Variable expression of cysteinyl leukotriene type I receptor splice variants in asthmatic females with different promoter haplotypes

Background Cysteinyl leukotrienes are potent inflammatory mediators implicated in the pathogenesis of asthma. Human cysteinyl leukotriene receptor 1 (CYSLTR1) gene contains five exons that are variably spliced. Within its promoter few polymorphisms were described. To date, there has been no evidence about the expression of different splice variants of CysLT1 in asthma and their association with CYSLTR1 promoter polymorphisms. The goal of our study was to investigate CysLT1 alternative transcripts expression in asthmatic patients with different CYSLTR1 promoter haplotypes. The study groups consisted of 44 patients with asthma, diagnosed according to GINA 2008 criteria and 18 healthy subjects. Genomic DNA and total RNA was extracted from peripheral blood mononuclear cells. Real-time PCR was performed with specific primers for transcript I [GenBank:DQ131799] and II [GenBank:DQ131800]. Fragments of the CYSLTR1 promoter were amplified by PCR and sequenced directly to identify four single nucleotide polymorphisms: C/T [SNP:rs321029], A/C [SNP:rs2637204], A/G [SNP:rs2806489] and C/T [SNP:rs7066737]. Results The expression of CysLT1 transcript I and II in asthma did not differ from its expression in healthy control group. However, in major alleles homozygotic CAAC/CAAC women with asthma we found significantly higher expression of transcript I as compared to heterozygous CAAC/TCGC women in that loci. CysLT1 transcript I expression tended to negative correlation with episodes of acute respiratory infection in our asthmatic population. Moreover, expression of CysLT1 transcript II in CAAC/CAAC homozygotic women with asthma was significantly lower than in CAAC/CAAC healthy control females. Conclusions Genetic variants of CYSLTR1 promoter might be associated with gender specific expression of CysLT1 alternative transcripts in patients with asthma. CysLT1 splice variants expression might also correlate with the susceptibility to infection in asthmatic population.


Background
Asthma is a complex disease influenced by genetic and environmental factors [1], with highly variable clinical spectrum, but with the consistent presence of airway inflammation. Among many important inflammatory mediators involved in its pathogenesis are cysteinyl leukotrienes. They are potent lipid mediators derived from the lipooxygenase pathway of arachidonic acid metabolism [2,3]. They are products of activated eosinophils, mast cells, basophils and macrophages [3,4]. Originally identified as potent mediators of bronchoconstriction, mucus secretion, and airway hyperresponsiveness [2,5], they are now also described as important factors of innate and adaptive immune responses [6], as well as in the effector phase of inflammation, tissue repair and fibrosis [7,8]. So far, three cysteinyl leukotrienes receptors have been identified: CysLT 1 [9,10], CysLT 2 [11][12][13] and recently described GPR17 [14]. All of them are seven transmembrane-spanning receptors that couple to G proteins (GPCR) and activate various intracellular signaling pathways [15]. The distribution of these three types of CysLTs receptors is diverse and depends on the cells and tissues as well as on the pathophysiologic conditions. In human lungs CysLT 1 protein was demonstrated in the smooth muscle cells at all levels of the respiratory tract, interstitial lung macrophages and in the epithelial cells as well as in infiltrating inflammatory cells such as monocytes/macrophages, mast cells, eosinophils, CD 34+ cells, neutrophils and B lymphocytes [9,10,16,17]. CysLT 1 mRNA was observed in normal and asthmatic human lungs [10,16]. However, recent studies have shown a significant increase in CysLT 1 positive cells in the bronchi of stable asthmatics in comparison to healthy, non smoking controls and further elevation of CysLT 1 mRNA and protein in acute severe exacerbations of asthma [18]. Nevertheless, response to leukotriene antagonists (LTRA) in asthma is known to be variable and it still has not been determined which of the phenotypes benefits most from this type of treatment [19][20][21]. Moreover, there is still a discrepancy about the precise location of the CYSLTR1 promoter region and the exon/ intron structure of this gene [22,23]. Therefore, there is a possibility that at least two alternative promoters exist and might initiate different transcripts of the receptor gene in various pathophysiological conditions [22]. Furthermore, functional and case-control studies of the CYSLTR1 gene contribution to asthma have shown inconsistent results [22,24,25]. We have also previously observed [26] that some clinical features of severe asthma were associated with the minor [TCGC] haplotype of CYSLTR1 promoter SNPs in severe asthmatic women. However, we have found no significant differences in gene reporter activity among all tested promoter SNPs constructs. Therefore in this study we hypothesized that CYSLTR1 gene promoter polymorphisms might influence on the expression of CysLT 1 alternative transcripts, which might further influ-ence on the course of the disease. To test this hypothesis, we analyze the expression of CysLT 1 alternative transcripts in patients with asthma and in healthy control group with the relevance to the CYSLTR1 gene promoter polymorphisms. We demonstrate here that there is no difference in the expression of CysLT 1 alternative transcripts between asthmatic and healthy population analyzed without focus on genetic background and sex stratification.
However, there is a difference in the CysLT 1 alternative transcripts expression in women with asthma with different promoter haplotypes. Furthermore, we also demonstrate that the expression of certain splice variants correlates with the episodes of acute infections.

