Type-2 CD8+ T lymphocytes responsive to PGD2 and LTE4 in severe eosinophilic asthma

The functions and in vivo roles of type-2 CD8+ T cells in humans have not been well defined and this cell type has been largely overlooked in models of disease. We investigated this in the context of severe asthma with persistent airway eosinophilia - a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.


Introduction
Type-2 cytokines (IL-4/5/9/13) orchestrate allergic inflammation, driving type-2 CD4 + T helper (Th2) cell differentiation, IgE production, mucus hypersecretion and airway hyperresponsiveness (AHR). Specifically, IL-5 activates and is chemotactic to eosinophils and prolongs their survival. Anti-type-2 cytokine therapies, notably mepolizumab, an anti-IL-5 antibody, are effective in severe eosinophilic asthma by reducing circulating eosinophils and asthma exacerbations [1][2][3] . The major sources of such type-2 cytokines are Th2, group 2 innate lymphoid cells (ILC2) 4 and type-2 CD8 + T cells (Tc2). Of these, most attention has been paid to CD4 + T cells and more recently ILC2s, especially in human disease. Although, it has been known that type-2 CD8 + T cell populations exist, their overall functionality, transcriptional machinery and the mechanisms by which they are triggered have not been defined. This is important to address as recent data in other contexts have revealed previously overlooked functional diversity of human CD8 + T cells in inflammatory diseases 5 .
Eosinophilic asthma constitutes an important clinical phenotype, defined by increased airway eosinophils 6,7 which release granule-derived basic proteins, lipid mediators, cytokines and chemokines, driving inflammation and exacerbations 8,9 . In some patients with severe asthma, airway eosinophils persist despite use of high-dose inhaled corticosteroids, suggesting relative steroid-insensitivity 10 . This phenotype is commonly associated with co-morbid rhinosinusitis, nasal polyposis and aspirin-induced bronchoconstriction 11 . Eosinophilic asthma is commonly considered as a Th2 disorder based on human data in mild asthma 12,13 and animal models 14 . Recently ILC2s have been implicated in murine airway inflammation 15 , and increased ILC2s are reported in human asthma 16,17 . In contrast, although some data exists for overall involvement of CD8 + cells in asthma in both human 18,19 and murine 20 studies, the specific functional role of Tc2 cells remains largely unexplored, particularly in defined asthma phenotypes. Improved understanding of the pathogenic roles of Tc2 in this specific phenotype is important for therapeutic advances.
These lipid mediators and their receptors have not been studied in relation to CD8 + cells.
To investigate this, we first analysed type-2 CD8 + T cell frequencies and functional profiles in blood, bronchoalveolar lavage (BAL) and bronchial biopsies (BB) in well-defined patient cohorts, and further evaluated whether the airway environment is conducive to Tc2 activation via CRTH2 by measuring airway PGD 2 and LTE 4 . We then defined the potent and broad activity of these lipids on Tc2 cells in vitro and investigate a mechanistic link between Tc2 cell activation and airway eosinophilia. Our observations provide compelling evidence of innate-like activation of Tc2 cells by pro-inflammatory lipids, a diverse range of functions of this cell population, and a potential role in severe eosinophilic asthma.

Tc2 cell migration induced by PGD 2 and LTE 4
To explore the potential pathogenic role of Tc2 cells and these lipid mediators, we isolated and cultured human Tc2 cells for further in vitro investigation ( Supplementary Fig. 7b). These cells are CCR7 -CD62Leffectors ( Supplementary Fig. 7c) and showed higher baseline type-2 gene expression signatures (IL17RB, GPR44, CLECL1, IL9R, NAMPT, AF208111, HPGDS, P2RY14, RG4, IRS2 and GATA3) but lower type-1 (IFNG, AIF1, LTA, TXK and IL18RAP) and PGD 2 and cysLTs are chemotactic agents for many types of immune cells 24,27 . To investigate airway Tc2 cell recruitment, we examined the effect of these lipids in chemotaxis assays. Both lipids caused cell migration in a typical bell-shaped dose-dependent manner, peaking at ~30 nM for PGD 2 and ~10 nM for LTE 4 (Fig. 4a). The maximum response induced by PGD 2 was higher (2.4 fold) than that by LTE 4 . Cell migration was synergistically enhanced by combined stimulation (Fig. 4b). The contribution of PGD 2 and LTE 4 on the cell migration was blocked by the CRTH2 antagonist TM30089 and the CysLT 1 antagonist montelukast.

