Magali Savignac

Protein kinase C-mediated calcium entry dependent upon dihydropyridine sensitive channels: a T cell receptor-coupled signaling pathway involved in IL-4 synthesis

Signaling events induced by T‐cell receptor (TCR) engagement involve a cascade of tyrosine phosphorylation events leading to activation of several downstream pathways and resulting in cytokine production. TCR‐dependent interferon γ (IFN‐γ) production by Th1 cells has been shown to require tyrosine phosphorylation of numerous proteins, intracellular Ca2+ mobilization, and mitogen‐activated protein kinase activation. In contrast, the signaling pathways responsible for TCR‐dependent interleukin (IL) 4 production remain poorly understood. By using a T‐cell hybridoma that displays a hierarchized production of IL‐4 and IFN‐γ following TCR engagement (IL‐4 being produced at a lower threshold of activation than IFN‐γ), we showed that IL‐4 can be produced in spite of the absence of tyrosine phosphorylation of phospholipase Cγ1. However, protein kinase C (PKC) was found to be translocated to the cell membrane, and an increase in intracellular Ca2+ concentration was observed. The PKC‐dependent Ca2+ response and IL‐4 expression were accounted for by a dihydropyridine‐sensitive Ca2+ entry, which could occur through L‐type calcium channels. This pathway was also functional in the D10G4.1 Th2 clone. The fact that this pathway, allowing IL‐4 production, did not require optimal activation might explain why low doses of peptides or altered peptide ligands favor Th2 responses.

Weak TCR stimulation induces a calcium signal that triggers IL-4 synthesis, stronger TCR stimulation induces MAP kinases that control IFN-γ production

Th1 and Th2 cells produce different cytokines and have distinct functions. Th1/Th2 cell differentiation is influenced, among other factors, by the nature of TCR‐MHC interactions. However, how the TCR transduces a signal resulting in IFN‐γ or IL‐4 production is a matter of debate. For example, some authors reported a loss of calcium signaling pathway in Th2 cells. We used a T cell hybridoma producing IL‐4 upon weak TCR stimulation and both IL‐4 and IFN‐γ for strong TCR engagement as a model to study how TCR signaling pathways are differentially activated in both conditions of stimulation and how this influences the production of cytokines. We show that: (1) the calcium response is identical following weak and strong TCR stimulation; (2) mitogen‐activated protein kinase(MAPK) activation is a gradual phenomenon depending upon the strength of TCR activation; (3) a calcium response, even weak, triggers IL‐4 expression; (4) IFN‐γ synthesis requires not only a calcium response but also MAPK activation. The MAPK pathway is dispensable for IL‐4 production, although it amplifies IL‐4 synthesis upon strong TCR stimulation; (5) TCR‐induced IL‐4 production also dependson calcium signaling in Th2 cells, while IFN‐γ synthesis is dependent, in addition, on MAPK activation in Th1 cells.

Dihydropyridine Receptors Are Selective Markers of Th2 Cells and Can Be Targeted to Prevent Th2-Dependent Immunopathological Disorders

Th1 cells that produce IFN-γ are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4+ T cells from BN rats into (LEW × BN)F1 hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.

Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma.

RATIONALE Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions. OBJECTIVES Because dihydropyridines bind to and modulate voltage-dependent calcium (Ca(v)1) channel in excitable cells, we aimed at showing that Th2 cells selectively express functional Ca(v)1-related channels, the inhibition of which may prevent asthma. METHODS We looked for Ca(v)1 channel expression in Th2 and Th1 cells by real-time polymerase chain reaction and Western blotting. We sequenced the isoforms expressed by Th2 cells and tested whether Ca(v)1 antisense oligodeoxynucleotides (Ca(v)1AS) affected Ca(2+) signaling and cytokine production. Finally, we tested the effect of Ca(v)1AS in the passive asthma model by injection of ovalbumin-specific Th2 cells transfected with Ca(v)1AS into BALB/c mice challenged with intranasal ovalbumin and in the active model of asthma by intranasal delivery of Ca(v)1AS together with soluble ovalbumin in BALB/c mice previously immunized with ovalbumin in alum. MEASUREMENTS AND MAIN RESULTS We show that mouse Th2 but not Th1 cells expressed Ca(v)1.2 and Ca(v)1.3 channels. Th2 cells transfected with Ca(v)1AS had impaired Ca(2+) signaling and cytokine production, and lost their ability to induce airway inflammation on adoptive transfer. Furthermore, intranasal administration of Ca(v)1AS suppressed airway inflammation and hyperreactivity in an active model of asthma. CONCLUSIONS These results indicate that Th2 cells selectively express Ca(v)1 channels that may be efficiently targeted in T lymphocytes to prevent experimental asthma.

Androgen signaling negatively controls group 2 innate lymphoid cells

Prevalence of asthma is higher in women than in men, but the mechanisms underlying this sex bias are unknown. Group 2 innate lymphoid cells (ILC2s) are key regulators of type 2 inflammatory responses. Here, we show that ILC2 development is greatly influenced by male sex hormones. Male mice have reduced numbers of ILC2 progenitors (ILC2Ps) and mature ILC2s in peripheral tissues compared with females. In consequence, males exhibit reduced susceptibility to allergic airway inflammation in response to environmental allergens and less severe IL-33–driven lung inflammation, correlating with an impaired expansion of lung ILC2s. Importantly, orchiectomy, but not ovariectomy, abolishes the sex differences in ILC2 development and restores IL-33–mediated lung inflammation. ILC2Ps express the androgen receptor (AR), and AR signaling inhibits their differentiation into mature ILC2s. Finally, we show that hematopoietic AR expression limits IL-33–driven lung inflammation through a cell-intrinsic inhibition of ILC2 expansion. Thus, androgens play a crucial protective role in type 2 airway inflammation by negatively regulating ILC2 homeostasis, thereby limiting their capacity to expand locally in response to IL-33.