Study population
The study involved 44 patients with persistent asthma randomly selected from asthmatic group treated in the Outpatient Unit of the Department of Immunology, Rheumatology and Allergy in Lodz, Poland. The sex matched healthy individuals group consisted of 18 unrelated, non-atopic subjects with a negative family history of allergy and asthma. Asthma was defined by a specialist of respiratory diseases according to GINA 2008 criteria [27]. Atopy was defined as one or more positive reactions in the skin prick tests with the battery of inhaled allergens. The aspirin intolerant asthma phenotype was defined in patients having a proven history of asthma aggravation on exposure to aspirin/NSAIDs and a positive response in a lysine-aspirin bronchoprovocation test. Exacerbations were defined as acute worsening of asthma symptoms with decrease of FEV 1 or PEF ≥ 20% diagnosed by the doctor and put in the case history as a word "exacerbation", with hospitalization or urgent visit for asthma or stated aggravation of asthma symptoms requiring at least doubling the dose of ICS or addition or periodic increase of oral GCS. Episodes of acute respiratory tract infection were defined as acute aggravation of patient's health with fever, yellow sputum and need for antibiotic usage with or without asthma exacerbation. Nocturnal dyspnoea was characterised and stated in the case history by the doctor according to the interview with the patient. Chronic rhinosinusitis was diagnosed on the basis of patients' symptoms and CT scans. Allergic rhinitis was defined according to ARIA 2008 criteria [28]. GERD was diagnosed according to American College of Gastroenterology 2005 recommendations [29]. The doses of ICS were calculated as the budesonide equivalent within the whole period of observation in the Outpatient Unit and divided by the number of observation days and presented as micrograms per day. Some patients received ICS by nebulization. The doses of systemic GCS were calculated as a prednisone equivalent. GCS, montelukast and zafirlukast were also divided by the number of whole days of observation and presented as milligrams per day. These drugs were prescribed to the patients for certain periods e.g. 2-3 weeks in regards of GCS in decreasing dosage in case of exacerbation or 3-4 months in regards of leukotriene receptor antagonists in case of worse asthma control and there were periods free of certain drugs. Therefore, median dose of certain drug per day includes periods without this drug usage. Thus, the dose of the drug could be lower than available and commonly used doses.
Clinical features of patients were analysed retrospectively over the whole period of observation using individually self-created database software, with the possibility of calculations of studied variables.
Characteristics of the studied groups are presented in Table 1. The study was approved by the Ethics Committee of the Medical University of Lodz and informed consent was obtained from every subject prior to the study.

