How About Role of CRAC Channel in Allergic Asthma

Title: The effect of CRAC inhibition on asthma models of inflammation

How about Role of CRAC channel in allergic asthma

Short Title: CRAC channels in asthma

Authors: Manminder Kaur1, Mark A Birrell2,3, Bilel Dekkak2 Sophie Reynolds1, Sissie Wong2, Jorge De Alba2, Kristof Raemdonck2, Simon Hall4, Karen Simpson4, Malcolm Begg4, Maria G Belvisi2,3, Dave Singh1

1 University of Manchester, Institute of Inflammation and Repair, University Hospital of South Manchester Foundation Trust, Southmoor Road, Manchester, M23 9LT UK. .

2. Respiratory Pharmacology Group, Pharmacology and Toxicology Section, NHLI

Sir Alexander Fleming Building (SAF), South Kensington Campus, Exhibition Road, Imperial College London, London SW7 2AZ, UK

3MRC-Asthma UK Centre in Allergic Mechanisms of Asthma.4 Refractory Respiratory Inflammation DPU, Respiratory TAU, GSK Medicines Research Centre, Stevenage, UK

Funding support – MAB and MGB received a research grant from GSK

Corresponding author: Dr Manminder Kaur, University of Manchester, Institute of Inflammation and Repair, University Hospital of South Manchester Foundation Trust, Southmoor Road, Manchester, UK, M23 9LT.

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Key words:Asthma, Lymphocytes, CRAC channel NF-AT

Abstract

How about something like this?

The incidence of asthma is increasing globally and whilst current treatments manage a proportion of patients, the drugs have unwanted side effects and fail to adequately control a sub-group of the more severe asthmatics. Thus there is an urgent need to develop new therapies. Lymphocytes are thought to play a central role in the pathophysiology of asthma through the production of inflammatory mediators. A key step in this is thought to be via the transcription factor NFAT which in turn can be activated though Ca2+ release-activated Ca2+ (CRAC) channelsThe aim of this work was to investigate the role of CRAC in clinical and pre-clinical models of allergic asthma.

Initial data demonstrated that the NFAT pathway is increased in stimulated lymphocytes collected from asthmatics and in a pre-clinical asthma model. To confirm a role for the channel we showed that a selective inhibitor, Synta 66, blocked mediator production from lymphocytes. Further confirmative data in the allergic asthma model demonstrated that CRAC played a central role in the airway inflammation and late asthmatic response (LAR).

In conclusion, our data package strongly suggests that targeting CRAC channels could be of therapeutic value for asthma sufferers.

ted the effects of CRAC channel inhibitor Synta 66 on human asthma lung lymphocytes and in an OVA model of allergic asthma. We show in human lung lymphocytes Synta 66 inhibits CD2/3/28 induced IL-2, IL-17, IL-13, IFNγ and IL-10 in a concentration dependent manner in healthy and severe asthma donors, with over 60% inhibition observed for all cytokines. In a late asthmatic response in an allergic mouse model Synta 66 successfully blocked OVA induced mRNA levels of TNFα, IL-1β, IL-5 & IL-6 and cytokine production for exotoxin & IL-6. Synta 66 also repressed OVA induced airway cellular recruitment showing a reduction in neutrophils and eosinophils. In addition Synta 66 reduced late asthmatic response bronchoconstriction but had no effect on the immediate bronchoconstriction observed in the early asthmatic response. Finally CRAC inhibitor Synta 66 acts via NFAT to reduce in vivo and in vitro asthma associated responses. The fact that there was no effect on the early asthmatic response suggests that Synta 66 failed to impact antigen induced release of preformed mediators in mast cells. These data provide evidence to suggest that Synta 66 may have therapeutic potential in the treatment of severe asthma.

Introduction

Asthma is characterised by airway inflammation, cough, bronchial hyperresponsiveness, and variable airflow obstruction (e.g. EAR and LAR) [1]. Inhaled corticosteroids (ICS) are the mainstay of anti-inflammatory therapy for asthma. ICS, however, are associated with unwanted side effects and many asthma patients have persistent symptoms despite taking high ICS doses [2, 3]. Thus there is a need for novel anti-inflammatory therapies for the treatment of asthma.

