-)/HCO(-) 3anion exchanger 2 (AE2) is known to be involved in intracellular pH (pHi) regulation and transepithelial acid-base transport. Early studies showed that AE2 gene expression is reduced in liver biopsies and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic non-suppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. Microfluorimetric analysis of the Cl(-)/HCO(-) 3 anion exchange (AE) in isolated cholangiocytes showed that the cAMP-stimulated AE activity is diminished in PBC compared to both healthy and diseased controls. More recently, it was found that miR-506 is upregulated in cholangiocytes of PBC patients and that AE2 may be a target of miR-506. Additional evidence for a pathogenic role of AE2 dysregulation in PBC was obtained with Ae2 (-/-) a,b mice, which develop biochemical, histological, and immunologic alterations that resemble PBC (including development of serum AMA). Analysis of HCO(-) 3 transport systems and pHi regulation in cholangiocytes from normal and Ae2 (-/-) a,b mice confirmed that AE2 is the transporter responsible for the Cl(-)/HCO(-) 3exchange in these cells. On the other hand, both Ae2 (+/+) a,b and Ae2 (-/-) a,b mouse cholangiocytes exhibited a Cl(-)-independent bicarbonate transport system, essentially a Na(+)-bicarbonate cotransport (NBC) system, which could contribute to pHi regulation in the absence of AE2.

Approaches to the pathogenesis of primary biliary cirrhosis through animal models.

Primary biliary cirrhosis (PBC) is a chronic and progressive cholestatic liver disease of unknown etiopathogenesis that mainly affects middle-aged women. Patients show non-suppurative cholangitis with damage and destruction of the small- and medium-sized intrahepatic bile ducts. Characteristically, the disease is strongly associated with autoimmune phenomena such as the appearance of serum antimitochondrial autoantibodies (AMA) and portal infiltrating T cells against the inner lipoyl domain in the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Here we review the major characteristics of a series of inducible and genetically modified animal models of PBC and analyze the similarities and differences to PBC features in humans.