Real-Time PCR analysis of alternative transcripts expression
Peripheral blood mononuclear cells were isolated from the blood using Histopaque 1077 (Sigma-Aldrich, Poland) density gradient centrifugation. Total RNA was isolated from peripheral blood mononuclear cells using the Total RNA Extraction Kit (A&A) according to manufacturer's protocol in the presence of RQ1 RNase-Free DNase (Promega, Madison, WI, USA). Total RNA (2 μg) was reverse-transcribed using the random hexamers and ImProm-II™ reverse transcription system (Promega) in a volume of 25 μl at 37°C for 90 min. All real-time PCR reactions were optimized to obtain the best amplification kinetics. Amplification reactions were performed in a final volume of 20 μl using qPCRTM Core Kit for SybrTM Green I w/o dUTP (EUROGENTEC, Seraing, Belgium), 250 nM primers and 25 ng cDNA template per reaction. Nuclease free water was used as a no-template control. The following primer pairs were used: for CysLT 1 transcript I [GenBank:DQ131799]: forward 5'-AACG-CAAAAGGACAGTAAACTGTG-3' and reverse 5'-ATCAATGCCTTTTACGGTGTAATATTAG-3'; for CysLT 1 transcript II [GenBank:DQ131800]: forward 5'-TCGAATTTACTGAAGACTTGGAGCTTGCTTC-3' and reverse 5'-TCTCTACGAATGTCTGCTTTGTGCCTGC-3', for RPS17: 5'-ACCCCAATGTCAAGGAGATCAAGGTC- To calculate the relative expression ratios of single samples a mathematical model was used which included an efficiency correction for the real-time PCR efficiency of the individual transcripts [30]. Relative expression was calculated using REST (Corbett Research, Australia) software with the two reference genes mentioned above. Each experiment was conducted in triplicate.

Statistical analysis
The data from the study were analyzed using Statistica (v. 6.0; StatSoft, Tulsa, OK). The distribution of all examined variables was checked for normality. Because of the deviation from normal distribution, in the analysis of alternative transcripts expression U-Mann-Whitney test was performed to analyze differences between variables. Data are expressed as mean relative expression ± SEM. Relationship between alternative transcripts expression and studied asthmatic features were evaluated utilizing Spearman's rank correlation test. Haplotype frequencies and linkage disequilibria were estimated using the HaploView 4.0 program [31]. Haplotypes of each individual were inferred by using the algorithm developed by Stephens et al. (PHASE version 2.1) [32]. The post-hoc analysis of the quantitative variables with the Bonferroni correction was performed to account for multiple testing.

Study population
We analyzed the expression of CysLT 1 alternative transcript I and II in 44 patients with asthma and 18 healthy controls. Characteristics of study populations are shown in Table 1

Alternative Transcripts-Haplotype Association Study
We hypothesized that the different expression of CysLT 1 alternative transcripts might depend on the polymorphisms in the CYSLTR1 gene promoter region. Thus, we performed an analysis concerning association of the CysLT 1 splice variants expression with the haplotypes in each locus within the whole study population. We analyzed the differences between the two main haplotypes CAAC and TCGC as they accounted for nearly 90% of the studied populations, which was shown in Table 2. The other haplotypes were too rare to perform appropriate analysis. In women with asthma who are homozygotes in Table 2: Haplotypes frequency in the asthmatic population (n = 44) and healthy subjects (n = 18)

Healthy subjects (n = 18)
T C A C 0 major alleles in each analyzed locus CAAC/CAAC we found significantly higher expression of transcript I 37.22 ± 14.11 (p cor = 0.0169) and transcript I/II 13.38 ± 6.10 ratio (p cor = 0.009) as compared to heterozygous CAAC/ TCGC women in that loci, 4.08 ± 1.06 and 1.49 ± 0.65, respectively (Figure 1, 2). We did not observe any differences in alternative transcripts expression in asthmatic men with different promoter genotypes or in both sexes of healthy patients.

CysLTR1 alternative transcripts correlations
We analyzed here correlations of CysLT 1 alternative transcripts expression with different features of asthma such as FEV 1 , PEF, exacerbations, episodes of acute infection, hospitalization for asthma, nocturnal dyspnoe and intake of ICS, systemic GCS and LTRA in the whole asthmatic population. We observed weak but statistically significant negative correlation of CysLT 1 transcript I expression with episodes of acute infection (R = -0,52, p cor = 0.0009) (Figure 3). Relative expression of CysLT 1 transcript II did not correlate with any of studied asthmatic features.

