PhD Nicolas Rosa

PhD Emily Triffaux

PhD Virginie Robert

MSc Marion Mars

PhD Martin Klein

PhD Gregory Bouchaud

MSc Astrid Canivet

MD Antoine Magnan

Jean-Charles Guéry

MD Lucette Pelletier
email: lucette.pelletier@inserm.fr.

Jean-Charles Gu

PhD Magali Savignac
email: magali.savignac@inserm.fr.

The β

Keywords: Asthma, T H 2, voltage-gated calcium channel 1, calcium, cytokines

The b and a2d auxiliary subunits of voltage-gated calcium channel 1 (Ca v 1) are required for T H 2 lymphocyte function and acute allergic airway inflammation Allergic diseases, including rhinitis, atopic dermatitis, asthma, and food allergies, are induced by T H 2 lymphocytes. T H 2-type responses are characterized by production of IL-4, IL-5, and IL-13, which contribute to mucus production, eosinophilia, and high levels of antigen-specific IgE. At present, treatments for allergic asthma are often symptomatic, even if in some cases specific allergenic immunotherapy and treatments targeting the cytokines (or their receptors) involved in type 2 inflammation can be beneficial. [START_REF] Holgate | Asthma[END_REF][START_REF] Becerra-Diaz | New perspectives on the regulation of type II inflammation in asthma[END_REF] The main immunosuppressants (eg, cyclosporine and tacrolimus) have also been proposed for use in patients with severe asthma resistant to glucocorticoids. However, because they decrease the activity of T cells and therefore the overall immune response by acting on calcium signaling, they have adverse effects.

Calcium is a second messenger that plays specific and key roles in various cellular functions, such as activation, differentiation, proliferation, and death. The role of store-operated Ca [START_REF] Canti | The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of alpha2delta subunits is key to trafficking voltage-gated Ca21 channels[END_REF] entry is well described, implicating the sensing of T-cell receptor (TCR)-driven endoplasmic reticulum (ER) Ca [START_REF] Canti | The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of alpha2delta subunits is key to trafficking voltage-gated Ca21 channels[END_REF] depletion by stromal interaction molecule 1, its oligomerization, and its localization in the vicinity of calcium release-activated calcium modulator 1 (ORAI1) channels at the plasma membrane, permitting sustained Ca 21 entry. [START_REF] Hogan | Molecular basis of calcium signaling in lymphocytes: STIM and ORAI[END_REF] In addition to these channels, the role of voltage-gated calcium channel (Ca v ) 1 channels (defined as voltage activated in excitable cells) in T lymphocytes is now accepted. [START_REF] Savignac | Protein kinase C-mediated calcium entry dependent upon dihydropyridine sensitive channels: a T cell receptor-coupled signaling pathway involved in IL-4 synthesis[END_REF][START_REF] Gomes | Lymphocyte calcium signaling involves dihydropyridine-sensitive L-type calcium channels: facts and controversies[END_REF][START_REF] Badou | Critical role for the beta regulatory subunits of Cav channels in T lymphocyte function[END_REF][START_REF] Cabral | Knocking-down Cav1 calcium channels implicated in Th2-cell activation prevents experimental asthma[END_REF][START_REF] Omilusik | The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis[END_REF][START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF][START_REF] Davenport | Signature channels of excitability no more: L-type channels in immune cells[END_REF][START_REF] Matza | T cell receptor mediated calcium entry requires alternatively spliced Cav1.1 channels[END_REF] In excitable cells, Ca v 1 channels are composed of the ion-forming pore a1 and auxiliary b and a2d subunits, with each subunit being encoded by 4 genes. Ca v 1.1 to Ca v 1.4 a1 form the ion pore and support the biophysical and pharmacologic properties of the channel, [START_REF] Catterall | Structure and regulation of voltage-gated Ca21 channels[END_REF][START_REF] Hofmann | L-Type Cav1.2 calcium channels: from in vitro findings to in vivo function[END_REF][START_REF] Zamponi | The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential[END_REF] whereas auxiliary b and a2d subunits increase Ca v currents by enhancing the number of channels at the cell membrane and favoring channel opening. [START_REF] Neely | Potentiation by the b Subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel[END_REF][START_REF] Canti | Evidence for two concentration-dependent processes for b-subunit effects on a1B calcium channels[END_REF][START_REF] Richards | Ca21 channel beta-subunits: structural insights AID our understanding[END_REF][START_REF] Hofmann | Emerging alternative functions for the auxiliary subunits of the voltage-gated calcium channels[END_REF][START_REF] Savalli | The a2d-1 subunit remodels CaV1.2 voltage sensors and allows Ca21influx at physiological membrane potentials[END_REF] Ca v b would act by facilitating the correct folding of a1 and promoting its exit from the ER, [START_REF] Bichet | The I-II loop of the Ca21 channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit[END_REF] whereas Ca v a2d increases insertion of the channel into the cell membrane by favoring the trafficking of the channel from the post-Golgi apparatus and decreasing its turnover. [START_REF] Canti | The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of alpha2delta subunits is key to trafficking voltage-gated Ca21 channels[END_REF][START_REF] Bernstein | Kinetics of internalization and degradation of N-type voltage-gated calcium channels: role of the alpha2/delta subunit[END_REF][START_REF] Dolphin | Calcium channel auxiliary a2d and b subunits: trafficking and one step beyond[END_REF] While Ca v b2 deletion [START_REF] Jha | Essential roles for Cavb2 and Cav1 channels in thymocyte development and T cell homeostasis[END_REF] inhibited thymocyte development, Ca v b3 and Ca v b4 were found to be important for calcium influx, nuclear factor of activated T cells (NFAT) nuclear translocation, and cytokine production by peripheral CD4 1 T lymphocytes. Previously, we reported that Ca v 1.2 was expressed and functional in human and mouse T H 2 cells. [START_REF] Cabral | Knocking-down Cav1 calcium channels implicated in Th2-cell activation prevents experimental asthma[END_REF][START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF][START_REF] Gomes | Calcium channel blocker prevents T helper type 2 cell-mediated airway inflammation[END_REF] In contrast, T H 1 and T H 17 lymphocytes in both species lacked Ca v 1.2 expression.