Enhancement of type-2 cytokine production in Tc2 cells by PGD 2 and LTE 4
We investigated the effects of PGD 2 or LTE 4 on type-2 cytokine production by Tc2 cells (Fig.   5). After treatment with PGD 2 or LTE 4 , IL-5 and IL-13 production was elevated at both transcriptional ( Fig. 5a) and translational (Fig. 5b) levels in a dose-dependent manner with EC 50 =17.3 nM (6.1 ng/ml) at mRNA or 17.9 nM (6.3 ng/ml) at protein on IL-5, and 21.1 nM (7.4 ng/ml) at mRNA or 16 nM (5.6 ng/ml) at protein on IL-13 for PGD 2 ; and EC 50 =4.5 nM (2 ng/ml) at mRNA or 13.5 nM (5.9 ng/ml) at protein on IL-5, and 7.4 nM (3.3 ng/ml) at mRNA or 9 nM (4 ng/ml) at protein on IL-13 for LTE 4 , which were close to the concentrations of these lipids detected in patients' sputa (Fig. 3b). Responses to PGD 2 were significantly stronger than to LTE 4 . Compared with Th2 cells 22,30 , the effect of PGD 2 and LTE 4 on Tc2 is much more potent (Fig. 5c).
We then further examined type-2 cytokine production by Tc2 cells in response to the lipids alone or combination (Fig. 5d,e). Both PGD 2 and LTE 4 increased type-2 cytokine production. Their combination enhanced the response synergistically. Using PrimeFlow assays to analyse IL-5/IL-13 mRNA at individual cell level confirmed these data (Fig. 5f). IL-5/13 positive cells were increased from 2.88% (control) to 23.17%, 12.23% or 28.8% after treatment with PGD 2 , LTE 4 or their combination respectively. Among these positive cells, some expressed IL-5 dominantly, some IL-13 dominantly, and only some produced IL-5/13 simultaneously, although >90% of these cells were capable of producing both following PMA/ionomycin stimulation ( Supplementary Fig. 7e).

Effect of PGD 2 and LTE 4 on the gene expression profile of Tc2 cells
Using microarrays we investigated Tc2 transcriptional responses to PGD 2 and LTE 4 . 1104, 3360, and 4593 gene transcriptions were significantly modulated (P<0.05) (including up/downregulation) by LTE 4 , PGD 2 , or their combination respectively ( Supplementary Fig.   8a). The effect of PGD 2 was much broader and stronger than that of LTE 4 , and the effect of the combination treatment was mainly contributed by PGD 2 (Supplementary Fig. 8b). We next focused on the genes encoding cytokines, chemokines, their receptors, and CD molecules ( Fig. 6a; Supplementary Table 2). About 90 such genes were significantly modulated, mostly upregulated, and most obviously cytokines (IL3, IL5, IL8, IL13, IL22, CSF2, TNF and XCL1). Although a few of these were induced by LTE 4 alone, most were driven by PGD 2 alone or the combination. Some transcriptional changes were regulated only by the combination treatment (eg, IL21, IL22, LASS1, CCL3, CCL4, IL1RL1, CD1E and CCL21).
Microarray data were largely confirmed by PCRarray on human common cytokines although some significant effects (IL9 and CSF1) were detected only in PCRarray (Fig. 6b).
For verification, we assayed selected cytokines by q-PCR (Fig. 6c) and Luminex ( Fig.   6d). At mRNA level, most of genes (except CSF1) showed synergistic effects of PGD 2 and

