Abstract

Background

   Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and
   comorbid asthma have more severe disease and are difficult to treat.
   However, the molecular endotypes associated with CRSwNP with comorbid
   asthma (CRSwNP + AS) are not clear. This study aimed to investigate the
   characteristics of type 2 inflammation and the molecular signatures
   associated with CRSwNP + AS.

Methods

   A total of 195 subjects; including 65 CRSwNP + AS patients, 99
   CRSwNP-alone patients, and 31 healthy control subjects; were enrolled
   in the study. Nasal tissues from patients with CRSwNP + AS,
   CRSwNP-alone and control subjects were assessed for infiltration of
   inflammatory cells and concentrations of total IgE. Whole-transcriptome
   sequencing was performed and differentially expressed (DE) mRNAs and
   long non-coding RNAs (lncRNAs) and their associated pathways were
   analyzed. The correlations between type 2 cytokines and local
   eosinophils, tissue IgE, and transcriptome signatures were evaluated.

Results

   Significantly higher local eosinophil infiltration and higher levels of
   total IgE were found in nasal tissues from CRSwNP + AS patients than in
   nasal tissues from CRSwNP-alone patients. Furthermore, atopy and
   recurrence were significantly more frequent in patients with
   CRSwNP + AS than in patients with CRSwNP-alone (62.5% vs 28.6% and
   66.7% vs 26.9%, respectively). RNA sequencing analysis identified 1988
   common DE-mRNAs, and 176 common DE-lncRNAs shared by CRSwNP + AS versus
   control and CRSwNP-alone versus control. Weighted gene coexpression
   network analysis (WGCNA) identified LINC01146 as hub lncRNA
   dysregulated in both subtypes of CRSwNP. Overall, 968 DE-mRNAs and 312
   DE-lncRNAs were identified between CRSwNP + AS and CRSwNP-alone. Both
   pathway enrichment analysis and WGCNA indicated that the phenotypic
   traits of CRSwNP + AS were mainly associated with higher activities of
   arachidonic acid metabolism, type 2 cytokines related pathway and
   fibrinolysis pathway, and lower activity of IL-17 signalling pathway.
   Furthermore, the expression of type 2 cytokines; IL5 and IL13, was
   positively correlated with local eosinophil infiltration, tissue IgE
   level, and the expression of DE-mRNAs that related to arachidonic acid
   metabolism. Moreover, WGCNA identified HK3-006 as hub lncRNA in yellow
   module that most positively correlated with phenotypic traits of
   CRSwNP + AS.

Conclusions

   Patients with CRSwNP + AS have distinct type 2-high
   inflammation-associated molecular signatures in nasal tissues compared
   to patients with CRSwNP-alone.

   Keywords: Chronic rhinosinusitis with nasal polyps, Asthma, Type 2
   inflammation, Molecular endotype, Transcriptome sequencing

Background

   Chronic rhinosinusitis (CRS), a disease characterized by chronic
   inflammation of the sinonasal tissue, affects 5.5–28% of the general
   population [[39]1]. CRS with nasal polyps (CRSwNP) accounts for
   approximately 20% of all CRS and has greater severity of clinical
   disease [[40]2]. Asthma is one of the most common chronic inflammatory
   disorders of the lower airway worldwide with increasing morbidity.
   Studies have reported that up to 60% of CRSwNP patients have comorbid
   asthma (CRSwNP + AS), which is one of the most challenging CRS subtypes
   to treat [[41]3]. Patients with CRSwNP + AS have greater disease
   severity, higher recurrence rates of nasal polyps after surgery, poorer
   asthma control and higher costs [[42]4–[43]6].

   There is evidence that eosinophilic CRSwNP tends to have comorbid
   asthma more frequently [[44]7]. Formation of IgE, which is independent
   of the presence of allergy in nasal polyp tissue, is also associated
   with asthmatic condition in patients with CRSwNP [[45]8]. However,
   there is still no clear explanation for the association between CRSwNP
   and asthma, and likewise the pathogenic mechanisms leading to CRSwNP
   and asthma are uncertain. The united airway concept suggests that the
   upper and lower airway inflammation share common pathogenic mechanisms
   and influence each other [[46]9, [47]10]. There is evidence that
   several features of inflammatory pattern, disrupted epithelial barrier
   and airway remodelling are similar in CRSwNP and asthma
   [[48]11–[49]13]. Thus, understanding the molecular relationship between
   CRSwNP and comorbid asthma may help to reveal the mechanisms that
   underlie airway chronic inflammation.

   CRSwNP is a heterogeneous inflammatory condition with different
   endotypes [[50]14]. The majority of white patients with CRSwNP in
   western countries have a type 2 pattern of inflammation characterized
   by pronounced eosinophilia and high levels of interleukin-4 (IL-4),
   IL-5 and IL-13 cytokines [[51]15]. In contrast, Chinese patients with
   CRSwNP have lower type 2 inflammation and show higher degree of type
   1/type 3 inflammation [[52]16, [53]17]. Furthermore, evidence from
   previous studies on epidemiology and clinical characteristics, suggests
   that CRSwNP + AS may be considered a subtype of CRSwNP [[54]18–[55]20].
   Thus, this study aimed to investigate the characteristics of type 2
   inflammation and molecular endotypes associated with CRSwNP + AS by
   whole-transcriptome sequencing. Distinct type 2-high inflammation and
   its associated transcriptome signatures, indicated by coding mRNAs and
   long non-coding RNAs (lncRNAs), were found in patients with CRSwNP + AS
   compared to patients with CRSwNP-alone.