Here we address whether Ca v auxiliary subunits are required for channel functions in T H 2 lymphocytes. We show that Ca v b1, Ca v b3, and a2d2 subunits are expressed in mouse and human T H 2 cells. Knocking down Ca v b promoted degradation of Ca v 1.2 a1, which was at least partly rescued by adding MG132, a proteasome inhibitor, whereas gabapentin, an inhibitor of a2d1/2 subunits, decreased a2d2 protein levels in T H 2 lymphocytes. In both cases, it was associated with a decreased TCR-dependent intracellular Ca 21 concentration ([Ca 21 ] i ) increase and T H 2 cytokine production. In accordance with our in vitro results, targeting either Ca v b or a2d in vivo was beneficial in a model of acute allergic airway inflammation.

METHODS

More details are provided in the Methods section in this article's Online Repository at www.jacionline.org.

Mice and model of acute airway allergic inflammation

Eight-to 12-week-old female BALB/c mice were obtained from Janvier (Le Genest St Isle, France), and TCR ovalbumin (OVA) transgenic DO11.10 mice were maintained in our pathogen-free animal facility. The INSERM U1043 Institutional Review Board for animal experimentation approved protocols. BALB/c mice immunized intraperitoneally with OVA (100 mg) in alum (2 mg) were 15 days later administered intranasal OVA (50 mg/d) in PBS for 5 days, as previously described, [START_REF] Cabral | Knocking-down Cav1 calcium channels implicated in Th2-cell activation prevents experimental asthma[END_REF] with or without Ca v b scrambled (Ca v bS) or Ca v b antisense (Ca v bAS) oligonucleotides (200 mg/d) or gabapentin (400 mg/L in drinking water) that was renewed every other day. [START_REF] Warnier | CACNA2D2 promotes tumorigenesis by stimulating cell proliferation and angiogenesis[END_REF] For T H 2 transfer experiments, BALB/c mice (Janvier) were transferred intravenously with 3 3 10 6 D011.10 T H 2 cells transfected with Ca v bS or Ca v bAS and given intranasal OVA (50 mg/d) for 5 days. Twenty-four hours after the final OVA administration, serum, bronchoalveolar lavage (BAL) fluid, lungs, and draining lymph nodes were collected and processed, as described in the Methods section in this article's Online Repository.

Cell culture

Mouse T H 1 and T H 2 cells were generated by weekly stimulation of DO11.10 CD4 1 T cells with antigen-presenting cells and the 323-339 OVA peptide plus appropriate differentiation cocktails: IL-12 (5 ng/mL) and anti-IL-4 antibody (11B11, 10 mg/mL) for T H 1 and IL-4 (5 ng/mL) and T and human T H 1 cells were obtained and expanded, as previously reported. [START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF] Cells were transfected with 8 mmol/L oligodeoxynucleotides (Eurogentec, Angers, France) by using TurboFect transfection reagent (Fermentas, Waltham, Mass) and collected 3 days after transfection.

Single-cell calcium imaging

Single-cell calcium imaging was done, as previously described, [START_REF] Cabral | Knocking-down Cav1 calcium channels implicated in Th2-cell activation prevents experimental asthma[END_REF][START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF] in cells loaded with Fura-2 AM before and after stimulation with biotinylated anti-CD3 cross-linked with streptavidin or anti-CD3/CD28-coated beads (1 bead per cell) for mouse or human T cells, respectively.

Confocal microscopy

Cells were stained with anti-CD2 rat IgG antibody (Abcam, Cambridge, United Kingdom) or anti-a2d2 rabbit antibody (Alomone Labs, Jerusalem, Israel) fixed, permeabilized and the Ca v channel subunit staining was performed by using anti-Ca v 1.2 mouse IgG 2b (NeuroMab, Davis, Calif), anti-Ca v b1 (Alomone Labs), or anti-Ca v b3 rabbit IgG (Santa Cruz Labs, Santa Cruz, Calif).

Real-time quantitative PCR

RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, Calif), and mRNA transcripts were measured by using real-time quantitative PCR with a LightCycler 480 Instrument (Roche, Mannheim, Germany).

ELISA

For in vitro cytokine assays, 5 3 10 4 T H 1 and T H 2 cells were seeded onto plates coated with anti-CD3 antibodies (3 mg/mL) and stimulated with soluble anti-CD28 antibodies (1 mg/mL). Supernatants were collected 24 hours later. For in vivo IgE assays, sera were isolated from blood collected into the mouse retro-orbital sinus. Cytokine and immunoglobulin levels were quantified by using the sandwich ELISA method.

Statistical analysis

Groups were compared by using the paired or unpaired t test as needed with GraphPad Prism software (GraphPad Software, La Jolla, Calif).

RESULTS

Knocking down Ca v b subunits decreases cytokine production induced by TCR engagement in mouse and human T H 2 cells

Murine T H 2 cells expressed principally Ca v b1 and Ca v b3 transcripts (Fig 1,A), as did human T H 2 cells. [START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF] The proteins Ca v b1 and Ca v b3 were detected by Western blotting with the same apparent molecular weights as the control neuronal forms (Fig 1,B) and by immunofluorescence (see Fig E1, A andB, in this article's Online Repository at www.jacionline.org). Although T H 1 cells did not express Ca v a1 subunits, they did express Ca v b1 and Ca v b3 at both the transcriptional (Fig 1,A) and protein levels, with lower amounts compared with those in T H 2 cells (see Fig E1, A andB).