Effect of mast cell-derived PGD 2 and LTE 4 on the activation of Tc2 cells
To investigate the mechanism of Tc2 activation under more physiological conditions, we evaluated the impact of endogenously synthesized eicosanoids on Tc2 function. In vitro derived human mast cells were stimulated with IgE followed by crosslinking using an anti-IgE antibody (Fig. 7a). Only low levels of PGD 2 (~0.1 ng/ml) and LTE 4 (~6 ng/ml) were detected in mast cell supernatants before stimulation (supernatant UM). Supernatant from activated mast cells (supernatant SM) contained high levels of PGD 2 (~11.5 ng/ml) and LTE 4 (~86.8 ng/ml), which were similar to concentrations in sputum from asthmatic patients (Fig. 3b). Supernatant SM induced Tc2 cell migration (Fig. 7b) and marked cytokine (IL-5/13) production (Fig. 7c).
Both effects were reduced by TM30089 and montelukast, and completely inhibited by their combination.
Recombinant human IL-5 (rhIL-5) induced eosinophil shape change, a biomarker of eosinophil migration, in fresh blood in a dose-dependent manner ( Supplementary Fig. 11a), which was inhibited by anti-IL-5 neutralizing antibody. Supernatant 3/28 more potently induced eosinophil shape change ex vivo than supernatant UT (Fig. 8b). The reaction was inhibited partially by IL-5 antibody, implicating IL-5 and other eosinophil-active factors.
We examined the influence of GM-CSF released by Tc2 cells on eosinophil survival by measuring protection from serum starvation-induced apoptosis (Fig. 8c). The increase of Annexin V-positive (apoptotic) eosinophils after serum withdrawal was inhibited by rhGM-CSF, which was reversed by anti-GM-CSF neutralizing antibody ( Supplementary Fig. 11b). Supernatant 3/28 provide similar protection, which was reduced by anti-GM-CSF antibody ( Fig. 8c).
These observations were confirmed in HBECs, although eotaxin-1/2 levels were not detected in these cells (Fig. 8f). Eotaxin induction by Tc2 media was significantly inhibited by anti-IL-4/13 neutralizing antibodies.

