Altered X-chromosome inactivation predisposes to autoimmunity.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Cav1.4 calcium channels control cytokine production by human peripheral TH17 cells and psoriatic skin-infiltrating T cells.

BACKGROUND: Type-17 inflammation characterizes psoriasis, a chronic skin disease. Because several inflammatory cytokines contribute to psoriasis pathogenesis, inhibiting the simultaneous production of these cytokines in TH17 cells may be beneficial in psoriasis. We found that Cav1.4, encoded by CACNA1F, was the only Cav1 calcium channel expressed in TH17 cells. OBJECTIVE: We sought to investigate the role of Cav1.4 expression in early TH17-activation events and effector functions, as well as its association with TH17 signature genes in lesional psoriatic (LP) skins. METHODS: Transcriptional gene signatures associated with CACNA1F expression were examined in LP skins by RT-PCR and in situ hybridization. Cav1 inhibitor and/or shRNA lentivectors were used to assess the contribution of Cav1.4 in TH17 activation and effector functions in a 3-dimensional skin reconstruction model. RESULTS: CACNA1F expression correlated with inflammatory cytokine expression that characterizes LP skins and was preferentially associated with RORC expression in CD4+ and CD4- cells from LP biopsies. Nicardipine, a Cav1 channel antagonist, markedly reduced inflammatory cytokine production by TH17 cells from blood or LP skin. This was associated with decreased TCR-induced early calcium events at cell membrane and proximal signaling events. The knockdown of Cav1.4 in TH17 cells impaired cytokine production. Finally, Cav1 inhibition reduced the expression of the keratinocyte genes characteristic of TH17-mediated psoriasis inflammation in human skin equivalents. CONCLUSIONS: Cav1.4 channels promote TH17-cell functions both at the periphery and in inflammatory psoriatic skin.

Separation of the CaV 1.2-CaV 1.3 calcium channel duo prevents type 2 allergic airway inflammation.

BACKGROUND: Voltage-gated calcium (Cav 1) channels contribute to T-lymphocyte activation. Cav 1.2 and Cav 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. METHODS: We generated mice deleted for Cav 1.2 in T cells or Cav 1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Cav 1.2 or Cav 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav 1.2 and Cav 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. RESULTS: We demonstrated non-redundant and synergistic functions of Cav 1.2 and Cav 1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Cav 1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav 1.2 and Cav 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav 1.2 and Cav 1.3 must be co-expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav 1.2 and Cav 1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2-cytokine transcription. CONCLUSIONS: Thus, Cav 1.2 and Cav 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.

Involvement of ion channels in allergy.

Allergic asthma is a complex disease, often characterized by an inappropriate Th2 response to normally harmless allergens. Epithelial cells damaged or activated by the allergen produce IL-33, TSLP and IL-25, activating ILC2 and dendritic cells. The latter migrate into lymph nodes where they induce Th2-cell commitment. Th2 and other type 2 innate inflammatory cells trigger inflammation and airway hyper-reactivity. The toolbox consisting of the ion channels varies from one cellular type to another and depends on its activation state, offering the possibility to design novel drugs in the field of allergy. We will discuss about some channels as calcium, nonselective cation, potassium and chloride channels that appear as good candidates in allergy.

The ß and α2δ auxiliary subunits of voltage-gated calcium channel 1 (Cav1) are required for TH2 lymphocyte function and acute allergic airway inflammation.

BACKGROUND: T lymphocytes express not only cell membrane ORAI calcium release-activated calcium modulator 1 but also voltage-gated calcium channel (Cav) 1 channels. In excitable cells these channels are composed of the ion-forming pore α1 and auxiliary subunits (ß and α2δ) needed for proper trafficking and activation of the channel. Previously, we disclosed the role of Cav1.2 α1 in mouse and human TH2 but not TH1 cell functions and showed that knocking down Cav1 α1 prevents experimental asthma. OBJECTIVE: We investigated the role of ß and α2δ auxiliary subunits on Cav1 α1 function in TH2 lymphocytes and on the development of acute allergic airway inflammation. METHODS: We used Cavß antisense oligonucleotides to knock down Cavß and gabapentin, a drug that binds to and inhibits α2δ1 and α2δ2, to test their effects on TH2 functions and their capacity to reduce allergic airway inflammation. RESULTS: Mouse and human TH2 cells express mainly Cavß1, ß3, and α2δ2 subunits. Cavß antisense reduces T-cell receptor-driven calcium responses and cytokine production by mouse and human TH2 cells with no effect on TH1 cells. Cavß is mainly involved in restraining Cav1.2 α1 degradation through the proteasome because a proteasome inhibitor partially restores the α1 protein level. Gabapentin impairs the T-cell receptor-driven calcium response and cytokine production associated with the loss of α2δ2 protein in TH2 cells. CONCLUSIONS: These results stress the role of Cavß and α2δ2 auxiliary subunits in the stability and activation of Cav1.2 channels in TH2 lymphocytes both in vitro and in vivo, as demonstrated by the beneficial effect of Cavß antisense and gabapentin in allergic airway inflammation.

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.

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, ß-, 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.

DREAM controls the on/off switch of specific activity-dependent transcription pathways.

Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory.

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/ß 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.