To investigate whether b subunits were important for T H 2 functions, we used antisense oligonucleotides targeting the control T H 2 cells (98% 6 2%) did (P < .005). Moreover, among responsive cells, Ca v bAS-transfected T H 2 cells had a delayed calcium response compared with control cells (Fig 2 ,C). However, ionomycin application at the end of the experiment induced a similar [Ca 21 ] i increase in T H 2 cells, irrespective of whether they were transfected with Ca v bS or Ca v bAS (Fig 2,A), indicating that Ca v bAS did not affect loading with the Fura-2 probe.

Ca v βΑS Ca v βS Ca v βΑS Ca v βS Ca v 1.2 α1 Ca v 1.2 α1 staining Ca v βΑS Ca v βS Ca v 1.2 α1 staining Ca v βΑS Ca v βS CD2 Ca v 1.2 α1
Ca v bAS had the same effect on the TCR-driven [Ca 21 ] i increase of human T H 2 lymphocytes with a delayed and reduced [Ca 21 ] i increase (Fig 2 , D-F). Conversely, transfection of mouse (see Fig 2, G-I) or human (Fig 2 , J-L) T H 1 cells with Ca v bAS had no effect on the shape and the amplitude of the calcium response induced by TCR stimulation, which is in agreement with the lack of an effect of Ca v bAS on IFN-g synthesis by T H 1 cells (Fig 1 , D andF).

An inhibitor of the proteasome protects the Ca v 1.2 a1 subunit from degradation in the absence of Ca v b but does not restore cytokine production One possible role of Ca v b subunits could be to prevent Ca v 1.2 degradation by the proteasome. [START_REF] Altier | The Cavbeta subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels[END_REF] In agreement with this hypothesis, we showed that transfection of T H 2 cells with Ca v bAS did not affect levels of Ca v 1.2 a1 transcripts (Fig 3,A [START_REF] Link | Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart[END_REF][START_REF] Catterall | Voltage-gated calcium channels[END_REF][START_REF] Brunet | Phosphorylation sites in the Hook domain of CaVb subunits differentially modulate CaV1.2 channel function[END_REF]

Intranasal delivery of Ca v bAS alleviates T H 2-dependent airway inflammation

With the goal to investigate the role of Ca v b in vivo, we evaluated whether Ca v bAS modified the course of allergic airway inflammation. Immunization with OVA in alum followed by inhalation of OVA (Fig 5,A) triggered T H 2-dependent airway inflammation marked by predominant eosinophil infiltration

Gabapentin, an inhibitor of Ca v a2d, impairs T H 2 function and reduces allergic airway inflammation

Mouse T H 2 cells expressed mainly Ca v a2d2 (Fig 7,A), as did human T H 2 cells. [START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF] Blotting and probing T H 2 cell or brain lysates with anti-a2d2 antibodies showed similar bands of around 140 kDa (Fig 7 ,B). To investigate the role of a2d in Ca v 1-dependent T H 2 function, we used gabapentin, a ligand binding to Ca v a2d1 and a2d2 but not to the other a2d subunits. [START_REF] Marais | Calcium channel alpha(2)delta subunits-structure and gabapentin binding[END_REF] Chronic in vitro treatment with this drug is known to inhibit Ca v currents. Accordingly, treatment of T H 2 cells with gabapentin for 72 hours decreased and delayed the TCR-driven calcium response ( Because gabapentin was reported to induce loss of the Ca v 2 complex at the cell membrane, [START_REF] Heblich | Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin[END_REF][START_REF] Hendrich | Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin[END_REF] we tested its effect on Ca v 1.2 protein expression in T H 2 cells. The amount of a2d2 protein was strongly reduced in gabapentin-treated T H 2 cells ( We then examined the effect of gabapentin administration on the development of allergic airway inflammation (Fig 8,A). Administration of gabapentin during OVA challenge partially reduced airway inflammation, as shown by the reduction in the number of inflammatory cells, including eosinophils in BAL fluid (Fig 8 ,B) and histologic examination of lung sections (Fig 8 , C andD). Expression of T H 2 cytokines and mucins 5ac and 5b, which are the major components of mucus in the lungs, [START_REF] Curran | Advances in mucous cell metaplasia: a plug for mucus as a therapeutic focus in chronic airway disease[END_REF] was decreased in the lungs of gabapentin-treated mice (Fig 8 ,E), supporting a beneficial effect of gabapentin on T H 2-mediated inflammation.

DISCUSSION

Previously, we reported that knocking down Ca v 1.2 and Ca v 1.3 a1 subunits in T H 2 cells impaired their functions and protected against asthma. [START_REF] Cabral | Knocking-down Cav1 calcium channels implicated in Th2-cell activation prevents experimental asthma[END_REF] These subunits are voltage-gated calcium ion-forming pores in excitable cells. However, we and other groups showed that they are not voltage gated in lymphocytes. We then investigated whether auxiliary subunits, such as Ca v b and Ca v a2d, are required for T H 2 cell functions and can be targeted beneficially in acute allergic airway inflammation. Our results now show that (1) knocking down Ca v b expression with Ca v bAS designed to target the 4 b-subunits reduces the TCR-driven [Ca 21 ] i increase and T H 2 cytokine production by mouse and human T H 2 cells while sparing T H 1 cell responses;

(2) this is associated with the loss of Ca v 1.2 a1 protein, which can be partially overcome by a proteasome inhibitor;

(3) intranasal delivery of Ca v bAS strongly diminishes T H 2-mediated immunopathology in a model of acute allergic airway inflammation; and (4) the Ca v a2d1/2 inhibitor gabapentin impairs calcium signaling and reduces OVA-induced asthma.