Lymphocytes play a central role in the pathophysiology of asthma. Allergic asthma is mediated by CD4 T helper (Th) 2 cells [4] through the secretion of cytokines including interleukin (IL)-4, IL-5 and IL-13; these cause diverse effects including eosinophil recruitment, B-cell IgE synthesis and fibroblast activation [4] [5]. Patients with severe asthma may have neutrophilic airway inflammation and skew towards a more Th1 type response [6] characterised by the production of cytokines such as IFNγ and IL-2. More recently, Th17 lymphocytes have also been implicated in severe asthma [7], as these cells produce IL-17 which stimulates bronchial epithelial cells, smooth muscle cells and fibroblasts to secrete neutrophil chemoattractants such as CXCL8 [8].

NFAT (nuclear factor activated by T cells) regulates the transcription of pro-inflammatory T cell cytokines. NFAT proteins in resting cells are phosphorylated and reside in the cytoplasm [9]. NFAT is activated by calcium released activated channel (CRAC) signalling via store operated calcium entry (SOCE) which activates calcineurin; this in turns dephosphorylates NFAT, allowing nuclear translocation and transcription of inflammatory genes [10]. ORAI1 is a pore subunit in CRAC channels; ORAI1 knockout mice show reduced T cell cytokine production, highlighting the role of CRAC channels in T cell mediated inflammation [11].

The aim of this project was to investigate the role of the CRAC/NFAT axis in clinical and pre-clinical models of asthma. The data showed that the NFAT pathway is increased in stimulated lymphocytes harvested from human asthmatics and in a model of allergic asthma. The selective inhibitor, Synta 66 (add ref), then confirmed a central role for CRAC channels in mediator release from primary human lymphocytes and in airway inflammation and asthma symptoms in a pre-clinical model system.

Methods

Study subjects

We recruited 11 patients with moderate to severe asthma and 7 healthy non-smoking (HNS) controls (demographics shown in table I). Samples from these subjects were also used as part of a separate study to evaluate corticosteroid effects on lymphocyte cytokine production. All subjects were required to be non-smokers. The inclusion criteria for the asthma patients were FEV1<80% predicted, ICS use > 800 mcg beclomethasone equivalent / day, and asthma control questionnaire (ACQ) score > 1. All asthma patients were required to demonstrate reversibility of 200 mls or 12% to salbutamol, or a methacholine PD20 <16 mg/ml. Skin prick testing using house dust mite, cat, and grass allergens and exhaled nitric oxide (eNO) at 50 ml/s (Niox, Aerocrine, Sweden) were also performed. The study was approved by the local South Manchester research ethics committee (08/51006/54 & 06/Q1403/156) and all subjects provided written informed consent.

Cell collection

Bronchoalveolar lavage (BAL) was collected from the upper lobes; the bronchoscope was wedged in the bronchus and a maximum of 4 × 60ml aliquots of pre-warmed sterile 0.9 % NaCl solution were instilled into each lobe (480ml total). The aspirated fluid was stored on ice before filtration (100m filter, Becton Dickenson). The filtrate was centrifuged (400 g/10min at 4°C) and the cell pellet resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Viable cell counts were determined by trypan blue exclusion (Neubauer hemocytometer). Total cell count was adjusted to 1 × 106 cells/ml in supplemented RPMI 1640 medium with additional 10 % (v/v) foetal calf serum and used for cell culture. Peripheral blood samples from healthy non-smoking donors (n=3) were collected to obtain peripheral blood mononuclear cells (PBMCs). Blood was layered onto sterile media (Ficoll-Paque Plus; Amersham Biosciences; Buckinghamshire, UK), centrifuged (400g/30 min at 18°C), and PBMCs were collected. Viable cell counts were determined by Trypan blue exclusion (Neubauer hemocytometer). Total cell count was adjusted to 1 × 106 cells/ml in supplemented RPMI 1640 medium with additional 10 % (v/v) foetal calf serum and used for cell culture