Alternative Transcripts-Haplotype-Phenotype Association Study
We analyzed here if there are differences in CysLT 1 splice variants I and II expression in carriers of the same haplotypes between asthmatic patients and healthy controls with subsequent sex stratification. Again, we took into consideration two main haplotypes CAAC and TCGC. We observed that relative expression of CysLT 1 transcript II in CAAC/CAAC homozygotic women with asthma (3.86 ± 1.06) was significantly lower than in CAAC/CAAC healthy control females (14.48 ± 6.90) (p cor = 0.019) (Figure 4).
There were no differences in case of CysLT 1 transcript I expression in either of analyzed configurations.

Discussion
Here we showed that there were no differences in CysLT 1 alternative transcripts expression in the blood mononuclear cells between asthmatic patients and controls, when they were evaluated as genetically homogeneous populations and without sex stratification. Figueroa et al found that 20% of peripheral blood leukocytes showed the presence of CysLT 1 . These are cells of particular relevance to CysLT 1 transcript I relative expression in women with asthma with two main CYSLTR1 haplotypes asthma and atopy, such as monocytes/macrophages, eosinophils, pregranulocytic CD34+ cells, neutrophils, and subsets of B lymphocytes [33] and probably also T lymphocytes [17]. According to our best knowledge there has been no data published comparing CysLT 1 mRNA or CysLT 1 alternative transcripts expression in peripheral blood mononuclear cells between asthmatic and healthy subjects. There might be a few reasons of lack of differences in the overall CysLT 1 mRNA expression at least in the blood mononuclear cells. First of all, our patients were in the stable period of the disease, without a trace of exacerbation in the moment of blood collection. Therefore, inflammation might be present only locally in the bronchi, not in the peripheral blood, mainly because of the persistent influx of inflammatory cells from the blood to the bronchi. Zhu and colleagues found that there is an increase of distinct inflammatory cells expressing the CysLT 1 receptor in the bronchial mucosa of mild asthmatic patients as compared to healthy subjects and further increase when there is a severe exacerbation of asthma [18]. However they did not analyze peripheral blood [18].
Nevertheless, it might also confirm our main hypothesis of indirect promoter SNPs influence on CYSLTR1 gene transcription and/or alternative splicing in a sex related manner. This might further correlate with the level of CysLT 1 protein expression and finally with the disease phenotype. Several lines of evidence support this hypothesis. First of all, we found that heterozygotic females CAAC/TCGC with asthma had significantly lower expression of CysLT 1 transcript I as compared to major female asthmatic homozygotes CAAC/CAAC (Figure 1, 2). This correlation was not observed in healthy women neither in asthmatic and healthy men. In our previous study on the association of these CYSLTR1 promoter SNPs with severe asthma [26] in the population of 93 severe asthmatics, 110 non-severe asthmatics and 100 healthy controls we showed that heterozygotic CAAC/TCGC females with severe asthma had significantly more episodes of infection per year, more exacerbations per year, more hospitalizations for asthma per year, and they used more ICS than homozygotic female carriers of the main haplotype CAAC/CAAC. However, we found no evidence on the influence of these promoter SNPs on the gene expression by means of reporter gene studies. Therefore, our current findings might at least partially link this association with the functional effects. Moreover, we showed here that CysLT 1 alternative transcripts expression might slightly affect factors associated with the course of asthma. We have observed the weak tendency that the more episodes of acute respiratory infection per year in patients with asthma ( Figure 3) the lower expression of CysLT 1 transcript I. However, the feeble distribution of data limits any potential conclusions. Nevertheless, it could indicate on the probability of impaired innate and afferent mechanisms of acquired immune response in patients with asthma, probably through altered CysLT 1 receptor expression [6]. CysLTs and their receptors were proven to be critical determinants of dendritic cells homing to regional lymph nodes and the repertoire of cytokines required to induce T cell responses [34,35]. It was shown that maturation of dendritic cells with LPS, a major endotoxin of Gram-negative bacteria and a classic Toll-like receptor 4 agonist, reduced CysLT 1 receptor expression by 50% [36]. Moreover, generation of leukotrienes from white blood cells was enhanced during sepsis, and further increased after stimulation but only in patients who survived this massive infection [37]. This data might indicate the importance of leukotrienes and their receptors not only in the development of chronic inflammation in asthma but also in episodes of acute respiratory infections, which are one of the most important determinants of asthma exacerbations.