Although Ca v b3 can regulate calcium signaling independently of a1 by limiting inositol 1, 4, 5-triphosphate generation in pancreatic cells, [START_REF] Berggren | Removal of Ca21 channel beta3 subunit enhances Ca21 oscillation frequency and insulin exocytosis[END_REF] this is unlikely in T H 2 cells because knocking down Ca v b decreases and not enhances [Ca 21 ] i level. In addition, Ca v bAS does not affect the calcium signal in T H 1 cells that express Ca v b3 but no Ca v 1 a1 subunits. Moreover, our results favor a role of Ca v b related to its interaction with Ca v 1 a1, as supported by the loss of expression of Ca v 1.2 a1 in T H 2 cells transfected with Ca v bAS. In T H 2 cells, Ca v b prevents the degradation of Ca v 1.2 a1, which is consistent with findings showing that b1b or b2a might be required for Ca v 1.2 or Ca v 2.1 a1 protein expression. [START_REF] Altier | The Cavbeta subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels[END_REF][START_REF] Waithe | Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation[END_REF] Surprisingly, transfection of ventricular myocytes with the same Ca v bAS we used in our study resulted in Ca v 1 a1 sequestration without increased cardiac Ca v 1.2 a1 degradation. [START_REF] Leach | Antisense oligonucleotide against the Ca channel beta subunit decreases L-type Ca current in rat ventricular myocytes[END_REF] This apparent discrepancy could be related to differences in Ca v b subunit equipment and/or regulation in these cell types. Along this line, it has been shown that the absence of Ca v b4 resulted in decreased expression of Ca v 1.1 but not Ca v 1.2 a1 protein in CD4 1 T cells. [START_REF] Badou | Critical role for the beta regulatory subunits of Cav channels in T lymphocyte function[END_REF] Two mechanisms have been proposed to explain how Ca v b can protect the a1 subunit from degradation. First, interaction of b2 with Ca v 1.2 a1 might limit degradation of the channel in lysosomes. [START_REF] Catalucci | Akt regulates L-type Ca21 channel activity by modulating Cavalpha1 protein stability[END_REF] Second, interaction of b1 or b2 with Ca v 1.2 or Ca v 2.2 a1 prevents a1 being addressed to and degraded by the proteasome. [START_REF] Altier | The Cavbeta subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels[END_REF][START_REF] Waithe | Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation[END_REF] This second mechanism is in agreement with our data in T H 2 cells. Indeed, we show that knocking down Ca v b diminished the total amount of endogenous Ca v 1.2 a1, which was partially restored on proteasome inhibition in T H 2 cells. Whether b subunits are also required for the export of a1 subunits to reach the cell surface is controversial. [START_REF] Dolphin | Calcium channel auxiliary a2d and b subunits: trafficking and one step beyond[END_REF][START_REF] Altier | The Cavbeta subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels[END_REF][START_REF] Fang | Mechanism of auxiliary beta-subunit-mediated membrane targeting of L-type (Ca(V)1.2) channels[END_REF] Knocking down Ca v b resulted in lower expression of Ca v 1.2 a1 at the cell membrane, which was restored in part by the proteasome inhibitor, indicating that Ca v b was not absolutely required for the channel to exit from the ER or that the remaining Ca v b subunits were sufficient to promote trafficking of the channel at the cell membrane. The absence of Ca v b, even in the presence of proteasome inhibitor, did not allow restoration of cytokine production by T H 2 cells, which could be due to the incomplete restoration of Ca v 1.2 a1 and/or a role of Ca v b on function of the channel at the cell membrane.

Mouse T H 2 cells express predominantly both b1 and b3 subunits, as do human T H 2 cells. [START_REF] Robert | Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions[END_REF] It was reported previously that the b1 variant preferentially interacts with Ca v 1.1 a1, allowing interaction of Ca v 1 with ER-localized type 1 ryanodine receptors [START_REF] Schredelseker | The beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle[END_REF] and leading to calcium depletion in the ER. It would be interesting to investigate the respective roles of Ca v b1 and Ca v b3 in Ca v 1.2 function. Interestingly, Ca v b can interact with G proteins, scaffolding proteins, [START_REF] Buraei | The beta subunit of voltage-gated Ca21 channels[END_REF] and especially AHNAK, which might be required for plasma membrane localization of the channel, as described for Ca v 1.1 a1 channels in T cells. [START_REF] Matza | A scaffold protein, AHNAK1, is required for calcium signaling during T cell activation[END_REF] We found predominantly a2d2 at the mRNA level in T H 2 cells. We also detected the protein by using Western blotting with the same apparent weight as in neurons and by means of immunofluorescence. Nerve injury can be responsible for chronic pain caused by a2d1 overexpression in injured sensory neurons. [START_REF] Li | Spinal dorsal horn calcium channel 2-1 subunit upregulation contributes to peripheral nerve injury-induced tactile allodynia[END_REF][START_REF] Dolphin | Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology[END_REF] Gabapentin is beneficial because this drug, which binds to a2d1 and a2d1 but not the other a2d isoforms, [START_REF] Hendrich | Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin[END_REF] might decrease expression of Ca v 2 channels at the cell membrane, [START_REF] Heblich | Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin[END_REF][START_REF] Hendrich | Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin[END_REF][START_REF] Cassidy | Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary alpha2delta-1 subunits[END_REF] leading to a reduction in Ca v calcium currents. However, the authors did not report an effect of gabapentin on the total amount of a2d and Ca v a1 subunits. Unexpectedly, T H 2 cells treated with gabapentin show reduced total amounts of a2d2, which are restored by an inhibitor of the vacuolar H 1 ATPAse. This suggests that gabapentin favors a2d2 degradation in an acidic compartment, such as lysosomes. Along this line, gabapentin was shown to prevent recycling of a2d2 from Rab11-positive recycling endosomes to the plasma membrane. [START_REF] Tran-Van-Minh | The alpha2delta ligand gabapentin inhibits the Rab11-dependent recycling of the calcium channel subunit alpha2delta-2[END_REF] We show here that gabapentin neither affects the total amount of Ca v 1.2 protein nor its localization at the plasma membrane in T H 2 cells. However, the calcium response was decreased on TCR activation, suggesting that a2d2 can contribute to the function of Ca v 1.2 a1, as described for a2d1. [START_REF] Segura | Proteolytic cleavage of the hydrophobic domain in the CaValpha2delta1 subunit improves assembly and activity of cardiac CaV1.2 channels[END_REF] Altogether, our results obtained with gabapentin and knockdown of Ca v b in the presence of MG132 indicate that the level of Ca v 1.2 a1 is not the only parameter limiting the calcium response and suggest that both auxiliary subunits are required in addition to Ca v 1.2 a1 for T H 2 functions.