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes.

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.

SERCA2 Dysfunction in Darier Disease Causes Endoplasmic Reticulum Stress and Impaired Cell-to-Cell Adhesion Strength: Rescue by Miglustat

Darier disease (DD) is a severe dominant genetic skin disorder characterized by the loss of cell-to-cell adhesion and abnormal keratinization. The defective gene, ATP2A2, encodes sarco/endoplasmic reticulum (ER) Ca2+ -ATPase isoform 2 (SERCA2), a Ca2+ -ATPase pump of the ER. Here we show that Darier keratinocytes (DKs) display biochemical and morphological hallmarks of constitutive ER stress with increased sensitivity to ER stressors. Desmosome and adherens junctions (AJs) displayed features of immature adhesion complexes: expression of desmosomal cadherins (desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3)) and desmoplakin was impaired at the plasma membrane, as well as E-cadherin, β-, α-, and p120-catenin staining. Dsg3, Dsc3, and E-cadherin showed perinuclear staining and co-immunostaining with ER markers, indicative of ER retention. Consistent with these abnormalities, intercellular adhesion strength was reduced as shown by a dispase mechanical dissociation assay. Exposure of normal keratinocytes to the SERCA2 inhibitor thapsigargin recapitulated these abnormalities, supporting the role of loss of SERCA2 function in impaired desmosome and AJ formation. Remarkably, treatment of DKs with the orphan drug Miglustat, a pharmacological chaperone, restored mature AJ and desmosome formation, and improved adhesion strength. These results point to an important contribution of ER stress in DD pathogenesis and provide the basis for future clinical evaluation of Miglustat in Darier patients.

Gold is a T cell polyclonal activator in BN and LEW rats but favors IL‐4 expression only in autoimmune prone BN rats

Gold salts are beneficial in the treatment of rheumatoid arthritis but may induce immune‐mediated disorders in predisposed patients. Gold salts induce Th2‐dependent autoimmunity in Brown‐Norway (BN) rats but not in Lewis (LEW) rats. The aim of this study was to define molecular targets of gold salts and to approach why LEW rats are resistant. Gold salts act on early steps of transductionin T cells from BN and LEW rats since they trigger tyrosine phosphorylation of numerous proteins including p56lck and a calcium signal which results in IL‐4 and IFN‐γ expression by BN and LEW T cells. However, the IL‐4 response was favored in BN spleen cells in vitro and in vivo. IFN‐γ, produced in part by CD8+ cells, contributes to the resistance of LEW rats since gold salt‐injected LEW rats receiving anti‐CD8 or anti‐IFN‐γ mAb displayed the parameters characteristics of gold salt‐induced Th2 autoimmunity although to a lesser extent than in BN rats. Gold salts transduce a signal in BN and LEW spleen cells resulting in IL‐4 and IFN‐γ gene transcription with a preferential IL‐4 response in BN rats, a Th2‐prone strain, while IFN‐γ contributes to the resistance of LEW rats.

Protein kinase C–dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions

BACKGROUND In addition to calcium release-activated calcium channel/ORAI calcium channels, the role of voltage-gated calcium (Cav1) channels in T-cell calcium signaling is emerging. Cav1 channels are formed by α1 (CaV1.1 to CaV1.4) and auxiliary subunits. We previously demonstrated that mouse TH2 cells selectively overexpressed CaV1.2 and CaV1.3 channels. Knocking down these channels with Cav1 antisense (AS) oligonucleotides inhibited TH2 functions and experimental asthma. OBJECTIVE We investigated the expression profile and role of Cav1 channels in human T-cell subsets, with a focus on TH2 cells. METHODS We compared the profile of CaV1 channel subunit expression in T-cell subsets isolated ex vivo from the blood of healthy donors, as well as in vitro-polarized T-cell subsets, and tested the effect of the Cav1 inhibitors nicardipine and Cav1.2AS on their functions. RESULTS CaV1.4 expression was detectable in CD4(+) T cells, ex vivo TH1 cells, and TH17 cells, whereas Cav1.2 channels predominated in TH2 cells only. T-cell activation resulted in Cav1.4 downregulation, whereas Cav1.2 expression was selectively maintained in polarized TH2 cells and absent in TH1 or TH9 cells. Nicardipine and CaV1.2AS decreased Ca(2+) and cytokine responses in TH2, but not TH1, cells. Protein kinase C (PKC) α/β inhibition decreased Ca(2+) and cytokine responses, whereas both calcium and cytokine responses induced by PKC activation were inhibited by nicardipine or Cav1.2AS in TH2 cells. CONCLUSION This study highlights the selective expression of Cav1.2 channels in human TH2 cells and the role of PKC-dependent Cav1.2 channel activation in TH2 cell function. Blocking PKC or Cav1.2 channel activation in TH2 cells might represent new strategies to treat allergic diseases in human subjects.

Increased B Cell Proliferation and Reduced Ig Production in DREAM Transgenic Mice

DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum Ig levels. In vitro assays show that reduced Ig secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation, class switch recombination, or Ig transcription. Surprisingly, transgenic B cells show an accelerated entry in cell division. Transcriptomic analysis of transgenic B cells revealed that hyperproliferative B cell response could be correlated with a reduced expression of Klf9, a cell-cycle regulator. Pulse-chase experiments demonstrated that the defect in Ig production is associated with reduced translation rather than with increased protein degradation. Importantly, transgenic B cells showed reduced expression of the Eif4g3 gene, which encodes a protein related to protein translation. Our results disclose, to our knowledge, a novel function of DREAM in proliferation and Ig synthesis in B lymphocytes.