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.