Discussion
Tc2 cells are type-2 cytokine secreting CD8 + T lymphocytes that highly express CRTH2. We found enrichment of Tc2 cells in both peripheral blood and airways specifically in patients with severe asthma and persistent corticosteroid-insensitive eosinophilia in two independent cohorts. Importantly, the same conclusion was achieved using different methodologies, either surface CRTH2 expression or type-2 cytokine production as markers identifying Tc2 cells.
Airway PGD 2 and LTE 4 were also increased in eosinophilic asthma. The interaction of Tc2 cells with these lipids induced strong activation of Tc2 cells, leading to cell migration and proinflammatory protein production, which in turn promoted eosinophil recruitment and survival directly or indirectly, suggesting an important role for Tc2 cells in the pathogenesis of eosinophilic asthma and their potential contribution to airway eosinophilia.
Research has long focussed on Th2 cells in asthma 13 although blood Th2 cells are not associated with eosinophilia 33 , and bronchial Th2 cells are elevated in mild, but not severe asthma 29 . We found no significant differences in Th2 numbers between eosinophilic and noneosinophilic asthma and healthy controls. Conversely Tc2 cells were significantly enriched, and associated with asthma severity, nasal polyposis, AHR and airway type-2 cytokines. This is consistent with reports in atopic asthma and non-atopic eosinophilic diseases 18,34 , of peripheral blood CD8 + T cell activation in severe asthma 35 and observed associations between submucosal bronchial CD8 + cells and eosinophilia 36 . Our findings are also consistent with immunohistochemical findings of increased activation and IL-4 secretion by submucosal CD8 + T cells in fatal, virus-associated acute asthma 19 . Onset of severe eosinophilic asthma commonly follows a viral respiratory tract infection, which could promote CD8 + T cell activation including Tc2 cells leading to enhanced type-2 immunity in this phenotype. Furthermore, in murine models Th2 cell-derived IL-4 can reprogram virus specific CD8 + T cells to secrete type-2 cytokines 20,37 . Other murine data suggest preventing Tc2 cell differentiation reduces development of allergic airways disease 38,39 . Therefore, our findings strongly support the hypothesis that Tc2 cells, a previously unappreciated lymphocyte population, are key drivers contributing to type-2 immunity, particularly in severe eosinophilic asthma and should stimulate further investigation of these mechanisms.
Airway eosinophilia is associated with asthma exacerbations 40 and airway remodelling 41 . In asthma, eosinophil granule proteins are cytotoxic and disrupt the protective pulmonary epithelial barrier triggering inflammation 42 . The importance of eosinophilic inflammation in asthma is best informed by response to therapies. Sputum eosinophilia is associated with responsiveness to corticosteroid therapy 43 . Other strategies such as anti-IgE, anti-IL-5 and CRTH2 antagonism aimed to normalize sputum eosinophils reduced exacerbation frequency and severity in clinical trials in asthma 2,3,25,26,40 . Our in vitro data have indicated that Tc2 activation can contribute to airway eosinophilia. Tc2-derived IL-5 and GM-CSF promote eosinophil migration and survival. Other Tc2 cytokines including IL-4/13 induce airway epithelial cells to produce eotaxins, the dominant chemokines for eosinophils in severe asthma, acting via CCR3 44 . Furthermore, in murine models, CD8 + T or virus-specific Tc2 cells mediate virus-induced lung eosinophilia and AHR 20,45 . All this evidence suggests an important role of Tc2 cells in eosinophil-mediated lung inflammation. PGD 2 and cysLTs are predominantly derived from mast cells in an IgE-dependent mechanism, which are abundant at sites of allergic responses, promoting airway inflammation and deterioration in lung function 46 . We observed increased airway PGD 2 in eosinophilic and non-eosinophilic severe asthma, while LTE 4 was enriched only in severe eosinophilic asthma, consistent with other reports 47 . Since many patients with severe eosinophilic asthma are nonatopic with low levels of IgE 6 , the key source of these lipids remains unclear. We previously demonstrated synergistic pro-inflammatory effects of PGD 2 and LTE 4 in human Th2 and ILC2s, which inhibited by TM30089 and montelukast 27,28,30 . This also occurs in Tc2 cells, indicating that these CD8 cells can be activated by lipid mediators in an antigen-independent manner. Of relevance, we observed higher potency of PGD 2 and LTE 4 on Tc2 than on Th2 cells. Synergistic effects of PGD 2 and LTE 4 in Tc2 were replicated using endogenous lipid mediators from activated mast cells. These findings provide a potential mechanistic insight into the clinical efficacy of CRTH2 antagonism in severe eosinophilic asthma 26 .
Transcriptional analysis suggested a broad range of effects of these lipids beyond type-2 cytokine induction in Tc2 cells, with >2000 upregulated transcripts including genes for bioactive cytokines and chemokines for eosinophils: IL-3, IL-5 and GM-CSF promote eosinophil differentiation and migration 48 ; TNFα delays eosinophil apoptosis 49 ; overexpression of IL-25 resulted in eosinophilia in murine models 50 ; and CCL3, CCL4 and CCL7 are chemokines for eosinophils 51 . One notable finding was the impact of these lipids on IL-5 expression by Tc2 cells. The EC 50 for PGD 2 induced IL-5 production was 17.9 nM, compared to 63 nM for Th2 cells 22 . Thus Tc2 cells stimulated in this way generate a strong and early IL-5 response, which could be relevant to the clear clinical benefit of anti-IL-5 in severe eosinophilic asthma 3,40,52 .
In conclusion, Tc2 represent an important cell type with innate-like characteristics and substantial pro-inflammatory potential. In vivo there is significant enrichment of Tc2 cells in both peripheral blood and airways in eosinophilic asthma, particularly in severe disease, associated with increases in airway PGD 2 and LTE 4 . These lipid mediators function potently and synergistically to recruit and activate Tc2 cells to produce type-2 and multiple other proinflammatory cytokines and chemokines, which sufficient to contribute to airway eosinophilia.
Taken together these data suggest that Tc2 cells constitute potentially important therapeutic targets in severe eosinophilic asthma. RNeasy Mini kit and Omniscript reverse transcription kit were supplied from Qiagen; Real time quantitative PCR (qPCR) Master Mix and probes were from Roche; Primers were synthesized by Eurofins MWG Operon; rhSCF, rhIL-6 and anti-IL-4/5/13 neutralizing antibodies were purchased from Bio-techne; goat anti-human CD3 was from Santa Cruz; rabbit anti-human CD8 was from Abcam; peroxidase polymer-conjugated anti-rabbit and peroxidase polymer-conjugated anti-goat antibodies were from Vector Laboratories; Fluorescein-tyramide and Cy5-tyramide were from PerkinElmer; human myeloma IgE was from Calbiochem; Antihuman GM-CSF antibody, Annexin V-APC and Human GM-CSF ELISA kit were obtained from BioLegend; IC fixation buffer, Permeabilisation Buffer, human IL-4, IL-5 and IL-13 ELISA kit were from eBioscience; and rhIL-2, rhIL-4 and rhGM-CSF were from PeproTech and other chemicals were from Sigma-Aldrich.