Singularities of calcium signaling in effector T-lymphocytes.

CD4(+) helper T (Th) lymphocytes orchestrate the immune response and include several types of effectors such as Th1, Th17 and Th2 cells. They fight against intracellular, extracellular pathogens and parasites respectively. They may also cause distinct immunopathological disorders. Th1 and Th17 are implicated in the development of autoimmune diseases while Th2 cells can initiate allergic diseases. These subsets differ by their TCR-associated signaling. In addition, the regulation of intracellular calcium concentration is not the same in Th1, Th2 and 17 cells. Our group showed that Th2 cells selectively overexpressed voltage-activated calcium (Cav1)-related channels. An increasing number of groups report the presence of Cav1-related products in T-lymphocyte subsets. This is a matter of debate since these calcium channels are classically defined as activated by high cell membrane depolarization in excitable cells. However, the use of mice with ablation of some Cav1 subunits shows undoubtedly an immune phenotype raising the question of how Cav1 channels are regulated in lymphocytes. We showed that knocking down Cav1.2 and/or Cav1.3 subunits impairs the functions of Th2 lymphocytes and is beneficial in experimental models of asthma, while it has no effect on Th1 cell functions. Beyond the role of Cav1 channels in T-lymphocytes, the identification of key components selectively implicated in one or the other T cell subset paves the way for the design of new selective therapeutic targets in the treatment of immune disorders while preserving the other T-cell subsets. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

[Calcium signaling in T lymphocytes]. / La signalisation calcique dans les lymphocytes T.

Calcium signaling is essential for all the functions of T lymphocytes, including those of Th2 cells. Th2 lymphocytes producing interleukins 4, 5 and 13 orchestrate allergic diseases including asthma. T-cell activation induces an influx of Ca(2+) from the external medium through ORAI calcium channels although other calcium channels are likely to be involved. Among them, voltage-gated calcium (Ca(v)1) channels have been reported in some T-cell subsets including Th2 cells. The inhibition of Ca(v)1 channels abrogates T-cell receptor-driven calcium influx and interleukin production by Th2 cells. From a therapeutic point of view, the inhibition of Ca(v)1 channels prevents Th2-dependent experimental allergic asthma. In this review, we will discuss the singularities of calcium responses depending upon the T-cell subset and its state of activation.

Darier disease : a disease model of impaired calcium homeostasis in the skin.

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca(2+) from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca(2+) transport by SERCA2 resulting in decreased ER Ca(2+) concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca(2+) signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

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.

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.

Ca2+-operated transcriptional networks: molecular mechanisms and in vivo models.

Calcium is the most universal signal used by living organisms to convey information to many different cellular processes. In this review we present well-known and recently identified proteins that sense and decode the calcium signal and are key elements in the nucleus to regulate the activity of various transcriptional networks. When possible, the review also presents in vivo models in which the genes encoding these calcium sensors-transducers have been modified, to emphasize the critical role of these Ca(2+)-operated mechanisms in many physiological functions.

Calcium channel blocker prevents T helper type 2 cell-mediated airway inflammation.

RATIONALE: Ca(2+) signaling controls the production of T helper (Th) type 2 cytokines known to be deleterious in asthma. Recently, we showed that Ca(2+) signaling was dihydropyridine (DHP)-sensitive in Th2 lymphocytes and that the DHP derivate, nicardipine, used in the treatment of cardiovascular pathologies, prevents Th2-dependent B cell polyclonal activation. OBJECTIVES: We tested the effect of nicardipine in experimental allergic asthma. METHODS: BALB/c mice immunized with ovalbumin (OVA) in alum and challenged with intranasal OVA were treated with nicardipine once the Th2 response, or even airway inflammation, was induced. We also tested the effect of nicardipine in asthma induced by transferring OVA-specific Th2 cells in BALB/c mice exposed to intranasal OVA. We checked the impact of nicardipine on T-cell responses and airway inflammation. MEASUREMENTS AND MAIN RESULTS: Nicardipine inhibited in vitro Ca(2+) response in Th2 cells. In vivo, it impeded the development of Th2-mediated airway inflammation and reduced the capacity of lymphocytes from lung-draining lymph nodes to secrete Th2, but not Th1, cytokines. Nicardipine did not affect antigen presentation to CD4(+) T lymphocytes, nor the initial localization of Th2 cells into the lungs of mice exposed to intranasal OVA; however, it reduced the production of type 2 cytokines and the amplification of the Th2 response in mice with asthma. Conversely, nicardipine had no effect on Th1-mediated airway inflammation. CONCLUSIONS: Nicardipine improves experimental asthma by impairing Th2-dependent inflammation. This study could provide a rationale for developing drugs selectively targeting DHP receptors of Th2 lymphocytes, potentially beneficial in the treatment of asthma.

Calcium-dependent transcription of cytokine genes in T lymphocytes.

The increase in intracellular calcium ion concentration is a general signaling mechanism used in many biological systems. In T lymphocytes, calcium is essential for activation, differentiation, and effector functions. In this study, we will summarize recent developments of how intracellular calcium concentrations are modified in T cells to affect the activity of three major calcium-dependent transcriptional effectors, i.e., NFAT, MEF2, and DREAM, involved in cytokine gene expression.