Altogether, these data stress the role of auxiliary subunits (Ca v b and a2d) in Ca v 1.2 channel function in T H 2 cells and show that the auxiliary subunits of Ca v 1 channels represent potential new therapeutic targets in allergy. Consistently, Ca v bAS has a marked effect on T H 2 functions, airway inflammation, and hyperreactivity. CD4 1 T cells infiltrating the lungs express the Ca v 1.2 a1 channel, and this expression is diminished in mice during Ca v bAS treatment, suggesting that Ca v bAS acts in a similar way in vitro and in vivo. Chronic administration of gabapentin decreased T H 2 functions and inflammation. Because Ca v bAS and gabapentin act in different ways to decrease Ca 21 signaling and T H 2 function, it is tempting to propose a combination of drugs targeting Ca v b and Ca v a2d. In that respect, gabapentin is already used for the treatment of chronic cough, [START_REF] Ji | Neuroimmune interactions in itch: do chronic itch, chronic pain, and chronic cough share similar mechanisms?[END_REF] a symptom that is currently found in asthmatic patients. Interestingly, peptides stapling the Ca v a1 domain interacting with Ca v b subunits and shown to inhibit Ca v functions have been recently described [START_REF] Findeisen | Stapled voltage-gated calcium channel (CaV) alpha-interaction domain (AID) peptides act as selective protein-protein interaction inhibitors of CaV function[END_REF] and could be tested for their efficacy in allergic diseases.

METHODS

Oligonucleotide sequences

The sequences for Ca v bS (59-gaagtaggtcttggtggtgg-39) and Ca v bAS (59-accagccttccgatccaccagtcatt-3'; Eurogentec) were chosen from Bichet et al. E1 Ca v bAS has 92% complementarity to mouse or human b1 and b3 mRNA.

Model of acute allergic airway inflammation

BAL fluid was collected, and the content in inflammatory cells was determined, as previously described. E2 Lung tissue was fixed in 10% buffered formalin for 24 hours and then placed in 70% ethanol before embedding in paraffin. Sections (4 mm) were stained with hematoxylin and eosin. Histologic disease scores of 0 to 3 were attributed based on the severity of peribronchial, perivascular, and interstitial immune cell infiltration and thickening of peribronchial epithelium, resulting in a maximum score of 12. Lung draining lymph nodes were harvested, and 2 3 10 5 cells were stimulated with OVA (100 mg/mL) for 3 days. Cytokine concentrations were measured in supernatants by means of ELISA. In some experiments CD4 1 T cells were purified from the lungs, stained with anti-Ca v 1.2 antibodies, and analyzed by using confocal microscopy.

Airway hyperresponsiveness

Anesthetized and ventilated mice were connected through an endotracheal cannula to a whole-body plethysmograph for ventilation (Emka Technologies, Paris, France). After initiating mechanical ventilation, the mouse was paralyzed with an intraperitoneal injection of 0.1 mL of a 10 mg/mL solution of rocuronium bromide. The animal was ventilated at a respiratory rate of 150 breaths/min and a tidal volume of 10 mL/kg against a positive end-expiratory pressure of 3 cm of H 2 O. Then mice were exposed to increasing doses of methacholine aerosol generated through a nebulizer containing 0 to 20 mg/mL methacholine in PBS. Dynamic lung resistance was measured with iox software (Emka Technologies).

Cell culture

Cells were cultured in complete medium (RPMI 1640 supplemented with 10% FCS [Lonza, Allendale, NJ], 1% pyruvate, 1% nonessential amino acids, 2 mmol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mmol/L b-mercaptoethanol).

Single-cell calcium imaging

Cells were loaded with Fura-2 AM, as previously described, E2,E3 in culture medium containing 5% heat-inactivated FBS for 30 minutes at 378C. Cells were then washed, plated, and excited in 10-second intervals by using 340-and 380-nm excitation filters. Emission was recorded with 510/540-nm bandpass filters by using a CCD camera before and after stimulation with biotinylated anti-CD3 cross-linked with streptavidin-or anti-CD3/CD28coated beads (1 bead per cell) for mouse or human T cells, respectively.

Fluorescence ratio images at wavelengths of 340 and 380 nm were then calculated with Metafluor software. The curve represents the mean 1 SEM of fluorescence ratio of 40 to 60 cells. Each experiment was repeated 3 or 4 times. For each cell, we calculated the area under the curve between TCR stimulation and ionomycin application with the GraphPad Prism software. The time of response was defined as the delay for which the fluorescence ratio becomes superior to the mean basal value 1 1 SD.