Human clinical samples
For the Oxford cohort, patients meeting the ATS/ERS definition of severe asthma 10 with a sputum eosinophil count of >3% (eosinophilic, n=26) or <3% (non-eosinophilic, n=14), and 16 healthy control subjects were recruited from Churchill Hospital, Oxford ( Peripheral blood was collected and used directly for flow cytometry. Sputum was induced with nebulized saline solution (3-5%) after pre-treatment with salbutamol. Selected sputum plugs were dispersed with 0.2% DTT, filtered for cells for flow cytometry analysis or microarray, and supernatants for ELISA analysis. BB and BAL were collected under bronchoscopy 29 . Biopsies were dispersed with collagenase for 1 h, and BALs were treated with 0.1% DTT and filtered for flow cytometry. For microarray, CD3 + sputum cells were FACS sorted using a FACS Aria II TM cell sorter (BD Biosciences).

Human CD8 + CRTH2 + Tc2 cell preparation and treatment
Human Tc2 cells were isolated from CD Leucocyte Cones (National Blood Service, UK).
Briefly, CD8 + cells were isolated from PBMC using MACS CD8 + T cell isolation kit, followed by CRTH2-positive selection using anti-human CD294 MicroBeads, and further amplified in X-VIVO 15 medium containing 10% human serum and 50 U/ml rhIL-2.
For gene or protein analysis, Tc2 cells were treated with conditions as indicated in the results for 4 h. Cell supernatants were collected for ELISA or Luminex assays, and cell pallets for qPCR, RNAarray or microarray.
For Tc2-conditioned supernatants, cells were treated with immobilized anti-CD3/anti-CD28 antibodies or PGD 2 (100 nM)/LTE 4 (50 nM) for 4 h, and then supernatants were harvested for ELISA, or used as Tc2-conditioned media for the treatment of fresh blood, eosinophils, A549 cells or primary human bronchial epithelial cells (HBECs).

Human Mast cell culture and treatment
Human mast cells were cultured and treated as described previously 30 . Briefly, CD34 + progenitor cells were isolated from human cord blood (National Blood Service, Oxford, UK) by using a human CD34 MicroBead kit. The cells were cultured with Iscove's modified Dulbecco's medium containing 10% human serum, 0.55 µM 2-ME, penicillin/streptomycin, human recombinant stem cell factor (100 ng/ml) and human rIL-6 (50 ng/ml) for 14-15 weeks.
Half the culture medium was replaced twice weekly with fresh medium containing the same concentration of cytokines. The cells were pre-treated with 5 mg/ml purified human myeloma IgE for 4 days, washed and then sensitized passively with fresh IgE (5 mg/ml) for 2 h. After washing, the cells were incubated with medium or challenged with goat anti-human IgE (1 µg/ml) for 1 h. The supernatants of the cells were collected and measured for PGD 2 and LTE 4 , and used as mast cell supernatants for the treatment of Tc2 cells.

PrimeFlow RNA assay
The levels of transcription for IL-5 and IL-13 in individual Tc2 cells in whole blood or Tc2 cultures were analysed with a PrimeFlow RNA Assay kit (eBioscience) according to the manufacturer's instructions. Briefly, fresh blood or purified Tc2 cells were treated with conditions indicated for 4 h, and then were stained with the antibodies (Table E3) and viability dye followed by fixation and permeabilisation. Then the cells were hybridised with RNA probes for IL-5 and IL-13. The signals were amplified and labelled with fluorescent probes.
The results were analysed with a BD LSRFortessa flow cytometer (BD Biosciences).

Microarrays
For CD3 + cells sorted from sputa, RNAs were isolated using an Absolutely RNA Nanoprep

PCRarray
The levels of mRNA for cytokines in the RNA samples from the Tc2 cells after treatments were assessed with PCRarray by using an RT 2 Profiler PCRarray Human Common Cytokines PCR Array kit (Qiagen) in a LightCycler 480 Real-Time PCR System (Roche).