Cell culture

1 × 105 total BAL cells were seeded in a 96 well plate in RPMI-1640 media supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and 10 % FCS (v/v). Cells were incubated Synta 66 (10, 1, 0.1 & 0.01 μM) for 1 hour at 37°C, 5 % CO2 prior to the addition of CD2/3/28 activation beads (Miltenyi Biotec) to specifically activate the lymphocytes for 24 hours at a ratio of 1 bead to 2 cells (ratio selected from preliminary data – see Supplementary Figure 1A). Supernatants were harvested by plate centrifugation 10 min, 400g, and 4ºC and stored at -20oC prior to ELISA & Luminex analysis. Synta 66 was provided by GSK, Stevenage, UK.

2.5 x 106 PBMCs were seeded per well of a 6 well plate in RPMI-1640 media supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and 10 % FCS. Cells were incubated with Synta 66 (10µM) for 1 hour at 37°C, 5 % CO2 prior to the addition of CD2/3/28 activation beads 1 hour at a ratio of 1 bead to 2 cells. Nuclear protein was extracted from the lung tissue using a CelLytic NuCLEAR Extraction kit (Sigma Aldrich, UK) according to the manufacturer’s instructions. NFAT assay?

Cytokine Quantification

Release of IL-2 was assayed on cell culture supernatants (either undiluted or up to 1:5 dilution with RPMI containing 10 % FCS as required) using R&D systems (Abingdon, UK) ELISA duo sets according to manufacturer’s instructions (lower level of detection 15.25 pg/ml). Release of IFNγ, IL-13, IL-17, (lower level of detection 0.17 pg/ml) were measured using the Luminex 100 system (Luminex, Madison WI, USA).

Cell viability

Potential effects on cell viability were assessed using 3-(4,5 dimethylthiazol-2-yl)-2,5,diphenyltetrazoliumbromide (MTT). After removal of culture media, BAL cells were incubated at 37˚C with 1mg/ml solution. After 30 min MTT reagent was removed and cells dissolved in DMSO. Optical density was determined at 550 nm. There were no effects on cell viability at any concentration of the drugs used in this study (data not shown)

Rodent model of allergic asthma

A rat model of allergic asthma was used as previously described (1). Briefly, male Brown Norway rats (Harlan, UK, 200-250g) were sensitised on day 0, 14 and 21 with chicken ovalbumin (OVA) (100 µg/rat, i.p., Grade V, Sigma, UK.) administered with Alum (20 mg/rat aluminium hydroxide and 20 mg/rat magnesium hydroxide, i.p., AlumTM Thermo Scientific, UK). On day 28 rats were challenged with vehicle (saline, aerosolised for 30 minutes) or OVA (1% w/v). Groups (n = 8) received vehicle (0.5% methyl cellulose and 0.2% tween80 in saline, 2 ml/kg, intraperitoneal, both from Sigma, UK), Synta 66 (30 mg/kg – dose selected from pilot study/DMPK data) or clinically relevant, positive control, budesonide (3 mg/kg, Sigma, UK) 1 hour before and 0.5 hour after challenge. Changes in airway obstruction (late asthmatic response, LAR) was monitored in the conscious animals from 1 to 6 hours after challenge as previously described (2, 3). Six hours after the challenge the animals were euthanised with pentobarbitone (200 mg/kg, i.p., Centaur Services, UK). Bronchoalveolar lavage (BAL) was carried out by injecting 3 ml of RPMI culture medium (Invitrogen, UK) via a cannula inserted into the trachea, waiting 30 second and then removing it. This was repeated and the collected BALF pooled. Immediately after BAL, the lungs were removed, cleaned, flash frozen in liquid nitrogen and stored at -80oC.

Proof of target engagement in the lung.