Confocal microscopy

Cells were seeded onto glass slides coated with poly-L-lysine (Sigma-Aldrich, St Louis, Mo) and then fixated with 4% paraformaldehyde. Staining was performed with anti-CD2 rat IgG antibody (Abcam) or anti-a2d2 rabbit antibody (Alomone Labs) and revealed with the appropriate secondary antibodies (Molecular Probes, Eugene, Ore). Cells were then permeabilized with PBS containing 0.1% saponin and 0.5% BSA. Ca v channel subunit staining was performed with anti-Ca v 1.2 mouse IgG 2b (NeuroMab), anti-Ca v b1 (Alomone Labs), or anti-Ca v b3 rabbit IgG (Santa Cruz Labs) overnight and revealed with the appropriate secondary antibodies (Molecular Probes). Slides were analyzed by using an LSM 710 confocal microscope (Zeiss, Oberkochen, Germany). Staining with control isotypes was constantly negative. The total fluorescence intensity of each cell was determined on the sum of overall Z sections with ImageJ software (National Institutes of Health, Bethesda, Md). For membrane Ca v 1.2 staining, a membrane surface was created by using CD2 staining, and this surface was duplicated on Ca v 1.2 staining to measure the intensity of Ca v 1.2 at the membrane with the Imaris software (8.4.1; Bitplane) module called Surface. Each measurement was done for each cell individually.

Real-time quantitative PCR

T H 1 and T H 2 cells were lysed, and RNA was extracted with the RNeasy Mini Kit (Qiagen). Reverse transcription was performed with the SuperScript III Reverse Transcriptase (Invitrogen). Transcripts were measured by using real-time quantitative PCR with a LightCycler 480 Instrument (Roche) and the LightCycler 480 SYBR Green I Master (Roche). We used the following primers: Ca v b1, 59-gcagagcgccaggcctta-39 and 59-tgatggccactccctgtaca-39; Ca v b2, 59-cgaacagagagccaagcaa-39 and 59-gggcactatgtcacccaaa-39; Ca v b3, 59-tgtggcatttgctgtgagga-39 and 59-ccgatccaccagtcattgc-39; Ca v b4, 59-gagcga gaacagcaagcag-39 and 59-tcaaaggagatggctgtgc-39; Ca v a2d1, 39 acgccaactggtt gaaattg 59 and 39 cttgcaaaatcttccctcca 59; Ca v a2d2, 39 atttccagaaagcccatcg 59 and 39 gtcttggatcccctctcca 59; Ca v a2d3, 39 cattttggtggacgtcagtg 59 and 39 ttccgttcagacaaggttcc 59; Ca v a2d4, 39 ggaaagtcgaggctgttgag 59 and 39 ctccacgtagttgcccttgt 59; and hypoxanthine-guanine phosphoribosyltransferase (HPRT), 59-ctggtgaaaaggacctctcg-39 and 59-tgaagtactcattatagtcaagggca-39. Primers for Il4, Il5, Il13, Muc5ac, and Muc5b were purchased from Qiagen. Quantification of target gene expression was calculated by normalizing values relative to expression of the housekeeping gene HPRT. Amounts of mRNA were expressed as arbitrary units relative to HPRT as follows:

2 2½Ct interest gene2Ct HPRT 310 23 :

Immunoblotting T H 2 lymphocytes or brain cells were lysed in RIPA buffer containing protease inhibitors (Roche). Lysates (30 mg/lane for T-cell extracts and 10 mg/lane for brain lysates) were submitted to 10% SDS electrophoresis, transferred to Hybond-C Extra nitrocellulose membranes (Amersham, Piscataway, NJ), and then blotted with anti-Ca v b1 (Alomone Labs), anti-Ca v b3 (Santa Cruz Biotechnology, Santa Cruz, Calif), or anti-a2d2 (Alomone Labs) primary antibodies. We then stained blots with horseradish peroxidasecoupled secondary antibodies (Cell Signaling Technology, Danvers, Mass) before detection and visualization with the ECL Prime (Amersham) using a ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, Calif). The specificity of anti-Ca v b1 and b3 antibodies was verified on human embryonic kidney cells transfected with the corresponding plasmids using Western blotting.

ELISA

Supernatants or sera were incubated into 96-well plates coated with anti-IL-4, anti-IL-5, anti-IL-13 (eBioscience, San Diego, Calif), anti-IFN-g, or anti-IgE (Serotec). Bound cytokines and IgE were then labeled with biotinylated anti-IL-4 (eBioscience), anti-IL-5 (eBioscience), anti-IL-13 (eBioscience), anti-IFN-g (eBioscience), or anti-IgE (BD PharMingen, San Jose, Calif). Biotinylated antibodies were revealed by means of incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pa) and subsequent addition of the alkaline phosphatase substrate pNPP disodium salt hexahydrate (Sigma-Aldrich). Absorbance was measured at 405 to 650 nm by using an EMax Microplate Reader (Molecular Devices, Sunnyvale, Calif). Cytokine and immunoglobulin concentrations were calculated from standard curves generated by means of titration of recombinant mouse cytokines and purified IgE. Human cytokine ELISAs were performed, as previously described. E3

FIG 1 .

 1 FIG 1. Ca v bAS diminishes cytokine production in mouse and human T H 2 but not in T H 1 cells. A and B, OVA-specific DO11.10 T H 2 and T H 1 cells were tested for Ca v b at the transcript (n 5 4; Fig 1, A) or protein level (Fig 1, B). C-F, Mouse DO11.10 T H 2 (Fig 1, C), mouse T H 1 (Fig 1, D), human CRTH2 1 (Fig 1, E), or human T H 1 (Fig 1, F) cells were transfected with Ca v bS or Ca v bAS for 72 hours, then stimulated for 24 hours with anti-CD3/C28 antibodies, and cytokine production was measured. Each pair represents a separate experiment in Fig 1, C and D, and a donor in Fig 1, E and F. *P < .02, **P < .01, ***P < .005, and ****P < .001.

FIG 2 .