Quantitative RT-PCR (qPCR)
qPCR was conducted as described previously 30 . Briefly, total RNA of the cells was extracted using an RNeasy Mini kit. cDNA of the samples was prepared from the same starting amount of RNA using a Omniscript RT kit. qPCR was conducted using Master Mix and Probe in a LightCycler 480 Real-Time PCR System (Roche). GAPDH was used as control gene. Primers and probes (Roche) used are listed in Table E4.

Flow cytometry
For blood samples, fresh blood was labelled using antibodies (Table E3) followed by red blood cell lysis with a BD FACS Lysing Solution. For cells from blood, sputum, BAL or BB, the cells were stained with antibody cocktail and live/dead dye (Table E3)

Chemotaxis assays
Tc2 cells were resuspended with RPMI 1640 media; 25 mL of cell suspension and 29-mL test compounds as indicated in the results or mast cell supernatants were applied to upper and lower chambers, respectively, in a 5-µm pore sized 96-well ChemoTx plate (Neuro Probe). After incubation (37°C for 60 minutes), the migrated cells in the lower chambers were collected and mixed with a Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) and quantified by using an EnVision Multilable Reader.

Paraffin-embedded sections of BB were prepared by Oxford Centre for Histopathology
Research. After deparaffin and rehydation, the sections were boiled in a Target Retrieval Solution (Dako), followed by incubation with peroxidase blocking reagent (Bio-Rad) and normal horse serum. The sections were then labelled with primary (goat anti-human CD3 and rabbit anti-human CD8) and secondary (peroxidase polymer-conjugated anti-rabbit followed by Cy5-tyramide) antibodies. After treatment with peroxidase blocking reagent again, the sections were further incubated with another secondary antibody (peroxidase polymerconjugated anti-goat followed by Fluorescein-tyramide) and then 5 µg/ml DAPI solution.
Images were acquired on an Olympus FV1200 inverted confocal microscope, and processed with ImageJ.

Eosinophil shape change assay
Fresh human blood was incubated with an equal volume of Tc2 conditioned media in the presence or absence of anti-IL-5 neutralizing antibody or other reagents as indicated for 1 h.
The samples were fixed with a Cytofix Fixation Buffer (BD Biosciences) followed by red blood cell lysis by using RBC Lysis Solution (Gentra Systems). Eosinophils were gated from granulocytes according to their autofluorescence during the analysis in a BD LSRFortessa flow cytometer. The eosinophil shape change was determined by the position shifting of the cells in forward scatter.

Eosinophil apoptosis assay
Eosinophils were prepared from human blood. Erythrocyte/granulocyte pellet was collected after Ficoll-Hypaque gradient, and then incubated with 3% dextran saline solution for sedimentation. Granulocytes were harvested from the supernatant of the sedimentation and further purified by lysis of the remaining erythrocytes with 0.6 M KCl hypotonic water, and followed by labelling with anti-CD16 microbeads. Unlabelled eosinophils were negatively selected and then resuspended in RPMI 1640 medium. After treatments with diluted Tc2conditioned media in the presence or absence of anti-GM-CSF neutralizing antibody or other reagents as indicated for 12 h, the eosinophils were labelled with Annexin V and then analysed with a BD LSRFortessa flow cytometer.

Statistics
Data for clinical samples in Figures 1, 2, 3, and Supplementary Fig. 1, 4, 5 are presented as medians with interquartile range (IQR) and other data are presented as means with SEM. Data were analysed using one-way ANOVA followed by the Tukey's test or Student's t-tests.
Groups ranked according to disease severity were tested for linear trends using Jonckheere-Terpstra tests. Values of p<0.05 were considered statistically significant.

Study approval
The studies were approved by Leicestershire, Nottinghamshire Rutland Ethics Committee, UK   samples were treated as 1-fold. e The cytokine production induced by PGD 2 and LTE 4 were inhibited by TM30089 and montelukast. *p < 0.05, **p < 0.005, ***p < 0.001, (n=1 for a; n=3 for b and c; n=6 for d; n=4 for e).