Discussion
This is the first study to demonstrate the transcriptomic and epigenomic profiles of patients with AERD. To discover the distinct signature of AERD, integrated analysis of transcriptomes and epigenomes was performed with the nasal tissue from asthmatics. We identified the genes related to vesicle transport, sphingolipid regulation, T helper cell and mast cell activation were dysregulated in AERD. In addition, the mRNA expression levels of these genes were significantly associated with the sputum eosinophil counts. Collectively, we identified the distinct transcriptomic and epigenomic signature of AERD, which may contribute to the pathogenesis of eosinophilic inflammation of AERD.
AERD is characterized by persistent eosinophil activation and overproduction of cyteinyl leukotrienes. The most important cells involved in the pathogenesis of AERD are eosinophils and mast cells.17 These cells contribute to airway inflammation and hyper-responsiveness through the release of diverse mediators (e.g. cytokines, chemokines, and growth factors).18 Bidirectional cytokine release induces reciprocal activation of eosinophils, mast cells, Th2 cells, and group 2 innate lymphoid cells. Preformed mediators which are stored in intracellular granules are released to the outside of the cells by several distinct mechanisms; classic exocytosis, compound exocytosis, piecemeal degranulation, and cytolysis.19 This degranulation process involves multiple steps of membrane fusion. Most of the membrane fusion events are mediated by solubleN -ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins.20 The SNARE complex is originally composed of v-SNAREs (e.g. vesicle-associated membrane protein) associated with the vesicle and t-SNARE (e.g. syntaxins) associated with the target compartment. Previous studies have reported the mRNA and protein expressions of SNAREs (e.g. syntaxins or vesicle-associated membrane protein) in diverse inflammatory cells of humans.21 In the present study, we found theSTX2 gene was differentially correlated between patients with AERD and those with ATA. In addition, the mRNA expression levels ofSTX2 significantly affected the sputum eosinophil counts. These findings collectively suggest that hypomethylation resulted in the up-regulation of the STX gene and then induced eosinophil inflammation in a target tissue of AERD patients.
Rab proteins, which belong to the GTP-binding protein superfamily, also participate in intracellular vesicle trafficking. They regulate the targeting/docking/fusion process of vesicle transport and the assembly of SNARE proteins.22, 23 The release of granules from eosinophils, neutrophils, and platelets is facilitated by these Rab proteins.22, 23 In a previous study, a common variant in RAB27A has been identified to have association with FeNO levels in adults.24 Another study reported the association of single-nucleotide polymorphisms on the RAB1A gene with the risk of AERD and with the responsiveness of airways to aspirin.25 The RAB1A gene has been suggested to play a role in the development of AERD.25 Consistent with these previous studies, we observed the RAB3B gene was a more significant DCG which was hypo-methylated and up-regulated in patients with AERD compared to those with ATA. Furthermore, the sputum eosinophil counts were affected by the levels of mRNA expression of theRAB3B gene. Taken together, genes related to vesicle transport were significantly hypo-methylated and up-regulated in patients with AERD, which may promote eosinophilic inflammation in a target tissue.
Dysregulation of sphingolipids in AERD and severe asthma has been reported in several studies.6, 26-28 Increased levels of ceramides enhanced the release of asthma-related cytokines and chemokines in an asthma mouse model.29 Ceramides can be generated via the de novo pathway by serine palmitoyl transferase and sphingomyelinase pathways regulated by sphingomyelinases, or the recycling pathway.30Increased levels of serine palmitoyl transferase, long-chain base subunit 2,28 and SMPD1 31 have been suggested to induce ceramide increase, and augment eosinophilic inflammation in AERD.28, 31 In this context, we found the SMPD3 gene was hypo-methylated and up-regulated in patients with AERD compared to those with ATA. This finding indicates that sphingolipids may play a role in the pathogenesis of AERD, which is strengthened by previous study results.
Type 2 (T2) inflammatory responses are known to play a central role in patients with AERD. Th2 and Th17 inflammation mutually affect each other, resulting in the augmentation of T2 immune responses.32-34 An IL-17-deficient allergic asthma mice model showed significant reductions in both Th17 and Th2 immune responses. RORγt is a member of the nuclear receptor superfamily that regulates Th17 differentiation. RORγt inhibitors also diminished Th17 and Th2 immune responses in an animal model of allergic asthma.33 TGF-β plays an important role in the development of regulatory T (Treg) cells. The defective regulatory function of Treg cells has been noticed in pollen-allergic subjects.35 Based on these previous studies, up-regulated RORγt and down-regulated TGF-β may play a role in the augmentation of T2 inflammation in patients with AERD.
Mast cell activation as well as release of cysteinyl leukotrienes, and prostaglandin D2 is an essential component of AERD pathogenesis. Mast cell activation is initiated by cross-linking of the high-affinity Fc receptor for IgE (FcɛRI) with IgE-bound antigen.36, 37Based on definitive evidence for mast cell activation in airway inflammation of AERD, omalizumab treatment has been reported to successfully reduce the levels of leukotrienes and eosinophils in AERD patients.8 Omalizumab, a monoclonal anti-IgE antibody, binds to circulating free IgE, disrupts FcɛRI: IgE complex and decreases FcɛRI expression, resulting in the regulation of allergic reactions. In the present study, up-regulation of the FCER1A gene expression was observed which is compatible with the results of previous studies, suggesting the role of FcɛRI in AERD pathogenesis.
This study has two limitations. One is that nasal tissues were used to profile the transcriptome and epigenome. Expression profiling using target tissues, such as bronchial airway epithelial cells obtained by endobronchial brushing, may have stronger power that reflects airway genetic signatures.38 However, in actual clinical practice, it is difficult to perform invasive bronchoscopy for collecting target tissues. Nasal cytology of nasal curette specimens, a simple and non-invasive procedure which allows for assessing the pathologic features of nasal mucosa, is a good alternative method.39, 40 Moreover, strong correlations between bronchial and nasal airway gene expression profiles have been reported in previous studies.38, 41 The other is that the number of the study subjects was too small to generalize the study results. Further studies are needed with a larger sample size.
In conclusion, our results demonstrated the distinct omics signature that is related to vesicle transport, sphingolipid regulation, and T helper cell/mast cell activation in AERD, which may play an important role in the pathogenesis of AERD.