 2 FIG 2. Knocking down Ca v b subunits reduces the TCR-driven [Ca] i increase in mouse and human T H 2 cells without any effect on T H 1 cells. Mouse DO11.10 T H 2 (A-C), human CRTH2 1 (D-F), mouse T H 1 (G-I), or human T H 1 (J-L) cells were transfected with Ca v bAS or Ca v bS and loaded with Fura-2 AM. The fluorescence ratio at 340-and 380-nm excitation wavelengths was recorded at the single-cell level before and after TCR stimulation; ionomycin (iono) was added at the end of the experiment. Results in Fig 2, A, D, G, and J are expressed as the mean of at least 30 cells, and bars represent SEMs. The area under the curve between the time of stimulation and the time at which ionomycin is added is shown in Fig 2, B, E, H, and K. The time of response (delay between stimulation and the time showing an increase in ratio over the mean ratio plus 1 SD recorded before stimulation) is shown in Fig 2, C, F, I, and L. One representative experiment of 4 to 6 is shown. ****P < .001.

FIG 3 .FIG 4 .

 34 FIG 3. Knocking down Ca v b subunits decreases Ca v 1 a1 expression in mouse and human T H 2 cells. Cells were transfected with Ca v bS or Ca v bAS for 72 hours. A, Amount of Ca v 1.2 a1 transcripts in mouse D011.10 T H 2 cells. B-E, Staining with anti-CD2 (green) and anti-Ca v 1.2 a1 (red) mAbs in mouse D011.10 T H 2 cells (Fig 3, B and C) and human CRTH2 1 cells (Fig 3, D and E). The Ca v 1.2 cell amount over all the Z-stack sections is quantified in Fig 3, C and E. One representative experiment of 3 is shown. ****P < .001.

  ) but strongly reduced the amount of Ca v 1.2 a1 protein in mouse (Fig 3, B and C) and human (Fig 3, D and E) cells. The proteasome inhibitor MG132 had no effect on amounts of Ca v b1 (Fig 4, A) and Ca v b3 (Fig 4, B) proteins, but it partially restored Ca v 1.2 a1 protein expression in murine T H 2 cells (Fig 4, C). Analysis of colocalization of Ca v 1.2 a1 with CD2 used as a cell membrane marker shows that Ca v bAS decreases the amount of protein localized at the cell membrane, which was partially restored by MG132 (see Fig E2 in this article's Online Repository at www.jacionline.org). However, MG132 treatment did not allow restoration of cytokine production by T H 2 cells transfected with Ca v bAS (Fig 4, D), suggesting that the Ca v b subunit could also contribute to regulate channel function, as previously reported.

FIG 5 .

 5 FIG 5. Ca v bAS administration strongly diminishes allergic airway inflammation. A, BALB/c mice were immunized with OVA in alum and challenged 15 days later with intranasal OVA only or OVA plus scrambled Ca v b (Ca v bS) or OVA plus Ca v b antisense (Ca v bAS) every day for 5 days. i.p., Intraperitoneal. B, Number of cells in BAL fluid. C, Hematoxylin and eosin-stained sections from mice were scored on a 0-to 12-point scale. D, Serum IgE concentrations were measured at the time of death. E, Mediastinal lymph nodes were collected, cell suspensions were recalled for 72 hours with OVA, and cytokine production was measured by ELISA. Results in Fig 5, B-E, are means of 5 mice and representative of 2 independent experiments. *P < .05, **P < .01, and ****P < .001.

Fig 7 ,

 7 C), as shown by quantification of the area under the curve and time of response, respectively (Fig 7, D and E). This decrease in calcium signaling was associated with reduced T H 2 cytokine production (Fig 7, F). However, gabapentin had no effect on calcium response and IFN-g production in T H 1 lymphocytes (see Fig E5 in this article's Online Repository at www.jacionline.org).

FIG 6 .

 6 FIG 6. T H 2 cells transfected with Ca v bAS have impaired ability to induce type 2 airway inflammation. A, T H 2 cells transfected with Ca v bS or Ca v bAS were injected into BALB/c mice given intranasal OVA. i.v., Intravenous. B, Lung-infiltrating cells were purified and analyzed by means of flow cytometry to enumerate CD3 1 and CD3 1 KJ1.26 1 cells. C, Inflammatory cells were enumerated in BAL fluid. D, Lung sections were scored for inflammation. E, Serum IgE concentrations were measured at the time of death. F, Lung draining lymph node cells were recalled with OVA for determination of cytokine production. Each group comprised 5 mice. *P < .05, **P < .01, and ***P < .005.

Fig 7 ,

 7 G, and see Fig E6 in this article's Online Repository at www.jacionline.org), with no effect on Ca v 1.2 expression (Fig 7, H, and see Fig E6). Moreover, Bafilomycin A1, an inhibitor of the vacuolar H 1 ATPase that inhibited acidification and protein degradation in lysosomes, partially restored the amount of a2d2 in gabapentin-treated T H 2 lymphocytes (Fig 7, G, and see Fig E6), demonstrating that gabapentin targeted a2d2 in lysosomes for its degradation.

FIG 7 .