To demonstrate that Synta 66 has blocked its target in the lung, NF-AT pathway activation (amount of the transcription factor in the nucleus) was measured using a TransAMTM ELISA-based plate assay according to the manufacturer’s instructions (Active Motif, Belgium). Briefly, an oligonucleotide containing a consensus sequence for NF-AT was immobilized to the bottom of each well in a 96-well plate. Nuclear protein was extracted from the lung tissue using a CelLytic NuCLEAR Extraction kit (Sigma Aldrich, UK) according to the manufacturer’s instructions. Samples were added to the assay plate and incubated for 1 hour before the addition of primary antibodies specific for NF-AT. Each well was then incubated with a horseradish peroxidase-antibody conjugate for 1 hour and developed with 3,3′,5,5′-Tetramethylbenzidine. The reaction was then stopped using an acidic stop solution and absorption was determined at 450 nm on a spectrophotometer (Biotek PowerWave XS Plate Reader, UK).

Role of CRACi in inflammatory status after allergen challenge

Quantitative RT-PCR (TNF, IL-1, IL-5 and IL-6) was used to measure mRNA levels from the lung samples using TaqMan® reagents and protocols (Applied Biosystems) as previously described (4). Mediators were measured at the protein level (Eotaxin and IL-6) using ELISAs (R&D systems, UK) in the BALF. Total and type of white cells in the BALF was determined as previously described (1).

Role of CRACi in EAR

To determine the role of CRACi in the EAR response we employed a rat model as previously described (5). Briefly, male Brown Norway rats were sensitised as above. Rats were anaesthetised with ketamine HCl and xylazine HCl (144 and 10 mg/kg respectively, i.p., Sigma, UK) and connected, via a tracheal cannula, to a ventilator (Ugo Basil; Comerio, Varese, Italy) set at 90 breaths/min. Pump volume was adjusted to give a tidal volume of 2 ml. Measurement of airflow was measured by whole body plethysmography. Water filled, oesophageal cannula was placed such that it estimated transpulmonary pressure. Resistance (RL) (cmH2O/ml/s) was continuously computed on a Buxco XA-analyser (USA). After recording basal airway resistance the rats were challenged with OVA (intravenous, 1 mg/ml, 1 ml/rat with saline flush via a vein). The analyser will monitor airways resistance for 10 minutes after challenge. Rats received vehicle (2 ml/kg, i.p., n = 6), Synta 66 (30 mg/kg), methysergide (10 mg/kg)/Montelukast (30 mg/kg) (positive control - (5) or the negative control, budesonide (3 mg/kg) prior to OVA challenge.

Data Analysis

For the in vivo work data are expressed as mean ± S.E.M.. Statistical significance was determined using either single or multiple comparisons (specific tests used are described in the Figure legends). For the human work the sample size for this study was based on previous publications of the effects of anti-inflammatory drugs on airway lymphocytes that have used similar sample sizes [13, 14]. The percentage inhibition of the stimulated cytokine levels was normally distributed. Cytokine levels with and without CRAC channel inhibitor were compared using ???? and statistical significance was indicated on the graphs showing the percentage inhibition. Maximum inhibition was defined as the effect of the highest concentration of each drug. The maximum inhibition of drug between subject groups was compared using by unpaired t-tests. P <0.05 was considered significant. Analysis was carried out using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA).

Results

Asthma lung lymphocytes

Patients with asthma were all taking ICS, and the majority (10 out of 11) were also using a long-acting β2 adrenergic agonist. The FEV1 (1.96 ±0.5) and ACQ (2.08 ± 0.76) scores were consistent with symptomatic, moderate to severe asthma. CD2/3/28 stimulation of the lymphocytes harvested from HNS controls and patients with asthma caused a significantly increased cytokine production; this observation has been previously reported (add ref) and are shown in on-line supplement (Sup Figure 1B). There was no significant difference in bead- induced cytokine levels between lymphocytes harvested from HNS and asthmatics (Sup Figure 1B). Preliminary data showed that CD2/3/28 activation of the lymphocytes increased NFAT DNA association suggesting an increase in the activation of this pathway (Figure 1A). Co-incubation with the CRAC channel inhibitor attenuated the NFAT signal indicating target engagement in these primary cells (Figure 1A).

