Here we showed that there were no differences in CysLT1 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 CysLT1. These are cells of particular relevance to asthma and atopy, such as monocytes/macrophages, eosinophils, pregranulocytic CD34+ cells, neutrophils, and subsets of B lymphocytes  and probably also T lymphocytes . According to our best knowledge there has been no data published comparing CysLT1 mRNA or CysLT1 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 CysLT1 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 CysLT1 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 . However they did not analyze peripheral blood .
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 CysLT1 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 CysLT1 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  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 CysLT1 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 CysLT1 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 CysLT1 receptor expression . 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 CysLT1 receptor expression by 50% . 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 . 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 [38–40] 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 CysLT1 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  or demonstrated no influence on the CYSLTR1 basic promoter activity . Nowadays, there is an emerging consensus suggesting that splicing is in most cases initiated cotranscriptionally and that introns are removed while the nascent transcript is still tethered to the DNA . Some transcriptional coregulators, brought to the promoters by transcription factors, either harbor splicing activity e.g. hnRNP  or interact with spliceosome components . Inversely, some proteins like SKIP described initially as RNA-binding splicing factors were later shown to be present at promoters and/or to function as transcriptional regulators . Four different mRNA CysLT1 transcripts were observed in human cells, with the dominant expression of transcript I in both unstimulated and IL-4 stimulated cells and far less abundant expression of transcript II. Transcript III and IV were found only in THP1 cell lines. However, the role of alternative transcripts of CysLT1 receptor has not been elucidated . 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 . 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 CysLT1 plays a role in asthma and its exacerbation as shown Zhu et al. 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 .
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 CysLT1 alternative transcripts 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 . Moreover, in accordance of our observations very recently Pergola et al showed higher expression of 5-LO, 15-H(P)ETE and CysLTs in stimulated whole blood and neutrophils in females comparing to males accompanied with the differences in 5-LO cellular localization and trafficking . These effects were further abolished by male hormones testosterone and 5-α-dihydrotestosterone . Moreover, it has been shown that estradiol modulates the pulmonary influx of inflammatory cells, increases generation of IL-4 and mediates mast cell degranulation  while progesterone upregulates IL-4 production . All of these effects might increase the number of cells with CysLT1 receptor in the bronchial mucosa of female subjects. Expression of CYSLTR1 gene may be also affected by exposure to different proinflammatory cytokines. It has been shown that CysLT1 may be upregulated in different cells by proasthmatic cytokines such as IL-4, IL-5 and IL-13  and the influence of analyzed haplotypes on gene expression and alternative splicing may only be present under specific inflammatory conditions.
In summary, still little is known about factors involved in the processes of CYSLTR1 transcription, alternative splicing, and their influence on the final CysLT1 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 CysLT1 alternative transcripts in various haplotypes and in sex-related manner, but the exact mechanisms remain to be clarified.