Episodes of acute respiratory infection in our study population by means of symptoms and reaction to antibiotic treatment fulfilled the definition of bacterial infection. The limitation of this definition is lack of exact evidence on bacterial or viral involvement. On the other hand, it has been proven that viral infections, like respiratory syncytial virus or parainfluenza virus, increase the level of cysteinyl leukotrienes [38][39][40] and that antileukotriene drugs alleviate symptoms of viral upper respiratory tract infections [41,42]. The last but not least argument for our initial hypothesis is that we have noticed that in CAAC/ CAAC women with asthma CysLT 1 transcript II is differentially expressed as compared to CAAC/CAAC healthy control women (Figure 4). Together with our previous findings that promoter SNPs did not influence its basal activity, these data might suggest that there are other factors e. g. splicing factors, sex hormones or the effects of some inflammatory cytokines that influence the CYSLTR1 gene transcription and/or splicing. It stays in agreement with primarily contradictory findings of our and other groups. Minor alleles of CYSLTR1 promoter SNPs increased [24,25] or decreased [22] or demonstrated no influence on the CYSLTR1 basic promoter activity [26].  [23]. They all form the same protein structure as the coding region and ORF is included in the fifth exon, present in each transcript. The putative CYSLTR1 promoter was found to contain two STAT6 consensus response elements, GATA and AP1 binding sites [23]. Four analyzed here CYSLTR1 promoter SNPs do not change any known transcription factor binding sites, but they might influence on the splicing binding factors. Altered ratio of main transcripts I and II might further influence on the expression status of structurally unchanged receptor protein, not only in blood leukocytes but also in inflammatory infiltration of the bronchi. This subsequently could correlate with the phenotype. Elevated expression of CysLT 1 plays a role in asthma and its exacerbation as shown Zhu et al [18]. Alternative splicing of pre mRNA represents a widespread mechanism for increasing variability of eukaryotic gene expression and has been associated with human pathologies, such as cancer, Alzheimer's, amyotrophic lateral sclerosis, ataxia teleangiectasia, cystic fibrosis, and other [47,48]. For instance, non functional alpha subunit of the epithelial sodium channel alternatively spliced forms have been proposed to serve as negative regulatory components for its activity in humans [49].
Finally, our previous and current data indicated the gender differences in cysteinyl leukotrienes and their receptors metabolic pathways. Female sex and a certain haplotype might be a risk factor of the disease. Lack of the TCGC haplotype effect on the CysLT 1 alternative tran-scripts expression in both asthmatic and healthy men might suggest an additional effect of sex hormones or some behavioral/environmental differences. Recently, it has been shown in the large European multi-centered cohort of patients that severe asthma occurs four times more often in women that in men In summary, still little is known about factors involved in the processes of CYSLTR1 transcription, alternative splicing, and their influence on the final CysLT 1 receptor protein expression and further correlation with the asthma phenotype. However, our data suggest that indeed CYSLTR1 gene might be implicated in asthma pathogenesis at least by differential expression of CysLT 1 alternative transcripts in various haplotypes and in sex-related manner, but the exact mechanisms remain to be clarified.

Conclusion
Genetic variants of CYSLTR1 promoter might be associated with gender specific expression of CysLT 1 transcripts in patients with asthma. CysLT 1 splice variants expression might also correlate with the susceptibility to infection in asthmatic population.

Authors' contributions
MS participated in the study design, performed experiments, interpreted the data and wrote the manuscript. KWN and KS performed experiments and drafted the manuscript. AKB and RP contributed funding and materials to the study, helped to design the experiments, analyzed the data and critically revised the manuscript adding important issues. MC enrolled patients to the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.