In the main part of the study Synta 66 caused a significant, concentration dependent inhibition of all 5 (only 4 in the figure???) cytokines. The magnitude and potency of the inhibition was similar in lymphocytes from asthma patients compared to healthy controls (Figure 1B-E). This would suggest that the CRAC channel plays a central role in CD2/3/28 driven production of cytokines in human lung lymphocytes.

Proof of target engagement in the lung.

To demonstrate that Synta 66 is active in the target tissue, the lungs, we measured the activation status of the NF-AT pathway as a downstream surrogate marker of inhibiting iCRAC. Antigen challenge caused a significant increase in the activation status of the NF-AT pathway (i.e. levels of NFAT in the nuclear fraction), and this signal was attenuated by Synta 66 (Figure 2). Interestingly, the glucocorticosteroid, budesonide, failed to impact on antigen activation of NFAT pathway activation (Figure 2).

Role of CRACi in inflammatory status after allergen challenge

To determine the impact of inhibiting iCRAC on the rodent allergic response, we measured the levels of inflammatory mediators in the lung. From previous data we selected a range of mediators to measure at the mRNA and protein level (1). Figure 3 shows that antigen challenge increased the expression of mRNA for TNF, IL-1, IL-5 and IL-6 in the lung tissue, this signal was significantly attenuated by treatment with Synta 66 and budesonide. Antigen challenge dramatically increased BALF levels of eotaxin and IL-6; again these levels were significantly reduced in the animals treated with Synta 66 or glucocortoid (Figure 4). The Synta 66 induced reduction in inflammatory mediators was associated with a significant reduction in antigen induced BAL eosinophilia and neutrophilia (Figure 5).

Role of CRACi in LAR and EAR

To explore whether CRACi plays a role in asthmatic symptoms, we recorded LAR and EAR in the model system. As previously shown (2, 3) antigen challenge caused a marked increase in breathing distress several hours after the antigen challenge (visual and audible signs correlating with changes in penH) (Figure 6). This model of LAR was significantly attenuated by both Synta 66 and the glucocorticosteroid (Figure 6). The immediate change in lung function observed after antigen challenge (EAR) was inhibited by a combination of 5-HT and cys-LT antagonist (Figure 7), as previously reported (6). As expected Synta 66 and acutely administered budesonide failed to impact on the EAR signal (Figure 8).

Discussion

Currently over 200 million people suffer from asthma and it is responsible for around 250,000 deaths each year (ref – GINA). There is therefore an urgent need to develop safe and effective treatments (add ref). The data suggests that blockade of the CRAC channel may be an effective therapy for reducing airway inflammation associated with asthma and combating changes in lung function These experiments showed that in primary human lung lymphocytes, harvested from healthy subjects and patients with severe asthma, blocking the CRAC channel inhibited the production of cytokines known to be associated with the clinical disease. Furthermore we go on to show the same tool is capable of attenuating the airway inflammation and compromised lung function in a pre-clinical model of allergic asthma.

Lymphocytes are thought to be central to the pathogenesis and symptoms associated with asthma (general refs). The CD4 Th-2 subtype have been reported to be increased in the airway of mild/moderate asthmatics (refs); whereas in the more severe end of the disease spectrum reports show the presences of Th-1 and Th-17 subtypes (refs). In pre-clinical model systems various groups have shown the central from of lymphocytes in the development and maintenance of the allergic phenotype (refs). This has led to a great deal of research effort geared towards targeting this cell type in the search for new asthma therapies (refs). As discussed the CRAC-NFAT axis has been shown to be central to mediator production by lymphocytes. Indeed CRAC channel inhibition has previously been shown to inhibit IFN-γ, IL-6, IL-17 and TNFα in human T cells [12], and cytokine release from COPD lung lymphocytes. [13]. Our data in primary human lung lymphocytes confirms previous data and extends the findings to cells from asthma patients with the more severe disease. Indeed, we observed a relatively large effect of the CRAC channel inhibitor on IL-17 production. We have previously shown that corticosteroids have a relatively limited effect on IL-17 production in lymphocytes from these moderate to severe asthma patients (ref). Thus it is possible that this class of compound may be a potential therapeutic option in the context of targeting IL-17 associated neutrophilic airway inflammation.