Materials and methods

Subjects

   A total of 195 subjects, including 65 CRSwNP patients with comorbid
   asthma (CRSwNP + AS), 99 patients with CRSwNP-alone and 31 healthy
   control subjects were enrolled in series from June 2017 to March 2018
   in the Rhinology Department of Beijing TongRen Hospital. Patients with
   CRSwNP were diagnosed according to the European Position Paper on
   Rhinosinusitis and Nasal Polyps 2020 guidelines [[56]1]. The diagnosis
   of comorbid asthma was based on the Global Initiative for Asthma 2019
   guidelines. The diagnosis of allergic rhinitis was according to
   Allergic Rhinitis and Its Impact on Asthma 2016 guidelines. Atopy was
   confirmed based on positive test for serum antigen-specific IgE
   (cut-off value, 0.35kUA/L), measured by Immuno-CAP 100 system
   (Pharmacia, Uppsala, Sweden). Patients undergoing septoplasty because
   of anatomic variations and without other sinonasal diseases were
   recruited as control subjects. All subjects were aged 18 to 70 years.
   Subjects with immunodeficiency, fungal sinusitis, coagulation disorder,
   neoplasia, pregnancy and aspirin-exacerbated respiratory disease (AERD)
   were excluded. None of the patients had been treated with
   corticosteroids, antibiotics or biologics within the 4-week period
   before surgery, and no patient had symptoms of infection at the time of
   sampling, apart from symptoms of chronic rhinosinusitis or asthma.
   Recurrence was defined as the presence of nasal polyps observed under
   nasal endoscopy, together with at least one symptom (nasal obstruction,
   rhinorrhea, headache/facial pain, reduction or loss of smell, sleep
   disturbance/fatigue) lasting at least 1 week, despite appropriate
   intranasal corticosteroid treatment. The postoperative follow-up period
   was 18 months for assessment of recurrence/non-recurrence of nasal
   polyps [[57]1, [58]21]. All patients were followed-up after weeks 1, 2,
   4, and 12, and then once every 3 months for up to 18 months by the same
   surgeon, who was blinded to all laboratory data [[59]21]. Nasal tissue
   samples were collected from the inferior turbinate of control subjects
   and the nasal polyps of CRSwNP patients during surgery. The tissue
   samples were processed for staining with haematoxylin and eosin, RNA
   sequencing and ELISA as previously described [[60]22].The Ethics
   Committee of Beijing Tongren Hospital approved this study, and all
   subjects signed informed consent forms prior to enrolment in the study.

Histological evaluation of polyp tissue

   Nasal polyp tissues were immediately formalin fixed after surgery, and
   then dehydrated and embedded in paraffin. Paraffin sections were
   stained with haematoxylin and eosin (H&E) and processed for
   histological evaluation. All sections were examined by optical
   microscopy at × 400 magnification. The absolute numbers and percentages
   of infiltrating inflammatory cells; including eosinophils, neutrophils,
   plasma cells, and lymphocytes; were recorded as mean of six
   non-overlapping regions in each section by two independent
   pathologists, who were blinded to the study design and clinical
   background of the patients.

Assessment of total IgE in nasal tissues

   Concentrations of total IgE in nasal tissues were assayed using the
   Human IgE ELISA Kit (Arigo Biolaboratories Corporation, Taiwan).
   Briefly, fresh nasal tissues were placed into RIPA lysis buffer with 1%
   protease inhibitor cocktail (Thermo Fisher Scientific) and homogenized
   using a standard bench-top homogenizer (Qiagen, Valencia, CA). The
   tissue homogenates were centrifuged and supernatants were collected for
   IgE analysis, according to the manufacturer’s instructions. All samples
   were tested in duplicate.

RNA isolation and RNA sequencing

   Nasal tissue samples of CRSwNP + AS (n = 10), CRSwNP-alone (n = 10),
   and control (n = 9) were randomly selected for whole-transcriptome
   sequencing. The nasal tissue samples collected after surgery were
   freshly preserved in RNAlater solution (Qiagen, Hilden, Germany) and
   processed for extraction and purification of total RNA using the RNeasy
   Kit (Qiagen, Hilden, Germany), according to the manufacturer’s
   instructions. The quantity and quality of the isolated RNA was
   determined with NanoDrop 2000 Spectrophotometer (Thermo Fischer
   Scientific) and 2100 TapeStation Automated Electrophoresis System
   (Agilent Technologies), and samples with an RNA integrity number of
   greater than 8.0 were chosen for sequencing. Ribosomal RNA was removed
   and sequencing libraries were prepared using the rRNA-depleted RNA by
   NEBNext UltraTM Directional RNA Library Prep Kit (New England Biolabs,
   USA), following the manufacturer’s instructions. RNA sequencing was
   performed on the Illumina Hiseq platform and 150 bp paired-end reads
   were generated by Novogene Bioinformatics Technology Cooperation
   (Beijing, China).

RNA sequencing data analysis

   Adapters and low-quality tail were trimmed from reads prior to read
   alignment. Clean sequence reads were aligned to the human genome with
   Hisat2 (v2.0.5). Cufflinks (v2.2.1) was used to assemble transcripts,
   estimate the abundance of these transcripts, and detect differential
   expression among samples. For mRNA analyses, the reference genome build
   GRCh37 was chosen as the annotation references. For lncRNA analyses,