 7 FIG 7. Gabapentin (gaba), an inhibitor of Ca v a2d auxiliary subunits, reduces calcium signaling and cytokine production by mouse T H 2 cells. A, Mouse DO11.10 T H 2 cells were analyzed for expression of a2d transcripts (n 5 4). B, T H 2 and brain (positive control) lysates were blotted and stained with anti-a2d2 antibodies. IB, Immunoblotting. T H 2 cells were preincubated with gabapentin (1 mmol/L) for 72 hours in Fig 7, C-H. C-E, Cells were then loaded with the calcium probe Fura-2 AM, and the fluorescence ratio at 340-and 380-nm excitation wavelengths was recorded at the single-cell level. Results are expressed as the mean of at least 50 cells, and bars represent SEMs. Fig 7, D and E, show, respectively, the area under the curve and the time of response for each cell defined as in Fig 3. Data are representative of 3 experiments. F, T H 2 cells were stimulated in the presence or not of gabapentin for an additional 24 hours with anti-CD3/anti-CD28 antibodies. Cytokine content was then measured. G and H, T H 2 cells were incubated or not with bafilomycin (bafilo; 0.5 mmol/L) for the last 24 hours. Cells were stained with anti-a2d2 (Fig 7, G) or anti-Ca v 1.2 a1 (Fig 7, H) antibodies. Immunofluorescence was summed all over the Z-stack sections. One representative experiment of 2 is shown. **P < .01, ***P < .005, and ****P < .001.

FIG 8 .

 8 FIG 8. Gabapentin (gaba), an inhibitor of Ca v a2d subunits, decreases type 2 airway inflammation. A, BALB/c mice were immunized with OVA in alum and then challenged with intranasal OVA for 5 days. Gabapentin was or was not given in drinking water for the last 5 days. i.p., Intraperitoneal. B, The next day, mice were killed, and BAL fluid was collected and analyzed for inflammatory cell content. C, Hematoxylin and eosin histologic sections were scored on a 0-to 12-point scale. D, Lung inflammation in mice given control (nt) and gabapentin. E, Expression of Il4, Il5, Il13, Muc5ac, and Muc5b was quantified in lungs by using quantitative PCR and normalized to housekeeping gene expression. Each experiment includes 6 to 7 mice per group. One representative experiment of 2 is shown. *P < .05, **P < .01, and ***P < .001.

  FIG E1. Ca v bAS targets Ca v b1 and Ca v b3 subunits in mouse and human T H 2 cells. A, OVA-specific DO11.10 T H 2 and T H 1 cells were stained with anti-Ca v b1 and anti-Ca v b3 antibodies. B, Expression of Ca v b per cell. One experiment of 3 was depicted, and each point represents 1 cell. C, Cell lysates were prepared from mouse T H 2 cells transfected with Ca v bS or Ca v bAS after 3 days of transfection. One representative blot is shown. D, Protein bands were quantified by using Image Lab software. The ratio between the protein of interest and b-actin was calculated. Results are expressed as the relative protein level to Ca v bS and shown as separate paired experiments. E and F, T H 2 cells transfected with Ca v bS or Ca v bAS were stained with anti-Ca v b1 and anti-Ca v b3 antibodies. Expression of Ca v b per cell is shown in Fig E1, F. One experiment of 3 is depicted, and each point represents 1 cell. G, CRTH2 1 cells transfected with Ca v bS or Ca v bAS were analyzed for Ca v b1 and Ca v b3 transcript expression. Results are shown as separate paired experiments. **P < .01, ***P < .005, and ****P < .0001.

FIG E6 .

 E6 FIG E6. Gabapentin (gaba) reduces protein levels of the a2d2 auxiliary subunit in T H 2 cells. T H 2 cells were incubated with gabapentin (1 mmol/L) for 96 hours with or without bafilomycin (bafilo; 0.5 mmol/L) for the last 24 hours. Cells were stained with anti-a2d2 or anti-Ca v 1.2 a1 antibodies. Immunofluorescence was summed over all the Z-stack sections. One representative experiment of 2 is shown. A white line surrounds each cell.

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We thank F. E. L'Faqihi-Olive, V. Duplan-Eche, and A. L. Iscache for technical assistance at the Flow cytometry facility of INSERM UMR 1043, CPTP and also the imaging platforms (INSERM U1043, CPTP), the animal facility, and the histology platforms (Anexplo Toulouse). We also thank Joris Hien-Turpin for his technical assistance and P. Lory for the gift of plasmids that encode Ca v b subunits. Helpful discussion was provided by the BM1406 COST Action.

Clinical implications: The demonstration that auxiliary subunits are involved in calcium signaling through Ca v 1 channels and the function of mouse and human T H 2 lymphocytes supports their potential beneficial effect on allergic asthma.

*These authors contributed equally to this work. Supported by the French National Institute for Health and Medical research (INSERM) and grants from the French Society of Allergology and the Conseil R egional Midi-Pyr en ees. N.R. was supported by a fellowship from Conseil Regional of Midi-Pyr en ees and INSERM. . Rosa's, E. Triffaux's, V. Robert's, M. Mars', J.-C. Gu ery's, L. Pelletier's, and M. Savignac's institutes received grants from the French Society of Allergology and Conseil R egional de Midi-Pyr en ees for this work.


Kathy Bru

Solène Touzé

Pascal Auger

Daniel B Parvaz

Recycling of ultra-high performance fibre-reinforced concrete with a high voltage electric pulse fragmentation process

Abstract

This study deals with the recycling of a particular Ultra-High Performance Fibre Reinforced Concrete (UHPFRC) called Ductal®. This material, characterized by a high mechanical resistance and a high steel fibre content, represent a challenge for the conventional recycling processes. The high voltage electric pulse fragmentation (EPF) technology was investigated here as a potential breakthrough technology for liberating the steel fibres from the sand/cement paste, with the objective to recycle both fractions into new concrete products. The EPF technology relies on highly energetic electrical pulses to selectively fragment composite materials i.e. to generate cracks along grain boundaries. Tests were performed at lab-scale and showed that the steel fibres are liberated in the 0/2 mm size fraction. The influence of the specific energy on EPF performances was investigated. Good recovery rates of the steel fibres were obtained, confirming the potential of EPF for the recycling of UHPFRC.

  BRGM, Water, Environment and Ecotechnologies Division, F-45060 Orléans, France (2) BRGM, Laboratories Division, F-45060 Orléans, France (3) SELFRAG AG, Kerzers, Switzerland